CHARACTERIZATION	
  OF	
  THE	
  DEVELOPMENT	
  OF	
  THE	
  TONSILLAR	
  MICROBIOME	
  IN	
  PIGS	
  
	
  
By	
  
	
  
Luis	
  Carlos	
  Peña	
  Cortes	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
A	
  DISSERTATION	
  
Submitted	
  to
	
  
Michigan	
  State	
  University
	
  
in	
  partial	
  fulfillment	
  of	
  the	
  requirements	
  	
  
for	
  the	
  degree	
  of	
  
	
  
Comparative	
  Medicine	
  and	
  Integrative	
  Biology	
  –	
  Doctor	
  of	
  Philosophy	
  
	
  
2017	
  
	
  	
  

ABSTRACT	
  
	
  
CHARACTERIZATION	
  OF	
  THE	
  DEVELOPMENT	
  OF	
  THE	
  TONSILLAR	
  MICROBIOME	
  IN	
  PIGS	
  
	
  
By	
  
	
  
Luis	
  Carlos	
  Peña	
  Cortes	
  
	
  
Pig	
   tonsils	
   are	
   identified	
   as	
   a	
   potential	
   reservoir	
   for	
   many	
   bacterial	
   and	
   viral	
  
pathogens	
  that	
  can	
  survive	
  asymptomatically	
  in	
  this	
  location	
  and	
  may	
  have	
  a	
  high	
  potential	
  
of	
   being	
   zoonotic.	
   It	
   has	
   been	
   suggested	
   that	
   the	
   microbiome	
   plays	
   a	
   significant/	
  
substantial	
   role	
   in	
   host	
   colonization	
   by	
   pathogenic	
   microorganisms	
   and	
   also	
   exerts	
  
regulatory	
  roles	
  in	
  the	
  resistance	
  to	
  infection.	
  Despite	
  the	
  important	
  role	
  that	
  the	
  tonsillar	
  
microbiome	
  could	
  play	
  in	
  the	
  colonization	
  and	
  persistence	
  of	
  pathogens	
  in	
  the	
  host,	
  there	
  
are	
  no	
  in-­‐‑depth	
  studies	
  characterizing	
  the	
  development	
  of	
  tonsillar	
  microbiome	
  in	
  pigs	
  or	
  
how	
  this	
  microbiome	
  is	
  structured	
  over	
  time.	
  Surprisingly	
  a	
  similar	
  study	
  is	
  also	
  absent	
  in	
  
humans.	
  There	
  was	
  a	
  clear	
  need	
  to	
  investigate	
  the	
  development	
  of	
  the	
  tonsillar	
  microbiome	
  
in	
   pigs	
   to	
   lay	
   the	
   basis	
   for	
   future	
   studies	
   focused	
   on	
   more	
   complex	
   subjects	
   such	
   as	
   the	
  
relationship	
   between	
   the	
   normal	
   tonsillar	
   microbiome	
   and	
   pathogens	
   in	
   the	
   tonsils.	
  
Understanding	
  the	
  development	
  of	
  the	
  pig	
  tonsillar	
  microbiome	
  over	
  time	
  and	
  the	
  role	
  of	
  
the	
   tonsillar	
   microbiome	
   in	
   the	
   acquisition	
   and	
   persistence	
   of	
   a	
   pathogenic	
   microorganism	
  
will	
  lay	
  the	
  basis	
  for	
  the	
  design	
  of	
  novel	
  intervention	
  strategies	
  to	
  control	
  the	
  presence	
  of	
  
the	
   pathogen	
   and	
   reduce	
   the	
   risk	
   of	
   transmission	
   to	
   other	
   animals	
   or	
   humans.	
   Moreover,	
  
these	
   studies	
   are	
   expected	
   to	
   provide	
   an	
   animal	
   model	
   to	
   test	
   hypotheses	
   generated	
   by	
  
microbiome	
   data	
   that	
   cannot	
   be	
   tested	
   in	
   humans.	
   The	
   goal	
   of	
   this	
   study	
   was	
   to	
  
characterize	
   tonsillar	
   microbiome	
   development	
   in	
   pigs,	
   and	
   how	
   this	
   microbiome	
   is	
  
structured	
   and	
   how	
   the	
   structure	
   changes	
   through	
   different	
   times	
   in	
   the	
   life	
   of	
   pigs.	
   The	
  

chapters	
  in	
  this	
  thesis	
  will	
  present	
  pertinent	
  data	
  related	
  with	
  the	
  composition	
  of	
  the	
  pig	
  
tonsillar	
  microbiome	
  and	
  how	
  it	
  alters	
  through	
  the	
  life	
  of	
  pigs,	
  possible	
  maternal	
  sources	
  
for	
  some	
  of	
  the	
  identified	
  members	
  of	
  this	
  microbiome,	
  as	
  well	
  as	
  the	
  microbiome	
  structure	
  	
  
and	
   progressive	
   change	
   through	
   their	
   life.	
   Furthermore,	
   the	
   results	
   will	
   show	
   that	
  
challenges	
   associated	
   with	
   management	
   procedures	
   typically	
   present	
   in	
   swine	
   farms	
  
generate	
   prominent	
   changes	
   in	
   the	
   microbiome	
   composition	
   and	
   abundance	
   of	
   diverse	
  
bacterial	
   families.	
   Finally,	
   the	
   study	
   will	
   show	
   the	
   microscopic	
   structure	
   of	
   the	
   tonsillar	
  
epithelium	
  and	
  crypts	
  and	
  the	
  presence	
  of	
  diverse	
  bacterial	
  communities	
  on	
  the	
  surface	
  of	
  
pig	
  tonsils	
  throughout	
  different	
  time	
  points	
  of	
  their	
  life.	
  The	
  final	
  chapter	
  will	
  also	
  describe	
  
the	
  morphological	
  changes	
  of	
  the	
  tonsillar	
  surface	
  in	
  pigs	
  that	
  are	
  seen	
  and	
  are	
  associated	
  
with	
   changes	
   in	
   the	
   microbial	
   communities	
   observed	
   through	
   different	
   time	
   points	
   in	
   their	
  
life.	
   Taken	
   together,	
   the	
   results	
   presented	
   here	
   demonstrate	
   that	
   there	
   is	
   a	
   temporal	
  
succession	
  in	
  the	
  development	
  of	
  the	
  pig	
  tonsillar	
  microbiome	
  through	
  the	
  life	
  of	
  pigs.	
  
	
  

ACKNOWLEDGEMENTS	
  
	
  
To	
  God.	
  
I	
   would	
   like	
   to	
   thank	
   my	
   parents	
   Lucila	
   Elsa	
   Cortés	
   de	
   Peña,	
   Jairo	
   Peña	
   Nieto,	
   and	
  
brothers	
   Jairo	
   Ricardo	
   Peña	
   Cortés	
   and	
   Cesar	
   Augusto	
   Peña	
   Cortés.	
   Most	
   of	
   what	
   I	
   am	
   is	
  
because	
  of	
  your	
  help	
  and	
  guidance.	
  Thank	
  you	
  for	
  all	
  your	
  continued	
  blessings,	
  support	
  and	
  
encouragement	
   every	
   day	
   of	
   my	
   life.	
   To	
   my	
   sisters-­‐‑in-­‐‑law,	
   Aileen	
   Agüero	
   and	
   Luz	
   Angela	
  
Moreno,	
  for	
  joining	
  our	
  family	
  and	
  for	
  the	
  happiness	
  that	
  you	
  have	
  brought	
  to	
  me	
  and	
  my	
  
family.	
   And	
   to	
   my	
   “novia”,	
   Puja	
   Basu	
   “Pujita	
   Maria”	
   for	
   her	
  constant	
   nagging,	
   unconditional	
  
love	
  and	
  precious	
  years	
  we	
  have	
  spent	
  together	
  and	
  for	
  all	
  the	
  ones	
  coming.	
  
I	
  also	
  would	
  like	
  to	
  thank	
  my	
  sponsors:	
  Colciencias	
  programa	
  becas	
  Francisco	
  Jose	
  
de	
  Caldas	
  –	
  Convocatoria	
  519	
  –	
  2011,	
  for	
  making	
  this	
  dream	
  a	
  reality	
  and	
  supporting	
  me	
  
financially	
   during	
   my	
   studies	
   at	
   Michigan	
   State	
   University.	
   Universidad	
   de	
   Pamplona	
   -­‐‑	
  
Colombia,	
   for	
   the	
   support	
   and	
   helping	
   me	
   with	
   the	
   study	
   commission	
   which	
   allowed	
   me	
   to	
  
continue	
  my	
  professional	
  development.	
  
A	
  huge	
  thank	
  you	
  to	
  	
  Dr.	
  Martha	
  Mulks	
  for	
  her	
  guidance	
  and	
  support	
  for	
  allowing	
  me	
  
to	
   work	
   and	
   learn	
   at	
   a	
   pace	
   that	
   made	
   me	
   feel	
   comfortable	
   and	
   allowed	
   me	
   to	
   grow	
   as	
   a	
  
researcher	
  and	
  a	
  scientist.	
  For	
  pushing	
  me	
  to	
  learn	
  and	
  adapt	
  to	
  our	
  project’s	
  needs	
  and	
  for	
  
encouraging	
  me	
  to	
  think	
  out	
  of	
  the	
  box.	
  	
  
I	
   would	
   like	
   to	
   thank	
   Dr.	
   Vilma	
   Yuzbasiyan-­‐‑Gurkan	
   for	
   being	
   my	
   steadfast	
   anchor	
  
during	
  the	
  past	
  five	
  years	
  of	
  my	
  life,	
  here	
  at	
  Michigan	
  State.	
  I	
  cannot	
  thank	
  her	
  enough	
  for	
  
her	
  continuous	
  guidance,	
  generosity	
  and	
  	
  for	
  being	
  that	
  person	
  who	
  is	
  always	
  beside	
  us	
  in	
  

	
  

iv	
  

the	
   good	
   and	
   bad	
   moments.	
   Thank	
   you	
   for	
   all	
   you	
   do	
   because	
   I	
   wouldn’t	
   be	
   at	
   this	
  
important	
  juncture	
  without	
  	
  your	
  support.	
  
I	
   acknowledge	
   my	
   graduate	
   committee	
   members:	
   Dr.	
   Terence	
   Marsh,	
   Dr.	
   Julie	
   Funk,	
  
Dr.	
   Shannon	
   Manning,	
   Dr.	
   Matti	
   Kiupel.	
   Thank	
   you	
   for	
   your	
   continuous	
   help,	
   guidance,	
  
support	
  and	
  encouragement	
  during	
  these	
  past	
  few	
  years.	
  	
  
I	
   would	
   also	
   like	
   to	
   thank	
   my	
   current	
   and	
   past	
   lab	
   members,	
   Rachel	
   Greenberg,	
  
Rhiannon	
  LeVeque,	
  and	
  Scott	
  Kramer.	
  Much	
  of	
  my	
  field	
  work	
  was	
  made	
  easier	
  because	
  of	
  
you	
  guys.	
  Rachel,	
  thank	
  you	
  for	
  helping	
  me	
  out	
  in	
  the	
  lab.	
  
To	
   my	
   good	
   friend	
   Astrith	
   Rubiano,	
   thanks	
   for	
   everything,	
   specially	
   you	
  
encouragement	
  in	
  the	
  difficult	
  moments.	
  	
  
Finally,	
   I	
   would	
   like	
   to	
   thank	
   all	
   the	
   people	
   who	
   have	
   supported	
   me,	
   either	
   those	
  
who	
  work	
  in	
  different	
  departments	
  of	
  Michigan	
  State	
  University	
  who	
  have	
  helped	
  me	
  with	
  
my	
  project,	
  and/or	
  those	
  who	
  have	
  helped	
  to	
  enrich	
  my	
  stay	
  in	
  the	
  United	
  States.	
  Specially	
  I	
  
have	
   to	
   thank	
   Kevin	
   Turner,	
   Chris	
   Rowboom,	
   Kent	
   Ames,	
   Madonna	
   Benjamin,	
   Thomas	
  
Wood,	
  Amy	
  Porter,	
  Kathy	
  Joseph,	
  Amy	
  Albin,	
  Melinda	
  Frame,	
  Rheannon	
  Bateman,	
  Poorna	
  
Viswanathan,	
   Dimity	
   Palazzola,	
   Vicky	
   Maddox,	
   Marilia	
   Takada,	
   Paulo	
   Vilar,	
   Laura	
   Ortiz,	
  
Daniel	
  Parrell,	
  Jake	
  Baker,	
  Pallavi	
  Sing,	
  Robert	
  Parker,	
  Woonie	
  Cha,	
  Alessandra	
  Hunt,	
  	
  Laura	
  
Gualdron,	
  David	
  Tarazona,	
  Yenni	
  Bernal,	
  Lyonel	
  Laulie,	
  Javiera	
  Ortiz,	
  Sarah	
  Corner,	
  Kailey	
  
Vincent,	
  Tina	
  Yang,	
  Shikha	
  Sigh,	
  Ben	
  Jhonson,	
  Geoffrey	
  Severin,	
  Clarissa	
  Strieder-­‐‑Barbosa,	
  
Oscar	
   Benitez,	
   Marianna	
   Bacellar,	
   Vinicius	
   Galdinho,	
   Cristina	
   Venegas,	
   Phillip	
   Brooks,	
  
Maciej	
  Parys,	
  	
  Geoffrey	
  Grzesiak	
  and	
  Arianna	
  Smith.	
  	
  
	
  

	
  

v	
  

TABLE	
  OF	
  CONTENTS	
  
	
  
	
  
	
  
LIST	
  OF	
  TABLES…………………………………………………………………………………………………………….	
  ix	
  
	
  
LIST	
  OF	
  FIGURES………………………………………………………………………………………………………...….	
  x	
  	
  
	
  
KEY	
  TO	
  ABBREVIATIONS……………………………………………………………………………………………...	
  xii	
  
	
  
CHAPTER	
  1.	
  INTRODUCTION	
  AND	
  LITERATURE	
  REVIEW……………………………………………..…	
  1	
  
TONSILS	
  AND	
  MICROBIOME	
  IN	
  HUMAN	
  AND	
  PIGS………………………………………………………….	
  2	
  
ANIMAL	
  
MODELS	
  
AND	
  
THE	
  
STUDY	
  
OF	
  
HUMAN	
  
RESPIRATORY	
  
DISEASES………………………………………………………………………………………….…………………………...	
  3	
  
ANATOMY	
  AND	
  HISTOLOGY	
  OF	
  TONSILS…………….………………………………………………………….	
  6	
  
TONSIL	
  DEVELOPMENT………………………..…………….………………………………………….…………….	
  12	
  
Human	
  tonsil	
  development……………………………………………………………………………...…	
  12	
  
	
  
Pig	
  tonsil	
  development………………………………………………………………………………….……	
  14	
  
MICROBIOME	
  AND	
  TONSILS……………….……………………………………………………………………...…	
  15	
  
Human	
   tonsillar	
   microbiome:	
   culture-­‐‑dependent	
   vs	
   culture-­‐‑independent	
  	
  	
  	
  	
  	
  	
  	
  
approach	
  …………………………………………………………………………………………………………..	
  16	
  
Pig	
   tonsillar	
   microbiome:	
   culture-­‐‑dependent	
   vs	
   culture-­‐‑independent	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
approach	
  ……………………………………………………………………….…………………………………	
  20	
  
GOAL	
  OF	
  THIS	
  THESIS…………….……………………………………………………………………………………	
  23	
  
REFERENCES……………………………………………………………………………………………………………….	
  24	
  
	
  
CHAPTER	
  2.	
  DEVELOPMENT	
  OF	
  THE	
  TONSILLAR	
  MICROBIOME	
  IN	
  PIGS	
  FROM	
  NEWBORN	
  
THROUGH	
  WEANING……………………………………………………………………………………………………	
  37	
  
INTRODUCTION…………………………………………………………………………………………………………...	
  38	
  
MATERIAL	
  AND	
  METHODS…………………………………………………………………………………………...	
  40	
  
	
  
Animals……………………………………………………………………………………………………………..	
  40	
  
	
  
Collection	
  of	
  microbiome	
  samples………………………………………………………………………	
  41	
  
	
  
Isolation	
  of	
  community	
  DNA…………………………………………………………………………….…	
  42	
  
	
  
Illumina	
  sequencing	
  and	
  sequence	
  analysis…………………………………………………………	
  43	
  
	
  
Diversity	
  and	
  statistical	
  analysis…………………………………………………………………………	
  44	
  
	
  
Availability	
  of	
  supporting	
  data…………………………………………………………………………....	
  45	
  
RESULTS……………………………………………………………………………………………………………………...	
  46	
  
	
  
The	
  tonsillar	
  microbiome	
  found	
  in	
  PB	
  samples	
  clusters	
  by	
  litter….………………………	
  46	
  
A	
   significant	
   proportion	
   of	
   the	
   microbiome	
   of	
   the	
   piglets	
   came	
   from	
   maternal	
  
sources……………………………………………………………………………………………………………..	
  49	
  
Tonsil	
  communities	
  of	
  newborn	
  piglets	
  differed	
  initially	
  between	
  litters	
  but	
  by	
  three	
  
weeks	
  of	
  age	
  clustered	
  together	
  reflecting	
  similar	
  composition……………………………	
  51	
  
The	
   transition	
   between	
   third	
   and	
   fourth	
   week	
   represents	
   a	
   critical	
   period	
   for	
   the	
  
development	
  of	
  the	
  microbiome……………………………………………………………………..….	
  54	
  
DISCUSSION………………………………………………………………………………………………………………...	
  57	
  
CONCLUSIONS…………………………………………………………………………………………………………..…	
  64	
  
	
  

vi	
  

APPENDIX	
  ….………………………………………………………………………………………………………………..	
  65	
  
REFERENCES……………………………………………………………………………………………………………….	
  71	
  
	
  
CHAPTER	
  3.	
  DEVELOPMENT	
  OF	
  THE	
  TONSILLAR	
  MICROBIOME	
  IN	
  PIGS	
  FROM	
  NEWBORN	
  
TO	
  MARKET	
  AGE………………………………………………………………………………………………………….	
  77	
  
INTRODUCTION…………………………………………………………………………………………………………...	
  78	
  
MATERIAL	
  AND	
  METHODS…………………………………………………………………………………………...	
  80	
  
	
  
Animals……………………………………………………………………………………………………………..	
  80	
  
	
  
Collection	
  of	
  microbiome	
  samples……………………………………………………………………....	
  81	
  
	
  
Isolation	
  of	
  community	
  DNA…………………………………………………………………………….…	
  82	
  
	
  
Illumina	
  sequencing	
  and	
  sequence	
  analysis………………………………………………………...	
  82	
  
	
  
Diversity	
  and	
  statistical	
  analysis…………………………………………………………………………	
  83	
  
	
  
Availability	
  of	
  supporting	
  data…………………………………………………………………………....	
  84	
  
RESULTS…………………………………………………………………………………………………………………...…	
  85	
  
	
  
Management	
  practices	
  are	
  related	
  with	
  changes	
  in	
  population	
  diversity……………….	
  86	
  
Challenging	
   management	
   conditions	
   during	
   development	
   of	
   the	
   pigs	
   generated	
  
disruption	
  in	
  the	
  microbiome……………………………………………………………………………..	
  88	
  	
  
Tonsil	
  microbiome	
  membership	
  throughout	
  the	
  life	
  of	
  the	
  pigs…………………………….	
  92	
  	
  
Disruption	
  of	
  specific	
  OTUs	
  throughout	
  the	
  life	
  of	
  the	
  pigs……………………………………	
  98	
  
Aerobic,	
  anaerobic,	
  and	
  facultative	
  anaerobic	
  organisms	
  in	
  the	
  tonsils………………….	
  99	
  
DISCUSSION……………………………………………………………………………………………………………....	
  	
  101	
  	
  
CONCLUSIONS……………………………………………………………………………………………………………	
  111	
  
APPENDIX….……………………………………………………………………………………………………………...	
  	
  113	
  
REFERENCES…………………………………………………………………………………………………………..…	
  117	
  
	
  
CHAPTER	
   4.	
   SPATIOTEMPORAL	
   DEVELOPMENT	
   OF	
   THE	
   TONSIL	
   MICROBIOME	
   IN	
  
PIGS………………………………………………………………………………………………………………………..…	
  123	
  
INTRODUCTION…………………………………………………………………………………………………….…...	
  124	
  
MATERIAL	
  AND	
  METHODS…………………………………………………………………………………………	
  126	
  
	
  
Animals………………………………………………………………………………………………………......	
  126	
  
	
  
Tissue	
  collection………………………………………………………………………………………………	
  127	
  
	
  
Bacterial	
  strains	
  and	
  smears	
  on	
  slides………………………………………………………………	
  128	
  
	
  
Bacterial	
  strains	
  and	
  muscle	
  slides……………………………………………………………………	
  128	
  
	
  
Oligonucleotide	
  probes……………………………………………………………………………….……	
  129	
  
	
  
Fluorecent	
  In	
  Situ	
  Hibridization	
  (FISH)…………………………………………………………..…	
  129	
  
	
  
Confocal	
  Laser	
  Scan	
  Microscopy	
  (CLSM)……………………………………………………………	
  130	
  
	
  
Scanning	
  Electron	
  Microscopy	
  (SEM)……………………………………………………………..…	
  131	
  
RESULTS……………………………………………………………………………………………………………….......	
  131	
  
	
  
Validation	
  of	
  probes…………………………………………………………………………………………	
  132	
  
	
  
Spatial	
  structure	
  of	
  communities	
  on	
  the	
  tonsils	
  using	
  FISH……………………………..…	
  132	
  
	
  
Spatial	
  structure	
  of	
  communities	
  on	
  the	
  tonsils	
  using	
  SEM…………………………..........	
  137	
  
DISCUSSION……………………………………………………………………………………………………………….	
  155	
  
REFERENCES……………………………………………………………………………………………………………..	
  162	
  
	
  
CHAPTER	
  5.	
  SUMMARY	
  AND	
  FUTURE	
  DIRECTIONS…………………………………………………….	
  168	
  
SUMMARY………………………………………………………………………………………………………...............	
  169	
  
	
  

vii	
  

FUTURE	
  DIRECTIONS………………………………………………………………………………………………...	
  174	
  
REFERENCES………………………………………………………………………………………………………..……	
  175	
  
	
  
	
  

	
  

viii	
  

LIST	
  OF	
  TABLES	
  
	
  
	
  
	
  
Table	
  1.1.	
  Characteristics	
  from	
  the	
  main	
  tonsils	
  identified	
  in	
  different	
  species…………………..	
  7	
  
	
  
Table	
  2.1.	
  Identification	
  of	
  sows,	
  litters,	
  sample	
  collection	
  and	
  times	
  of	
  collection	
  ...…………	
  47	
  
	
  
Table	
  2.2.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  litters	
  and	
  sow	
  samples………………………………	
  66	
  
	
  
Table	
  2.3.	
  SIMPER	
  analysis	
  between	
  litters	
  through	
  different	
  sampling	
  times………………….	
  68	
  
	
  
Table	
  2.4.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  third	
  week………………………………………………….	
  69	
  
	
  
Table	
  2.5.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  fourth	
  week……………………………………………….	
  70	
  
	
  
Table	
  3.1.	
  Samples	
  processed	
  by	
  sampling	
  time	
  and	
  litter……………………………………………….	
  85	
  
	
  
Table	
  3.2.	
  Number	
  of	
  observed	
  OTUs	
  (sobs)	
  during	
  the	
  different	
  sampling	
  times…………….	
  88	
  
	
  
Table	
  3.3.	
  Top	
  20	
  most	
  abundant	
  families	
  per	
  sampling	
  time……………………………………….	
  114	
  
	
  	
  
Table	
  4.1.	
  Oligonucleotide	
  probes……………………………………………………………………………….	
  129	
  
	
  

	
  

ix	
  

LIST	
  OF	
  FIGURES	
  
	
  
Figure	
  2.1.	
  Unrooted	
  Bray-­‐‑Curtis	
  dendrogram	
  of	
  PB	
  and	
  sow	
  microbiomes……………………..	
  48	
  	
  

Figure	
  2.2.	
  Thirty	
  most	
  abundant	
  Operational	
  Taxonomic	
  Units	
  (OTUs)	
  for	
  piglets	
  and	
  sow	
  
samples……………………………………………………………………………………………………………………….	
  49	
  
	
  
Figure	
   2.3.	
   Twenty	
   most	
   abundant	
   families	
   identified	
   in	
   sows	
   and	
   PB	
   	
   microbiome	
  
samples……………………………………………………………………………………………………………………….	
  52	
  
	
  
Figure	
   2.4.	
   Principal	
   Coordinate	
   Analysis	
   (PCoA)	
   characterizing	
   the	
   tonsillar	
   microbiome	
  
from	
  PB	
  piglets	
  through	
  the	
  different	
  sampling	
  times…………………………………………………….	
  53	
  
	
  
Figure	
  2.5.	
  The	
  abundance	
  of	
  the	
  twenty	
  most	
  common	
  families	
  in	
  PB	
  piglets	
  sampled	
  from	
  
newborn	
  through	
  four	
  weeks…………..…………………………………………………………………………....	
  55	
  
	
  
Figure	
  3.1.	
  Significant	
  management	
  practices	
  at	
  the	
  swine	
  farm	
  during	
  the	
  life	
  of	
  the	
  pigs	
  in	
  
this	
  study……………………………………………………………………………………………………………………..	
  86	
  
	
  
Figure	
  3.2.	
  Unrooted	
  Bray-­‐‑Curtis	
  dendrogram	
  for	
  all	
  sampled	
  weeks………………………………	
  89	
  	
  
	
  
Figure	
  3.3.	
  Unrooted	
  Bray-­‐‑Curtis	
  dendrogram	
  for	
  three	
  pre-­‐‑transition	
  times	
  …………………	
  91	
  
	
  
Figure	
   3.4.	
   Twenty	
   most	
   abundant	
   families	
   identified	
   in	
   the	
   tonsillar	
   microbiome	
   of	
   pigs	
  
from	
  newborn	
  to	
  market	
  age	
  and	
  in	
  the	
  sows………………………………………………………………….	
  93	
  
	
  
Figure	
   3.5.	
   Forty	
   most	
   abundant	
   Operational	
   Taxonomic	
   Units	
   (OTUs)	
   for	
   pigs	
   through	
  
different	
  sampling	
  times…………………………………………..…………………………………………………...	
  99	
  
	
  
	
  
Figure	
   3.6.	
   Top	
   20	
   most	
   abundant	
   families	
   per	
   sampling	
   time	
   for	
   4	
   selected	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
pigs	
  ……………………………………………………..…………………………………..............................……………	
  115	
  
	
  
Figure	
  3.7.	
  Proportions	
  of	
  aerobes,	
  anaerobes	
  and	
  facultative	
  bacteria…………………………	
  116	
  
	
  
Figure	
   4.1.	
   Representative	
   images	
   for	
   the	
   validation	
   of	
   FISH	
   probes	
   and	
   identification	
   of	
  
Streptococcus	
  cells	
  on	
  the	
  tonsillar	
  surface………………………………………………………………..…	
  134	
  	
  
	
  
Figure	
  4.2.	
  Representative	
  images	
  of	
  bacterial	
  cells	
  and	
  cellular	
  debris	
  in	
  tonsils	
  using	
  FISH	
  
and	
  SEM…………………………………………………………………………………………………………………….	
  135	
  
	
  
Figure	
   4.3.	
   Representative	
   scanning	
   electron	
   microscopy	
   images	
   of	
   tonsil	
   tissue	
   from	
   one	
  
week	
  old	
  pigs.…………………………………………………………………………………………………………….	
  138	
  
	
  

	
  

x	
  

Figure	
   4.4.	
   Representative	
   scanning	
   electron	
   microscopy	
   images	
   of	
   tonsil	
   tissue	
   from	
   three	
  
week	
  old	
  pigs……………………………………………………………………………………………………………..	
  140	
  
	
  
Figure	
  4.5.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  from	
  four	
  
week	
  old	
  pigs……………………………………………………………………………………………………………..	
  143	
  
	
  
Figure	
  4.6.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  from	
  eight	
  
week	
  old	
  pigs……………………………………………………………………………………………………………...	
  145	
  
	
  
Figure	
   4.7.	
   Representative	
   scanning	
   electron	
   microscopy	
   images	
   of	
   tonsil	
   tissue	
   from	
   ten	
  
week	
  old	
  pigs…………………………………………………………………………………………………………...…	
  149	
  
	
  
Figure	
   4.8.	
   Representative	
   scanning	
   electron	
   microscopy	
   images	
   of	
   tonsil	
   tissue	
   from	
  
seventeen	
  week	
  old	
  pigs……………………………………………………………………………………………..	
  151	
  
	
  
Figure	
  4.9.	
  Representative	
  bacterial	
  micro-­‐‑colonies	
  identified	
  through	
  the	
  different	
  sampling	
  
times………………………………………………………………………………………………………………………….	
  153	
  
	
  
	
  

	
  

xi	
  

rRNA	
   	
  
	
  
OTUs	
   	
  
	
  
SIMPER	
  	
  
	
  
FISH	
   	
  
	
  
CLSM	
   	
  
	
  
SEM	
   	
  

	
  

KEY	
  ABBREVIATIONS	
  
	
  
	
  
	
  
ribosomal	
  Ribonucleic	
  acid	
  
Operational	
  Taxonomic	
  Units	
  
Similarity-­‐‑Percentage	
  
Fluorescent	
  In	
  Situ	
  Hibridization	
  
Confocal	
  Laser	
  Scanning	
  Microscopy	
  
Scanning	
  Electron	
  Microscopy	
  

xii	
  

CHAPTER	
  I.	
  INTRODUCTION	
  AND	
  LITERATURE	
  REVIEW	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

1	
  

TONSILS	
  AND	
  MICROBIOME	
  IN	
  HUMAN	
  AND	
  PIGS	
  
	
  
Recently	
   humans	
   have	
   become	
   more	
   aware	
   of	
   the	
   role	
   played	
   by	
   bacterial	
  
communities	
   in	
   human	
   and	
   animal	
   health.	
   	
   These	
   bacterial	
   communities	
   can	
   reduce	
  
acquisition	
   of	
   pathogens	
   by	
   competitive	
   exclusion,	
   improve	
   many	
   physiological	
   functions	
  
and	
  play	
  a	
  complex	
  role	
  in	
  shaping/regulating	
  tissue	
  homeostasis	
  and	
  health.	
  In	
  the	
  last	
  few	
  
years	
   since	
   the	
   advent	
   of	
   advanced	
   sequencing	
   technologies	
   and	
   increased	
   affordability,	
  
there	
  has	
  been	
  a	
  bloom	
  in	
  studies	
  identifying	
  the	
  presence	
  of	
  a	
  normal	
  bacterial	
  population	
  
in	
   different	
   hosts	
   and	
   host	
   tissues.	
   Sequencing	
   has	
   enabled	
   identification	
   of	
   complex	
  
relationships	
   between	
   diverse	
   bacterial	
   populations	
   in	
   host	
   tissues,	
   including	
  the	
  presence	
  
of	
  pathogenic	
  microbes	
  and	
  the	
  relationship	
  of	
  the	
  normal	
  microbiota	
  to	
  other	
  microbes	
  and	
  
disease	
  states.	
  For	
  example,	
  a	
  recent	
  study	
  showed	
  how	
  the	
  presence	
  of	
  bacterial	
  members	
  
of	
  order	
  Clostridiales	
  enhanced	
  the	
  resistance	
  of	
  mice	
  to	
  experimental	
  infections	
  with	
  enteric	
  
pathogens	
  such	
  as	
  Salmonella	
  enterica	
  	
  Typhimurium	
  and	
  Citrobacter	
  rodentium	
  [1].	
  
It	
   is	
   broadly	
   acknowledged	
   that	
   tonsils	
   act	
   as	
   a	
   first	
   line	
   of	
   defense,	
   continually	
  
surveilling	
  for	
  pathogens	
  entering	
  the	
  host	
  through	
  the	
  nasal	
  or	
  oral	
  routes.	
  Further,	
  tonsils	
  
function	
  as	
  a	
  site	
  of	
  colonization	
  and	
  primary	
  replication	
  for	
  bacterial	
  and	
  viral	
  pathogens	
  
that	
   can	
   be	
   host-­‐‑specific	
   or	
   zoonotic	
   organisms	
   transmitted	
   from	
   animals	
   to	
   humans	
   [2].	
  	
  
Currently,	
  there	
  are	
  no	
  studies	
  identifying	
  the	
  interaction	
  of	
  the	
  normal	
  microbial	
  population	
  
in	
  human	
  tonsils	
  with	
  pathogenic	
  microorganisms	
  and,	
  more	
  importantly,	
  no	
  worthy	
  animal	
  
models	
   to	
   study	
   and	
   test	
   hypotheses	
   about	
   relationships	
   between	
   normal	
   tonsillar	
  
microbiota	
  and	
  pathogenic	
  bacteria	
  in	
  humans	
  have	
  been	
  described.	
  	
  

	
  

2	
  

In	
   this	
   review,	
   we	
   will	
   briefly	
   discuss	
   the	
   use	
   and	
   role	
   of	
   animal	
   models	
   to	
   study	
  
bacterial	
   respiratory	
   diseases	
   in	
   humans,	
   particularly	
   the	
   use	
   of	
   pigs	
   to	
   study	
   diseases	
   in	
  
humans.	
  The	
  review	
  will	
  further	
  discuss	
  tonsils,	
  including	
  their	
  structure	
  and	
  their	
  role	
  and	
  
relevance	
  as	
  a	
  first	
  barrier	
  to	
  prevent	
  the	
  colonization	
  of	
  the	
  host	
  by	
  pathogenic	
  bacteria.	
  
Additionally,	
   the	
   review	
   will	
   elucidate	
   some	
   particularities	
   of	
   tonsillar	
   development	
   in	
  
human	
   and	
   pigs.	
   Finally,	
   the	
   review	
   will	
   summarize	
   the	
   current	
   base	
   of	
   knowledge	
   of	
  
microbial	
  populations	
  in	
  human	
  and	
  pig	
  tonsils.	
  
	
  
ANIMAL	
  MODELS	
  AND	
  THE	
  STUDY	
  OF	
  HUMAN	
  RESPIRATORY	
  DISEASES.	
  	
  
	
  
Animals	
  have	
  been	
  used	
  as	
  	
  models	
  to	
  study	
  human	
  diseases	
  for	
  many	
  years,	
  and	
  have	
  
helped	
  researchers	
  fathom	
  the	
  mechanisms	
  of	
  pathogenesis	
  of	
  some	
  diseases	
  as	
  well	
  as	
  to	
  
develop	
  therapies	
  to	
  cure	
  those	
  conditions	
  in	
  humans	
  [3,	
  4].	
  Many	
  of	
  the	
  “traditional”	
  models	
  
are	
   selected	
   based	
   on	
   simplicity	
   to	
   manage	
   and	
   celerity	
   to	
   conduct	
   studies,	
   sometimes	
  
leaving	
  aside	
  the	
  intricacies	
  of	
  certain	
  physiological	
  aspects	
  [4].	
  One	
  of	
  the	
  most	
  common	
  
animal	
  models	
  used	
  is	
  the	
  humanized	
  mouse	
  model;	
  however	
  this	
  model	
  frequently	
  does	
  not	
  
mimic	
   the	
   human	
   conditions	
   [3].	
   This	
   becomes	
   more	
   relevant	
   when	
   working	
   on	
  
microorganism-­‐‑associated	
  disease,	
  due	
  to	
  the	
  fact	
  that	
  mice	
  and	
  other	
  laboratory	
  species	
  are	
  
usually	
   not	
   infected	
   by	
   microorganisms	
   that	
   infect	
   humans	
   [5].	
   	
   Other	
   cheap	
   or	
   easier	
   to	
  
handle	
  models	
  broadly	
  used	
  in	
  comparison/translational	
  studies	
  are	
  the	
  rodent	
  models	
  such	
  
as	
   rat	
   and	
   guinea	
   pigs.	
   	
   Although	
   these	
   rodents	
   are	
   readily	
   available,	
   their	
   lack	
   of	
  
differentiated	
  tonsillar	
  tissue	
  [6,	
  7]	
  makes	
  them	
  a	
  very	
  poor	
  model	
  for	
  tonsillar	
  microbiome	
  
research.	
  	
  	
  

	
  

3	
  

There	
   is	
   not	
   an	
   animal	
   model	
   that	
   meets	
   all	
   the	
   specific	
   requirements	
   for	
   a	
  
translational	
   study	
   [8].	
   The	
   selection	
   of	
   a	
   good	
   animal	
   model	
   depends	
   on	
   the	
   special	
  
conditions	
  required	
  for	
  the	
  study.	
  The	
  pig	
  has	
  been	
  used	
  as	
  a	
  relevant	
  model	
  for	
  a	
  wide	
  range	
  
of	
   human	
   diseases,	
   such	
   as	
   atherosclerosis,	
   diabetes,	
   and	
   skin	
   disease,	
   because	
   of	
   the	
  
similarities	
  it	
  shares	
  with	
  humans,	
  including	
  anatomy,	
  physiology,	
  organ	
  development	
  and	
  
disease	
  progression	
  [3,	
  4,	
  9].	
  	
  	
  
Growing	
  interest	
  in	
  the	
  use	
  of	
  pigs	
  as	
  a	
  translational	
  model	
  have	
  been	
  the	
  object	
  of	
  
international	
   interest	
   represented	
   in	
   symposiums	
   to	
   discuss	
   their	
   prospective	
   uses,	
  
advantages	
   and	
   disadvantages	
   [8,	
   10,	
   11].	
   Examples	
   of	
   the	
   use	
   of	
   pigs	
   as	
   a	
   translational	
  
model	
   to	
   provide	
   new	
   understanding	
   of	
   the	
   pathogenesis	
   of	
   relevant	
   disease	
   affecting	
  
humans	
   are	
   multiple.	
   Pigs	
   have	
   been	
   genetically	
   modified	
   to	
   generate	
   models	
   of	
   cystic	
  
fibrosis	
   (an	
   inherited	
   and	
   fatal	
   disease	
   of	
   Caucasians),	
   providing	
   better	
   approaches	
   to	
  
understand	
   the	
   disease	
   mechanism,	
   especially	
   based	
   on	
   the	
   organs	
   involved	
   in	
   the	
  
pathology,	
  which	
  is	
  not	
  provided	
  by	
  the	
  existing	
  mouse	
  models	
  [12-­‐‑14].	
  Pigs	
  have	
  been	
  used	
  
as	
   model	
   to	
   study	
   organic	
   responses	
   to	
   Pseudomonas	
   aeruginosa	
   in	
   sepsis	
   [15]	
   and	
   in	
  
ventilator-­‐‑associated	
  pneumonia	
  [16]	
  as	
  well	
  as	
  in	
  lung	
  infections	
  [17].	
  Pigs	
  have	
  been	
  used	
  
to	
  study	
  the	
  pathogenesis	
  of	
  influenza	
  and	
  infection	
  with	
  secondary	
  bacterial	
  pathogens	
  such	
  
as	
   Bordetella	
   bronchiseptica	
   [18]	
   or	
   Staphylococcus	
   aureus	
   [19],	
   as	
   well	
   as	
   a	
   model	
   to	
   study	
  
the	
   pathogenesis	
   of	
   respiratory	
   infection	
   by	
   Bordetella	
   pertussis	
   [20]	
   and	
   Bordetella	
  
parapertussis	
  [21].	
  
Humans	
   and	
   pigs	
   share	
   significant	
   similarities	
   in	
   anatomical	
   features	
   of	
   the	
   upper	
  
respiratory	
   tract,	
   such	
   as	
   the	
   arrangement	
   of	
   the	
   pharyngeal	
   “Waldeyer´s	
   ring”	
   lymphoid	
  
tissue.	
   With	
   the	
   exception	
   of	
   primates,	
   the	
   pig	
   resembles	
   the	
   human	
   upper	
   respiratory	
  

	
  

4	
  

anatomy	
  most	
  closely	
  [2].	
  Further,	
  pigs	
  have	
  a	
  distinct	
  advantage	
  over	
  rodents	
  due	
  	
  to	
  the	
  
high	
  similarity	
  of	
  immune	
  parameters	
  with	
  humans	
  (higher	
  than	
  80%,	
  compared	
  with	
  less	
  
than	
  10%	
  of	
  similarity	
  that	
  has	
  been	
  reported	
  for	
  mice)	
  [9].	
  
Pigs	
   and	
   humans	
   share	
   common	
   pathogens	
   that	
   are	
   able	
   to	
   induce	
   disease	
   or	
   be	
  
transmitted	
  between	
  the	
  two	
  species.	
  Examples	
  of	
  such	
  pathogens	
  include	
  Streptococcus	
  suis,	
  
which	
  is	
  an	
  important	
  respiratory	
  and	
  neurologic	
  pathogen	
  of	
  pigs	
  that	
  is	
  now	
  identified	
  as	
  
a	
   cause	
   of	
   meningitis	
   in	
   humans	
   worldwide	
   and	
   an	
   important	
   emerging	
   disease	
   of	
   humans	
  
[22-­‐‑39],	
  and	
  influenza,	
  	
  a	
  viral	
  pathogen	
  shared	
  with	
  humans	
  [40-­‐‑49].	
  The	
  fact	
  that	
  many	
  
respiratory	
  pathogens	
  have	
  similar	
  pathogenic	
  mechanisms	
  in	
  humans	
  and	
  pigs	
  has	
  led	
  to	
  
increased	
  use	
  of	
  pigs	
  as	
  a	
  model	
  to	
  study	
  human	
  respiratory	
  pathogens	
  [9].	
  	
  
Tonsils	
  have	
  been	
  reported	
  as	
  a	
  primary	
  portal	
  of	
  entrance	
  and	
  colonization	
  for	
  some	
  
of	
   these	
   pathogens,	
   from	
   where	
   they	
   are	
   routinely	
   isolated	
   [50].	
   Studies	
   of	
   bacterial	
  
interactions	
  with	
  normal	
  tonsils	
  in	
  humans	
  are	
  complicated	
  due	
  to	
  the	
  reported	
   difficulties	
  
in	
   obtaining	
   normal	
   human	
   tonsillar	
   tissue	
   [51]	
   and	
   the	
   inability	
   to	
   study	
   the	
   effects	
   of	
  
acquisition	
   of	
   pathogens	
   in	
   humans.	
   Human	
   tonsil	
   explants	
   have	
   been	
   used	
   to	
   study	
   the	
  
interactions	
  of	
  human	
  tissue	
  with	
  diverse	
  infectious	
  agents	
  [52].	
  However,	
  this	
  model	
  lacks	
  
certain	
   aspects	
   present	
   in	
   normal	
   tissue	
   interactions,	
   such	
   as	
   the	
   presence	
   of	
   an	
   intact	
  
immune	
   system.	
   The	
   potential	
   contamination	
   inherent	
   to	
   the	
   non-­‐‑sterile	
   nature	
   of	
   these	
  
explants	
  is	
  another	
  serious	
  problem	
  [52].	
  Because	
  of	
  the	
  high	
  similarities	
  between	
  pig	
  and	
  
human	
   tonsils,	
   described	
   below,	
   it	
   is	
   reasonable	
   to	
   propose	
   the	
   use	
   of	
   pigs	
   as	
   a	
   good	
  
exploratory	
   translational	
   model	
   to	
   study	
   the	
   tonsillar	
   microbiome	
   and	
   to	
   test	
   hypotheses	
  
about	
  the	
  interaction	
  between	
  tonsils	
  and	
  the	
  microbiome.	
  
	
  

	
  

5	
  

ANATOMY	
  AND	
  HISTOLOGY	
  OF	
  TONSILS	
  	
  
	
  
The	
  pharyngeal	
  region	
  of	
  some	
  species	
  is	
  characterized	
  by	
  the	
  presence	
  of	
  lymphoid	
  
cell	
   aggregations	
   located	
   in	
   the	
   wall	
   of	
   its	
   lamina	
   propria.	
   These	
   aggregations	
   are	
   called	
  
tonsils	
   [53],	
   and	
   	
   include	
   the	
   lingual	
   tonsils,	
   palatine	
   tonsils,	
   paraepiglottic	
   tonsils,	
  
pharyngeal	
  tonsils,	
  tubal	
  tonsils	
  and	
  the	
  tonsils	
  of	
  soft	
  palate.	
  From	
  the	
  aforementioned,	
  the	
  
single	
   nasopharyngeal	
   tonsils	
   or	
   adenoid,	
   lingual	
   tonsil,	
   paired	
   palatine	
   and	
   tubal	
   tonsils,	
  
form	
  the	
  vast	
  majority	
  of	
  the	
  subepithelial	
  secondary	
  lymphoid	
  organs	
  located	
  in	
  pharynx	
  
known	
   as	
   Waldeyer´s	
   ring	
   [53],	
   which	
   have	
   been	
   suggested	
   to	
   play	
   a	
   role	
   in	
   local	
   B	
   cell	
  
dissemination	
   [54].	
   	
   The	
   number	
   of	
   tonsils	
   present	
   in	
   each	
   species	
   and	
   the	
   features	
   of	
   each	
  
one	
  can	
  vary	
  widely	
  (Table	
  1.1.)	
  
First,	
   it	
   is	
   important	
   to	
   mention	
   the	
   confusion	
   related	
   to	
   the	
   existence	
   of	
   palatine	
  
tonsils	
  in	
  pigs,	
  which	
  have	
  been	
  clarified	
  recently	
  by	
  Casteleyn	
  et	
  al	
  [55,	
  56]	
  to	
  actually	
  be	
  
tonsils	
  of	
  the	
  soft	
  palate	
  and	
  not	
  palatine	
  tonsils.	
  Previous	
  to	
  Casteleyn´s	
  detailed	
  report	
  on	
  
the	
  structure	
  of	
  tonsils	
  in	
  many	
  species	
  of	
  mammals,	
  some	
  reference	
  text	
  books	
  [57]	
  as	
  well	
  
as	
  research	
  papers	
  [50,	
  58-­‐‑63]	
  regularly	
  referred	
  to	
  the	
  use	
  of	
  palatine	
  tonsils	
  in	
  pigs	
  for	
  
their	
  research	
  work.	
  Because	
  of	
  that,	
  most	
  of	
  the	
  references	
  previous	
  to	
  Casteleyn´s	
  report	
  
in	
   2011	
   can	
   generate	
   a	
   confusion.	
   	
   We	
   will	
   use	
   the	
   term	
   tonsils	
   of	
   the	
   soft	
   palate	
   in	
   this	
  
review.	
  
Tonsils	
  as	
  aggregations	
  of	
  mucosa-­‐‑associated	
  lymphoid	
  tissue	
  (MALT)	
  are	
  formed	
  by	
  
areas	
  of	
  B-­‐‑cell	
  collections	
  (lymphoid	
  follicles)	
  surrounded	
  by	
  T-­‐‑cells	
  (interfollicular	
  regions)	
  
[53].	
  Approximately	
  90%	
  of	
  tonsils	
  are	
  comprised	
  of	
  	
  lymphoid	
  tissue	
  [64],	
  clustered	
  as	
  	
  
	
  

	
  

6	
  

Table	
  1.1.	
  Characteristics	
  from	
  the	
  main	
  tonsils	
  identified	
  in	
  different	
  species	
  

	
  
All	
  the	
  data	
  contained	
  in	
  this	
  table,	
  except	
  *	
  was	
  extracted	
  from	
  [56].	
  *	
  data	
  was	
  extracted	
  from	
  [53]	
  

	
  

7	
  

Table	
  1.1.	
  (cont´d).	
  	
  

	
  
All	
  the	
  data	
  contained	
  in	
  this	
  table,	
  except	
  *	
  was	
  extracted	
  from	
  [56].	
  *	
  data	
  was	
  extracted	
  from	
  [53]	
  

	
  
	
  
	
  
	
  
	
  

	
  

8	
  

lymphoid	
   follicles	
   	
   	
   where	
   	
   	
   B	
   	
   	
   cells	
   	
   mature	
   	
   	
   and	
   	
   	
   differentiate	
   	
   	
   and	
   	
   T	
   	
   cells	
   	
   are	
  	
  
activated,	
  	
  while	
  	
  the	
  interfollicular	
  regions	
  are	
  the	
  location	
  of	
  specialized	
  venules	
  (High	
  
Endothelial	
  Venules:	
  HEV)	
  which	
  play	
  a	
  role	
  in	
  the	
  migration	
  of	
  T	
  and	
  B	
  cell	
  	
  from	
  blood	
  
to	
  tonsils	
  [65].	
  	
  
The	
  	
  lymphoid	
  	
  follicles	
  	
  are	
  	
  spread	
  uniformly	
  in	
  	
  the	
  	
  subepithelial	
  	
  mucosal	
  
layer	
   [64]	
   and	
   have	
   been	
   classified	
   as	
   primary	
   lymphoid	
   follicles	
   and	
   secondary	
  
lymphoid	
  follicles.	
  Primary	
  follicles,	
  or	
  resting	
  follicles,	
  are	
  not	
  involved	
  in	
  response	
  to	
  
antigens	
  [66],	
  while	
  secondary	
  follicles	
  are	
  busily	
  involved	
  in	
  antigen-­‐‑driven	
  processes	
  
[67]	
  	
  Within	
  the	
  secondary	
  follicles,	
  immune	
  activated	
  B	
  cells	
  proliferate	
  centrally.	
  The	
  
B-­‐‑cells	
  also	
  form	
  zones	
  with	
  diverse	
  degrees	
  of	
  differentiation	
  and	
  stratification	
  known	
  
as	
  dark,	
  light	
  and	
  mantle	
  zones	
  [68].	
  The	
  anatomical	
  location	
  makes	
  the	
  palatine	
  tonsils	
  
easily	
  accessible	
  thereby	
  making	
  them	
  one	
  of	
  the	
  most	
  widely	
  studied	
  lymphoid	
  organs	
  
to	
  understand	
  interactions	
  of	
  immunological	
  cells	
  and	
  antigens	
  [65].	
  The	
  remainder	
  of	
  
this	
  review	
  will	
  primarily	
  focus	
  on	
  these	
  particular	
  tonsils	
  for	
  humans	
  and	
  the	
  tonsils	
  
of	
  the	
  soft	
  palate	
  for	
  pigs	
  and	
  their	
  resident	
  microbiome.	
  
Palatine	
  tonsils	
  play	
  an	
  important	
  role	
  in	
  immune	
  defense,	
  preventing	
  foreign	
  
antigens	
  from	
  entering	
  the	
  organism	
  via	
  the	
  respiratory	
  and	
  gastrointestinal	
  pathways	
  
[69,	
  70],	
  suggesting	
  an	
  active	
  role	
  as	
  one	
  of	
  the	
  first	
  components	
  of	
  the	
  immune	
  system	
  
to	
  be	
  exposed	
  to	
  antigenic	
  stimulation	
  after	
  birth	
  [71].	
  The	
  tonsil	
  surface	
  possesses	
  thin	
  
channels	
  known	
  as	
  	
  crypts,	
  which	
  facilitate	
  the	
  interaction	
  between	
  foreign	
  antigens	
  
and	
   the	
   immune	
   system	
   [72].Their	
   numbers	
   vary	
   between	
   species	
   and	
   type	
   of	
   tonsils.	
  
For	
  palatine	
  tonsils,	
  in	
  particular,	
  cats	
  do	
  not	
  have	
  crypts	
  [73],	
  rabbits	
  only	
  have	
  one	
  
[7],	
  ovine	
  have	
  1	
  –	
  3	
  [74],	
  and	
  humans	
  possess	
  10	
  –	
  30	
  [75],	
  while	
  the	
  tonsils	
  of	
  soft	
  

	
  

9	
  

palate	
  in	
  pigs	
  have	
  between	
  160	
  -­‐‑190	
  [76].	
  These	
  crypts	
  are	
  present	
  at	
  birth,	
  and	
  the	
  
length	
  and	
  size	
  of	
  tonsillar	
  crypts	
  continues	
  to	
  grow	
  after	
  birth	
  [77].	
  The	
  crypt	
  shape	
  
can	
   vary	
   between	
   species,	
   however	
   tonsils	
   of	
   human	
   and	
   pigs	
   have	
   characteristic	
  
tubular	
  crypts	
  [76,	
  78].	
  Tonsils	
  	
  act	
  	
  as	
  	
  a	
  first	
  	
  interacting	
  	
  organ	
  with	
  	
  antigens	
  	
  entering	
  	
  
by	
  the	
  oral	
  route	
  [79],	
  	
  where	
  the	
  epithelial	
  layer	
  lining	
  the	
  tonsillar	
  surface	
  and	
  crypts	
  
plays	
  a	
  critical	
  role	
  in	
  sensing	
  antigens	
  [80].	
  Because	
  tonsillar	
  lymphoid	
  follicles	
  lack	
  
afferent	
   lymphatic	
   vessels,	
   the	
   intraepithelial	
   passage-­‐‑ways	
   present	
   in	
   the	
   reticular	
  
epithelium	
   allow	
   the	
   flow	
   of	
   intercellular	
   fluid	
   and	
   lymphoid	
   cells,	
   which	
   will	
   finally	
  
reach	
  lymphatic	
  vessels	
  and	
  follicles	
  [81].	
  
Ultrastructural	
  studies	
  of	
  the	
  epithelial	
  surface	
  in	
  palatine	
  tonsils	
  have	
  shown	
  
that	
  there	
  are	
  two	
  distinguishable	
  types	
  of	
  epithelium	
  lining	
  the	
  tonsil	
  surface,	
  reticular	
  
and	
   non-­‐‑reticular	
   [80,	
   82].	
   The	
   non-­‐‑reticular	
   epithelium	
   is	
   composed	
   of	
   non-­‐‑
keratinized	
  stratified	
  squamous	
  cells	
  that	
  cover	
  the	
  tonsillar	
  surface	
  and	
  entrance	
  of	
  
the	
  crypts.	
  The	
  non-­‐‑reticular	
  epithelial	
  cells	
  have	
  characteristic	
  microscopic	
  folds	
  on	
  
the	
  surface,	
  whose	
  shape	
  varies	
  and	
  is	
  more	
  noticeable	
  through	
  the	
  crypt	
  extension,	
  
especially	
  in	
  the	
  deeper	
  parts	
  of	
  the	
  crypt.	
  	
  A	
  second	
  and	
  major	
  component,	
  covering	
  
most	
  of	
  the	
  crypt	
  is	
  a	
  reticular	
  epithelium	
  invaded	
  by	
  lymphoid	
  cells	
  [76,	
  82].	
  In	
  the	
  
junction	
   of	
   the	
   borders	
   of	
   both	
   epithelia,	
   the	
   borders	
   of	
   the	
   reticular	
   epithelial	
   are	
  
depressed	
  and	
  might	
  provide	
  a	
  surface	
  for	
  bacteria	
  to	
  adhere	
  to	
  [82].	
  	
  	
  
The	
  epithelium	
  lining	
  the	
  tonsil	
  surface	
  is	
  formed	
  by	
  polygonal	
  cells	
  whose	
  apical	
  
surface	
  is	
  covered	
  by	
  patterns	
  of	
  characteristic	
  microscopic	
  folds	
  (microridges)	
  [83],	
  or	
  
identified	
  as	
  microvilli	
  or	
  microplications	
  on	
  the	
  surface	
  of	
  epithelium	
  [76],	
  which	
  may	
  
assist	
   bacterial	
   attachment	
   [83],	
   since	
   it	
   has	
   been	
   demonstrated	
   that	
   the	
   bacteria,	
  

	
  

10	
  

Streptococcus	
  pyogenes,	
  use	
  the	
  crest	
  of	
  these	
  microridges	
  for	
  initial	
  attachment,	
  a	
  first	
  
step	
  in	
  the	
  colonization	
  process	
  [84].	
  	
  	
  Another	
  characteristic	
  cell,	
  the	
  fungiform	
  cell	
  
[82]	
  has	
  been	
  described	
  and	
  appears	
  to	
  be	
  the	
  same	
  cell	
  identified	
  by	
  other	
  authors	
  as	
  
the	
  specialized	
  surface	
  cell	
  (M	
  cell),	
  characterized	
  by	
  the	
  presence	
  of	
  microvilli	
  which	
  
suggest	
  a	
  critical	
  role	
  in	
  the	
  antigen	
  uptake	
  [85].	
  The	
  M	
  cells	
  vary	
  morphologically,	
  but	
  
frequently	
  are	
  characterized	
  by	
  large	
  and	
  developed	
  folds	
  of	
  the	
  apical	
  membrane	
  [76,	
  
80]	
  that	
  are	
  tightly	
  associated	
  to	
  intraepithelial	
  lymphocytes	
  [76].	
  The	
  characteristic	
  
shape	
   of	
   M	
   cells	
   seems	
   to	
   provide	
   a	
   large	
   contact	
   surface,	
   facilitating	
   the	
   uptake	
   of	
  
foreign	
  material	
  [80].	
  M	
  cells	
  were	
  found	
  disseminated	
  through	
  the	
  crypt	
  epithelium	
  
especially	
  at	
  projections	
  of	
  the	
  crypt	
  surface	
  and	
  comprise	
  up	
  to	
  35%	
  of	
  epithelial	
  cells	
  
[86]	
   in	
   the	
   tonsillar	
   crypts.	
   They	
   play	
   a	
   significant	
   role	
   in	
   mucosal	
   defense	
   [80],	
   being	
  
capable	
  to	
  transporting	
  antigens	
  across	
  the	
  epithelium	
  [87].	
  It	
  has	
  been	
  proposed	
  that	
  
tonsillar	
  M	
  cells	
  are	
  constantly	
  sampling	
  antigens,	
  such	
  as	
  bacteria	
  and	
  other	
  microbes	
  
which	
  are	
  continuous	
  tonsillar	
  colonizers,	
  	
  and	
  may	
  assist	
  with	
  tolerance	
  and	
  defense	
  
mechanisms,	
  either	
  local	
  or	
  systemic,	
  against	
  these	
  microorganisms	
  [88]	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
Tonsillar	
  crypts	
  provide	
  a	
  large	
  surface	
  area	
  for	
  antigen	
  contact	
  [75],	
  and	
  the	
  
stratified	
   squamous	
   epithelia	
   lining	
   the	
   crypts	
   of	
   palatine	
  tonsils	
   are	
   characterized	
   by	
  
the	
   presence	
   of	
   multiple	
   channels	
   infiltrated	
   by	
   diverse	
   cells	
   including	
   lymphocytes,	
  
plasma	
   cells	
   and	
   mononuclear	
   phagocytic	
   cells	
   [85].	
   Abundance	
   of	
   infiltrating	
   non-­‐‑
epithelial	
   cells	
   (specially	
   lymphocytes	
   B	
   and	
   T),	
   macrophages,	
   dendritic	
   langerhans	
  
cells	
  and	
  Natural	
  Killer	
  (NK)	
  cells	
  and	
  intraepithelial	
  small	
  vessels	
  scattered	
  through	
  
epithelial	
   layer	
   [89,	
   90]	
   have	
   been	
   reported,	
   as	
   well	
   as	
   the	
   presence	
   of	
   	
   neutrophils	
   in	
  
the	
  crypt	
  lumen	
  [90].	
  Goblet	
  cells,	
  characterized	
  by	
  the	
  presence	
  of	
  mucous	
  cytoplasmic	
  

	
  

11	
  

granules,	
   have	
   also	
   been	
   described	
   in	
   the	
   middle	
   and	
   terminal	
   regions	
   of	
   the	
   crypts,	
  
often	
  tightly	
  associated	
  to	
  M	
  cells	
  [76].	
  Also,	
  diverse	
  leucocytes,	
  epithelial	
  cells,	
  Periodic	
  
acid-­‐‑Shiff	
   (PAS)	
   positive	
   content	
   (polysaccharides	
   and	
   mucosubstances)	
   and	
   free	
   or	
  
phagocytosed	
  bacteria	
  have	
  been	
  observed	
  in	
  the	
  lumen	
  of	
  crypts	
  [91]	
  and	
  seems	
  to	
  be	
  
sloughed	
  off	
  from	
  the	
  area	
  [64].	
  	
  
	
  
TONSIL	
  DEVELOPMENT	
  	
  
	
  
Human	
   tonsil	
   development.	
   The	
   second	
   branchial	
   pouch,	
   known	
   to	
   be	
   the	
  
precursor	
  of	
  palatine	
  tonsils	
  in	
  the	
  fetus,	
  is	
  identifiable	
  in	
  the	
  first	
  month	
  of	
  gestation	
  
and	
  by	
  the	
  second	
  month	
  the	
  epithelium	
  begins	
  to	
  evaginate	
  to	
  form	
  the	
  early	
  tonsillar	
  
fossa	
  and	
  there	
  is	
  evidence	
  of	
  canalization	
  and	
  branching	
  into	
  early	
  crypts	
  present	
  in	
  
the	
   structure	
   [92].	
   By	
   the	
   seventh	
   month	
   of	
   gestation	
   there	
   is	
   evidence	
   of	
   lymphoid	
  
infiltration	
   into	
   the	
   lamina	
   propria,	
   and	
   by	
   the	
   last	
   trimester	
   of	
   gestation,	
   the	
   primary	
  
follicles	
   can	
   be	
   identified	
   [92].	
   However,	
   newborn	
   infants	
   lack	
   tonsillar	
   lymphoid	
  
follicles,	
  which	
  are	
  numerous	
  in	
  adults	
  [93].	
  	
  
The	
   tonsillar	
   crypt	
   structure	
   in	
   humans	
   has	
   been	
   reported	
   as	
   beginning	
   to	
  
appear	
  at	
  the	
  third	
  month	
  of	
  fetus	
  development	
  and	
  to	
  reach	
  an	
  approximately	
  constant	
  
number	
   at	
   birth	
   [77].	
   Minear	
   et	
   al.	
   showed	
   that	
   the	
   number	
   of	
   crypts	
   seems	
   to	
   be	
  
constant	
  even	
  in	
  adulthood,	
  reaching	
  a	
  maximum	
  of	
  thirty	
  eight	
  [77].	
  This	
  	
  is	
  in	
  contrast	
  
to	
  the	
  studies	
  done	
  by	
  Kassay	
  and	
  Sandor	
  which	
  reported	
  that	
  the	
  age	
  and	
  size	
  of	
  the	
  
tonsil	
   have	
   a	
   higher	
   influence	
   on	
   the	
   numbers	
   of	
   crypts	
   observed,	
   with	
   the	
   number	
   of	
  
crypts	
  varying	
  from	
  16	
  to	
  up	
  to	
  25,	
  depending	
  on	
  whether	
  it	
  is	
  a	
  small	
  or	
  a	
  giant	
  tonsil	
  

	
  

12	
  

[75].	
   However,	
   more	
   important	
   than	
   the	
   number	
   of	
   crypts	
   are	
   the	
   number	
   and	
  length	
  
of	
  the	
  crypt	
  branches,	
  which	
  expands	
  the	
  surface	
  area	
  of	
  the	
  tonsils	
  significantly	
  [75].	
  	
  
Tonsillar	
  germinal	
  centers	
  appear	
  only	
  after	
  birth	
  once	
  they	
  are	
  stimulated	
  by	
  
antigens	
  and	
  continue	
  to	
  proliferate	
  during	
  the	
  first	
  year	
  of	
  life	
  [92,	
  94],	
  reaching	
  their	
  
highest	
  abundance	
  during	
  the	
  first	
  2-­‐‑3	
  years	
  of	
  childhood	
  and	
  declining	
  around	
  8	
  –	
  14	
  
years	
   [95],	
   they	
   revert	
   with	
   age,	
   which	
   involves	
   a	
   decrease	
   in	
   the	
   proliferating	
   pool	
   of	
  
the	
   B	
   cell	
   population	
   [96].	
   It	
   has	
   been	
   shown	
   that	
   the	
   presence	
   of	
   immunoglobulin	
  
positive	
  cells	
  in	
  tonsils	
  is	
  negatively	
  correlated	
  with	
  age,	
  meaning	
  that	
  number	
  of	
  cells	
  
positive	
  for	
  immunoglobulin	
  decreases	
  as	
  age	
  increases	
  [69].	
  
Human	
   tonsils	
   are	
   characterized	
   by	
   the	
   absence	
   of	
   sinuses	
   and	
   the	
   presence	
   of	
  
epithelial	
  infiltrates	
  of	
  macrophages	
  and	
  lymphoid	
  cells	
  [97],	
  where	
  the	
  luminal	
  side	
  of	
  
the	
  mucosa	
  is	
  particularly	
  populated	
  by	
  B	
  cells	
  and	
  the	
  apical	
  side	
  by	
  mature	
  plasma	
  
cells	
  predominantly	
  positive	
  for	
  IgG	
  and	
  in	
  lower	
  numbers	
  positive	
  to	
  IgA,	
  IgM	
  and	
  IgD	
  
[90].	
   This	
   epithelial	
   cell	
   infiltration	
   and	
   the	
   presence	
   of	
   pores	
   in	
   the	
   basement	
  
membrane	
   of	
   the	
   tonsillar	
   crypt	
   epithelium	
   is	
   a	
   natural	
   developmental	
   finding	
   in	
  
prenatal	
  and	
  postnatal	
  human	
  crypts	
  [98]	
  
Immunoglobulin	
   production	
   by	
   cells	
   from	
   normal	
   human	
   palatine	
   tonsils	
   was	
  
initially	
   investigated	
   by	
   Brandtzaeg	
   et	
   al.	
   [99]	
   and	
   they	
   demonstrated	
   that	
   B	
   cells	
  
differentiate	
  into	
  IgG	
  blast-­‐‑cells	
  in	
  the	
  germinal	
  centers	
  of	
  tonsils,	
  and	
  that	
  this	
  B	
  cell	
  
system	
  is	
  highly	
  activated	
  throughout	
  human	
  life.	
  The	
  B	
  cell	
  system	
  plays	
  a	
  significant	
  
role	
  in	
  identifying	
  foreign	
  material	
  and	
  driving	
  clonal	
  expansion	
  and	
  differentiation,	
  in	
  
order	
  to	
  control	
  antigenic	
  agents	
  [98,	
  99].	
  It	
  has	
  been	
  demonstrated	
  that	
  tonsils	
  have	
  a	
  

	
  

13	
  

role	
  in	
  the	
  generation	
  of	
  local	
  and	
  disseminated	
  antibody	
  responses,	
  and	
  contribute	
  to	
  
the	
  development	
  of	
  immunological	
  memory	
  [100].	
  	
  	
  
Pig	
  tonsil	
  development.	
  The	
  pig	
  tonsils	
  also	
  arise	
  from	
  the	
  pharyngeal	
  pouches	
  
[101],	
  as	
  in	
  humans.	
  The	
  first	
  evidence	
  of	
  formation	
  of	
  tonsillar	
  tissue	
  appears	
  in	
  fetuses	
  
of	
  	
  90	
  mm	
  	
  (approximately	
  70/114	
  days	
  of	
  gestation),	
  evidence	
  of	
  crypts	
  appears	
  in	
  the	
  
142	
   mm	
   stage	
   (approximately	
   80/114	
   days	
   of	
   gestation),	
   and	
   defined	
   lymphoid	
  
nodules	
  appear	
  in	
  the	
  275	
  mm	
  stage	
  (approximately	
  110/114	
  days	
  of	
  gestation)	
  	
  [101].	
  
Germinal	
  centers	
  in	
  tonsils	
  are	
  absent	
  in	
  germ-­‐‑free	
  piglets	
  under	
  4	
  weeks	
  of	
  age	
  [102].	
  
However,	
  the	
  presence	
  of	
  bacterial	
  antigens	
  is	
  able	
  to	
  induce	
  the	
  formation	
  of	
  germinal	
  
centers	
  in	
  tonsillar	
  tissue	
  [103].	
  In	
  tonsils,	
  the	
  lymphoid	
  follicles	
  develop	
  as	
  piglets	
  age,	
  
where	
  1	
  week	
  old	
  piglets	
  	
  have	
  less	
  developed	
  lymphoid	
  follicles	
  when	
  compared	
  with	
  	
  
6	
  month	
  old	
  pigs	
  [79].	
  	
  
Tonsil	
   dimensions	
   vary	
   with	
   age	
   and	
   can	
   range	
   	
   from	
   3.3	
   x	
   1.8	
   cm	
   for	
   pigs	
  
between	
  6	
  –	
  10	
  weeks	
  old	
  [76]	
  to	
  approximate	
  4.5	
  x	
  2	
  cm	
  in	
  adult	
  animals	
  [101].	
  Pig	
  
tonsils	
   have	
   been	
   reported	
   to	
   have	
   160	
   to	
   190	
   oval	
   to	
   circular	
   surface	
   openings,	
  
corresponding	
   to	
   the	
   entrances	
   of	
   tonsillar	
   crypts	
   [76].	
   Tonsillar	
   crypts	
   in	
   pigs	
   are	
  
covered	
   by	
   a	
   non-­‐‑keratinized	
   squamous	
   epithelium,	
   supported	
   by	
   dense	
   connective	
  
tissue	
   and	
   with	
   a	
   parenchyma	
   uniformly	
   populated	
   with	
   lymphocytes	
   [103].	
   They	
   are	
  
characterized	
  by	
  the	
  presence	
  of	
  intraepithelial	
  mucous	
  cells,	
  which	
  suggests	
  a	
  possible	
  
role	
   in	
   the	
   elimination	
   of	
   debris	
   due	
   to	
   its	
   secretory	
   role	
   [79].	
   Tonsillar	
   lymphoid	
  
follicles	
  are	
  characterized	
  by	
  an	
  oval	
  shape	
  with	
  the	
  long	
  axis	
  oriented	
  perpendicular	
  
to	
  the	
  epithelial	
  layer	
  of	
  crypts	
  [79].	
  	
  

	
  

14	
  

Studies	
  show	
  that	
  during	
  the	
  development	
  of	
  tonsils	
  in	
  pigs,	
  follicles	
  are	
  formed	
  
mainly	
  by	
  B-­‐‑cells	
  and	
  T-­‐‑cells	
  present	
  in	
  the	
  interfollicular	
  area.	
  IgM+	
  cells	
  are	
  found	
   in	
  
follicles	
  of	
  new	
  born	
  to	
  9	
  month	
  old	
  animals,	
  while	
  IgG+	
  and	
  IgA+	
  cells	
  are	
  only	
  seen	
  after	
  
4	
  weeks	
  of	
  age	
  [104].	
  Activation	
  of	
  tonsillar	
  T	
  cell	
  increases	
  with	
  age,	
  and	
  suggests	
  a	
  
direct	
   relation	
   of	
   levels	
   of	
   activation	
   and	
   differentiation	
   of	
   T	
   cells	
   with	
   colonization	
   of	
  
microbial	
  flora.	
  There	
  is	
  an	
  association	
  of	
  microbial	
  colonization	
  with	
  changes	
  in	
  the	
  
size	
  and	
  structure	
  of	
  follicles,	
  which	
  increased	
  with	
  the	
  age,	
  when	
  compared	
  pigs	
  from	
  
birth	
  to	
  4	
  weeks	
  of	
  age	
  [104].	
  
	
  As	
   can	
   be	
   seen	
   in	
   the	
   above	
   paragraphs,	
   pig	
   and	
   human	
   tonsils	
   share	
   many	
  
similarities	
  and	
  the	
  possibilities	
  to	
  use	
  pigs	
  as	
  translational	
  models	
  for	
  studies	
  involving	
  
tonsils	
  are	
  promising.	
  The	
  development	
  of	
  such	
  important	
  tissue	
  as	
  the	
  tonsils	
  involves	
  
many	
   important	
   features	
   that	
   are	
   not	
   the	
   objective	
   of	
   this	
   brief	
   review.	
   For	
   readers	
  
interested	
   in	
   a	
   more	
   extensive	
   review	
   of	
   pig	
   and	
   human	
   tonsils,	
   the	
   articles	
   by	
   Horter	
  
et	
  al	
  [2]	
  and	
  Perry	
  and	
  White	
  [53]	
  are	
  good	
  sources.	
  
	
  
MICROBIOME	
  AND	
  TONSILS	
  	
  	
  
	
  
It	
   was	
   mentioned	
   previously	
   that	
   in	
   recent	
   years	
   there	
   has	
   been	
   a	
   bloom	
   in	
  
microbiome	
  publications	
  in	
  different	
  sources	
  using	
  a	
  sequencing	
  approach.	
  The	
  study	
  
of	
  the	
  human	
  microbiome	
  has	
  been	
  led	
  by	
  a	
  giant	
  consortium	
  of	
  scientists	
  that	
  want	
  to	
  
study	
  the	
  various	
  aspects	
  of	
  the	
  human	
  microbiome,	
  such	
  as	
  	
  the	
  structure,	
  function	
  and	
  
diversity	
   in	
   healthy	
   populations	
   and	
   how	
   disease	
   correlates	
   with	
   changes	
   in	
   the	
  
microbiome	
   [105,	
   106].	
   However,	
   very	
   few	
   studies	
   focus	
   particularly	
   on	
   the	
   tonsil	
  

	
  

15	
  

microbiome.	
  Most	
  available	
  studies	
  are	
  focused	
  on	
  the	
  oral	
  cavity	
  as	
  a	
  whole	
  [107-­‐‑110]	
  
or	
  studied	
  multiple	
  body	
  sites	
  without	
  a	
  particular	
  focus	
  on	
  the	
  tonsils	
  [111].	
  The	
  only	
  
available	
   study	
   focused	
   particularly	
   on	
   human	
   tonsils	
   analyzed	
   the	
   microbiota	
   of	
  
tonsillar	
   crypts	
   in	
   children	
   and	
   healthy	
   adult	
   	
   patients	
   diagnosed	
   with	
   tonsillar	
  
hyperplasia	
  compared	
  to	
  patients	
  with	
  recurrent	
  tonsillitis	
  [112].	
  However	
  there	
  are	
  
no	
  studies	
  characterizing	
  the	
  development	
  of	
  the	
  tonsil	
  microbiome	
  in	
  humans,	
  using	
  a	
  
sequencing	
  approach.	
  Similarly,	
  there	
  are	
  multiple	
  studies	
  focused	
  on	
  the	
  microbiome	
  
of	
  pigs	
  in	
  different	
  body	
  regions,	
  particularly	
  the	
  intestinal	
  microbiome,	
  using	
  a	
  culture-­‐‑
independent	
  approach.	
  	
  There	
  are	
  a	
  few	
  studies	
  focused	
  on	
  the	
  tonsillar	
  microbiome	
  of	
  
pigs	
  [113-­‐‑115]	
  	
  but	
  none	
  have	
  studied	
  the	
  development	
  of	
  tonsillar	
  microbiome	
  of	
  pigs.	
  	
  
Use	
   of	
   a	
   pig	
   model	
   as	
   a	
   model	
   of	
   human	
   infants	
   has	
   been	
   suggested,	
   where	
   pigs	
  
inoculated	
   with	
   human	
   microbiota	
   have	
   the	
   potential	
   to	
   simulate	
   closely	
   an	
   overall	
  
human	
   system	
   behavior,	
   especially	
   because	
   of	
   the	
   similarities	
   shared	
   between	
   both	
  
species	
  [116].	
  Further,	
  it	
  has	
  been	
  reported	
  that	
  92%	
  of	
  the	
  bacterial	
  families	
  identified	
  
in	
  human	
  tonsils	
  are	
  also	
  present	
  in	
  pig	
  tonsils	
  [117],	
  although	
  different,	
  often	
  host-­‐‑
specific	
  species	
  are	
  found.	
  The	
  following	
  section	
  will	
  review	
  what	
  is	
  known	
  about	
  the	
  
tonsillar	
  microbiome	
  of	
  humans	
  and	
  pigs,	
  focusing	
  on	
  the	
  different	
  methodologies	
  used.	
  
	
  
Human	
  tonsillar	
  microbiome:	
  culture-­‐‑dependent	
  vs	
  culture-­‐‑independent	
  
approach.	
   The	
   initial	
   studies	
   characterizing	
   the	
   human	
   tonsil	
   microbiome	
   were	
  
performed	
  using	
  a	
  culture-­‐‑dependent	
  approach	
  in	
  order	
  to	
  establish	
  the	
  members	
  of	
  
the	
   tonsillar	
   flora.	
   Predominantly	
   these	
   studies	
   were	
   focused	
   on	
   characterizing	
   the	
  
aerobic	
   tonsillar	
   bacterial	
   population	
   while	
   a	
   smaller	
   number	
   of	
   studies	
   have	
  

	
  

16	
  

characterized	
   the	
   anaerobic	
   population.	
   Most	
   of	
   these	
   studies	
   were	
   based	
   on	
   young	
  
patients,	
  mainly	
  children	
  between	
  1	
  and	
  13	
  years	
  old	
  (usually	
  after	
  tonsillectomy)	
  [118-­‐‑
120],	
  while	
  a	
  few	
  others	
  have	
  also	
  been	
  done	
  in	
  older	
  patients	
  [121,	
  122].	
  
Studies	
  done	
  in	
  1	
  -­‐‑	
   3	
  years	
  old	
  children	
  [118]	
  as	
  well	
  as	
  between	
  5	
  –	
  13	
  years	
  
[119],	
  identified	
  members	
  of	
  the	
  genera	
  Streptococcus	
  and	
  Staphylococcus	
  	
  as	
  the	
  main	
  
aerobic	
  organisms	
  isolated	
  and	
  members	
  of	
  genera	
  Bacteroides	
  and	
  Fusobacterium	
  as	
  
the	
   predominant	
   anaerobic	
   organisms	
   isolated.	
   β-­‐‑haemolytic	
   Streptococcus,	
  
Streptococcus	
   pneumoniae	
   and	
   Haemophilus	
   influenzae	
   were	
   the	
   prevalent	
   aerobic	
  
bacteria	
  [118,	
  121],	
  followed	
  by	
  Staphylococcus	
  aureus	
  [121],	
  	
  while	
  Bacteroides	
  fragilis	
  
was	
  the	
  most	
  common	
  anaerobe	
  isolated	
  from	
  the	
  surface	
  and	
  core	
  of	
  tonsils	
  of	
  very	
  
young	
   children	
   [118].	
   H	
   influenzae	
   was	
   most	
   prevalent	
   in	
   youngsters	
   	
   vs	
   adults	
   and	
   β-­‐‑	
  
haemolytic	
  	
  Streptococcus	
  was	
  less	
  prevalent	
  in	
  younger	
  children	
  (2-­‐‑7)	
  years,	
  compared	
  
to	
   older	
   adults	
   [121].	
   A	
   predominance	
   of	
   anaerobic	
   bacteria	
   was	
   found	
   in	
   adults	
  
compared	
   to	
   younger	
   ages,	
   	
   identifying	
   higher	
   proportions	
   of	
   isolated	
   anaerobes	
   in	
  
adults	
  (older	
  than	
  15	
  years)	
  vs	
  younger	
  children	
  [121].	
  
Using	
  a	
  culture-­‐‑dependent	
  approach,	
  Kasenõmm	
  et	
  al.	
  found	
  mixed	
  anaerobic	
  
and	
   aerobic	
   bacteria	
   as	
   core	
   microorganisms	
   in	
   adult	
   patients	
   with	
   acute	
   tonsillitis	
  
[123].	
   	
   The	
   authors	
   reported	
   the	
   frequent	
   isolation	
   of	
   α-­‐‑	
   and	
   β-­‐‑haemolytic	
  
Streptococcus,	
   Staphylococcus	
   aureus	
   and	
   coagulase	
   negative	
   Staphylococci,	
   and	
  
Corynebacterium	
   spp	
   as	
   the	
   aerobic	
   population	
   and	
   members	
   of	
   Peptostreptococcus,	
  
Propionibacterium,	
  Actinomyces,	
  Prevotella,	
  Bacteroides	
  and	
  Fusobacterium	
  species	
  as	
  
the	
  most	
  frequent	
  anaerobes.	
  	
  	
  

	
  

17	
  

	
  Although	
  what	
  is	
  known	
  about	
  microbiome	
  of	
  tonsils	
  by	
  the	
  culture-­‐‑dependent	
  
approach	
  is	
  based	
  on	
  studies	
  of	
  samples	
  of	
  patients	
  with	
  acute	
  tonsillitis,	
  it	
  has	
  been	
  
reported	
  that	
  there	
  is	
  similarity	
  in	
  the	
  organisms	
  that	
  form	
  the	
  tonsillar	
  microbial	
  flora	
  
of	
  normal	
  patients	
  and	
  patients	
  with	
  recurrent	
  history	
  of	
  tonsillitis.	
  However,	
  a	
  higher	
  
concentration	
  of	
  some	
  bacterial	
  members	
  was	
  present	
  in	
  the	
  patients	
  with	
  tonsillitis.	
  
[119].	
  
The	
  use	
  of	
  culture-­‐‑independent	
  approaches	
  to	
  identify	
  the	
  tonsillar	
  microbiome	
  
in	
  humans	
  has	
  produced	
  new	
  insights.	
  Using	
  clone	
  libraries	
  of	
  16S	
  rRNA	
  genes,	
  Aas	
  et.	
  
al.	
   studied	
   the	
   microbiome	
   of	
   5	
   different	
   patients/subjects	
   in	
   nine	
   different	
   oral	
  
locations	
   including	
   the	
   tonsils	
   [108].	
   They	
   were	
   able	
   to	
   identify	
   that	
   there	
   is	
   diversity	
  
in	
  the	
  bacteria	
  identified	
  in	
  the	
  study	
  subjects	
  where	
  some	
  bacterial	
  species	
  were	
  found	
  
only	
   in	
   one	
   subject,	
   while	
   others	
   were	
   present	
   in	
   all	
   or	
   most	
   of	
   the	
   subjects.	
   However,	
  
they	
  emphasized	
  that	
  the	
  tonsils	
  had	
  the	
  highest	
  numbers	
  of	
  different	
  species	
  compared	
  
to	
  the	
  other	
  oral	
  sites	
  sampled.	
  	
  
Other	
   studies	
   have	
   also	
   examined	
   the	
   tonsillar	
   microbiome,	
   but	
   using	
   a	
   454	
  
pyrosequencing	
  approach.	
  Liu	
  et	
  al	
  [124]	
  studied	
  the	
  composition	
  of	
  tonsillar	
  bacterial	
  
microbiota	
  in	
  a	
  8	
  year	
  old	
  patient	
  suffering	
  chronic	
  serous	
  otitis	
  media.	
  The	
  authors	
  
detected	
   9	
   different	
   families	
   in	
   the	
   tonsillar	
   microbiome	
   (Pseudomonadaceae,	
  
Streptococcaceae,	
  Fusobacteriaceae,	
  Pasteurellaceae,	
  Prevotellaceae,	
  Flavobacteriaceae,	
  
Bacillales	
   family	
   XI,	
   Carnobacteriaceae	
   and	
   Neisseriaceae),	
   and	
   identified	
  
Streptococcaceae	
   as	
   the	
   most	
   dominant,	
   with	
   a	
   relative	
   abundance	
   of	
   approximately	
  
69.2	
  %.	
  Similarly,	
  other	
  authors	
  have	
  reported	
  that	
  multiple	
  OTUs	
  assigned	
  to	
  the	
  genus	
  
Streptococcus	
   were	
   abundant	
   in	
   the	
   tonsils,	
   as	
   well	
   as	
   others	
   assigned	
   to	
   the	
   genera	
  

	
  

18	
  

Prevotella,	
   Fusobacterium,	
   and	
   Neisseria	
   [111].	
   Some	
   OTUs	
   that	
   could	
   not	
   be	
   classified	
  
further	
  than	
  family	
  level	
  and	
  belonging	
  to	
  the	
  families	
  Neisseriaceae,	
  Pasteurellaceae	
  
and	
   Prevotellaceae	
   were	
   also	
   identified	
  [111].	
   Segata	
   et	
   al	
   [125]	
   studied	
   the	
   microbial	
  
communities	
   in	
   ten	
   different	
   sites	
   of	
   the	
   digestive	
   tract	
   (seven	
   mouth	
   surfaces,	
   throat,	
  
palatine	
   tonsils	
   and	
   colon	
   (stools)),	
   and	
   they	
   found	
   that	
   the	
   community	
   structure	
  
allowed	
   them	
   to	
   group	
   the	
   samples	
   in	
   four	
   distinct	
   community	
   types,	
   where	
   saliva,	
  
tongue,	
   tonsils	
   and	
   throat	
   formed	
   one	
   group.	
   This	
   group	
   was	
   characterized	
   by	
   a	
  
decreased	
   relative	
   abundance	
   of	
   the	
   phylum	
   Firmicutes	
   and	
   increased	
   presence	
   of	
  
members	
   of	
   the	
   phyla	
   Bacteroidetes,	
   Fusobacteria,	
   Actinobacteria	
   and	
   TM7.	
   Also,	
  
members	
  of	
  the	
  genera	
  Streptococcus,	
  Veillonella,	
  Prevotella,	
  Neisseria,	
  Fusobacterium,	
  
Actinomyces	
   and	
   Leptotrichia	
   were	
   identified	
   evenly	
   distributed	
   and	
   present	
   at	
   an	
  
abundance	
  of	
  ≥2%	
  on	
  average,	
  while	
  members	
  of	
  the	
  genus	
  Moraxella	
  were	
  detected	
  in	
  
low	
  relative	
  abundance.	
  In	
  contrast,	
  Jensen	
  et	
  al	
  [112],	
  identified	
  bacteria	
  from	
  different	
  
genera,	
   including	
   Prevotella,	
   	
   Streptococcus,	
   Haemophilus,	
   Fusobacterium,	
  
Porphyromonas,	
   Gemella,	
   Neisseria,	
   Veillonella,	
   Capnocytophaga,	
   Parvimonas,	
   Rothia,	
  
Actinomyces	
  and	
  Treponema,	
  as	
  the	
  predominant	
  members	
  of	
  the	
  core	
  microbiota	
  in	
  
tonsillar	
  crypts,	
  which	
  include	
  both	
  Gram	
  (+)	
  and	
  Gram	
  (-­‐‑)	
  bacteria,	
  as	
  well	
  as	
  aerobic	
  
and	
  anaerobic	
  organisms	
  .	
  	
  
Finally,	
   another	
   approach	
   used	
   to	
   investigate	
   the	
   components	
   of	
   the	
   human	
  
tonsillar	
  microbiome	
  was	
  the	
  use	
  of	
  a	
  selective	
  isolation	
  combined	
  with	
  the	
  use	
  of	
  	
  PCR	
  
denaturing	
   gradient	
   gel	
   electrophoresis	
   [126].	
   The	
   authors	
   profiled	
   the	
   microbiota	
  
from	
  10	
  healthy	
  adults	
  and	
  identified	
  three	
  phyla	
  as	
  predominant	
  in	
  tonsils	
  (Firmicutes,	
  

	
  

19	
  

Proteobacteria	
   and	
   Fusobacteria),	
   and	
   further	
   identified	
   members	
   of	
   the	
   genus	
  
Streptococcus	
  as	
  abundant	
  in	
  tonsils.	
  	
  	
  
	
  Pig	
   tonsillar	
   microbiome:	
   culture-­‐‑dependent	
   vs	
   culture-­‐‑independent	
  
approach.	
  The	
  majority	
  of	
  what	
  is	
  known	
  about	
  tonsillar	
  microbiome	
  of	
  pigs	
  based	
  on	
  
culture-­‐‑dependent	
  approach	
  studies	
  is	
  based	
  on	
  studies	
  focused	
  on	
  identifying	
  tonsillar	
  
bacterial	
   isolates	
   from	
   specific	
   genera.	
   Devriese	
   et	
   al	
   [127]	
   studied	
   the	
   tonsillar	
  
bacterial	
  population	
  of	
  pigs	
  brought	
  for	
  necropsy	
  suffering	
  from	
  a	
  variety	
  of	
  conditions	
  
distinct	
  from	
  streptococcal	
  infection,	
  and	
  yet	
  isolated	
  Streptococcus	
  suis	
  predominantly,	
  
followed	
   by	
   S.	
   dysgalactiae	
   and	
   Enterococcus	
   faecalis.	
   In	
   another	
   study	
   directed	
   to	
  
identify	
  the	
  tonsillar	
  Gram	
  (+)	
  flora	
  of	
  piglets	
  before	
  and	
  after	
  weaning,	
  Baele	
  et	
  al	
  [128]	
  
showed	
   that	
   the	
   most	
   prevalent	
   bacterial	
   genera	
   found	
   were	
   Streptococcus	
  
(Streptococcus	
   suis,	
   S.	
   dysgalactiae),	
   as	
   well	
   as	
   Staphylococcus	
   (S.	
   hyicus,	
   S.	
   aureus).	
  
Arcanobacterium	
  pyogenes	
  and	
  Actinomyces	
  hyovaginalis	
  were	
  also	
  frequently	
  isolated.	
  
The	
   authors	
   found	
   marginal	
   numbers	
   of	
   members	
   of	
   the	
   genus	
   Lactobacillus	
   that	
  
appeared	
   post-­‐‑weaning.	
   MacInnes	
   et	
   al	
   [129]	
   studied	
   the	
   prevalence	
   of	
   important	
  
respiratory	
  bacterial	
  pathogens	
  in	
  swine	
  herds	
  in	
  Ontario,	
  using	
  a	
  culture-­‐‑dependent	
  
approach	
   but	
   also	
   using	
   serological	
   and	
   PCR-­‐‑based	
   tests,	
   and	
   identified	
   a	
   high	
  
prevalence	
   of	
   Haemophilus	
   parasuis,	
   Actinobacillus	
   pleuropneumoniae,	
   Actinobacillus	
  
suis,	
  and	
  S.	
  suis	
  .	
  O´Sullivan	
  et	
  al	
  [130]	
  identified	
  the	
  prevalence	
  of	
  porcine	
  pathogens	
  
from	
   pig	
   tonsils	
   collected	
   at	
   slaughter	
   houses.	
   This	
   study	
   determined	
   that	
   most	
  
prevalent	
   bacteria	
   isolated	
   from	
   pig	
   tonsils	
   were	
   members	
   of	
   the	
   genus	
   Streptococcus	
  
(S.	
   suis,	
   S.	
   equisimilis,	
   S.	
   porcinus),	
   followed	
   by	
   Arcanobacterium	
   (Arcanobacterium	
  
pyogenes),	
  Pasteurella	
  (Pasteurella	
  multocida),	
  Staphylococcus	
  (S.	
  hyicus,	
  S.	
  aureus),	
  as	
  

	
  

20	
  

well	
   as	
   a	
   lower	
   prevalence	
   of	
   other	
   pathogenic	
   bacteria	
   such	
   as	
   Actinobacillus	
  
pleuropneumoniae,	
   Yersinia	
   enterocolitica,	
   Haemophilus	
   parasuis,	
   Erysipelothrix	
   spp,	
  
Listeria	
   monocytogenes,	
   Actinomyces	
   sp	
   and	
   Salmonella	
   spp..	
   Finally,	
   a	
   culture-­‐‑
dependent	
  approach	
  to	
  identify	
  the	
  microbial	
  communities	
  in	
  pig	
  tonsils,	
  was	
  done	
  by	
  
Lowe	
  et	
  al	
  [115]	
  and	
  by	
  using	
  aerobic	
  cultivation,	
  they	
  isolated	
  Pasteurella	
  multocida,	
  
Actinobacillus	
  spp.,	
  Staphylococcus	
  (S.	
  aureus,	
  S.	
  epidermidis),	
  Streptococcus	
  (S.	
  suis,	
  S.	
  
dysgalactiae),	
   and	
   Escherichia	
   coli	
   in	
   half	
   of	
   the	
   tonsil	
   samples	
   collected	
   from	
   two	
  
different	
  herds.	
  The	
  same	
  study	
  also	
  had	
  a	
  culture-­‐‑independent	
  approach	
  using	
  clone	
  
libraries	
  of	
  16s	
  rRNA	
  genes.	
  This	
  study	
  concluded	
  that	
  ~74%	
  of	
  the	
  members	
  of	
  the	
  
microbial	
   communities	
   in	
   pigs	
   sampled	
   from	
   two	
   different	
   herds	
   belonged	
   to	
   the	
  
families	
  Pasteurellaceae,	
  Porphyromonadaceae,	
  Bacteroidaceae	
  and	
  Prevotellaceae.	
  The	
  
authors	
   reported	
   members	
   of	
   the	
   genus	
   Actinobacillus,	
   Haemophilus,	
   Pasteurella,	
  
Porphyromonas,	
   Fusobacterium,	
   Bacteroides,	
   and	
   Prevotella	
   as	
   the	
   dominant	
   genera	
  
identified	
  in	
  both	
  herds.	
  	
  It	
  was	
  highlighted	
  from	
  this	
  study	
  that	
  although	
  there	
  were	
  
strong	
  similarities	
  between	
  the	
  herds,	
  there	
  were	
  also	
  unique	
  genera	
  that	
  differentiated	
  
them.	
  	
  
Two	
  other	
  studies	
  have	
  been	
  published	
  using	
  a	
  culture-­‐‑independent	
  approach	
  
to	
   identify	
   the	
   bacterial	
   population	
   in	
   pig	
   tonsils.	
   Lowe	
   et	
   al	
   [114]	
   used	
   a	
   454-­‐‑
pyrosequencing	
  approach	
  to	
  identify	
  the	
  core	
  microbial	
  communities	
  in	
  tonsils,	
  using	
  
samples	
   from	
   18	
   –	
   20	
   week	
   old	
   pigs,	
   which	
   included	
   the	
   same	
   samples	
   from	
   their	
  
previous	
  study	
  in	
  2011	
  [115]	
  as	
  well	
  as	
  new	
  samples	
  which	
  were	
  used	
  to	
  validate	
  a	
  non-­‐‑
invasive	
  approach	
  to	
  collect	
  samples	
  to	
  study	
  the	
  pig	
  tonsillar	
  communities,	
  since	
  the	
  
previous	
   study	
   involved	
   the	
   euthanasia	
   of	
   the	
   animals	
   and	
   collection	
   of	
   tonsils.	
   In	
   this	
  

	
  

21	
  

study,	
   the	
   authors	
   defined	
   a	
   core	
   microbiome	
   for	
   pig	
   tonsils	
   [114].	
   This	
   core	
  
microbiome	
  was	
  defined	
  as	
  composed	
  of	
  5	
  phyla	
  (out	
  of	
  17	
  identified),	
  which	
  are	
  in	
  
order	
   of	
   abundance	
   Proteobacteria,	
   Firmicutes,	
   Fusobacteria,	
   Actinobacteria	
   and	
  
Bacteroidetes,	
  which	
  comprised	
  ~98.8%	
  of	
  the	
  identified	
  organisms.	
  At	
  the	
  family	
  level,	
  
the	
   authors	
   identified	
   that	
   8	
   families	
   (out	
   of	
   61	
   identified)	
   	
   comprised	
   90.4%	
   of	
   the	
  
members	
   of	
   the	
   microbiome,	
   and	
   were	
   in	
   order	
   of	
   abundance	
   Pasteurellaceae,	
  
Moraxellaceae,	
  Fusobacteriaceae,	
  Veillonellaceae,	
  Neisseriaceae,	
  Peptostreptococcaceae,	
  
Enterobacteriaceae	
  and	
  Streptococcaceae.	
  Finally,	
  at	
  the	
  genus	
  level,	
  8	
  out	
  of	
  101	
  genera	
  
formed	
   the	
   core	
   microbiome	
   and	
   were	
   identified	
   as	
   Actinobacillus,	
   Pasteurella,	
  
Alkanindiges,	
   Fusobacterium,	
   Haemophilus,	
   Veillonella,	
   Peptostreptococcus	
   and	
  
Streptococcus,	
  comprising	
  85.1%	
  of	
  the	
  identified	
  genera.	
  	
  
The	
   most	
   recent	
   identified	
   study	
   on	
   the	
   tonsillar	
   microbiome	
   of	
   pigs	
   was	
  
performed	
  by	
  	
  Mann	
  et	
  al	
  [113],	
  who	
  studied	
  the	
  metabolically	
  active	
  bacteria	
  in	
  tonsils	
  
from	
  slaughter	
  pigs	
  and	
  identified	
  members	
  of	
  five	
  phyla	
  (Bacteroidetes,	
  Proteobacteria,	
  
Firmicutes,	
   Spirochaetes	
   and	
   Fusobacterium)	
   as	
   the	
   most	
   abundant	
   in	
   the	
   sampled	
  
animals,	
  comprising	
  	
  ~95.7	
  %	
  of	
  the	
  identified	
  phyla.	
  Within	
  those	
  five	
  phyla,	
  members	
  
of	
  14	
  different	
  genera	
  had	
  the	
  highest	
  relative	
  abundance	
  (Prevotella,	
  Porphyromonas,	
  
Campylobacter,	
   Treponema,	
   Streptococcus,	
   Serratia,	
   Paraprevotella,	
   Bacteroides,	
  
Fusobacterium,	
  Herbaspirillum,	
  Flavitalea,	
  Dehalospirillum,	
  Alysiella	
  and	
  Pseudomonas).	
  
Taken	
  together,	
  the	
  information	
  summarized	
  above	
  allows	
  us	
  to	
  conclude	
  that	
  
there	
   are	
   multiple	
   members	
   of	
   the	
   microbiome	
   shared	
   between	
   humans	
   and	
   pigs	
   at	
  
phylum,	
   family	
   and	
   even	
   genus	
   level.	
   Further,	
   there	
   are	
   multiple	
   similarities	
   shared	
  
between	
  humans	
  and	
  pigs	
  at	
  the	
  level	
  of	
  tonsil	
  structure	
  as	
  well	
  as	
  the	
  development	
  of	
  

	
  

22	
  

the	
   tonsils	
   as	
   lymphoid	
   organs.	
   	
   This	
   data	
   leads	
   us	
   to	
   believe	
   that	
   the	
   pig	
   can	
   be	
   an	
  
excellent	
  translational	
  model	
  to	
  study	
  and	
  test	
  hypotheses	
  about	
  relationships	
  between	
  
normal	
  tonsillar	
  microbiota	
  and	
  pathogenic	
  bacteria	
  in	
  humans.	
  
	
  
GOAL	
  OF	
  THIS	
  THESIS	
  
	
  
	
  

Prior	
  research	
  in	
  the	
  Mulks’	
  laboratory	
  developed	
  and	
  validated	
  a	
  non-­‐‑invasive	
  

method	
   to	
   collect	
   tonsil	
   samples	
   and	
   used	
   this	
   method	
   to	
   describe	
   the	
   core	
   tonsil	
  
microbiome	
   in	
   grower-­‐‑finisher	
   pigs	
   [114].	
   One	
   goal	
   of	
   this	
   thesis	
   project	
   was	
   to	
  
characterize	
   the	
   development	
   of	
   the	
   tonsil	
   microbiome	
   in	
   pigs	
   from	
   birth	
   to	
   market	
  
weight,	
   by	
   sequencing	
   the	
   16s	
   rRNA	
   gene	
   to	
   classify	
   or	
   categorize	
   the	
   members	
   of	
   the	
  
microbiome	
  and	
  determine	
  how	
  those	
  members	
  change	
  over	
  time.	
  Another	
  goal	
  was	
  to	
  
use	
  microscopy,	
  both	
  confocal	
  laser	
  and	
  scanning	
  electron	
  microscopy,	
  to	
  examine	
  the	
  
physical	
  structure	
  of	
  tonsils	
  and	
  their	
  attached	
  microbial	
  communities.	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

23	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
REFERENCES	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

24	
  

REFERENCES	
  
	
  
	
  
	
  
1.	
  

	
  
2.	
  
	
  
3.	
  
	
  
4.	
  
	
  
5.	
  
	
  
6.	
  
	
  
7.	
  
	
  
8.	
  
	
  
9.	
  
	
  
10.	
  
	
  
11.	
  

	
  
12.	
  

	
  

Kim	
  Y-­‐‑G,	
  Sakamoto	
  K,	
  Seo	
  S-­‐‑U,	
  Pickard	
  JM,	
  Gillilland	
  MG,	
  III,	
  Pudlo	
  NA,	
  Hoostal	
  
M,	
   Li	
   X,	
   Wang	
   TD,	
   Feehley	
   T,	
   et	
   al:	
   Neonatal	
   acquisition	
   of	
   Clostridia	
   species	
  
protects	
   against	
   colonization	
   by	
   bacterial	
   pathogens.	
   Science	
   2017,	
  
356:312-­‐‑315.	
  
Horter	
  DC,	
  Yoon	
  KJ,	
  Zimmerman	
  JJ:	
  A	
  review	
  of	
  porcine	
  tonsils	
  in	
  immunity	
  
and	
  disease.	
  Anim	
  Health	
  Res	
  Rev	
  2003,	
  4:143-­‐‑155.	
  
Lunney	
   JK:	
   Advances	
   in	
   swine	
   biomedical	
   model	
   genomics.	
   International	
  
Journal	
  of	
  Biological	
  Sciences	
  2007,	
  3:179-­‐‑184.	
  
Bassols	
  A,	
  Costa	
  C,	
  Eckersall	
  PD,	
  Osada	
  J,	
  Sabria	
  J,	
  Tibau	
  J:	
  The	
  pig	
  as	
  an	
  animal	
  
model	
  for	
  human	
  pathologies:	
  a	
  proteomics	
  perspective.	
  Proteomics	
  Clinical	
  
Applications	
  2014,	
  8:715-­‐‑731.	
  
Shultz	
   LD,	
   Ishikawa	
   F,	
   Greiner	
   DL:	
   Humanized	
   mice	
   in	
   translational	
  
biomedical	
  research.	
  Nature	
  Reviews	
  Immunology	
  2007,	
  7:118-­‐‑130.	
  
Kingsbury	
   BF,	
   Rogers	
   WM:	
   The	
   development	
   of	
   the	
   palatine	
   tonsil	
   calf	
   (Bos	
  
taurus).	
  American	
  Journal	
  of	
  Anatomy	
  1927,	
  39:379-­‐‑435.	
  
Slipka	
   J:	
   Palatine	
   tonsils:	
   their	
   evolution	
   and	
   ontogeny.	
   Acta	
   Oto-­‐‑
Laryngologica	
  1988:18-­‐‑22.	
  
Dodds	
   WJ:	
   The	
   pig	
   model	
   for	
   biomedical	
   research.	
   Federation	
   Proceedings	
  
1982,	
  41:10.	
  
Meurens	
  F,	
  Summerfield	
  A,	
  Nauwynck	
  H,	
  Saif	
  L,	
  Gerdts	
  V:	
  The	
  pig:	
  a	
  model	
  for	
  
human	
  infectious	
  diseases.	
  Trends	
  in	
  Microbiology	
  2012,	
  20:50-­‐‑57.	
  
Bustad	
   LK,	
   McClellan	
   RO:	
   Swine	
   in	
   biomedical	
   research.	
   Science	
   1966,	
  
152:1526-­‐‑1528;1530.	
  
Schook	
   L,	
   Beattie	
   C,	
   Beever	
   J,	
   Donovan	
   S,	
   Jamison	
   R,	
   Zuckermann	
   F,	
   Niemi	
   S,	
  
Rothschild	
  M,	
  Rutherford	
  M,	
  Smith	
  D:	
  Swine	
  in	
  biomedical	
  research:	
  Creating	
  
the	
   building	
   blocks	
   of	
   animal	
   models.	
   Animal	
   Biotechnology	
   2005,	
   16:183-­‐‑
190.	
  
Klymiuk	
  N,	
  Mundhenk	
  L,	
  Kraehe	
  K,	
  Wuensch	
  A,	
  Plog	
  S,	
  Emrich	
  D,	
  Langenmayer	
  
MC,	
  Stehr	
  M,	
  Holzinger	
  A,	
  Kroner	
  C,	
  et	
  al:	
  Sequential	
  targeting	
  of	
  CFTR	
  by	
  BAC	
  
vectors	
  generates	
  a	
  novel	
  pig	
  model	
  of	
  cystic	
  fibrosis.	
  J	
  Mol	
  Med	
  (Berl)	
  2012,	
  
90:597-­‐‑608.	
  
25	
  

	
  
13.	
  
	
  
14.	
  
	
  
15.	
  

	
  
16.	
  

	
  
17.	
  

	
  
18.	
  

	
  
19.	
  

	
  
20.	
  

	
  
21.	
  
	
  
22.	
  

	
  

Rogers	
  CS,	
  Stoltz	
  DA,	
  Meyerholz	
  DK,	
  Ostedgaard	
  LS,	
  Rokhlina	
  T,	
  Taft	
  PJ,	
  Rogan	
  
MP,	
  Pezzulo	
  AA,	
  Karp	
  PH,	
  Itani	
  OA,	
  et	
  al:	
  Disruption	
  of	
  the	
  CFTR	
  gene	
  produces	
  
a	
  model	
  of	
  cystic	
  fibrosis	
  in	
  newborn	
  pigs.	
  Science	
  2008,	
  321:1837-­‐‑1841.	
  
Meyerholz	
  DK,	
  Stoltz	
  DA,	
  Pezzulo	
  AA,	
  Welsh	
  MJ:	
  Pathology	
  of	
  gastrointestinal	
  
organs	
  in	
  a	
  porcine	
  model	
  of	
  cystic	
  fibrosis.	
  American	
  Journal	
  of	
  Pathology	
  
2010,	
  176:1377-­‐‑1389.	
  
Mustard	
  RA,	
  Fisher	
  J,	
  Hayman	
  S,	
  Matlow	
  A,	
  Mullen	
  JB,	
  Odumeru	
  J,	
  Roomi	
  MW,	
  
Schouten	
   BD,	
   Swanson	
   HT:	
   Cardiopulmonary	
   responses	
   to	
   Pseudomonas	
  
septicemia	
  in	
  swine:	
  an	
  improved	
  model	
  of	
  the	
  adult	
  respiratory	
  distress	
  
syndrome.	
  Lab	
  Anim	
  Sci	
  1989,	
  39:37-­‐‑43.	
  
Li	
  Bassi	
  G,	
  Rigol	
  M,	
  Marti	
  JD,	
  Saucedo	
  L,	
  Ranzani	
  OT,	
  Roca	
  I,	
  Cabanas	
  M,	
  Munoz	
  L,	
  
Giunta	
   V,	
   Luque	
   N,	
   et	
   al:	
   A	
   novel	
   porcine	
   model	
   of	
   ventilator-­‐‑associated	
  
pneumonia	
   caused	
   by	
   oropharyngeal	
   challenge	
   with	
   Pseudomonas	
  
aeruginosa.	
  Anesthesiology	
  2014,	
  120:1205-­‐‑1215.	
  
Chevaleyre	
  C,	
  Riou	
  M,	
  Brea	
  D,	
  Vandebrouck	
  C,	
  Barc	
  C,	
  Pezant	
  J,	
  Melo	
  S,	
  Olivier	
  M,	
  
Delaunay	
   R,	
   Boulesteix	
   O,	
   et	
   al:	
   The	
   pig:	
   a	
   relevant	
   model	
   for	
   evaluating	
   the	
  
neutrophil	
   serine	
   protease	
   activities	
   during	
   acute	
   Pseudomonas	
  
aeruginosa	
  lung	
  infection.	
  PLoS	
  One	
  2016,	
  11:17.	
  
Loving	
   CL,	
   Brockmeier	
   SL,	
   Vincent	
   AL,	
   Palmer	
   MV,	
   Sacco	
   RE,	
   Nicholson	
   TL:	
  
Influenza	
   virus	
   coinfection	
   with	
   Bordetella	
   bronchiseptica	
   enhances	
  
bacterial	
   colonization	
   and	
   host	
   responses	
   exacerbating	
   pulmonary	
  
lesions.	
  Microb	
  Pathog	
  2010,	
  49:237-­‐‑245.	
  
Smith	
   EA,	
   Kumar	
   SR,	
   Deventhiran	
   J,	
   Cecere	
   TE,	
   Leroith	
   T,	
   McGilliard	
   M,	
  
Elankumaran	
   S,	
   Mullarky	
   IK:	
   A	
   time	
   course	
   for	
   susceptibility	
  
to	
  Staphylococcus	
  aureus	
  respiratory	
  infection	
  during	
  Influenza	
  in	
  a	
  swine	
  
model.	
  Influenza	
  Res	
  Treat	
  2011,	
  2011:10.	
  
Elahi	
   S,	
   Brownlie	
   R,	
   Korzeniowski	
   J,	
   Buchanan	
   R,	
   O'Connor	
   B,	
   Peppler	
   MS,	
  
Halperin	
  SA,	
  Lee	
  SF,	
  Babiuk	
  LA,	
  Gerdts	
  V:	
  Infection	
  of	
  newborn	
  piglets	
  with	
  
Bordetella	
  pertussis:	
  a	
  new	
  model	
  for	
  pertussis.	
  Infect	
  Immun	
  2005,	
  73:3636-­‐‑
3645.	
  
Elahi	
  S,	
  Thompson	
  DR,	
  Strom	
  S,	
  O'Connor	
  B,	
  Babiuk	
  LA,	
  Gerdts	
  V:	
  Infection	
  with	
  
Bordetella	
  parapertussis	
  but	
  not	
  Bordetella	
  pertussis	
  causes	
  pertussis-­‐‑like	
  
disease	
  in	
  older	
  pigs.	
  J	
  Infect	
  Dis	
  2008,	
  198:384-­‐‑392.	
  
Koch	
  E,	
  Fuentes	
  G,	
  Carvajal	
  R,	
  Palma	
  R,	
  Aguirre	
  V,	
  Cruz	
  C,	
  Henriquez	
  R,	
  Calvo	
  M:	
  
Streptococcus	
   suis	
   meningitis	
   in	
   pig	
   farmers:	
   report	
   of	
   first	
   two	
   cases	
   in	
  
Chile.	
  Revista	
  Chilena	
  De	
  Infectologia	
  2013,	
  30:557-­‐‑561.	
  
26	
  

	
  
23.	
  
	
  
24.	
  
	
  
25.	
  
	
  
26.	
  

	
  
27.	
  
	
  
28.	
  
	
  
29.	
  
	
  
30.	
  
	
  
31.	
  
	
  
32.	
  
	
  
33.	
  
	
  
34.	
  
	
  

	
  

Lun	
   ZR,	
   Wang	
   QP,	
   Chen	
   XG,	
   Li	
   AX,	
   Zhu	
   XQ:	
   Streptococcus	
   suis:	
   an	
   emerging	
  
zoonotic	
  pathogen.	
  Lancet	
  Infectious	
  Diseases	
  2007,	
  7:201-­‐‑209.	
  
Robertson	
   ID,	
   Blackmore	
   DK:	
   Prevalence	
   of	
   Streptococcus	
   suis	
   type	
   1	
   and	
  
type	
   2	
   in	
   domestic	
   pigs	
   in	
   Australia	
   and	
   New	
   Zealand.	
   Veterinary	
   Record	
  
1989,	
  124:391-­‐‑394.	
  
Perch	
  B,	
  Kristjan.P,	
  Skadhaug.K:	
  Group	
  R	
  Streptococci	
  pathogenic	
  for	
  man	
  -­‐‑	
  2	
  
cases	
   of	
   meningitis	
   and	
   one	
   fatal	
   case	
   of	
   sepsis.	
   Acta	
   Pathologica	
   Et	
  
Microbiologica	
  Scandinavica	
  1968,	
  74:69-­‐‑76.	
  
Huong	
  VTL,	
  Ha	
  N,	
  Huy	
  NT,	
  Horby	
  P,	
  Nghia	
  HDT,	
  Thiem	
  VD,	
  Zhu	
  XT,	
  Hoa	
  NT,	
  Hien	
  
TT,	
  Zamora	
  J,	
  et	
  al:	
  Epidemiology,	
  clinical	
  manifestations,	
  and	
  outcomes	
  of	
  
Streptococcus	
   suis	
   infection	
   in	
   humans.	
   Emerging	
   Infectious	
   Diseases	
   2014,	
  
20:1105-­‐‑1114.	
  
Rajahram	
   GS,	
   Hameed	
   AA,	
   Menon	
   J,	
   William	
   T,	
   Tambyah	
   PA,	
   Yeo	
   TW:	
   Case	
  
report:	
   two	
   human	
   Streptococcus	
   suis	
   infections	
   in	
   Borneo,	
   Sabah,	
  
Malaysia.	
  Bmc	
  Infectious	
  Diseases	
  2017,	
  17:3.	
  
Gustavsson	
   C,	
   Ramussen	
   M:	
   Septic	
   arthritis	
   caused	
   by	
   Streptococcus	
   suis	
  
serotype	
  5	
  in	
  pig	
  farmer.	
  Emerging	
  Infectious	
  Diseases	
  2014,	
  20:489-­‐‑490.	
  
Haleis	
  A,	
  Alfa	
  M,	
  Gottschalk	
  M,	
  Bernard	
  K,	
  Ronald	
  A,	
  Manickam	
  K:	
  Meningitis	
  
caused	
   by	
   Streptococcus	
   suis	
   serotype	
   14,	
   North	
   America.	
   Emerging	
  
Infectious	
  Diseases	
  2009,	
  15:350-­‐‑352.	
  
Rao	
   SS,	
   Mariathas	
   A,	
   Teare	
   L:	
   Meningitis	
   in	
   a	
   butcher.	
   Emergency	
   Medicine	
  
Journal	
  2008,	
  25:607-­‐‑608.	
  
Taipa	
   R:	
   Streptococcus	
   suis	
   meningitis:	
   first	
   case	
   report	
   from	
   Portugal.	
  
Journal	
  of	
  Infection	
  2008,	
  56:482-­‐‑483.	
  
van	
   de	
   Beek	
   D,	
   Spanjaard	
   L,	
   de	
   Gans	
   J:	
   Streptococcus	
   suis	
   meningitis	
   in	
   the	
  
Netherlands.	
  Journal	
  of	
  Infection	
  2008,	
  57:158-­‐‑161.	
  
Nguyen	
   THM,	
   Ngo	
   TH,	
   Tran	
   VTN,	
   Le	
   DL,	
   Tran	
   THC,	
   Dinh	
   XS,	
   Nguyen	
   HP,	
   Ly	
   VC,	
  
To	
   SD,	
   James	
   C,	
   et	
   al:	
   Streptococcus	
   suis	
   meningitis	
   in	
   adults	
   in	
   Vietnam.	
  
Clinical	
  Infectious	
  Diseases	
  2008,	
  46:659-­‐‑667.	
  
Willenburg	
   KS,	
   Sentochnik	
   DE,	
   Zadoks	
   RN:	
   Human	
   Streptococcus	
   suis	
  
meningitis	
   in	
   the	
   United	
   States.	
   New	
   England	
   Journal	
   of	
   Medicine	
   2006,	
  
354:1325-­‐‑1325.	
  

27	
  

35.	
  
	
  
36.	
  
	
  
37.	
  

	
  
38.	
  
	
  
39.	
  
	
  
40.	
  

	
  
41.	
  

	
  
42.	
  
	
  
43.	
  

	
  
44.	
  
	
  
45.	
  

	
  

Lee	
  GT,	
  Chiu	
  CY,	
  Haller	
  BL,	
  Denn	
  PM,	
  Hall	
  CS,	
  Gerberding	
  JL:	
  Streptococcus	
  suis	
  
meningitis,	
  United	
  States.	
  Emerging	
  Infectious	
  Diseases	
  2008,	
  14:183-­‐‑185.	
  
Strangmann	
   E,	
   Froleke	
   H,	
   Kohse	
   KP:	
   Septic	
   shock	
   caused	
   by	
   Streptococcus	
  
suis:	
  case	
  report	
  and	
  investigation	
  of	
  a	
  risk	
  group.	
  International	
  Journal	
  of	
  
Hygiene	
  and	
  Environmental	
  Health	
  2002,	
  205:385-­‐‑392.	
  
Chang	
   B,	
   Wada	
   A,	
   Ikebe	
   T,	
   Ohnishi	
   M,	
   Mita	
   K,	
   Endo	
   M,	
   Matsuo	
   H,	
   Asatuma	
   Y,	
  
Kuramoto	
  S,	
  Sekiguchi	
  H,	
  et	
  al:	
  Characteristics	
  of	
  Streptococcus	
  suis	
  isolated	
  
from	
  patients	
  in	
  Japan.	
  Japanese	
  Journal	
  of	
  Infectious	
  Diseases	
  2006,	
  59:397-­‐‑
399.	
  
Prince-­‐‑David	
   M,	
   Salou	
   M,	
   Marois-­‐‑Crehan	
   C,	
   Assogba	
   K,	
   Plainvert	
   C,	
   Balogou	
   KA,	
  
Poyart	
  C,	
  Tazi	
  A:	
  Human	
  meningitis	
  due	
  to	
  Streptococcus	
  suis	
  in	
  Lome,	
  Togo:	
  
a	
  case	
  report.	
  Bmc	
  Infectious	
  Diseases	
  2016,	
  16:4.	
  
Halaby	
  T,	
  Hoitsma	
  E,	
  Hupperts	
  R,	
  Spanjaard	
  L,	
  Luirink	
  M,	
  Jacobs	
  J:	
  Streptococcus	
  
suis	
   meningitis,	
   a	
   poacher's	
   risk.	
   European	
   Journal	
   of	
   Clinical	
   Microbiology	
  
&	
  Infectious	
  Diseases	
  2000,	
  19:943-­‐‑945.	
  
Rith	
  S,	
  Netrabukkana	
  P,	
  Sorn	
  S,	
  Mumford	
  E,	
  Mey	
  C,	
  Holl	
  D,	
  Goutard	
  F,	
  Bunthin	
  Y,	
  
Fenwick	
   S,	
   Robertson	
   I,	
   et	
   al:	
   Serologic	
   evidence	
   of	
   human	
   influenza	
   virus	
  
infections	
  in	
  swine	
  populations,	
  Cambodia.	
  Influenza	
  and	
  Other	
  Respiratory	
  
Viruses	
  2013,	
  7:271-­‐‑279.	
  
Killian	
   ML,	
   Swenson	
   SL,	
   Vincent	
   AL,	
   Landgraf	
   JG,	
   Shu	
   B,	
   Lindstrom	
   S,	
   Xu	
   X,	
  
Klimov	
  A,	
  Zhang	
  Y,	
  Bowman	
  AS:	
  Simultaneous	
  infection	
  of	
  pigs	
  and	
  people	
  
with	
   triple-­‐‑reassortant	
   swine	
   influenza	
   virus	
   H1N1	
   at	
   a	
   U.S.	
   county	
   fair.	
  
Zoonoses	
  and	
  Public	
  Health	
  2013,	
  60:196-­‐‑201.	
  
Holyoake	
  PK,	
  Kirkland	
  PD,	
  Davis	
  RJ,	
  Arzey	
  KE,	
  Watson	
  J,	
  Lunt	
  RA,	
  Wang	
  J,	
  Wong	
  
F,	
  Moloney	
  BJ,	
  Dunn	
  SE:	
  The	
  first	
  identified	
  case	
  of	
  pandemic	
  H1N1	
  influenza	
  
in	
  pigs	
  in	
  Australia.	
  Australian	
  Veterinary	
  Journal	
  2011,	
  89:427-­‐‑431.	
  
Vincent	
   AL,	
   Swenson	
   SL,	
   Lager	
   KM,	
   Gauger	
   PC,	
   Loiacono	
   C,	
   Zhang	
   Y:	
  
Characterization	
   of	
   an	
   influenza	
   A	
   virus	
   isolated	
   from	
   pigs	
   during	
   an	
  
outbreak	
  of	
  respiratory	
  disease	
  in	
  swine	
  and	
  people	
  during	
  a	
  county	
  fair	
  
in	
  the	
  United	
  States.	
  Veterinary	
  Microbiology	
  2009,	
  137:51-­‐‑59.	
  
Thompson	
  RL,	
  Sande	
  MA,	
  Wenzel	
  RP,	
  Hoke	
  CH,	
  Gwaltney	
  JM:	
   Swine	
  influenza	
  
infection	
  in	
  civilians:	
  report	
  of	
  2	
  cases.	
  New	
  England	
  Journal	
  of	
  Medicine	
  1976,	
  
295:714-­‐‑715.	
  
Gray	
  GC,	
  McCarthy	
  T,	
  Capuano	
  AW,	
  Setterquist	
  SF,	
  Olsen	
  CW,	
  Alavania	
  MC,	
  Lynch	
  
CF:	
  Swine	
  workers	
  and	
  swine	
  influenza	
  virus	
  infections.	
  Emerging	
  Infectious	
  
Diseases	
  2007,	
  13:1871-­‐‑1878.	
  
28	
  

	
  
46.	
  
	
  
47.	
  
	
  
48.	
  
	
  
49.	
  

	
  
50.	
  
	
  
51.	
  

	
  
52.	
  
	
  
53.	
  
	
  
54.	
  
	
  
55.	
  
	
  
56.	
  

	
  

	
  

Schnurrenberger	
   PR,	
   Woods	
   GT,	
   Martin	
   RJ:	
   Serologic	
   evidence	
   of	
   human	
  
infection	
   with	
   swine	
   influenza	
   virus.	
   American	
   Review	
   of	
   Respiratory	
   Disease	
  
1970,	
  102:6.	
  
Wentworth	
   DE,	
   McGregor	
   MW,	
   Macklin	
   MD,	
   Neumann	
   V,	
   Hinshaw	
   VS:	
  
Transmission	
   of	
   swine	
   influenza	
   virus	
   to	
   humans	
   after	
   exposure	
   to	
  
experimentally	
  infected	
  pigs.	
  Journal	
  of	
  Infectious	
  Diseases	
  1997,	
  175:7-­‐‑15.	
  
Gregory	
  V,	
  Bennett	
  M,	
  Thomas	
  Y,	
  Kaiser	
  L,	
  Wunderli	
  W,	
  Matter	
  H,	
  Hay	
  A,	
  Lin	
  YP:	
  
Human	
   infection	
   by	
   a	
   swine	
   influenza	
   A	
   (H1N1)	
   virus	
   in	
   Switzerland.	
  
Archives	
  of	
  Virology	
  2003,	
  148:793-­‐‑802.	
  
Wells	
   DL,	
   Hopfensperger	
   DJ,	
   Arden	
   NH,	
   Harmon	
   MW,	
   Davis	
   JP,	
   Tipple	
   MA,	
  
Schonberger	
  LB:	
  Swine	
  influenza	
  virus	
  infections:	
  transmission	
  from	
  ill	
  pigs	
  
to	
   humans	
   at	
   a	
   Wisconsin	
   agricultural	
   fair	
   and	
   subsequent	
   probable	
  
person	
   to	
   person	
   transmission.	
   Jama-­‐‑Journal	
   of	
   the	
   American	
   Medical	
  
Association	
  1991,	
  265:478-­‐‑481.	
  
Williams	
   DM,	
   Lawson	
   GHK,	
   Rowland	
   AC:	
   Streptococcal	
   infection	
   in	
   piglets-­‐‑
palatine	
   tonsils	
   as	
   portals	
   of	
   entry	
   for	
   Streptococcus	
   suis.	
   Research	
   in	
  
Veterinary	
  Science	
  1973,	
  15:352-­‐‑362.	
  
Surjan	
  L,	
  Brandtzaeg	
  P,	
  Berdal	
  P:	
  Immunoglobulin	
  systems	
  of	
  human	
  tonsils.	
  
II.	
  Patients	
  with	
  chronic	
  tonsillitis	
  or	
  tonsillar	
  hyperplasia:	
  quantification	
  
of	
  Ig-­‐‑producing	
  cells,	
  tonsillar	
  morphometry	
  and	
  serum	
  Ig	
  concentrations.	
  
Clinical	
  and	
  Experimental	
  Immunology	
  1978,	
  31:382-­‐‑390.	
  
Grivel	
   J-­‐‑C,	
   Margolis	
   L:	
   Use	
   of	
   human	
   tissue	
   explants	
   to	
   study	
   human	
  
infectious	
  agents.	
  Nature	
  Protocols	
  2009,	
  4:256-­‐‑269.	
  
Perry	
   M,	
   Whyte	
   A:	
   Immunology	
   of	
   the	
   tonsils.	
   Immunology	
   Today	
   1998,	
  
19:414-­‐‑421.	
  
Brandtzaeg	
   P:	
   Regionalized	
   immune	
   function	
   of	
   tonsils	
   and	
   adenoids.	
  
Immunology	
  Today	
  1999,	
  20:383-­‐‑384.	
  
Casteleyn	
   C,	
   Simoens	
   P,	
   Van	
   den	
   Broeck	
   W:	
   Terminology	
   of	
   the	
   tonsils.	
  
Anatomia	
  Histologia	
  Embryologia	
  2011,	
  40:204-­‐‑209.	
  
Casteleyn	
   C,	
   Breugelmans	
   S,	
   Simoens	
   P,	
   Van	
   den	
   Broeck	
   W:	
   The	
   tonsils	
  
revisited:	
   review	
   of	
   the	
   anatomical	
   localization	
   and	
   histological	
  
characteristics	
  of	
  the	
  tonsils	
  of	
  domestic	
  and	
  laboratory	
  animals.	
  Clinical	
  &	
  
Developmental	
  Immunology	
  2011:1-­‐‑14.	
  

29	
  

57.	
  
	
  
58.	
  
	
  
59.	
  
	
  
60.	
  
	
  
61.	
  
	
  
62.	
  

	
  
63.	
  
	
  
64.	
  
	
  
65.	
  
	
  
66.	
  
	
  
67.	
  
	
  
68.	
  
	
  
69.	
  

Banks	
   WJ:	
   Applied	
   veterinary	
   histology.	
   Third	
   edn:	
   St.	
   Louis,	
   Mo.	
   :	
   Mosby	
   -­‐‑	
  
Year	
  Book;	
  1993.	
  
Effat	
   KG,	
   Milad	
   M:	
   Comparative	
   study	
   of	
   palatine	
   tonsil	
   histology	
   in	
  
mammals,	
   with	
   special	
   reference	
   to	
   tonsillar	
   salivary	
   glands.	
   Journal	
   of	
  
Laryngology	
  and	
  Otology	
  2007,	
  121:468-­‐‑471.	
  
Arends	
   JP,	
   Hartwig	
   N,	
   Rudolphy	
   M,	
   Zanen	
   HC:	
   Carrier	
   rate	
   of	
   Streptococcus	
  
suis	
  capsular	
  type	
  2	
  in	
  palatine	
  tonsils	
  of	
  slaughtered	
  pigs.	
  Journal	
  of	
  Clinical	
  
Microbiology	
  1984,	
  20:945-­‐‑947.	
  
Williams	
  DM:	
  Palatine	
  tonsils	
  of	
  pig.	
  Journal	
  of	
  Anatomy	
  1971,	
  110:156.	
  
Williams	
  DM,	
  Rowland	
  AC:	
  Palatine	
  tonsils	
  of	
  pig:	
  afferent	
  route	
  to	
  lymphoid	
  
tissue.	
  Journal	
  of	
  Anatomy	
  1972,	
  113:7.	
  
Wilson	
   SM,	
   Norton	
   P,	
   Haverson	
   K,	
   Leigh	
   J,	
   Bailey	
   M:	
   Interactions	
   between	
  
Streptococcus	
   suis	
   serotype	
   2	
   and	
   cells	
   of	
   the	
   myeloid	
   lineage	
   in	
   the	
  
palatine	
  tonsil	
  of	
  the	
  pig.	
  Veterinary	
  Immunology	
  and	
  Immunopathology	
  2007,	
  
117:116-­‐‑123.	
  
Wood	
  RL,	
  Rose	
  R:	
  Populations	
  of	
  Salmonella	
  typhimurium	
  in	
  internal	
  organs	
  
of	
   experimentally	
   infected	
   carrier	
   swine.	
   American	
   Journal	
   of	
   Veterinary	
  
Research	
  1992,	
  53:653-­‐‑658.	
  
Kumar	
  P,	
  Timoney	
  JF:	
  Histology,	
  immunohistochemistry	
  and	
  ultrastructure	
  
of	
   the	
   equine	
   palatine	
   tonsil.	
   Anatomia	
   Histologia	
   Embryologia-­‐‑Journal	
   of	
  
Veterinary	
  Medicine	
  Series	
  C	
  2005,	
  34:192-­‐‑198.	
  
Nave	
   H,	
   Gebert	
   A,	
   Pabst	
   R:	
   Morphology	
   and	
   immunology	
   of	
   the	
   human	
  
palatine	
  tonsil.	
  Anatomy	
  and	
  Embryology	
  2001,	
  204:367-­‐‑373.	
  
Howard	
   JC,	
   Hunt	
   SV,	
   Gowans	
   JL:	
   Identification	
  of	
   marrow-­‐‑derived	
   and	
  
thymus-­‐‑derived	
  small	
  lymphocytes	
  in	
  lymphoid-­‐‑tissue	
  and	
  thoracic-­‐‑duct	
  
lymph	
  of	
  normal	
  rats.	
  Journal	
  of	
  Experimental	
  Medicine	
  1972,	
  135:200-­‐‑219.	
  
Maclennan	
   ICM:	
   Germinal	
   centers.	
   Annual	
   Review	
   of	
   Immunology	
   1994,	
  
12:117-­‐‑139.	
  
Brandtzaeg	
  P:	
  The	
  B-­‐‑cell	
  development	
  in	
  tonsillar	
  lymphoid	
  follicles.	
  Acta	
  
Oto-­‐‑Laryngologica	
  1996:55-­‐‑59.	
  
Kataura	
   A,	
   Harabuchi	
   Y,	
   Matsuyama	
   H,	
   Yamanaka	
   N:	
   Immunohistological	
  
studies	
   on	
   immunocompetent	
   cells	
   in	
   palatine	
   tonsil.	
   Tonsils:	
   a	
   clinically	
  
oriented	
  update	
  1992,	
  47:97-­‐‑100.	
  

	
  
	
  

30	
  

70.	
  
	
  
71.	
  
	
  
72.	
  

	
  
73.	
  
	
  
74.	
  
	
  
75.	
  
	
  
76.	
  
	
  
77.	
  
	
  
78.	
  
	
  
79.	
  
	
  
80.	
  
	
  
81.	
  
	
  
82.	
  

	
  

Tohya	
   K,	
   Kimura	
   M:	
   Scanning,	
   transmission	
   and	
   immunoelectron	
  
microscopic	
   studies	
   on	
   crypt	
   epithelium	
   of	
   palatine	
   tonsil	
   in	
   laboratory	
  
musk	
  shrew	
  (Suncus	
  murinus).	
  Electron	
  Microscopy	
  II	
  1992,	
  2:256-­‐‑257.	
  
van	
  Buchem	
  FL,	
  Kuijpers	
  W:	
  On	
  the	
  origin	
  of	
  lymphoid	
  cells	
  in	
  the	
  palatine	
  
tonsil.	
  Acta	
  Otolaryngol	
  1973,	
  75:527-­‐‑534.	
  
Ruco	
   LP,	
   Uccini	
   S,	
   Stoppacciaro	
   A,	
   Pilozzi	
   E,	
   Morrone	
   S,	
   Gallo	
   A,	
   Devincentiis	
   M,	
  
Santoni	
   A,	
   Baroni	
   CD:	
   The	
   lymphoepithelial	
   organization	
   of	
   the	
   tonsil:	
   an	
  
immunohistochemical	
   study	
   in	
   chronic	
   recurrent	
   tonsillitis.	
   Journal	
   of	
  
Pathology	
  1995,	
  176:391-­‐‑398.	
  
Ramsay	
  AJ:	
  The	
  development	
  of	
  the	
  palatine	
  tonsil	
  (cat).	
  American	
  Journal	
  of	
  
Anatomy	
  1935,	
  57:171-­‐‑203.	
  
Casteleyn	
  C,	
  Van	
  den	
  Broeck	
  W,	
  Simoens	
  P:	
  Histological	
  characteristics	
  and	
  
stereological	
   volume	
   assessment	
   of	
   the	
   ovine	
   tonsils.	
   Veterinary	
  
Immunology	
  and	
  Immunopathology	
  2007,	
  120:124-­‐‑135.	
  
Kassay	
   D,	
   Sandor	
   A:	
   The	
   crypt	
   system	
   of	
   the	
   palatine	
   tonsils.	
   Archives	
   of	
  
Otolaryngology	
  1962,	
  75:144-­‐‑155.	
  
Belz	
  GT,	
  Heath	
  TJ:	
  Tonsils	
  of	
  the	
  soft	
  palate	
  of	
  young	
  pigs:	
  crypt	
  structure	
  
and	
  lymphoepithelium.	
  Anatomical	
  Record	
  1996,	
  245:102-­‐‑113.	
  
Minear	
   WL,	
   Arey	
   LB,	
   Milton	
   JT:	
   Prenatal	
   and	
   postnatal	
   development	
   and	
  
form	
   of	
   crypts	
   of	
   human	
   palatine	
   tonsil.	
   Archives	
   of	
   Otolaryngology	
   1937,	
  
25:487-­‐‑519.	
  
Sato	
   Y,	
   Wake	
   K,	
   Watanabe	
   I:	
   Differentiation	
   of	
   crypt	
   epithelium	
   in	
   human	
  
palatine	
   tonsils:	
   the	
   microenvironment	
   of	
   crypt	
   epithelium	
   as	
   a	
  
lymphoepithelial	
  organ.	
  Archives	
  of	
  Histology	
  and	
  Cytology	
  1990,	
  53:41-­‐‑54.	
  
Ramos	
  JA,	
  Ramis	
  AJ,	
  Marco	
  A,	
  Domingo	
  M,	
  Rabanal	
  R,	
  Ferrer	
  L:	
  Histochemical	
  
and	
  immunohistochemical	
  study	
  of	
  the	
  mucosal	
  lymphoid	
  system	
  in	
  swine.	
  
American	
  Journal	
  of	
  Veterinary	
  Research	
  1992,	
  53:1418-­‐‑1426.	
  
Belz	
   GT,	
   Heath	
   TJ:	
   The	
   epithelium	
   of	
   canine	
   palatine	
   tonsils.	
   Anatomy	
   and	
  
Embryology	
  1995,	
  192:189-­‐‑194.	
  
Belz	
   GT,	
   Heath	
   TJ:	
   Intercellular	
   and	
   lymphatic	
   pathways	
   of	
   the	
   canine	
  
palatine	
  tonsils.	
  Journal	
  of	
  Anatomy	
  1995,	
  187:93-­‐‑105.	
  
Olah	
  I,	
  Everett	
  NB:	
  Surface	
  epithelium	
  of	
  the	
  rabbit	
  palatine	
  tonsil:	
  scanning	
  
and	
   transmission	
   electron	
   microscopic	
   study.	
   J	
   Reticuloendothel	
   Soc	
   1975,	
  
18:53-­‐‑62.	
  
31	
  

	
  
83.	
  
	
  
84.	
  
	
  
85.	
  
	
  
86.	
  
	
  
87.	
  
	
  
88.	
  
	
  
89.	
  
	
  
90.	
  
	
  
91.	
  

	
  
92.	
  
	
  
93.	
  
	
  
94.	
  

	
  

Fredriksen	
  F,	
  Raisanen	
  S,	
  Myklebust	
  R,	
  Stenfors	
  LE:	
  Bacterial	
  adherence	
  to	
  the	
  
surface	
   and	
   isolated	
   cell	
   epithelium	
   of	
   the	
   palatine	
   tonsils.	
   Acta	
   Oto-­‐‑
Laryngologica	
  1996,	
  116:620-­‐‑626.	
  
Lilja	
   M,	
   Silvola	
   J,	
   Raisanen	
   S,	
   Stenfors	
   LE:	
   Where	
   are	
   the	
   receptors	
   for	
  
Streptococcus	
   pyogenes	
   located	
   on	
   the	
   tonsillar	
   surface	
   epithelium?	
  
International	
  Journal	
  of	
  Pediatric	
  Otorhinolaryngology	
  1999,	
  50:37-­‐‑43.	
  
Howie	
   AJ:	
   Scanning	
   and	
   transmission	
   electron-­‐‑microscopy	
   on	
   the	
  
epithelium	
  of	
  human	
  palatine	
  tonsils.	
  Journal	
  of	
  Pathology	
  1980,	
  130:8.	
  
Gebert	
   A:	
   M	
   cells	
   in	
   the	
   rabbit	
   palatine	
   tonsil:	
   the	
   distribution,	
   spatial	
  
arrangement	
   and	
   membrane	
   subdomains	
   as	
   defined	
   by	
   confocal	
   lectin	
  
histochemistry.	
  Anatomy	
  and	
  Embryology	
  1997,	
  195:353-­‐‑358.	
  
Gebert	
  A,	
  Pabst	
  R:	
  M	
  cells	
  at	
  locations	
  outside	
  the	
  gut.	
  Seminars	
  in	
  Immunology	
  
1999,	
  11:165-­‐‑170.	
  
Gebert	
   A:	
   Identification	
   of	
   M	
   cells	
   in	
   the	
   rabbit	
   tonsil	
   by	
   vimentin	
  
immunohistochemistry	
   and	
   in	
   vivo	
   protein	
   transport.	
   Histochemistry	
   and	
  
Cell	
  Biology	
  1995,	
  104:211-­‐‑220.	
  
Perry	
   ME:	
   The	
   specialized	
   structure	
   of	
   crypt	
   epithelium	
   in	
   the	
   human	
  
palatine	
   tonsil	
   and	
   its	
   functional	
   significance.	
   Journal	
   of	
   Anatomy	
   1994,	
  
185:111-­‐‑127.	
  
Tang	
   XY,	
   Hori	
   S,	
   Osamura	
   RY,	
   Tsutsumi	
   Y:	
   Reticular	
   crypt	
   epithelium	
   and	
  
intraepithelial	
  lymphoid	
  cells	
  in	
  the	
  hyperplastic	
  human	
  palatine	
  tonsil	
  -­‐‑	
  
an	
  immunohistochemical	
  analysis.	
  Pathology	
  International	
  1995,	
  45:34-­‐‑44.	
  
Baykan	
   M,	
   Celik	
   I,	
   Gezici	
   M,	
   Donmez	
   HH,	
   Eken	
   E,	
   Sur	
   E,	
   Ozkan	
   Y:	
   A	
   light	
  
microscopic	
   study	
   on	
   the	
   uptake	
   and	
   transportation	
   route	
   of	
   carbon	
  
particles	
  in	
  the	
  canine	
  palatine	
  tonsil.	
  Revue	
  De	
  Medecine	
  Veterinaire	
  2001,	
  
152:709-­‐‑715.	
  
Isaacson	
   G,	
   Parikh	
   T:	
   Developmental	
   anatomy	
   of	
   the	
   tonsil	
   and	
   its	
  
implications	
   for	
   intracapsular	
   tonsillectomy.	
   International	
   Journal	
   of	
  
Pediatric	
  Otorhinolaryngology	
  2008,	
  72:89-­‐‑96.	
  
Muretto	
   P:	
   Immunohistochemical	
   study	
   of	
   tonsils	
   from	
   newborn	
   infants	
  
with	
   emphasis	
   on	
   follicular	
   dendritic	
   reticulum	
   cells.	
   European	
   Journal	
   of	
  
Histochemistry	
  1998,	
  42:189-­‐‑195.	
  
Brandtzaeg	
  P,	
  Halstensen	
  TS:	
  Immunology	
  and	
  immunopathology	
  of	
  tonsils.	
  
Tonsils	
  :	
  a	
  Clinically	
  Oriented	
  Update	
  1992,	
  47:12.	
  
32	
  

	
  
95.	
  
	
  
96.	
  

	
  
97.	
  
	
  
98.	
  
	
  
99.	
  

Vallesayoub	
  Y,	
  Govan	
  HL,	
  Braun	
  J:	
  Evolving	
  abundance	
  and	
  clonal	
  pattern	
  of	
  
human	
  germinal	
  center	
  B-­‐‑cells	
  during	
  childhood.	
  Blood	
  1990,	
  76:17-­‐‑23.	
  
Siegel	
  G,	
  Linse	
  R,	
  Macheleidt	
  S:	
  Factors	
  of	
  tonsillar	
  involution:	
  age-­‐‑dependent	
  
changes	
   in	
   B-­‐‑cell	
   activation	
   and	
   Langerhans	
   cell	
   density.	
   Archives	
   of	
   Oto-­‐‑
Rhino-­‐‑Laryngology-­‐‑Archiv	
   Fur	
   Ohren-­‐‑Nasen-­‐‑Und	
   Kehlkopfheilkunde	
   1982,	
  
236:261-­‐‑269.	
  
Morente	
   M,	
   Piris	
   MA,	
   Orradre	
   JL,	
   Rivas	
   C,	
   Villuendas	
   R:	
   Human	
   tonsil	
  
intraepithelial	
   B-­‐‑cells:	
   a	
   marginal	
   zone-­‐‑related	
   subpopulation.	
   Journal	
   of	
  
Clinical	
  Pathology	
  1992,	
  45:668-­‐‑672.	
  
Choi	
  G,	
  Suh	
  YL,	
  Lee	
  HM,	
  Jung	
  KY,	
  Hwang	
  SJ:	
  Prenatal	
  and	
  postnatal	
  changes	
  of	
  
the	
  human	
  tonsillar	
  crypt	
  epithelium.	
  Acta	
  Oto-­‐‑Laryngologica	
  1996:28-­‐‑33.	
  
Brandtzaeg	
  P,	
  Surjan	
  L,	
  Berdal	
  P:	
  Immunoglobulin	
  systems	
  of	
  human	
  tonsils	
  
.1.	
  Control	
  subjects	
  of	
  various	
  ages	
  -­‐‑	
   quantification	
  of	
  Ig-­‐‑producing	
  cells,	
  
tonsillar	
   morphometry	
   and	
   serum	
   Ig	
   concentrations.	
   Clinical	
   and	
  
Experimental	
  Immunology	
  1978,	
  31:367-­‐‑381.	
  

	
  
100.	
   Quidingjarbrink	
   M,	
   Granstrom	
   G,	
   Nordstrom	
   I,	
   Holmgren	
   J,	
   Czerkinsky	
   C:	
  
Induction	
   of	
   compartmentalized	
   B-­‐‑cell	
   responses	
   in	
   human	
   tonsils.	
  
Infection	
  and	
  Immunity	
  1995,	
  63:853-­‐‑857.	
  
	
  
101.	
   Levin	
   PM:	
   The	
   development	
   of	
   the	
   tonsil	
   of	
   the	
   domestic	
   pig.	
   Anatomical	
  
Record	
  1930,	
  45:189-­‐‑201.	
  
	
  
102.	
   Anderson	
  JC:	
  The	
  use	
  of	
  germ-­‐‑free	
  piglets	
  in	
  the	
  study	
  of	
  lymphoid	
  tissue	
  
and	
  germinal	
  centre	
  formation.	
  Advances	
  in	
  experimental	
  medicine	
  and	
  biology	
  
1973,	
  29:643-­‐‑649.	
  
	
  
103.	
   Anderson	
   JC:	
   The	
   response	
   of	
   the	
   tonsil	
   and	
   associated	
   lymph	
   nodes	
   of	
  
gnotobiotic	
  piglets	
  to	
  the	
  presence	
  of	
  bacterial	
  antigen	
  in	
  the	
  oral	
  cavity.	
  J	
  
Anat	
  1974,	
  117:191-­‐‑198.	
  
	
  
104.	
   Wilson	
  S,	
  Norton	
  P,	
  Haverson	
  K,	
  Leigh	
  J,	
  Bailey	
  M:	
  Development	
  of	
  the	
  palatine	
  
tonsil	
  in	
  conventional	
  and	
  germ-­‐‑free	
  piglets.	
  Developmental	
  and	
  Comparative	
  
Immunology	
  2005,	
  29:977-­‐‑987.	
  
	
  
105.	
   Human	
   Microbiome	
   Project	
   C:	
   A	
   framework	
   for	
   human	
   microbiome	
  
research.	
  Nature	
  2012,	
  486:215-­‐‑221.	
  
	
  
106.	
   Human	
   Microbiome	
   Project	
   C:	
   Structure,	
   function	
   and	
   diversity	
   of	
   the	
  
healthy	
  human	
  microbiome.	
  Nature	
  2012,	
  486:207-­‐‑214.	
  
	
  
	
  

33	
  

107.	
   Eren	
  AM,	
  Borisy	
  GG,	
  Huse	
  SM,	
  Welch	
  JLM:	
  Oligotyping	
  analysis	
  of	
  the	
  human	
  
oral	
  microbiome.	
  Proceedings	
  of	
  the	
  National	
  Academy	
  of	
  Sciences	
  of	
  the	
  United	
  
States	
  of	
  America	
  2014,	
  111:E2875-­‐‑E2884.	
  
	
  
108.	
   Aas	
   JA,	
   Paster	
   BJ,	
   Stokes	
   LN,	
   Olsen	
   I,	
   Dewhirst	
   FE:	
   Defining	
   the	
   normal	
  
bacterial	
   flora	
   of	
   the	
   oral	
   cavity.	
   Journal	
   of	
   Clinical	
   Microbiology	
   2005,	
  
43:5721-­‐‑5732.	
  
	
  
109.	
   Dewhirst	
   FE,	
   Chen	
   T,	
   Izard	
   J,	
   Paster	
   BJ,	
   Tanner	
   ACR,	
   Yu	
   WH,	
   Lakshmanan	
   A,	
  
Wade	
   WG:	
   The	
   Human	
   Oral	
   Microbiome.	
   Journal	
   of	
   Bacteriology	
   2010,	
  
192:5002-­‐‑5017.	
  
	
  
110.	
   Chen	
  T,	
  Yu	
  WH,	
  Izard	
  J,	
  Baranova	
  OV,	
  Lakshmanan	
  A,	
  Dewhirst	
  FE:	
  The	
  human	
  
oral	
  microbiome	
  database:	
  a	
  web	
  accessible	
  resource	
  for	
  investigating	
  oral	
  
microbe	
   taxonomic	
   and	
   genomic	
   information.	
   Database-­‐‑the	
   Journal	
   of	
  
Biological	
  Databases	
  and	
  Curation	
  2010.	
  
	
  
111.	
   Huse	
   SM,	
   Ye	
   YZ,	
   Zhou	
   YJ,	
   Fodor	
   AA:	
   A	
   core	
   human	
   microbiome	
   as	
   viewed	
  
through	
  16s	
  rRNA	
  sequence	
  clusters.	
  Plos	
  One	
  2012,	
  7:12.	
  
	
  
112.	
   Jensen	
  A,	
  Fago-­‐‑Olsen	
  H,	
  Sorensen	
  CH,	
  Kilian	
  M:	
  Molecular	
  mapping	
  to	
  species	
  
level	
  of	
  the	
  tonsillar	
  crypt	
  microbiota	
  associated	
  with	
  health	
  and	
  recurrent	
  
tonsillitis.	
  Plos	
  One	
  2013,	
  8:15.	
  
	
  
113.	
   Mann	
   E,	
   Pinior	
   B,	
   Wetzels	
   SU,	
   Metzler-­‐‑Zebeli	
   BU,	
   Wagner	
   M,	
   Schmitz-­‐‑Esser	
   S:	
  
The	
  metabolically	
  active	
  bacterial	
  microbiome	
  of	
  tonsils	
  and	
  mandibular	
  
lymph	
  nodes	
  of	
  slaughter	
  pigs.	
  Frontiers	
  in	
  Microbiology	
  2015,	
  6:10.	
  
	
  
114.	
   Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Defining	
   the	
   "core	
   microbiome"	
   of	
   the	
   microbial	
   communities	
   in	
   the	
  
tonsils	
  of	
  healthy	
  pigs.	
  Bmc	
  Microbiology	
  2012,	
  12:14.	
  
	
  
115.	
   Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Microbial	
  communities	
  in	
  the	
  tonsils	
  of	
  healthy	
  pigs.	
  Veterinary	
  Microbiology	
  
2011,	
  147:346-­‐‑357.	
  
	
  
116.	
   Hvistendahl	
   M:	
   Pigs	
   as	
   Stand-­‐‑Ins	
   for	
   microbiome	
   studies.	
   Science	
   2012,	
  
336:1250-­‐‑1250.	
  
	
  
117.	
   Kernaghan	
   S,	
   Bujold	
   AR,	
   MacInnes	
   JI:	
   The	
   microbiome	
   of	
   the	
   soft	
   palate	
   of	
  
swine.	
  Animal	
  Health	
  Research	
  Reviews	
  2012,	
  13:110-­‐‑120.	
  
	
  
118.	
   Toner	
   JG,	
   Stewart	
   TJ,	
   Campbell	
   JB,	
   Hunter	
   J:	
   Tonsil	
   flora	
   in	
   the	
   very	
   young	
  
tonsillectomy	
  patient.	
  Clinical	
  Otolaryngology	
  1986,	
  11:171-­‐‑174.	
  
	
  

	
  

34	
  

119.	
   Brook	
  I,	
  Foote	
  PA:	
  Microbiology	
  of	
  normal	
  tonsils.	
  Annals	
  of	
  Otology	
  Rhinology	
  
and	
  Laryngology	
  1990,	
  99:980-­‐‑983.	
  
	
  
120.	
   Gaffney	
   RJ,	
   Timon	
   CI,	
   Freeman	
   DF,	
   Walsh	
   MA,	
   Cafferkey	
   MT:	
   Bacteriology	
   of	
  
tonsil	
   and	
   adenoid	
   and	
   sampling	
   techniques	
   of	
   adenoidal	
   bacteriology.	
  
Respiratory	
  Medicine	
  1993,	
  87:303-­‐‑308.	
  
	
  
121.	
   Gaffney	
   RJ,	
   Freeman	
   DJ,	
   Walsh	
   MA,	
   Cafferkey	
   MT:	
   Differences	
   in	
   tonsil	
   core	
  
bacteriology	
  in	
  adults	
  and	
  children	
  -­‐‑	
   a	
  prospective	
  study	
  of	
  262	
  patients.	
  
Respiratory	
  Medicine	
  1991,	
  85:383-­‐‑388.	
  
	
  
122.	
   Gaffney	
   RJ,	
   Cafferkey	
   MT:	
   Bacteriology	
   of	
   normal	
   and	
   diseased	
   tonsils	
  
assessed	
   by	
   fine-­‐‑needle	
   aspiration:	
   Haemophilus	
   influenzae	
   and	
   the	
  
pathogenesis	
  of	
  recurrent	
  acute	
  tonsillitis.	
  Clin	
  Otolaryngol	
  Allied	
  Sci	
  1998,	
  
23:181-­‐‑185.	
  
	
  
123.	
   Kasenõmm	
   P,	
   Kull	
   M,	
   Mikelsaar	
   M:	
   Association	
   between	
   tonsillar	
   core	
  
microflora	
   and	
   post-­‐‑tonsillectomy	
   bacteremia.	
   Microbial	
   Ecology	
   in	
   Health	
  
and	
  Disease	
  2002,	
  14:122-­‐‑127.	
  
	
  
124.	
   Liu	
  CM,	
  Cosetti	
  MK,	
  Aziz	
  M,	
  Buchhagen	
  JL,	
  Contente-­‐‑Cuomo	
  TL,	
  Price	
  LB,	
  Keim	
  
PS,	
   Lalwani	
   AK:	
   The	
   otologic	
   microbiome	
   -­‐‑	
   a	
   study	
   of	
   the	
   bacterial	
  
microbiota	
   in	
   a	
   pediatric	
   patient	
   with	
   chronic	
   serous	
   otitis	
   media	
   using	
  
16S	
   rRNA	
   gene-­‐‑based	
   pyrosequencing.	
   Archives	
   of	
   Otolaryngology-­‐‑Head	
  
&	
  Neck	
  Surgery	
  2011,	
  137:664-­‐‑668.	
  
	
  
125.	
   Segata	
  N,	
  Haake	
  SK,	
  Mannon	
  P,	
  Lemon	
  KP,	
  Waldron	
  L,	
  Gevers	
  D,	
  Huttenhower	
  C,	
  
Izard	
   J:	
   Composition	
   of	
   the	
   adult	
   digestive	
   tract	
   bacterial	
   microbiome	
  
based	
   on	
   seven	
   mouth	
   surfaces,	
   tonsils,	
   throat	
   and	
   stool	
   samples.	
   Genome	
  
Biology	
  2012,	
  13:18.	
  
	
  
126.	
   Humphreys	
   GJ,	
   McBain	
   AJ:	
   Continuous	
   culture	
   of	
   sessile	
   human	
  
oropharyngeal	
   microbiotas.	
   Journal	
   of	
   Medical	
   Microbiology	
   2013,	
   62:906-­‐‑
916.	
  
	
  
127.	
   Devriese	
  LA,	
  Hommez	
  J,	
  Pot	
  B,	
  Haesebrouck	
  F:	
  Identification	
  and	
  composition	
  
of	
  the	
  streptococcal	
  and	
  enterococcal	
  flora	
  of	
  tonsils,	
  intestines	
  and	
  feces	
  
of	
  pigs.	
  Journal	
  of	
  Applied	
  Bacteriology	
  1994,	
  77:31-­‐‑36.	
  
	
  
128.	
   Baele	
   M,	
   Chiers	
   K,	
   Devriese	
   LA,	
   Smith	
   HJ,	
   Wisselink	
   HJ,	
   Vaneechoutte	
   M,	
  
Haesebrouck	
  F:	
  The	
  Gram-­‐‑positive	
  tonsillar	
  and	
  nasal	
  flora	
  of	
  piglets	
  before	
  
and	
  after	
  weaning.	
  Journal	
  of	
  Applied	
  Microbiology	
  2001,	
  91:997-­‐‑1003.	
  
	
  
129.	
   MacInnes	
   JI,	
   Gottschalk	
   M,	
   Lone	
   AG,	
   Metcalf	
   DS,	
   Ojha	
   S,	
   Rosendal	
   T,	
   Watson	
   SB,	
  
Friendship	
   RM:	
   Prevalence	
   of	
   Actinobacillus	
   pleuropneumoniae,	
  
Actinobacillus	
   suis,	
   Haemophilus	
   parasuis,	
   Pasteurella	
   multocida,	
   and	
  
	
  

35	
  

Streptococcus	
  suis	
  in	
  representative	
  Ontario	
  swine	
  herds.	
  Canadian	
  Journal	
  
of	
   Veterinary	
   Research-­‐‑Revue	
   Canadienne	
   De	
   Recherche	
   Veterinaire	
   2008,	
  
72:242-­‐‑248.	
  
	
  
130.	
   O'Sullivan	
   T,	
   Friendship	
   R,	
   Blackwell	
   T,	
   Pearl	
   D,	
   McEwen	
   B,	
   Carman	
   S,	
   Slavic	
   D,	
  
Dewey	
   C:	
   Microbiological	
   identification	
   and	
   analysis	
   of	
   swine	
   tonsils	
  
collected	
   from	
   carcasses	
   at	
   slaughter.	
   Canadian	
   Journal	
   of	
   Veterinary	
  
Research-­‐‑Revue	
  Canadienne	
  De	
  Recherche	
  Veterinaire	
  2011,	
  75:106-­‐‑111.	
  
	
  

	
  

36	
  

CHAPTER	
  2.	
  DEVELOPMENT	
  OF	
  THE	
  TONSILLAR	
  MICROBIOME	
  IN	
  PIGS	
  FROM	
  NEWBORN	
  
THROUGH	
  WEANING	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

37	
  

INTRODUCTION	
  
	
  
Tonsils	
  are	
  lympho-­‐‑epithelial	
  tissues	
  located	
  at	
  the	
  junction	
  of	
  the	
  oropharynx	
  and	
  
nasopharynx	
  that	
  play	
  a	
  key	
  role	
  in	
  surveillance	
  of	
  inhaled	
  or	
  ingested	
  pathogens	
  [1].	
  In	
  pigs,	
  
the	
   tonsils	
   are	
   colonized	
   by	
   numerous	
   microbes,	
   and	
   serve	
   as	
   a	
   reservoir	
   for	
   both	
   pig	
  
pathogens	
  and	
  zoonotic	
  pathogens	
  with	
  a	
  high	
  potential	
  of	
  transmission	
  to	
  humans	
  [1-­‐‑4].	
  
Bacterial	
   pathogens	
   such	
   as	
   Actinobacillus	
   pleuropneumoniae,	
   Streptococcus	
   suis,	
   and	
  
Salmonella	
   enterica	
   are	
   frequently	
   found	
   in	
   tonsils	
   of	
   asymptomatic	
   animals.	
   Under	
  
conditions	
   of	
   stress,	
   such	
   as	
   animal	
   transport,	
   these	
   pathogens	
   can	
   spread	
   to	
   the	
   lower	
  
respiratory	
   and	
   gastrointestinal	
   tracts	
   and	
   be	
   transmitted	
   to	
   other	
   animals,	
   including	
  
humans	
   [5].	
   It	
   has	
   been	
   suggested	
   that	
   the	
   microbiome	
   plays	
   a	
   preventive	
   role	
   in	
   host	
  
colonization	
   by	
   pathogenic	
   microorganisms	
   [6-­‐‑9]	
   and	
   also	
   exerts	
   regulatory	
   roles	
   in	
  
maintaining	
  immune	
  homeostasis,	
  providing	
  resistance	
  to	
  infection	
  [10,	
  11].	
  	
  
In	
  contrast	
  to	
  the	
  rising	
  number	
  of	
  studies	
  characterizing	
  the	
  intestinal	
  microbiome	
  
of	
  mammals,	
  there	
  are	
  few	
  available	
  studies	
  characterizing	
  the	
  tonsillar	
  microbiome	
  and	
  its	
  
development	
  in	
  those	
  species.	
  We	
  have	
  previously	
  characterized	
  the	
  core	
  microbiome	
  of	
  the	
  
tonsils	
   in	
   healthy	
   18-­‐‑20	
   week	
   old	
   pigs	
   [12].	
   In	
   that	
   study,	
   members	
   of	
   the	
   families	
  
Pasteurellaceae,	
   Moraxellaceae,	
   Streptococcaceae,	
   Fusobacteriaceae,	
   Veillonellaceae,	
  
Enterobacteriaceae,	
   Neisseriaceae,	
   and	
   Peptostreptococcaceae,	
   as	
   well	
   as	
   the	
   order	
  
Clostridiales,	
  constituted	
  the	
  core	
  tonsil	
  microbiome	
  in	
  these	
  grower-­‐‑finisher	
  pigs.	
  Tonsils	
  
remain	
   an	
   under-­‐‑explored	
   habitat	
   of	
   the	
   mammalian	
   microbiome,	
   and	
   how	
   the	
   tonsil	
  
microbiome	
   is	
   established,	
   how	
   the	
   structure	
   of	
   this	
   community	
   affects	
   acquisition	
   and	
  

	
  

38	
  

carriage	
  of	
  pathogens,	
  and	
  how	
  it	
  contributes	
  to	
  health	
  and	
  disease,	
  in	
  animals	
  or	
  in	
  humans,	
  
are	
  not	
  well	
  understood.	
  
Currently,	
  there	
  are	
  no	
  studies	
  following	
  the	
  development	
  of	
  tonsillar	
  microbiomes	
  in	
  
humans	
  or	
  in	
  pigs,	
  while	
  other	
  microbiomes,	
  such	
  as	
  the	
  intestinal	
  microbiome	
  in	
  mammals,	
  
have	
  been	
  more	
  broadly	
  studied	
  in	
  the	
  last	
  decade.	
  Studies	
  conducted	
  in	
  pigs	
  and	
  humans	
  
have	
   suggested	
   that	
   the	
   development	
   of	
   the	
   intestinal	
   microbiome	
   is	
   a	
   gradual	
   and	
  
successional	
  process	
  [13-­‐‑16]	
  with	
  a	
  significant	
  fluctuation	
  after	
  weaning	
  [17].	
  The	
  cessation	
  
of	
   milk	
   feeding	
   as	
   well	
   as	
   supplementation	
   of	
   other	
   diets	
   and	
   feeding	
   sources	
   generate	
  
changes	
  in	
  the	
  intestinal	
  microbiota	
  [18-­‐‑22].	
  It	
  is	
  unknown	
  if	
  a	
  similar	
  successional	
  process	
  
occurs	
  in	
  tonsils.	
  
It	
  has	
  been	
  shown	
  for	
  humans	
  and	
  pigs	
  that	
  in	
  the	
  development	
  of	
  intestinal	
  microbial	
  
communities	
  there	
  is,	
  initially,	
  substantial	
  individual	
  variation	
  in	
  community	
  composition	
  
that	
   tends	
   to	
   shift	
   over	
   time,	
   eventually	
   becoming	
   similar	
   in	
   the	
   major	
   phyla	
   across	
  
individuals	
  [15].	
  Environmental	
  factors	
  such	
  as	
  the	
  maternal	
  microbiota	
  can	
  play	
  a	
  relevant	
  
role	
   in	
   the	
   initial	
   development	
   of	
   the	
   intestinal	
   microbiota	
   [13,	
   15,	
   23].	
   Nevertheless,	
   the	
  
developing	
   microbiome	
   is	
   composed	
   of	
   interacting	
   bacteria	
   and	
   not	
   simply	
   randomly	
  
assembled	
  microorganisms	
  [14].	
  	
  
The	
   goal	
   of	
   the	
   current	
   study	
   was	
   to	
   utilize	
   culture-­‐‑independent,	
   high-­‐‑throughput	
  
sequencing	
   of	
   16S	
   rRNA	
   genes	
   to	
   follow	
   the	
   development	
   of	
   the	
   tonsillar	
   microbial	
  
communities	
  in	
  pigs	
  from	
  birth	
  through	
  weaning.	
  	
  	
  
	
  
	
  

	
  

	
  

39	
  

MATERIAL	
  AND	
  METHODS	
  
	
  
Animals.	
  The	
  Michigan	
  State	
  University	
  Institutional	
  Animal	
  Care	
  and	
  Use	
  Committee	
  
approved	
   this	
   study	
   and	
   the	
   animal	
   procedures.	
   Ten	
   crossbred	
   sows	
   (Yorkshire	
   x	
  
Hampshire)	
  from	
  a	
  high	
  health	
  status	
  herd	
  were	
  used	
  in	
  this	
  study.	
  	
  This	
  herd	
  has	
  no	
  history	
  
of	
   recent	
   respiratory	
   disease	
   and	
   is	
   considered	
   free	
   of	
   Actinobacillus	
   pleuropneumoniae,	
  
Mycoplasma	
  hyopneumoniae,	
  and	
  Porcine	
  Respiratory	
  and	
  Reproductive	
  Syndrome	
  virus	
  by	
  
medical	
  history.	
  	
  This	
  herd	
  experienced	
  a	
  recent	
  outbreak	
  of	
  porcine	
  epidemic	
  diarrhea	
  virus	
  
(PEDV),	
  which	
  was	
  under	
  control	
  at	
  the	
  time	
  this	
  study	
  was	
  initiated.	
  	
  The	
  herd	
  has	
  a	
  history	
  
of	
  vaccination	
  against	
  porcine	
  circovirus	
  type	
  2	
  (PCV2),	
  erysipelas	
  and	
  atrophic	
  rhinitis.	
  
This	
  herd	
  was	
  managed	
  as	
  a	
  farrow	
  to	
  finishing	
  facility	
  with	
  ~200	
  sows.	
  	
  Newborn	
  
piglets	
   received	
   a	
   single	
   intramuscular	
   injection	
   of	
   Iron-­‐‑Dextran.	
  	
   Piglets	
   were	
   weaned	
   at	
  
three	
  weeks	
  of	
  age	
  (21	
  to	
  24	
  days	
  –	
  average	
  weight	
  18	
  pounds)	
  and	
  moved	
  to	
  a	
  nursery	
  room,	
  
with	
   litters	
   maintained	
   as	
   pen	
   mates.	
   The	
   weaned	
   piglets	
   were	
   introduced	
   to	
   a	
   solid	
   diet	
  
based	
   on	
   a	
   pellet	
   ration	
   (Pig	
   1300 ,	
   Akey	
   Nutrition,	
   Brookville,	
   OH)	
   supplemented	
   with	
  
®

Carbadox 	
  at	
  a	
  dose	
  of	
  50	
  g/ton.	
  	
  
®

Individually	
  identified	
  sows	
  of	
  different	
  parity,	
  from	
  primiparous	
  (pregnant	
  for	
  the	
  
first	
  time)	
  to	
  multiparous	
  (pregnant	
  multiple	
  times),	
  were	
  purposely	
  selected	
  for	
  this	
  study	
  
and	
  included	
  two	
  first	
  parity	
  sows	
  (ear	
  tags	
  1700	
  and	
  1707,	
  respectively),	
  one	
  second	
  parity	
  
sow	
  (1631),	
  one	
  fifth	
  parity	
  sow	
  (1445),	
  one	
  sixth	
  parity	
  sow	
  (1402)	
  and	
  one	
  tenth	
  parity	
  
sow	
   (1711).	
   	
   Four	
   piglets	
   from	
   each	
   of	
   these	
   six	
   sows,	
   selected	
   randomly,	
   were	
   sampled	
  
within	
  a	
  period	
  no	
  longer	
  than	
  8	
  hours	
  post-­‐‑birth	
  (PB)	
  (PB	
  were	
  piglets	
  which	
  might	
  have	
  
interacted	
  with	
  other	
  piglets	
  or	
  the	
  sow	
  before	
  sampling)	
  	
  and	
  then	
  at	
  1,	
  2,	
  3	
  and	
  4	
  weeks	
  of	
  

	
  

40	
  

age.	
  	
  An	
  additional	
  four	
  freshly	
  delivered	
  piglets	
  were	
  randomly	
  selected	
  (N=16	
  piglets)	
  from	
  
each	
   of	
   4	
   crossbred	
   Yorkshire	
   x	
   Hampshire	
   sows	
   (one	
   second	
   parity	
   sow	
   (1785),	
   two	
   third	
  
parity	
  sows	
  (1604,	
  1760)	
  and	
  one	
  fourth	
  parity	
  sow	
  (1704)),	
  and	
  sampled	
  immediately	
  at	
  
birth	
  (AB)	
  (AB	
  piglets	
  were	
  sampled	
  before	
  they	
  had	
  any	
  contact	
  with	
  external	
  sources	
  other	
  
than	
  vaginal	
  sources),	
  avoiding	
  any	
  contact	
  of	
  the	
  piglet	
  with	
  either	
  the	
  sow	
  teats	
  or	
  the	
  pen	
  
environment	
   prior	
   to	
   sampling,	
   with	
   the	
   purpose	
   of	
   determining	
   the	
   status	
   of	
   the	
  
microbiome	
  right	
  at	
  birth.	
  The	
  litter	
  from	
  sow	
  1604	
  was	
  sampled	
  in	
  2014;	
  the	
  remaining	
  
three	
  litters	
  were	
  sampled	
  in	
  2015.	
  
Collection	
   of	
   microbiome	
   samples.	
   Samples	
   of	
   the	
   tonsil	
   microbiome	
   were	
  
collected	
  from	
  sows	
  and	
  piglets	
  using	
  either	
  cytology	
  brushes	
  (CytosoftTM,	
  Medical	
  Packaging	
  
Corporation,	
  Camarillo,	
  CA)	
  for	
  very	
  small	
  piglets	
  or	
  tonsil	
  brushes	
  developed	
  by	
  our	
  group	
  
and	
  validated	
  in	
  previous	
  studies	
   [12].	
  Collection	
  of	
  samples	
  was	
  done	
  by	
  the	
  same	
  person,	
  
and	
  as	
  previously	
  described	
  [12].	
  Briefly,	
  samples	
  were	
  collected	
  at	
  approximately	
  the	
  same	
  
time	
  of	
  day	
  for	
  each	
  sampling	
  time.	
  Sow	
  tonsillar	
  samples	
  were	
  collected	
  before	
  they	
  were	
  
fed.	
  The	
  pigs’	
  movement	
  was	
  restricted	
  either	
  by	
  holding	
  them	
  firmly	
  wrapped	
  with	
  a	
  towel,	
  
or	
  by	
  using	
  a	
  snare	
  on	
  larger	
  animals.	
  	
  The	
  mouth	
  was	
  held	
  open	
  with	
  a	
  mouth	
  speculum	
  
while	
  the	
  tonsils	
  were	
  brushed.	
  Right	
  and	
  left	
  tonsils	
  were	
  brushed	
  approximately	
  ten	
  times	
  
each,	
  rotating	
  the	
  brush	
  in	
  a	
  clockwise	
  fashion.	
  Brushes	
  were	
  removed	
  from	
  the	
  pig’s	
  mouth	
  
and	
   placed	
   into	
   a	
   50	
   ml	
   sterile	
   test	
   tube	
   containing	
   20	
   ml	
   of	
   80%	
   ice-­‐‑cold	
   ethanol.	
   Samples	
  
were	
  stored	
  at	
  -­‐‑20	
  °C	
  until	
  processed.	
  	
  
The	
   sow	
   vaginal	
   microbiome	
   was	
   sampled	
   by	
   introducing	
   a	
   sterile	
   cotton	
   swab	
  
approximately	
  8	
  cm	
  into	
  the	
  vaginal	
  tract	
  and	
  rubbing	
  the	
  vaginal	
  walls	
  with	
  the	
  swab	
  while	
  
turning	
   the	
   swab	
   in	
   a	
   clockwise	
   fashion.	
   The	
   teat	
   microbiome	
   was	
   also	
   collected	
   by	
   using	
   a	
  

	
  

41	
  

cotton	
  swab	
  and	
  rubbing	
  the	
  teat	
  surface	
  of	
  at	
  least	
  10	
  teats	
  per	
  sow.	
  Vaginal	
  and	
  teat	
  swabs	
  
were	
   placed	
   individually	
   into	
   50	
   ml	
   sterile	
   test	
   tubes	
   containing	
   20	
   ml	
   of	
   80%	
   ice-­‐‑cold	
  
ethanol.	
  Samples	
  were	
  stored	
  at	
  -­‐‑20	
  °C	
  until	
  processed.	
  
The	
  sow	
  fecal	
  microbiome	
  was	
  sampled	
  by	
  collecting	
  approximately	
  5	
  grams	
  of	
  feces	
  
directly	
   from	
   the	
   rectum.	
   Samples	
   were	
   placed	
   individually	
   into	
   50	
   ml	
   sterile	
   test	
   tubes	
  
containing	
  20	
  ml	
  of	
  80%	
  ice-­‐‑cold	
  ethanol.	
  Samples	
  were	
  stored	
  at	
  -­‐‑20	
  °C	
  until	
  processed.	
  
Isolation	
   of	
   community	
   DNA.	
   Sample	
   extraction	
   was	
   performed	
   as	
   previously	
  
described	
   [12].	
   Briefly,	
   the	
   20	
   ml	
   of	
   80%	
   ice-­‐‑cold	
   ethanol	
   containing	
   the	
   brushes,	
   swabs	
   or	
  
feces	
   with	
   the	
   microbiome	
   samples	
   were	
   thoroughly	
   vortexed	
   for	
   one	
   minute,	
   divided	
   into	
  
equal	
  volumes	
  and	
  transferred	
  to	
  two	
  sterile	
  acid	
  washed	
  Corex®	
  tubes,	
  and	
  centrifuged	
  in	
  a	
  
refrigerated	
   Sorvall	
   SS-­‐‑34	
   rotor	
   at	
   16,000	
   x	
   g	
   for	
   30	
   min.	
   After	
   centrifugation,	
   the	
  
supernatant	
  was	
  removed	
  and	
  discarded.	
  The	
  pellet	
  of	
  one	
  tube	
  was	
  suspended	
  in	
  5	
  ml	
  of	
  
ice-­‐‑cold	
  80%	
  ethanol	
  and	
  archived	
  at	
  -­‐‑20	
  °C.	
  The	
  second	
  pellet	
  was	
  suspended	
  in	
  0.25	
  ml	
  of	
  
phosphate	
  buffered	
  saline,	
  pH	
  7,	
  and	
  transferred	
  to	
  PowerBead	
  tubes	
  (MoBio	
  Laboratories,	
  
Carlsbad,	
   CA)	
   and	
   vigorously	
   shaken	
   for	
   approximately	
   2	
   min	
   at	
   room	
   temperature	
   using	
   a	
  
MiniBeadBeater-­‐‑16	
  (BioSpec	
  Products,	
  Inc.,	
  Bartlesville,	
  OK).	
  An	
  exception	
  to	
  this	
  protocol	
  
was	
  followed	
  with	
  fecal	
  samples,	
  where	
  both	
  pellets	
  were	
  suspended	
  in	
  0.5	
  ml	
  of	
  phosphate	
  
buffered	
  saline,	
  pH	
  7.	
  Community	
  DNA	
  was	
  then	
  extracted	
  using	
  a	
  PowerSoil	
  DNA	
  Isolation	
  
Kit	
   (MoBio	
   Laboratories,	
   Carlsbad,	
   CA)	
   following	
   the	
   manufacturer´s	
   instructions.	
   The	
  
concentration	
  of	
  extracted	
  community	
  DNA	
  was	
  determined	
  by	
  spectrophotometry,	
  using	
  a	
  
Nanodrop	
  (Thermo	
  Scientific,	
  Wilmington,	
  DE).	
  	
  Each	
  sample	
  was	
  then	
  split	
  in	
  two	
  vials;	
  one	
  
was	
  archived	
  at	
  -­‐‑80	
  °C	
  and	
  the	
  other	
  was	
  processed	
  for	
  sequencing.	
  

	
  

42	
  

Illumina	
   sequencing	
   and	
   sequence	
   analysis.	
   For	
   Illumina	
   sequencing,	
   samples	
  
were	
   processed	
   at	
   the	
   Michigan	
   State	
   University	
   Research	
   Technology	
   Support	
   Facility	
  
(RTSF)	
  using	
  an	
  Illumina	
  MiSeq	
  platform.	
  	
  Negative	
  controls	
  consisting	
  of	
  either	
  DNA-­‐‑free	
  
water	
  or	
  MoBio	
  C6	
  solution	
  (“blank	
  library	
  controls”,	
  [24])	
  and	
  positive	
  controls	
  consisting	
  
of	
   either	
   Escherichia	
   coli	
   DH5α	
   genomic	
   DNA	
   or	
   a	
   well-­‐‑characterized	
   activated	
   sludge	
  
polymicrobial	
  community	
  [25]	
  were	
  included	
  in	
  the	
  sequencing	
  runs.	
   Briefly,	
  the	
  V4	
  region	
  
of	
  the	
  16S	
  rRNA	
  gene	
  of	
  the	
  community	
  DNA	
  was	
  amplified	
  using	
  uniquely	
  indexed	
  primers	
  
for	
   each	
   sample,	
   as	
   described	
   by	
   Caporaso	
   [26].	
   After	
   PCR,	
   amplification	
   products	
   were	
  
normalized	
   using	
   an	
   Invitrogen	
   SequalPrep	
   normalization	
   plate.	
   The	
   normalized	
   samples	
  
were	
  pooled	
  and	
  PCR	
  reaction	
  cleanup	
  was	
  done	
  with	
  AMPure	
  XP	
  beads.	
  After	
  quality	
  control	
  
and	
  quantitation,	
  the	
  pool	
  was	
  loaded	
  on	
  an	
  Illumina	
  MiSeq	
  v2	
  flow	
  cell	
  and	
  sequenced	
  with	
  
a	
  500-­‐‑cycle	
  v2	
  reagent	
  kit	
  (PE250	
  reads).	
  Base	
  calling	
  was	
  performed	
  by	
  Illumina	
  Real	
  Time	
  
Analysis	
  Software	
  (RTA)	
  v1.18.54	
  and	
  output	
  of	
  RTA	
  demultiplexed	
  and	
  converted	
  to	
  FastQ	
  
files	
  with	
  Illumina	
  Bcl2fastq	
  v1.8.4.	
  	
  
Amplicon	
   analysis	
   was	
   performed	
   using	
   the	
   open-­‐‑source,	
   platform-­‐‑independent,	
  
community-­‐‑supported	
   software	
   program	
   mothur	
   v.1.38.0	
   (http://www.mothur.org)	
   [27].	
  
Processing	
  of	
  the	
  raw	
  sequencing	
  data	
  was	
  done	
  according	
  to	
  the	
  mothur	
  standard	
  operating	
  
procedure	
   (http://www.mothur.org/wiki/MiSeq_SOP)	
   [28].	
   Alignment	
   was	
   accomplished	
  
using	
  the	
  mothur-­‐‑formatted	
  version	
  123	
  of	
  Silva	
  16S	
  rRNA	
  gene	
  database	
  [29].	
  Sequences	
  
were	
   classified	
   and	
   any	
   sequences	
   classified	
   as	
   Chloroplast,	
   Mitochondria,	
   unknown,	
  
Archaea,	
  or	
  Eukaryota	
  were	
  removed	
  from	
  the	
  data	
  set.	
  Subsampling	
  at	
  5907	
  sequences	
  per	
  
sample	
  was	
  performed,	
  followed	
  by	
  a	
  preclustering	
  of	
  the	
  sequences	
  and	
  removal	
  of	
  chimeric	
  
sequences	
   using	
   a	
   mothur-­‐‑formatted	
   version	
   of	
   the	
   Ribosomal	
   Database	
   Project	
   (RDP)	
  

	
  

43	
  

training	
  set	
  version	
  14	
  and	
  uchime,	
  based	
  on	
  the	
  mothur	
  protocol.	
  Sequences	
  were	
  classified	
  
into	
   Operational	
   Taxonomic	
   Units	
   (OTUs)	
   of	
   ≥97%	
   sequence	
   identity.	
   Singleton	
   and	
  
doubleton	
   reads	
   were	
   removed,	
   followed	
   by	
   subsampling	
   at	
   3776	
   sequences	
   per	
   sample.	
  
Because	
  the	
  negative	
  controls	
  consistently	
  showed	
  high	
  levels	
  of	
  contaminants	
  that	
  skewed	
  
the	
   results	
   seen	
   with	
   some	
   of	
   the	
   low	
   biomass	
   samples	
   such	
   as	
   those	
   from	
   the	
   newborn	
  
piglets,	
  	
  especially	
  those	
  from	
  Litter	
  1700,	
  4	
  OTUs	
  (Ralstonia,	
  Bacillaceae	
  1,	
  Burkholderia,	
  and	
  
Brevundimonas)	
   were	
   also	
   removed	
   from	
   the	
   data	
   set	
   prior	
   to	
   final	
   analysis.	
   	
   A	
   SIMPER	
  
comparison	
  of	
  Litter	
  1700	
  piglet	
  and	
  sow	
  samples	
  to	
  the	
  relevant	
  negative	
  controls	
  showed	
  
66.1%	
  or	
  greater	
  dissimilarity	
  between	
  the	
  negative	
  controls	
  and	
  the	
  pig	
  samples,	
  rejecting	
  
the	
   hypothesis	
   that	
   the	
   sequence	
   data	
   for	
   Litter	
   and	
   sow	
   1700	
   samples	
   is	
   due	
   to	
  
contamination	
  only	
  [24].	
  	
  Therefore	
  these	
  samples	
  were	
  included	
  in	
  the	
  final	
  data	
  set.	
  For	
  
the	
   final	
   analysis	
   of	
   the	
   data,	
   samples	
   were	
   subsampled	
   to	
   1979	
   reads	
   per	
   sample.	
   The	
   full	
  
data	
  

set	
  

analyzed	
  

is	
  

available	
  

as	
  

a	
  

supplemental	
  

file	
  

at	
  

(https://figshare.com/s/1ac201b155d662dcc646).	
  
Diversity	
   and	
   statistical	
   analysis.	
   The	
   statistical	
   analysis	
   was	
   performed	
   using	
   a	
  
clustering	
   cutoff	
   of	
   3%	
   for	
   the	
   processed	
   sequences.	
   Mothur	
   output	
   files	
   were	
   used	
   to	
  
estimate	
   diversity	
   indexes	
   and	
   core	
   microbiomes.	
   PAST3	
   was	
   used	
   for	
   generation	
   of	
   the	
  
UPGMA	
  dendogram	
  file,	
  SIMPER,	
  coordinate	
  analysis	
  of	
  the	
  samples,	
  two	
  dimensional	
  scatter	
  
plot	
  and	
  95%	
  concentration	
  ellipses	
  [30].	
  ImageJ	
  was	
  used	
  to	
  measure	
  the	
  area	
  of	
  the	
  ellipses	
  
for	
   the	
   two	
   dimensional	
   scatter	
   plot	
   [31].	
   Dendogram	
   construction	
   was	
   performed	
   using	
  
FigTree	
   v.1.4.2.	
   (http://tree.bio.ed.ac.uk/software/figtree/).	
   RStudio	
   (Version	
   0.99.446;	
  
https://www.rstudio.com/)	
  

and	
  

libraries:	
  

gplots	
  

(https://CRAN.R-­‐‑

project.org/package=gplots),	
  plot3D	
  (https://CRAN.R-­‐‑project.org/package=plot3D),	
  and	
  rgl	
  

	
  

44	
  

(https://CRAN.R-­‐‑project.org/package=rgl)	
   were	
   used	
   to	
   generate	
   heatmaps	
   and	
  
tridimensional	
  

scatter	
  

plots,	
  

respectively.	
  

Inkscape	
  

0.91	
  

(https://inkscape.org/en/download/mac-­‐‑os/),	
  was	
  used	
  to	
  process	
  images	
  and	
  edit	
  labels.	
  
Availability	
  of	
  supporting	
  data.	
  Raw	
  sequence	
  data	
  is	
  available	
  at	
  the	
  NCBI	
  database	
  
(SRA	
   accession	
   number:	
   SRP110992)	
   and	
   the	
   code	
   for	
   the	
   mothur	
   analysis	
   is	
   available	
   at	
  
(https://figshare.com/s/2c98593a953cc9bb1366).	
  	
  

	
  

45	
  

	
  

RESULTS	
  
	
  
A	
   total	
   of	
   171	
   samples	
   derived	
   from	
   the	
   tonsillar	
   microbiome	
   of	
   piglets	
   and	
   sow	
  
tonsils,	
  teat	
  skin,	
  vagina	
  and	
  feces	
  as	
  well	
  as	
  control	
  samples	
  were	
  processed.	
  One	
  hundred	
  
forty-­‐‑four	
  pig	
  samples	
  were	
  collected	
  in	
  Spring	
  2014,	
  and	
  an	
  additional	
  12	
  pig	
  samples	
  were	
  
collected	
  in	
  Fall	
  2015.	
  A	
  total	
  of	
  26	
  samples	
  with	
  less	
  than	
  3776	
  final	
  reads	
  after	
  singleton	
  
and	
  doubleton	
  removal,	
  were	
  not	
  included	
  in	
  the	
  final	
  analysis.	
  	
  One	
  of	
  the	
  approaches	
  that	
  
we	
  used	
  was	
  comparative	
  analysis	
  of	
  core	
  microbiomes.	
  We	
  defined	
  the	
  core	
  microbiome	
  for	
  
a	
  specific	
  litter	
  as	
  the	
  OTU	
  members	
  of	
  the	
  microbiome	
  that	
  were	
  present	
  in	
  at	
  least	
  75%	
  of	
  
piglets	
   of	
   a	
   litter,	
   when	
   litters	
   had	
   at	
  least	
   4	
   samples;	
   otherwise	
   core	
   OTUs	
   were	
  defined	
   as	
  
OTUs	
   present	
   in	
   all	
   the	
   samples.	
   For	
   the	
   sow	
   samples,	
   where	
   6	
   samples	
   were	
   analyzed,	
   we	
  
considered	
  OTUs	
  present	
  in	
  66.6%	
  of	
  the	
  samples	
  to	
  be	
  core	
  OTUs.	
  Further,	
  core	
  OTUs	
  were	
  
defined	
  as	
  present	
  in	
  a	
  minimum	
  relative	
  abundance	
  equal	
  or	
  higher	
  than	
  1%	
  and/or	
  0.1%	
  
of	
  total	
  reads	
  for	
  a	
  selected	
  litter	
  and/or	
  period	
  (Table	
  2.2.).	
  
The	
  tonsillar	
  microbiome	
  found	
  in	
  PB	
  samples	
  clusters	
  by	
  litter.	
  	
  Among	
  the	
  156	
  
samples	
  collected,	
  40	
  samples	
  contain	
  the	
  tonsillar	
  microbiome	
  of	
  newborn	
  piglets	
  (Table	
  
2.1.).	
  Twenty	
  four	
  of	
  these	
  40	
  samples	
  were	
  collected	
  from	
  PB	
  piglets	
  and	
  the	
  remaining	
  16	
  
samples	
  were	
  collected	
  from	
  AB	
  piglets.	
  Six	
  samples	
  were	
  discarded	
  from	
  the	
  analysis	
  due	
  
to	
  low	
  number	
  of	
  reads.	
  	
  	
  
	
  

	
  

	
  

46	
  

Table	
  2.1.	
  Identification	
  of	
  sows,	
  litters,	
  sample	
  collection	
  and	
  times	
  of	
  collection	
  

	
  
PB.	
  Samples	
  were	
  collected	
  from	
  newborn	
  piglets	
  in	
  a	
  period	
  not	
  greater	
  than	
  8	
  hours	
  post	
  birth.	
  
AB.	
  Samples	
  were	
  collected	
  from	
  newborn	
  piglets	
  immediately	
  after	
  birth.	
  

	
  

A	
  dendrogram	
  clustering	
  of	
  PB	
  piglet	
  samples	
  and	
  sow	
  derived	
  samples,	
  based	
  on	
  the	
  
Bray-­‐‑Curtis	
  dissimilarity	
  index	
  (Figure	
  2.1.)	
  showed	
  that	
  samples	
  from	
  each	
  litter	
  clustered	
  
together	
  with	
  littermates.	
  Microbiomes	
  derived	
  from	
  piglets	
  from	
  primiparous	
  sows	
  (Litter	
  
1700	
  and	
  1707)	
  clustered	
  in	
  separated	
  clades,	
  as	
  did	
  the	
  piglets	
  from	
  sow	
  1402.	
  In	
  general,	
  
PB	
   piglet	
   samples	
   were	
   more	
   closely	
   associated	
   with	
   samples	
   derived	
   from	
   the	
   sow	
   teat	
  
microbiome	
   as	
   opposed	
   to	
   other	
   sow	
   samples.	
   However,	
   piglets	
   from	
   litter	
   1700	
   also	
  
clustered	
  with	
  the	
  sample	
  from	
  the	
  sow	
  vaginal	
  microbiome.	
  
Samples	
  from	
  the	
  sow	
  tonsils	
  and	
  sow	
  feces	
  clustered	
  together	
  by	
  source.	
  Samples	
  
from	
  the	
  vaginal	
  tract	
  of	
  multiparous	
  sows	
  clustered	
  together,	
  but	
  were	
  distinct	
  from	
  those	
  
from	
  the	
  two	
  primiparous	
  sows.	
  
A	
   heat-­‐‑map	
   representation	
   of	
   the	
   major	
   OTUs	
   reveals	
   the	
   differences	
   driving	
   the	
  
clustering	
   (Figure	
   2.2.).	
   	
   In	
   the	
   PB	
   piglets	
   from	
   sows	
   1445,	
   1631,	
   and	
   1711,	
   all	
   multiparous	
  
sows,	
  the	
  most	
  abundant	
  OTUs	
  were	
  Pasteurellaceae,	
  Streptococcus,	
  Moraxella,	
  Rothia,	
  and	
  
Staphylococcus	
  (OTUs	
  001,	
  002,	
  003,	
  007,	
  and	
  009,	
  respectively).	
  	
  PB	
  piglets	
  from	
  sow	
  1402	
  

	
  

47	
  

(parity	
   6)	
   contained	
   significant	
   numbers	
   of	
   Pasteurellaceae,	
   Streptococcus,	
   and	
   Moraxella	
  
(OTUs	
  001,	
  002,	
  and	
  003)	
  but	
  also	
  contained	
  large	
  numbers	
  of	
  Enterobacteriaceae	
  (OTU005)	
  
and	
  Clostridium	
  sensu	
  strictu	
  (OTU010).	
  In	
  contrast,	
  piglets	
  derived	
  from	
  primiparous	
  sows	
  
1700	
  and	
  1707	
  had	
  a	
  very	
  low	
  abundance	
  of	
  Pasteurellaceae,	
  Streptococcus,	
  and	
  Moraxella	
  
(OTUs	
   001,	
   002	
   and	
   003),	
   but	
   a	
   high	
   abundance	
   of	
   Staphylococcus	
   (OTU009)	
   as	
   well	
   as	
  
several	
  anaerobic	
  organisms.	
  	
  

	
  
Figure	
   2.1.	
   Unrooted	
   Bray-­‐‑Curtis	
   dendrogram	
   of	
   PB	
   and	
   sow	
   microbiomes.	
   The	
  
samples	
  are	
  color	
  coded	
  by	
  the	
  source.	
  Bootstrap	
  values	
  higher	
  than	
  70	
  %	
  at	
  1000	
  iterations	
  
are	
  shown.	
  	
  

	
  

48	
  

Parity 1

Parity 1

Parity 2

Parity 5

Parity 6

Parity 10

10A
11A
12A
13A
Litter 1700
Sow 1700 Teats
Sow 1700 Vagina
Sow 1700 Tonsils
Sow 1700 Feces
31A
34A
35A
Litter 1707
Sow 1707 Teats
Sow 1707 Vagina
Sow 1707 Tonsils
15A
16A
17A
18A
Litter 1631
Sow 1631 Teats
Sow 1631 Vagina
Sow 1631 Tonsils
36A
39A
40A
42A
Litter 1445
Sow 1445 Teats
Sow 1445 Vagina
Sow 1445 Tonsils
1A
4A
6A
Litter 1402
Sow 1402 Teats
Sow 1402 Vagina
Sow 1402 Tonsils
22A
23A
24A
26A
Litter 1711
Sow 1711 Teats
Sow 1711 Vagina
Sow 1711 Tonsils
Sow 1711 Feces
Sows Teats
Sows Vagina
Sows Tonsils
Sows Feces

Otu00001__Pasteurellaceae__
Otu00002__Streptococcaceae__Streptococcus
Otu00003__Moraxellaceae__Moraxella
Otu00004__Comamonadaceae__
Otu00005__Enterobacteriaceae__Escherichia/Shigella
Otu00007__Micrococcaceae__Rothia
Otu00009__Staphylococcaceae__Staphylococcus
Otu00010__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00012__Streptococcaceae__Streptococcus
Otu00016__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00018__Peptostreptococcaceae__Clostridium_XI
Otu00020__Erysipelotrichaceae__Turicibacter
Otu00021__Aerococcaceae__Aerococcus
Otu00023__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00025__Peptostreptococcaceae__Clostridium_XI
Otu00027__Veillonellaceae__Veillonella
Otu00030__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00034__Corynebacteriaceae__Corynebacterium
Otu00037__Clostridiales_Incertae_Sedis_XI__Tissierella
Otu00038__Micrococcaceae__Micrococcus
Otu00042__Ruminococcaceae__
Otu00045__Moraxellaceae__Acinetobacter
Otu00046__Comamonadaceae__
Otu00047__Streptococcaceae__Streptococcus
Otu00048__Lactobacillaceae__Lactobacillus
Otu00051__Corynebacteriaceae__Corynebacterium
Otu00054__Clostridiales_Incertae_Sedis_XI__Sedimentibacter
Otu00056__Enterobacteriaceae__Yersinia
Otu00070__Pasteurellaceae__Actinobacillus
Otu00080__Bifidobacteriaceae__Bifidobacterium

PB piglets, litter avg. and sow microbiome samples

0

10

20

30

40

50

60

% OTU relative abundance

Sow avg.

	
  

Figure	
  2.2.	
  Thirty	
  most	
  abundant	
  Operational	
  Taxonomic	
  Units	
  (OTUs)	
  for	
  piglets	
  and	
  
sow	
  samples.	
  Heat-­‐‑map	
  showing	
  the	
  relative	
  abundance	
  of	
  the	
  top	
  30	
  OTUs	
  identified	
  for	
  
all	
  samples.	
  The	
  figure	
  shows	
  the	
  relative	
  abundance	
  of	
  the	
  OTUs	
  for	
  each	
  PB	
  piglet	
  and	
  sow	
  
samples,	
  as	
  well	
  as	
  the	
  average	
  for	
  the	
  litters	
  and	
  sow	
  samples.	
  
	
  
A	
  significant	
  proportion	
  of	
  the	
  microbiome	
  of	
  the	
  piglets	
  came	
  from	
  maternal	
  
sources.	
   We	
   processed	
   samples	
   derived	
   from	
   teat	
   skin,	
   vagina	
   and	
   tonsil	
   from	
   six	
   sows	
  
belonging	
   to	
   the	
   PB	
   group	
   (Table	
   2.1.),	
   and	
   fecal	
   samples	
   from	
   two	
   of	
   these	
   sows.	
   We	
  
compared	
   and	
   traced	
   which	
   OTUs	
   were	
   identified	
   as	
   core	
   microbiome	
   for	
   each	
   litter	
   and	
   if	
  
they	
  were	
  identified	
  as	
  core	
  for	
  microbiomes	
  derived	
  from	
  the	
  different	
  sow	
  sources	
  (Table	
  
2.2).	
  	
  

	
  

49	
  

The	
  OTUs	
  identified	
  as	
  core	
  for	
  PB	
  litters	
  varied	
  between	
  litters.	
  As	
  was	
  seen	
  in	
  Figure	
  
2.2.,	
  Pasteurellaceae,	
  Streptococcus	
  and	
  Moraxella	
  (OTUs	
  001,	
  002	
  and	
  003)	
  were	
  identified	
  
as	
  core	
  with	
  a	
  minimum	
  relative	
  abundance	
  equal	
  or	
  higher	
  than	
  1%	
  and	
  were	
  found	
  in	
  high	
  
proportions	
   in	
   most	
   piglets	
   from	
   multiparous	
   sows	
   but	
   not	
   piglets	
   from	
   primiparous	
   sows.	
  	
  
When	
   sow	
   sources	
   for	
   these	
   three	
   OTUs	
   were	
   examined,	
   OTU001	
   (Pasteurellaceae)	
   was	
  
identified	
  as	
  a	
  core	
  organism	
  in	
  the	
  sow	
  vaginal	
  tract,	
  representing	
  on	
  average	
  20.1%	
  of	
  the	
  
vaginal	
   microbiome.	
   	
   However,	
   OTU001	
   was	
   present	
   in	
   only	
   low	
   amounts	
   in	
   the	
   vaginal	
  
tracts	
   from	
   primiparous	
   sows.	
   In	
   contrast,	
   OTU002	
   (Streptococcus)	
   was	
   found	
   in	
   both	
  
vaginal	
   and	
   teat	
   skin	
   samples,	
   while	
   OTU003	
   (Moraxella)	
   was	
   found	
   mainly	
   in	
   teat	
   skin	
  
samples;	
   both	
   were	
   more	
   prevalent	
   in	
   samples	
   from	
   multiparous	
   sows.	
   	
   In	
   addition,	
   Rothia	
  
(OTU007)	
  and	
  Staphylococcus	
  (OTU009)	
  were	
  identified	
  in	
  high	
  proportions	
  and	
  as	
  core	
  for	
  
most	
  PB	
  litters	
  regardless	
  of	
  sow	
  parity,	
  as	
  well	
  as	
  core	
  for	
  the	
  sow	
  teat	
  microbiome,	
  but	
  not	
  
as	
  part	
  of	
  the	
  core	
  microbiome	
  for	
  other	
  sow	
  samples.	
  	
  
These	
  results	
  suggested	
  that	
  the	
  PB	
  piglets	
  had	
  acquired	
  new	
  organisms	
  from	
  teat	
  
skin	
   or	
   other	
   sources	
   within	
   the	
   first	
   few	
   hours	
   of	
   life.	
   To	
   test	
  this	
   hypothesis,	
   we	
   collected	
  
samples	
  from	
  piglets	
  immediately	
  at	
  birth	
  (AB),	
  prior	
  to	
  any	
  contact	
  with	
  sources	
  other	
  than	
  
the	
   uterus	
   and	
   the	
   sow	
   vaginal	
   tract.	
   These	
   samples	
   were	
   very	
   sparse	
   in	
   content,	
   and	
  
unfortunately	
   the	
   resulting	
   sequence	
   data	
   was	
   dominated	
   by	
   organisms	
   known	
   to	
   be	
  
frequent	
   contaminants	
   in	
   DNA	
   extraction	
   kits	
   and	
   library	
   preparation	
   kits,	
   including	
  
Comamonadaceae,	
  Sphingomonadaceae	
  and	
  Xanthomonadaceae	
  [24].	
  	
  However,	
  when	
  these	
  
likely	
   contaminants	
   were	
   deleted	
   from	
   the	
   data,	
   it	
   was	
   clear	
   that	
   the	
   organisms	
   most	
  
frequently	
   seen	
   were	
   organisms	
   associated	
   mainly	
   with	
   the	
   sow	
   vaginal	
   tract,	
   including	
  
OTU002	
   Streptococcus,	
   Corynebacterium	
   (OTU034	
   and	
   051),	
   and	
   several	
   anaerobic	
  

	
  

50	
  

organisms	
   including	
   multiple	
   OTUs	
   of	
   Clostridiaceae	
   and	
   Peptostreptococcaceae.	
   OTU001	
  
(Pasteurellaceae)	
  was	
  also	
  found	
  although	
  in	
  low	
  numbers.	
  	
  
An	
  analysis	
  of	
  the	
  microbiome	
  derived	
  from	
  sow	
  sources	
  as	
  well	
  as	
  from	
  PB	
  piglets	
  at	
  
the	
  family	
  level	
  showing	
  the	
  20	
  most	
  abundant	
  taxa	
  is	
  presented	
  in	
  Figure	
  2.3.	
  These	
  taxa	
  
generally	
  include	
  multiple	
  OTUs.	
  	
  Streptococcaceae	
  (20	
  OTUs),	
  Staphylococcaceae	
  (4	
  OTUs),	
  
Micrococcaceae	
  (10	
  OTUs),	
  Pasteurellaceae	
  (21	
  OTUs)	
  Moraxellaceae	
  (29	
  OTUs)	
  and	
  families	
  
belonging	
  to	
  the	
  Clostridiales	
  (746	
  OTUs)	
  were	
  present	
  in	
  high	
  abundance	
  in	
  the	
  PB	
  piglets,	
  
and	
  were	
  also	
  found	
  in	
  the	
  sow	
  vagina	
  samples	
  and/or	
  the	
  sow	
  teat	
  skin	
  samples.	
  	
  
Tonsil	
  communities	
  of	
  newborn	
  piglets	
  differed	
  initially	
  between	
  litters	
  but	
  by	
  
three	
  weeks	
  of	
  age	
  clustered	
  together	
  reflecting	
  similar	
  composition.	
  We	
  inquired	
  if,	
  
over	
  time,	
  the	
  tonsillar	
  communities	
  of	
  piglets	
  reached	
  a	
  common	
  microbiome.	
  A	
  principal	
  
coordinate	
   analysis	
   (PCoA)	
   based	
   on	
   Bray-­‐‑Curtis	
   distances	
   for	
   the	
   PB	
   newborn	
   through	
  
week	
  4	
  samples	
  (Figure	
  2.4.A.	
  and	
  2.4.B.)	
  showed	
  a	
  dynamic	
  pattern	
  in	
  the	
  samples.	
  	
  The	
  PB	
  
newborns	
  were	
  widely	
  spread	
  although	
  two	
  distinct	
  clusters	
  were	
  detectable.	
  Over	
  the	
  next	
  
three	
   weeks	
   the	
   microbiomes	
   formed	
   increasingly	
   tighter	
   clusters	
   reflecting	
   greater	
  
similarity	
   as	
   the	
   animals	
   aged.	
   However,	
   in	
   the	
   fourth	
   week	
   there	
   was	
   a	
   dramatic	
   shift,	
  
where,	
  instead	
  of	
  continuing	
  to	
  cluster	
  together	
  more	
  tightly	
  as	
  observed	
  in	
  previous	
  weeks,	
  
samples	
  were	
  scattered.	
  
Further	
  analysis	
  using	
  a	
  SIMPER	
  approach	
  based	
  on	
  Bray-­‐‑Curtis	
  distances	
  (Table	
  2.3.)	
  
reinforced	
   what	
   we	
   observed	
   in	
   the	
   PCoA	
   plots.	
   Microbiome	
   samples	
   from	
   PB	
   piglets	
   were	
  
dissimilar	
   when	
   compared	
   with	
   others	
   and	
   the	
   value	
   of	
   dissimilarity	
   fluctuated,	
   with	
   the	
  
lowest	
  dissimilarity	
  being	
  45.8%	
  for	
  litter	
  1445	
  vs	
  1711	
  and	
  the	
  highest	
  being	
  94.04%	
  for	
  
litter	
   1631	
   vs	
   1707.	
   Although	
   there	
   was	
   a	
   substantial	
   variability	
   in	
   the	
   overall	
   dissimilarity	
  

	
  

51	
  

between	
   litters,	
   as	
   time	
   advanced	
   the	
   dissimilarity	
   value	
   between	
   litters	
   decreased	
   and	
  
reached	
  the	
  lowest	
  values	
  in	
  the	
  third	
  week,	
  when	
  values	
  were	
  as	
  low	
  as	
  23.5	
  %	
  for	
  litters	
  
1402	
  vs	
  1707	
  and	
  the	
  highest	
  was	
  37.3%	
  for	
  litters	
  1402	
  vs	
  1445.	
  	
  
100%

90%
Others
Xanthomonadaceae

80%

Moraxellaceae
Pasteurellaceae

70%

Enterobacteriaceae
Comamonadaceae
Veillonellaceae

60%

Erysipelotrichaceae
Ruminococcaceae
Peptostreptococcaceae

50%

Lachnospiraceae
Clostridiales_Incertae_Sedis_XI

40%

Clostridiaceae_1
Streptococcaceae
Lactobacillaceae

30%

Aerococcaceae
Staphylococcaceae
Prevotellaceae

20%

Porphyromonadaceae
Micrococcaceae

10%

0%

Corynebacteriaceae

Sow	
feces

Sow	
tonsils

Sow	
teat

Sow	
vagina

Litter	
1700

Litter	
1707

Litter	
1631

Litter	
1445

Litter	
1402

Litter	
1711

All	 PB	
piglets

	
  

Figure	
   2.3.	
   Twenty	
   most	
   abundant	
   families	
   identified	
   in	
   sows	
   and	
   PB	
   	
   microbiome	
  
samples.	
  Bar	
  plot	
  shows	
  the	
  mean	
  values	
  for	
  each	
  family	
  in	
  Sow	
  and	
  PB	
  samples,	
  including	
  
each	
   PB	
   litter	
   (percent	
   of	
   total	
   OTUs).	
   “Others”	
   represent	
   members	
   of	
   bacterial	
   families	
  
different	
  from	
  the	
  20	
  most	
  abundant	
  families	
  identified.	
  	
  
	
  
	
  
	
  

52	
  

	
  
Figure	
   2.4.	
   Principal	
   Coordinate	
   Analysis	
   (PCoA)	
   characterizing	
   the	
   tonsillar	
  
microbiome	
   from	
   PB	
   piglets	
   through	
   the	
   different	
   sampling	
   times.	
   Two	
   dimensional	
  
plotting	
  	
  	
  illustrating	
  	
  	
  the	
  	
  	
  distribution	
  	
  	
  of	
  	
  	
  microbiome	
  	
  	
  in	
  	
  	
  the	
  	
  	
  first	
  	
  	
  two	
  	
  	
  axes,	
  	
  the	
  	
  95%	
  

	
  

53	
  

Figure	
  2.4.	
  (cont´d).	
  concentration	
  ellipses	
  for	
  newborn	
  piglets	
  through	
  four	
  weeks	
  and	
  the	
  
relative	
  area	
  for	
  the	
  ellipses	
  (A).	
  Three-­‐‑dimensional	
  plot	
  illustrating	
  the	
  main	
  three	
  axes	
  for	
  
the	
  distribution	
  of	
  the	
  microbiome	
  of	
  PB	
  piglets	
  aged	
  newborn	
  through	
  four	
  weeks	
  (B).	
  	
  	
  
	
  
To	
  visualize	
  the	
  temporal	
  patterns	
  of	
  distribution	
  of	
  different	
  bacterial	
  families,	
  we	
  
charted	
   the	
   mean	
   value	
   for	
   the	
   20	
   most	
   commonly	
   identified	
   families	
   per	
   sampling	
   time	
  
(Figure	
  2.5.).	
  While	
  there	
  were	
  litter	
  to	
  litter	
  variations	
  in	
  the	
  microbiome	
  of	
  PB	
  newborn	
  
piglets,	
   as	
   shown	
   in	
   Figure	
   3,	
   the	
   differences	
   between	
   litters	
   disappeared	
   through	
   the	
  
successional	
  development	
  of	
  the	
  microbiome	
  in	
  the	
  following	
  weeks.	
  	
  
Members	
  of	
  the	
  families	
  Micrococcaceae	
  and	
  Staphylococcaceae	
  were	
  abundant	
  in	
  PB	
  
newborns	
   but	
   decreased	
   drastically	
   and	
   almost	
   disappeared	
   in	
   the	
   following	
   weeks.	
   By	
   1	
  
week	
   of	
   age,	
   the	
   tonsil	
   microbiome	
   was	
   dominated	
   by	
   members	
   of	
   the	
   Pasteurellaceae,	
  
Moraxellaceae,	
   and	
   Streptococcaceae	
   families,	
   a	
   dominance	
   that	
   remained	
   throughout	
   the	
  
lives	
   of	
   these	
   pigs	
   (unpublished	
   data).	
   	
   In	
   contrast,	
   some	
   members	
   of	
   the	
   microbiome	
  
appeared	
  and	
  disappeared	
  over	
  time,	
  such	
  as	
  Porphyromonadaceae	
  and	
  Flavobacteriaceae	
  
which	
  appeared	
  at	
  weeks	
  1	
  to	
  3,	
  as	
  well	
  as	
  Fusobacteriaceae	
  and	
  Leptotrichiaceae	
  that	
  were	
  
present	
  only	
  during	
  weeks	
  2	
  and	
  3.	
  	
  
The	
  transition	
  between	
  third	
  and	
  fourth	
  week	
  represents	
  a	
  critical	
  period	
  for	
  
the	
   development	
   of	
   the	
   microbiome.	
   According	
   to	
   MSU	
   farm	
   management	
   practices,	
  
piglets	
  were	
  weaned	
  between	
  the	
  third	
  and	
  fourth	
  week.	
  We	
  investigated	
  if	
  the	
  management	
  
practices	
  experienced	
  by	
  the	
  piglets	
  (weaning,	
  shift	
  to	
  a	
  nursery	
  room	
  and	
  introduction	
  to	
  a	
  
pelleted	
   ration	
   supplemented	
   with	
   Carbadox ),	
   correlated	
   with	
   the	
   timing	
   of	
   shifts	
   in	
  
®

community	
  structures.	
  

	
  

54	
  

100%

Others

90%

Moraxellaceae
Pasteurellaceae

80%

Enterobacteriaceae
Neisseriaceae

70%

Leptotrichiaceae
Fusobacteriaceae

60%

Veillonellaceae
Ruminococcaceae
Peptostreptococcaceae

50%

Lachnospiraceae
Clostridiales_Incertae_Sedis_XI

40%

Clostridiaceae_1
Streptococcaceae

30%

Lactobacillaceae
Staphylococcaceae
Bacillales_Incertae_Sedis_XI

20%

Flavobacteriaceae
Prevotellaceae

10%

Porphyromonadaceae
Micrococcaceae

0%

PB	
Newborn

First	
week

Second	
week

Third	
week

Fourth	
week

	
  

	
  
Figure	
  2.5.	
  The	
  abundance	
  of	
  the	
  twenty	
  most	
  common	
  families	
  in	
  PB	
  piglets	
  sampled	
  
from	
   newborn	
   through	
   four	
   weeks.	
   Bar	
   plot	
   shows	
   the	
   mean	
   values	
   for	
   the	
   twenty	
   most	
  
abundant	
  families	
  identified	
  over	
  the	
  first	
  four	
  weeks	
  of	
  life	
  (percent	
  of	
  total	
  OTUs).	
  
	
  
An	
   analysis	
   of	
   the	
   members	
   of	
   core	
   microbiome	
   (Table	
   2.4.	
   and	
   Table	
   2.5.)	
  
demonstrated	
  that	
  for	
  the	
  third	
  week,	
  OTU001	
  (Pasteurellaceae),	
  OTU002	
  (Streptococcus),	
  
OTU003	
   (Moraxella),	
   OTU006	
   (Porphyromonas),	
   OTU017	
   (Fusobacterium)	
   and	
   OTU026	
  
(Flavobacteriaceae),	
   identified	
   as	
   core	
   with	
   a	
   minimum	
   relative	
   abundance	
   equal	
   or	
   higher	
  

	
  

55	
  

than	
   1%,	
   were	
   shared	
   among	
   all	
   the	
   litters,	
   and	
   represented	
   on	
   average	
   70.1%	
   of	
   the	
  
identified	
   OTUs.	
   In	
   addition,	
   OTU012	
   (Streptococcus),	
   OTU15	
   (Prevotellaceae),	
   OTU018	
  
(Peptostreptococcaceae)	
  and	
  OTU024	
  (Bacillales	
  Incertae	
  Sedis	
  XI)	
  were	
  core	
  members	
  at	
  a	
  
minimum	
   relative	
   abundance	
   of	
   0.1%	
   or	
   higher.	
   OTU001	
   (Pasteurellaceae),	
   OTU002	
  
(Streptococcus)	
  and	
  OTU003	
  (Moraxella)	
  dominated	
  the	
  microbiome,	
  together	
  representing	
  
on	
  average	
  59.5%	
  of	
  the	
  identified	
  OTUs.	
  An	
  analysis	
  at	
  the	
  family	
  level	
  (Figure	
  2.5.)	
  for	
  the	
  
same	
   period	
   shows	
   the	
   families	
   Moraxellaceae	
   and	
   Pasteurellaceae	
   as	
   the	
   most	
   abundant	
  
families	
   with	
   an	
   abundance	
   of	
   almost	
   55.6%,	
   followed	
   by	
   members	
   of	
   the	
   families	
  
Streptococcaceae,	
  

Porphyromonadaceae,	
  

Fusobacteriaceae,	
  

Leptotrichiaceae	
  

and	
  

Flavobacteriaceae,	
  together	
  with	
  an	
  abundance	
  of	
  24.5%.	
  
The	
   fourth	
   week	
   showed	
   a	
   different	
   panorama,	
   where	
   only	
   two	
   OTUs,	
   OTU001	
  
(Pasteurellaceae)	
   and	
   OTU002	
   (Streptococcus),	
   were	
   identified	
   as	
   core	
   at	
   1%	
   for	
   all	
   the	
  
litters.	
   However,	
   in	
   contrast	
   to	
   the	
   third	
   week,	
   these	
   two	
   OTUs	
   represented	
   on	
   average	
  
67.6%	
  of	
  the	
  identified	
  OTUs.	
  In	
  the	
  fourth	
  week,	
  members	
  of	
  the	
  Streptococcaceae	
  family	
  
increased	
   dramatically	
   5-­‐‑6	
   fold	
   compared	
   to	
   week	
   3,	
   Pasteurellaceae	
   remained	
   at	
   the	
   same	
  
levels,	
  and	
  members	
  of	
  Moraxellaceae	
  decreased	
  4	
  fold.	
  Members	
  of	
  Porphyromonadaceae,	
  
Flavobacteriaceae,	
   Fusobacteriaceae,	
   and	
   Leptotrichiaceae	
   decreased	
   to	
   negligible	
   numbers	
  
in	
  week	
  4.	
  	
  	
  
Furthermore,	
   a	
   SIMPER	
   analysis	
   (Table	
   2.3.)	
   indicates	
   that	
   the	
   dissimilarity	
   in	
  
percentages	
  between	
  litters	
  showed	
  changes;	
  instead	
  of	
  continuing	
  the	
  downward	
  trend	
  as	
  
observed	
  in	
  previous	
  weeks,	
  the	
  dissimilarity	
  values	
  generally	
  increased	
  by	
  10-­‐‑20%,	
  with	
  
the	
  lowest	
  being	
  32.04%	
  between	
  litters	
  1631	
  and	
  1402	
  and	
  the	
  highest	
  47.98	
  %	
  between	
  
1700	
  and	
  1445.	
  	
  	
  

	
  

56	
  

	
  
DISCUSSION	
  
	
  
In	
  this	
  study,	
  we	
  followed	
  the	
  development	
  of	
  the	
  tonsillar	
  microbial	
  communities	
  in	
  
pigs	
   from	
   birth	
   through	
   weaning.	
   	
   We	
   focused	
   on	
   identification	
   of	
   the	
   source	
   of	
   bacteria	
  
found	
  in	
  the	
  tonsils,	
  the	
  successional	
  development,	
  and	
  the	
  apparent	
  effect	
  of	
  weaning	
  on	
  the	
  
tonsillar	
  microbiome.	
  
Analysis	
   of	
   the	
   microbiome	
   in	
   the	
   PB	
   piglets	
   showed	
   that	
   the	
   most	
   abundant	
  
organisms	
  were	
  Streptococcus,	
  Staphylococcus,	
  Moraxella,	
  Rothia,	
  and	
  Pasteurellaceae	
  (OTUs	
  
002,	
  009,	
  003,	
  007,	
  and	
  001	
  respectively).	
  Based	
  on	
  comparative	
  analysis	
  of	
  putative	
  source	
  
microbiomes	
  and	
  piglet	
  tonsil	
  microbiomes,	
  as	
  well	
  as	
  cultivation	
  studies,	
  we	
  concluded	
  that	
  
while	
  the	
  Pasteurellaceae	
  and	
  Streptococcus	
  were	
  most	
  likely	
  acquired	
  from	
  the	
  sow	
  vaginal	
  
tract	
   during	
   the	
   birth	
   process,	
   Moraxella,	
   Staphylococcus	
   and	
   Rothia	
   were	
   likely	
   acquired	
  
from	
  the	
  sow	
  teat	
  skin	
  (or	
  milk,	
  which	
  we	
  did	
  not	
  sample)	
  within	
  a	
  few	
  hours	
  after	
  birth.	
  	
  	
  
To	
   determine	
   what	
   organisms	
   were	
   actually	
   acquired	
   during	
   birth,	
   we	
   collected	
  
samples	
  from	
  piglets	
  immediately	
  after	
  they	
  were	
  born	
  (AB	
  piglets),	
  prior	
  to	
  contact	
  with	
  
teat	
  skin	
  or	
  other	
  sources	
  except	
  the	
  sow	
  vaginal	
  tract.	
  	
  As	
  described	
  in	
  results,	
  these	
  samples	
  
were	
   very	
   sparse	
   and	
   the	
   microbiome	
   identified	
   in	
   them	
   was	
   heavily	
   biased	
   by	
   likely	
  
contaminants	
  from	
  either	
  the	
  DNA	
  extraction	
  kit	
  or	
  library	
  construction	
  kit	
  used.	
  	
  This	
  has	
  
recently	
   become	
   a	
   recognized	
   problem	
   in	
   microbiome	
   studies	
   on	
   low	
   microbial	
   biomass	
  
samples	
  [24,	
  32,	
  33].	
  	
  While	
  we	
  ran	
  appropriate	
  positive	
  controls	
  and	
  no	
  template	
  library	
  
construction	
  controls,	
  we	
  did	
  not	
  run	
  DNA	
  extraction	
  controls	
  with	
  the	
  same	
  kit	
  used	
  in	
  this	
  
study.	
   We	
   have	
   done	
   this	
   with	
   a	
   different	
   lot	
   number	
   of	
   the	
   same	
   kit,	
   and	
   used	
   the	
  

	
  

57	
  

information	
  from	
  that	
  experiment	
  as	
  well	
  as	
  other	
  published	
  studies	
  [24]	
  to	
  help	
  analyze	
  the	
  
data	
  on	
  the	
  microbiome	
  in	
  AB	
  piglets.	
  	
  	
  
When	
   we	
   removed	
   what	
   we	
   considered	
   to	
   be	
   contaminants	
   from	
   the	
   data	
   set	
   for	
   AB	
  
piglets,	
   the	
   remaining	
   organisms	
   included	
   OTU002	
   Streptococcus	
   and	
   OTU001	
  
Pasteurellaceae,	
  both	
  of	
  which	
  colonized,	
  multiplied,	
  and	
  persisted	
  for	
  the	
  life	
  of	
  these	
  pigs.	
  
It	
  should	
  be	
  noted	
  that	
  we	
  have	
  cultured	
  Streptococcus	
  suis	
  from	
  at	
  least	
  half	
  of	
  these	
  AB	
  
piglet	
  samples,	
  as	
  well	
  as	
  from	
  sow	
  vaginal	
  samples,	
  providing	
  additional	
  support	
  for	
  the	
  
conclusion	
   that	
   this	
   organism	
   is	
   acquired	
   during	
   the	
   birth	
   process.	
   We	
   also	
   identified	
  
organisms	
  such	
  as	
  OTU016	
  Clostridium	
  sensu	
  strictu	
  and	
  two	
  OTUs	
  (034	
  and	
  051)	
  identified	
  
as	
  Corynebacterium	
  that	
  were	
  found	
  in	
  the	
  sow	
  vaginal	
  tract	
  and	
  in	
  AB	
  and	
  PB	
  piglets,	
  but	
  
not	
  older	
  piglets,	
  demonstrating	
  that	
  some	
  of	
  the	
  organisms	
  acquired	
  during	
  birth	
  do	
  not	
  
persist	
  in	
  the	
  tonsils.	
  	
  	
  
If	
  these	
  organisms	
  failed	
  to	
  adhere	
  to	
  the	
  tonsil	
  epithelium,	
  they	
  would	
  be	
  washed	
  out	
  
of	
   the	
   oropharynx,	
   and	
   other	
   organisms	
   acquired	
   from	
   the	
   vaginal	
   tract,	
   such	
   as	
  
Streptococcus,	
   which	
   do	
   adhere	
   to	
   the	
   tonsil	
   epithelium,	
   would	
   be	
   able	
   to	
   colonize	
   and	
  
multiply.	
  	
  Further,	
  once	
  the	
  piglets	
  had	
  contact	
  with	
  other	
  sow	
  sources	
  such	
  as	
  the	
  teat	
  skin	
  
or	
   milk,	
   new	
   colonizers	
   such	
   as	
   Staphylococcus	
   could	
   be	
   acquired,	
   leading	
   to	
   a	
   sequential	
  
development	
   of	
   the	
   tonsil	
   microbiome	
   starting	
   within	
   the	
   first	
   few	
   hours	
   of	
   life.	
   	
   The	
  
clustering	
  pattern	
  observed	
  in	
  Figure	
  2.1.,	
  where	
  samples	
  from	
  three	
  of	
  the	
  four	
  multiparous	
  
litters	
   clustered	
   together	
   as	
   a	
   group	
   with	
   sow	
   teat	
   skin	
   samples	
   and	
   litters	
   born	
   from	
  
primiparous	
  sows	
  clustered	
  with	
  the	
  cognate	
  teat	
  and	
  vaginal	
  or	
  just	
  teat	
  samples,	
  reinforce	
  
that	
  teat	
  and/or	
  vaginal	
  samples	
  from	
  sow	
  are	
  the	
  initial	
  source	
  of	
  the	
  microbiome	
  for	
  PB	
  
newborns.	
  Our	
  findings	
  are	
  supported	
  by	
  the	
  results	
  of	
  other	
  studies,	
  such	
  as	
  the	
  study	
  of	
  

	
  

58	
  

Mandar	
  and	
  Mikelsaar	
  [34],	
  which	
   characterized	
   the	
  initial	
  colonization	
  of	
  the	
  external	
   ear	
  
canal	
  in	
  newborn	
  humans	
  and	
  compared	
  the	
  microorganisms	
  found	
  with	
  the	
  vaginal	
  flora	
  of	
  
their	
  mothers	
  and	
  concluded	
  that	
  there	
  is	
  a	
  significant	
  influence	
  of	
  the	
  vaginal	
  microflora	
  in	
  
the	
  initial	
  microbial	
  population	
  found	
  in	
  the	
  newborns.	
  	
  Further,	
  Bokulich	
  et	
  al	
  [13],	
  studying	
  
the	
   effect	
   of	
   antibiotics,	
   birthmode	
   and	
   diet	
   in	
   the	
   development	
   of	
   fecal	
   microbiota	
   in	
  
children	
  during	
  early	
  life,	
  suggested	
  that	
  early	
  colonizers	
  are	
  transmitted	
  to	
  children	
  from	
  
maternal	
   microbiota.	
   They	
   found	
   that	
   children	
   delivered	
   vaginally	
   shared	
   more	
   OTUs	
  
derived	
  from	
  the	
  vagina	
  than	
  children	
  delivered	
  by	
  caesarean.	
  Additionally,	
  they	
  identified	
  
that	
   the	
   infant	
   fecal	
   microbiota	
   was	
   initially	
   associated	
   with	
   vaginal	
   and	
   rectal	
   maternal	
  
microbiota,	
  but	
  later	
  was	
  more	
  associated	
  with	
  maternal	
  fecal	
  microbiota.	
  Further,	
  a	
  study	
  
of	
   the	
   development	
   of	
   the	
   human	
   intestinal	
   microbiome	
   suggested	
   that	
   the	
   bacterial	
  
population	
  detected	
  in	
  human	
  infants	
  in	
  early	
  stages	
  of	
  life	
  might	
  be	
  determined	
  by	
  specific	
  
bacteria	
   to	
   which	
   infant	
   was	
   previously	
   exposed,	
   based	
   on	
   similarity	
   patterns	
   observed	
  
between	
  infant	
  samples	
  and	
  maternal	
  sources	
  as	
  breast	
  milk	
  and	
  vaginal	
  swabs	
  [15].	
  These	
  
authors	
   identified	
   Streptococcus	
   and	
   Staphylococcus	
   as	
   well	
   as	
   other	
   aerobes	
   as	
   first	
  
colonizers	
  [15].	
  Similarly,	
  we	
  observed	
  that	
  piglets	
  sampled	
  within	
  short	
  period	
  after	
  being	
  
born	
  had	
  high	
  proportions	
  of	
  Streptococcus,	
  Moraxella	
  and	
  Staphylococcus	
  in	
  their	
  tonsils.	
  
Our	
   results	
   demonstrated	
   a	
   strong	
   litter	
   effect	
   in	
   the	
   tonsillar	
   microbiome	
   in	
   PB	
  
piglets	
   (Figure	
   2.3.).	
   	
   However,	
   over	
   the	
   following	
   three	
   weeks	
   there	
   was	
   a	
   gradual	
  
successional	
  development	
  in	
  the	
  tonsil	
  microbiomes	
  of	
  all	
  piglets	
  and	
  by	
  the	
  third	
  week	
  the	
  
microbiomes	
  of	
  all	
  piglets	
  from	
  all	
  litters	
  were	
  highly	
  similar	
  (Figure	
  2.4.).	
  	
  This	
  was	
  true	
  
even	
  for	
  a	
  litter	
  that	
  did	
  not	
  share	
  the	
  same	
  farrowing	
  room,	
  as	
  was	
  the	
  case	
  for	
  piglets	
  from	
  
litter	
   1445	
   (data	
   not	
   shown).	
   	
   This	
   is	
   in	
   contrast	
   to	
   the	
   reported	
   development	
   of	
   the	
   pig	
  

	
  

59	
  

intestinal	
   microbiome	
   over	
   the	
   first	
   few	
   weeks	
   of	
   life,	
   where	
   no	
   obvious	
   effect	
   of	
   litter	
   was	
  
seen	
  [35].	
  	
  
In	
   this	
   successional	
   development,	
   some	
   organisms	
   such	
   as	
   Staphylococcus	
   and	
  
Micrococcaceae	
  that	
  were	
  found	
  in	
  high	
  proportions	
  in	
  the	
  PB	
  piglets	
  decreased	
  dramatically	
  
within	
   the	
   next	
   2	
   weeks,	
   suggesting	
   a	
   role	
   only	
   as	
   initial	
   weak	
   colonizers.	
   These	
   organisms	
  
are	
  commonly	
  isolated	
  from	
  multiple	
  skin	
  locations	
  [36,	
  37]	
  and	
  vagina	
  [38]	
  and	
  are	
  likely	
  
to	
   be	
   vertically	
   transmitted	
   from	
   sow	
   to	
   offspring.	
   Over	
   the	
   same	
   period	
   there	
   was	
   a	
  
concomitant	
   increase	
   in	
   stronger	
   colonizers,	
   particularly	
   members	
   of	
   the	
   families	
  
Pasteurellaceae	
  and	
  Moraxellaceae,	
  as	
  well	
  as	
  maintenance	
  of	
  the	
  levels	
  of	
  Streptococcaceae.	
  
These	
  three	
  families,	
  which	
  contain	
  both	
  commensals	
  and	
  pathogens	
  that	
  are	
  residents	
  of	
  
mucosal	
   surfaces	
   of	
   animals	
   and	
   humans,	
   comprise	
   a	
   large	
   proportion	
   of	
   the	
   tonsil	
  
microbiome	
   throughout	
   the	
   lives	
   of	
   pigs.	
   Members	
   of	
   the	
   order	
   Clostridiales	
   were	
   also	
  
identified	
   as	
   a	
   small	
   but	
   consistent	
   part	
   of	
   the	
   microbiome	
   throughout	
   the	
   first	
   3	
   weeks,	
  
which	
  is	
  not	
  surprising	
  since	
  they	
  were	
  identified	
  as	
  part	
  of	
  the	
  core	
  tonsillar	
  microbiome	
  of	
  
pigs	
  [12]	
  as	
  well	
  as	
  members	
  of	
  intestinal	
  microbiome	
  [16,	
  19,	
  35,	
  39].	
  
Over	
  this	
  period	
  we	
  also	
  found	
  several	
  transitory	
  OTUs	
  or	
  families	
  that	
  appeared	
  and	
  
disappeared	
  at	
  specific	
  time	
  points.	
  	
  For	
  example,	
  Porphyromonadaceae,	
  Prevotellaceae,	
  and	
  
Flavobacteriaceae	
   appeared	
   at	
   week	
   1	
   and	
   increased	
   slightly	
   over	
   the	
   next	
   2	
   weeks.	
  	
  
Similarly,	
  Fusobacteriaceae	
  and	
  Leptotrichiaceae	
  appeared	
  at	
  weeks	
  2	
  and	
  3.	
  	
  By	
  week	
  three,	
  
this	
   successional	
   development	
   of	
   the	
   tonsil	
   microbiome	
   of	
   all	
   of	
   the	
   piglets,	
   regardless	
   of	
  
litter,	
  led	
  to	
  a	
  distinct	
  common	
  consortium	
  of	
  bacterial	
  species.	
  	
  
Similarly,	
  Palmer	
  et	
  al	
  [15],	
  studied	
  the	
  microbiome	
  profiles	
  from	
  human	
  infant	
  stool	
  
samples	
   and	
   suggested	
   that	
   although	
   initially	
   the	
   microbiome	
   was	
   very	
   distinct	
   between	
  

	
  

60	
  

individuals,	
   over	
   time	
   it	
   converged	
   towards	
   a	
   common	
   profile.	
   The	
   authors	
   followed	
   the	
  
development	
   of	
   the	
   intestinal	
   microbiome	
   in	
   14	
   human	
   infants,	
   and	
   showed	
   that	
   there	
   was	
  
a	
   considerable	
   variation	
   in	
   the	
   colonization	
   process	
   among	
   individuals.	
   Each	
   infant	
   had	
   a	
  
distinct	
   arrangement	
   of	
   bacterial	
   species	
   that	
   it	
   acquired	
   and	
   maintained.	
   The	
   acquired	
  
microorganisms	
  had	
  a	
  temporal	
  pattern	
  in	
  which	
  they	
  appeared	
  and	
  disappeared;	
  however,	
  
they	
  reached	
  a	
  stable	
  population	
  over	
  time,	
  with	
  some	
  taxonomic	
  groups	
  persisting	
  while	
  
the	
   presence	
   of	
   other	
   taxa	
   was	
   only	
   transient	
   [15].	
   The	
   authors	
   reported	
   the	
   occurrence	
   of	
  
significant	
  shifts	
  in	
  the	
  population	
  assembly,	
  which	
  seemed	
  to	
  stabilize	
  over	
  time.	
  	
  	
  
At	
  3	
  weeks	
  of	
  age,	
  the	
  piglets	
  were	
  weaned	
  onto	
  a	
  solid	
  ration	
  containing	
  the	
  growth	
  
promoter	
  Carbadox 	
  and	
  were	
  moved	
  from	
  the	
  farrowing	
  room	
  to	
  the	
  nursery,	
  while	
  kept	
  in	
  
®

groups	
  with	
  their	
  littermates.	
  Comparison	
  of	
  the	
  microbiome	
  composition	
  for	
  the	
  third	
  and	
  
fourth	
  weeks	
  showed	
  a	
  major	
  shift	
  associated	
  with	
  the	
  significant	
  stress	
  event	
  of	
  weaning,	
  
with	
  its	
  environmental,	
  social	
  and	
  feed	
  changes.	
  Our	
  results	
  are	
  in	
  concordance	
  with	
  other	
  
studies	
   following	
   the	
   development	
   of	
   the	
   intestinal	
   microbiome	
   in	
   humans	
   and	
   pigs,	
   which	
  
reported	
  significant	
  changes	
  in	
  microbiome	
  composition	
  associated	
  with	
  life	
  events	
  [14,	
  15].	
  
In	
   pigs,	
   it	
   has	
   been	
   demonstrated	
   that	
   the	
   transition	
   from	
   nursery	
   to	
   weaning	
   is	
   associated	
  
with	
   a	
   significant	
   change	
   in	
   the	
   intestinal	
   microbiota	
   [19,	
   40].	
   However,	
   the	
   effect	
   of	
   this	
  
transition	
  on	
  the	
  tonsillar	
  microbiome	
  has	
  not	
  previously	
  been	
  studied	
  in	
  pigs	
  or	
  humans.	
  In	
  
this	
  study,	
  we	
  have	
  demonstrated	
  that	
  the	
  transition	
  from	
  farrowing	
  to	
  a	
  nursery	
  room	
  in	
  
parallel	
   with	
   weaning	
   and	
   supplementation	
   of	
   the	
   diet	
   with	
   Carbadox 	
   was	
   associated	
   with	
  
®

a	
  major	
  shift	
  in	
  the	
  tonsil	
  microbiota.	
  	
  
The	
   most	
   obvious	
   effect	
   of	
   weaning	
   was	
   the	
   5-­‐‑6	
   fold	
   increase	
   in	
   members	
   of	
   the	
  
Streptococcaceae,	
   from	
   an	
   average	
   of	
   8%	
   before	
   weaning	
   to	
   ~43%	
   of	
   the	
   total	
   identified	
  

	
  

61	
  

families	
   after	
   weaning.	
   These	
   organisms	
   were	
   primarily	
   members	
   of	
   the	
   genus	
  
Streptococcus.	
  There	
  was	
  a	
  concomitant	
  decrease	
  for	
  members	
  of	
  the	
  Moraxellaceae	
  family,	
  
which	
  decreased	
  approximately	
  4-­‐‑fold,	
  from	
  ~32%	
  before	
  weaning	
  to	
  ~8%	
  after	
  weaning.	
  
In	
  

addition,	
  

Fusobacteriaceae,	
  

Leptotrichiaceae,	
  

Porphyromonadaceae,	
  

and	
  

Flavobacteriaceae	
   decreased	
   dramatically.	
   However,	
   at	
   the	
   same	
   time,	
   the	
   proportion	
  
represented	
  by	
  Pasteurellaceae	
  remained	
  constant.	
  	
  	
  
It	
  is	
  not	
  clear	
  whether	
  a	
  single	
  stress,	
  such	
  as	
  change	
  in	
  food	
  or	
  change	
  in	
  environment	
  
or	
   application	
   of	
   antibiotic,	
   or	
   a	
   combination	
   of	
   stresses	
   was	
   responsible	
   for	
   the	
   major	
  
disruption	
  in	
  the	
  tonsil	
  microbiome	
  at	
  this	
  time.	
  It	
  has	
  been	
  demonstrated	
  in	
  multiple	
  studies	
  
that	
  the	
  intestinal	
  microbiota	
  composition	
  was	
  deeply	
  perturbed	
  when	
  the	
  host	
  was	
  treated	
  
with	
   antibiotics	
   [8,	
   13,	
   15,	
   41-­‐‑44].	
   In	
   our	
   study,	
   the	
   piglets	
   were	
   supplemented	
   with	
  
Carbadox 	
   in	
   food	
   at	
   the	
   time	
   of	
   weaning.	
   	
   	
   It	
   was	
   reported	
   by	
   Looft	
   et	
   al	
   [41]	
   that	
   the	
  
®

structure	
  and	
  composition	
  of	
  the	
  intestinal	
  community	
  of	
  pigs	
  supplemented	
  with	
  Carbadox 	
  
®

changed	
  significantly,	
  where	
  the	
  relative	
  abundance	
  of	
  Prevotella	
  increased	
  associated	
  with	
  
Carbadox 	
   administration	
   as	
   a	
   result	
   of	
   decreased	
   abundance	
   of	
   other	
   bacteria.	
   Another	
  
®

study	
  correlating	
  changes	
  in	
  microbiota	
  with	
  changes	
  of	
  diet	
  during	
  nursing	
  and	
  weaning	
  
found	
   that	
   the	
   fecal	
   population	
   of	
   Prevotellaceae	
   increased	
   ~50-­‐‑fold	
   in	
   weaned	
   pigs	
  	
  
compared	
   with	
   nursing	
   animals	
   [19].	
   In	
   our	
   study,	
   members	
   of	
   Prevotellaceae,	
   a	
   minor	
  
population	
  in	
  the	
  tonsils,	
  decreased	
  slightly	
  from	
  2.7	
  %	
  for	
  week	
  three	
  to	
  1.9%	
  for	
  week	
  four.	
  
We	
   can	
   only	
   speculate	
   about	
   the	
   opposite	
   results	
   identified	
   in	
   our	
   studies,	
   since	
   we	
   are	
  
comparing	
  different	
  niches	
  (feces	
  vs	
  tonsils)	
  and	
  we	
  did	
  not	
  have	
  controls	
  that	
  were	
  not	
  fed	
  
Carbadox 	
  as	
  this	
  was	
  not	
  a	
  goal	
  of	
  our	
  study.	
  
®

	
  

62	
  

It	
   has	
   been	
   suggested	
   that	
   the	
   environment	
   also	
   plays	
   a	
   relevant	
   role	
   in	
   the	
   initial	
  
acquisition	
  of	
  the	
  microbiome.	
  Mulder	
  et	
  al	
  [11],	
  followed	
  the	
  development	
  of	
  gut	
  microbiota	
  
and	
  the	
  potential	
  impacts	
  of	
  early	
  environmental	
  changes,	
  and	
  demonstrated	
  a	
  major	
  impact	
  
in	
  the	
  microbial	
  diversity	
  related	
  with	
  those	
  changes	
  and	
  that	
  the	
  impact	
  of	
  those	
  changes	
  
are	
   preserved	
   through	
   adulthood.	
   One	
   of	
   the	
   major	
   changes	
   experienced	
   by	
   the	
   weaned	
  
piglets	
  is	
  the	
  separation	
  from	
  the	
  sow	
  and	
  the	
  introduction	
  to	
  new	
  food.	
  A	
  deep	
  impact	
  in	
  the	
  
intestinal	
  microbial	
  composition	
  has	
  been	
  seen	
  associated	
  with	
  cessation	
  of	
  breast	
  feeding	
  
and	
  introduction	
  to	
  a	
  different	
  diet	
  [20].	
  	
  	
  	
  
We	
  observed	
  members	
  of	
  the	
  microbiota	
  that	
  were	
  present	
  all	
  the	
  time,	
  some	
  in	
  high	
  
relative	
  abundance	
  while	
  others	
  were	
  found	
  in	
  low	
  abundance.	
  Similarly,	
  there	
  were	
  also	
  
transient	
  members	
  whose	
  exact	
  role	
  in	
  the	
  development	
  of	
  the	
  tonsil	
  microbiome	
  is	
  unclear	
  
but	
   worth	
   investigating	
   in	
   future	
   studies.	
   It	
   is	
   possible	
   that	
   these	
   transient	
   organisms	
  
appeared	
   as	
   secondary	
   colonizers	
   but	
   were	
   displaced	
   by	
   other	
   members	
   of	
   the	
   microbiota,	
  
or	
   that	
   these	
   microorganisms	
   were	
   adversely	
   affected	
   by	
   the	
   stressful	
   event	
   of	
   weaning	
  
and/or	
   the	
   Carbadox 	
  supplementation	
  and	
  thus	
  disappeared.	
  We	
   have	
  an	
  imprecise	
  idea	
  
®

about	
  the	
  true	
  role	
  of	
  Carbadox 	
  in	
  the	
  development	
  of	
  the	
  tonsillar	
  microbiome,	
  since	
  our	
  
®

study	
  was	
  not	
  specifically	
  intended	
  to	
  answer	
  this	
  question.	
  We	
  unfortunately	
  did	
  not	
  collect	
  
sow	
  milk	
  samples,	
  or	
  samples	
  from	
  the	
  pen/cage	
  floor	
  which	
  might	
  have	
  given	
  us	
  a	
  better	
  
idea	
  of	
  the	
  possible	
  sources	
  of	
  the	
  PB	
  and	
  subsequent	
  microbiome.	
  	
  
There	
  are	
  many	
  questions	
  that	
  arise	
  from	
  our	
  work	
  that	
  will	
  be	
  the	
  subject	
  of	
  future	
  
research.	
   However,	
   this	
   study	
   lays	
   the	
   foundation	
   of	
   our	
   knowledge	
   of	
   how	
   the	
   tonsillar	
  
microbiome	
  develops	
  in	
  pigs	
  in	
  the	
  first	
  hours	
  and	
  weeks	
  of	
  age	
  and	
  how	
  weaning	
  affects	
  

	
  

63	
  

this	
  microbiome.	
  To	
  the	
  best	
  of	
  our	
  knowledge,	
  this	
  is	
  the	
  first	
  published	
  study	
  that	
  follows	
  
the	
  development	
  of	
  the	
  tonsillar	
  microbiome	
  in	
  any	
  mammal	
  during	
  the	
  first	
  weeks	
  of	
  life.	
  	
  
	
  
CONCLUSIONS	
  
	
  
Our	
  data	
  demonstrate	
  a	
  temporal	
  succession	
  in	
  the	
  development	
  of	
  the	
  pig	
  tonsillar	
  
microbiome	
   through	
   the	
   first	
   weeks	
   of	
   life.	
   	
   Many	
   of	
   the	
   organisms	
   found	
   in	
   the	
   piglet	
  
oropharynx	
  and	
  tonsils	
  immediately	
  after	
  birth	
  disappear	
  rapidly,	
  within	
  the	
  first	
  few	
  hours	
  
of	
  life,	
  while	
  other	
  organisms	
  acquired	
  from	
  the	
  vaginal	
  tract,	
  such	
  as	
  Streptococcaceae	
  and	
  
Pasteurellaceae,	
  colonize	
  and	
  multiply.	
  Additional	
  organisms	
  are	
  also	
  acquired	
  rapidly	
  from	
  
the	
  sow	
  teat	
  skin,	
  and	
  possibly	
  from	
  milk,	
  and	
  eventually	
  also	
  from	
  feces.	
  The	
  composition	
  
of	
   the	
   PB	
   newborn	
   piglet	
   tonsil	
   microbiome	
   initially	
   can	
   be	
   differentiated	
   by	
   litter	
   and	
  
clusters	
  mainly	
  with	
  the	
  sow	
  teat	
  skin	
  microbiome.	
  Nevertheless,	
  over	
  the	
  next	
  three	
  weeks,	
  
the	
   composition	
   and	
   structure	
   of	
   the	
   tonsil	
   microbiome	
   reaches	
   a	
   common	
   point	
   of	
  
development,	
   showing	
   a	
   high	
   degree	
   of	
   similarity	
   among	
   all	
   piglets,	
   regardless	
   of	
   litter	
   and	
  
just	
  prior	
  to	
  weaning.	
  However,	
  there	
  was	
  a	
  dramatic	
  change	
  in	
  the	
  post	
  weaning	
  tonsillar	
  
microbiome,	
   which	
   was	
   likely	
   engendered	
   by	
   a	
   combination	
   of	
   change	
   in	
   diet,	
   change	
   in	
  
environment,	
   and	
   addition	
   of	
   in-­‐‑	
   feed	
   antibiotic,	
   demonstrating	
   the	
   effect	
   that	
   weaning	
  
management	
   practices	
   exert	
   in	
   shaping	
   tonsillar	
   microbial	
   communities.	
   This	
   research	
  
demonstrates	
  the	
  need	
  for	
  further	
  studies	
  to	
  elucidate	
  the	
  role	
  of	
  antibiotic	
  supplementation	
  
of	
   feed	
   in	
   the	
   development	
   of	
   tonsillar	
   microbial	
   communities,	
   specifically	
   when	
  
administered	
  during	
  the	
  highly	
  susceptible	
  time	
  of	
  weaning.	
  	
  

	
  

64	
  

	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
APPENDIX	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

65	
  

Table	
  2.2.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  litters	
  and	
  sow	
  samples.	
  	
  

	
  
Numbers	
   in	
   different	
   colors	
   identify	
   core	
   OTUs	
   at	
   different	
   percentages	
   of	
   relative	
  
abundance.	
  Red	
  shows	
  core	
  OTUs	
  at	
  1%	
  relative	
  abundance.	
  Blue	
  identify	
  core	
  OTUs	
  at	
  0.1%	
  
relative	
  abundance.	
  Black	
  color	
  denotes	
  OTUs	
  that	
  were	
  not	
  core.	
  
	
  

	
  

66	
  

Table	
  2.2.	
  (cont´d).	
  	
  

	
  
Numbers	
   in	
   different	
   colors	
   identify	
   core	
   OTUs	
   at	
   different	
   percentages	
   of	
   relative	
  
abundance.	
  Red	
  shows	
  core	
  OTUs	
  at	
  1%	
  relative	
  abundance.	
  Blue	
  identify	
  core	
  OTUs	
  at	
  0.1%	
  
relative	
  abundance.	
  Black	
  color	
  denotes	
  OTUs	
  that	
  were	
  not	
  core.	
  
	
  
	
  
	
  
	
  
	
  
	
  

67	
  

Table	
  2.3.	
  SIMPER	
  analysis	
  between	
  litters	
  through	
  different	
  sampling	
  times	
  

	
  	
  
Data	
   displayed	
   represent	
   values	
   of	
   overall	
   average	
   dissimilarity,	
   when	
   a	
   comparison	
  
between	
  litter	
  was	
  performed	
  for	
  the	
  different	
  sampling	
  times.	
  
n.i.:	
   Number	
  of	
  samples	
  included	
  in	
  the	
  analysis	
  
parity:	
  Total	
  number	
  of	
  deliveries	
  that	
  the	
  sow	
  had	
  when	
  was	
  sampled	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

68	
  

Table	
  2.4.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  third	
  week	
  
OTUs
Otu00001__Pasteurellaceae__
Otu00002__Streptococcaceae__Streptococcus
Otu00003__Moraxellaceae__Moraxella
Otu00006__Porphyromonadaceae__
Otu00007__Micrococcaceae__Rothia
Otu00008__Moraxellaceae__
Otu00010__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00012__Streptococcaceae__Streptococcus
Otu00015__Prevotellaceae__
Otu00016__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00017__Fusobacteriaceae__Fusobacterium
Otu00018__Peptostreptococcaceae__Clostridium_XI
Otu00020__Erysipelotrichaceae__Turicibacter
Otu00023__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00024__Bacillales_Incertae_Sedis_XI__Gemella
Otu00025__Peptostreptococcaceae__Clostridium_XI
Otu00026__Flavobacteriaceae__
Otu00027__Veillonellaceae__Veillonella
Otu00028__Leptotrichiaceae__
Otu00030__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00035__Pasteurellaceae__Pasteurella
Otu00041__Neisseriaceae__Neisseria
Otu00042__Ruminococcaceae__
Otu00043__Flavobacteriaceae__
Otu00044__Neisseriaceae__
Otu00045__Moraxellaceae__Acinetobacter
Otu00048__Lactobacillaceae__Lactobacillus
Otu00050__Bacteroidaceae__Bacteroides
Otu00051__Corynebacteriaceae__Corynebacterium
Otu00064__Aerococcaceae__
Otu00070__Pasteurellaceae__Actinobacillus
Otu00071__Prevotellaceae__Prevotella
Otu00072__Ruminococcaceae__
Otu00076__Lachnospiraceae__
Otu00077__Carnobacteriaceae__
Otu00078__Ruminococcaceae__
Otu00079__Bacteroidaceae__Bacteroides
Otu00085__Streptococcaceae__Streptococcus
Otu00091__Lachnospiraceae__Roseburia
Otu00094__Streptococcaceae__Lactococcus
Otu00097__Alcaligenaceae__
Otu00098__Clostridiales_Incertae_Sedis_XI__Helcococcus
Otu00108__Lachnospiraceae__
Otu00122__Leptotrichiaceae__Leptotrichia
Otu00127__Fusobacteriaceae__Fusobacterium
Otu00130__Propionibacteriaceae__Propionibacterium
Otu00143__Campylobacteraceae__Campylobacter
Otu00147__Erysipelotrichaceae__
Otu00149__Bacteria_unclassified__
Otu00156__Leptotrichiaceae__Sneathia
Otu00159__Alcaligenaceae__
Otu00162__Succinivibrionaceae__Succinivibrio
Otu00174__Erysipelotrichaceae__
Otu00176__Dermabacteraceae__Brachybacterium
Otu00183__Actinomycetaceae__Actinomyces
Otu00195__Spirochaetaceae__
Otu00200__Moraxellaceae__Moraxella
Otu00204__Ruminococcaceae__
Otu00205__Lachnospiraceae__
Otu00209__Aerococcaceae__Facklamia
Otu00225__Leptotrichiaceae__
Otu00232__Fusobacteriaceae__Fusobacterium
Otu00280__Spirochaetaceae__
Otu00288__Dietziaceae__Dietzia
Otu00292__Bacteroidaceae__Bacteroides
Otu00311__Xanthomonadaceae__Stenotrophomonas
Otu00324__Lachnospiraceae__
Otu00338__Lachnospiraceae__
Otu00346__Leptotrichiaceae__
Otu00347__Ruminococcaceae__
Otu00510__Lachnospiraceae__
Otu00545__Cardiobacteriaceae__Suttonella
Otu00582__Lachnospiraceae__Clostridium_XlVa
Otu00653__Ruminococcaceae__
Otu00982__Clostridiales_Incertae_Sedis_XI__Anaerococcus
Otu01018__Oligosphaeraceae__Oligosphaera
Otu01058__Ruminococcaceae__Butyricicoccus
Otu01160__Ruminococcaceae__Faecalibacterium
Otu01326__Alcanivoracaceae__Alcanivorax
Otu01828__Lachnospiraceae__Clostridium_XlVa
Otu02516__Ruminococcaceae__
Otu03039__Moraxellaceae__Moraxella

Litter	
1700	
23.1
5.6
27.6
4.6
0.4
5.7
0.1
0.3
0.6
0.0
4.0
0.2
0.1
0.0
2.1
0.1
2.3
0.1
3.2
0.0
0.1
0.2
0.0
0.6
0.2
0.0
0.0
0.3
0.0
0.0
0.0
0.1
2.5
0.2
0.2
0.0
0.4
0.1
0.0
0.0
0.2
0.1
0.1
0.0
0.2
0.0
0.1
0.1
0.0
0.6
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

*Litter	
1707
16.5
10.3
40.7
2.6
0.5
3.8
0.6
0.6
3.2
0.2
1.1
0.1
0.1
0.0
1.0
0.0
2.3
0.2
0.3
0.0
0.0
0.0
0.8
0.0
0.0
0.1
0.0
0.0
0.3
0.0
0.0
0.4
2.0
0.0
0.6
0.0
0.0
0.3
0.0
0.3
0.3
0.0
0.0
0.3
0.0
1.9
0.0
0.2
0.0
0.0
0.3
0.0
0.2
1.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
2.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1

Litter	
1631
23.3
6.2
24.1
7.2
0.5
10.1
0.5
2.0
1.3
0.1
3.0
0.6
0.3
0.0
0.8
0.3
2.7
0.2
3.2
0.2
0.1
0.6
0.1
0.3
0.4
0.1
0.0
0.4
0.0
0.1
0.0
0.4
0.0
0.2
0.3
0.1
0.5
0.0
0.0
0.0
0.3
0.2
0.1
0.0
0.1
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.4
0.0
0.0
0.0
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

Litter	
1445
22.3
7.3
26.3
5.9
0.6
0.2
0.4
0.2
1.9
0.1
5.3
0.3
0.1
0.1
0.8
0.2
5.1
0.4
1.9
0.0
0.1
0.2
0.1
0.8
2.8
0.0
0.1
0.5
0.0
0.2
0.0
0.7
1.0
0.3
0.1
0.1
0.8
0.1
0.0
0.0
0.3
0.6
0.1
0.1
0.2
0.0
0.4
0.1
0.1
0.0
0.0
0.0
0.0
0.0
0.2
0.1
0.2
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

*Litter	
1402
18.5
5.6
38.9
2.8
0.7
5.5
3.6
1.1
0.7
0.2
1.4
1.6
1.0
0.0
0.8
2.0
1.2
0.0
0.1
0.8
0.3
0.4
0.3
0.4
0.0
0.0
0.1
0.0
0.0
0.3
0.2
0.0
0.3
0.0
0.8
0.1
0.2
0.1
0.5
0.0
0.0
0.0
0.3
0.0
0.0
0.1
0.0
0.0
0.2
0.0
0.0
0.4
0.0
0.0
0.0
0.0
0.0
0.1
0.1
0.3
0.0
0.2
0.1
0.0
0.0
0.1
0.2
0.1
0.1
0.2
0.3
0.0
0.3
0.4
0.3
0.1
0.2
0.4
0.3
0.1
0.1
0.0

Litter	
1711
23.7
8.6
28.4
4.2
0.1
1.8
0.2
0.4
1.9
0.1
4.4
0.1
0.1
0.0
0.6
0.0
3.8
0.1
3.9
0.0
0.1
0.2
0.0
0.9
0.1
0.0
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.5
0.4
0.0
1.3
0.0
0.0
0.0
0.0
0.7
0.0
0.6
0.1
0.0
0.3
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.2
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

Average
21.2
7.3
31.0
4.5
0.4
4.5
0.9
0.8
1.6
0.1
3.2
0.5
0.3
0.0
1.0
0.4
2.9
0.2
2.1
0.2
0.1
0.2
0.2
0.5
0.6
0.0
0.0
0.4
0.1
0.1
0.0
0.3
0.9
0.2
0.4
0.1
0.5
0.1
0.1
0.1
0.2
0.3
0.1
0.2
0.1
0.3
0.1
0.1
0.0
0.1
0.1
0.1
0.0
0.2
0.1
0.0
0.2
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.1
0.1
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.0

	
  

Numbers	
   in	
   different	
   colors	
   identify	
   core	
   OTUs	
   at	
   different	
   percentages	
   of	
   relative	
  
abundance.	
  Red	
  shows	
  core	
  OTUs	
  at	
  1%	
  relative	
  abundance.	
  Blue	
  identify	
  core	
  OTUs	
  at	
  
0.1%	
   relative	
   abundance.	
   Black	
   color	
   denotes	
   OTUs	
   that	
   were	
   not	
   core.	
   *	
   indicates	
   litters	
  
with	
  less	
  than	
  4	
  samples	
  for	
  this	
  specific	
  time.	
  
	
  
	
  

69	
  

Table	
  2.5.	
  Core	
  microbiome	
  at	
  OTU	
  level	
  for	
  fourth	
  week	
  
OTUs
Otu00001__Pasteurellaceae__
Otu00002__Streptococcaceae__Streptococcus
Otu00003__Moraxellaceae__Moraxella
Otu00005__Enterobacteriaceae__Escherichia/Shigella
Otu00007__Micrococcaceae__Rothia
Otu00008__Moraxellaceae__
Otu00010__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00012__Streptococcaceae__Streptococcus
Otu00018__Peptostreptococcaceae__Clostridium_XI
Otu00019__Sphingomonadaceae__Sphingomonas
Otu00024__Bacillales_Incertae_Sedis_XI__Gemella
Otu00026__Flavobacteriaceae__
Otu00027__Veillonellaceae__Veillonella
Otu00034__Corynebacteriaceae__Corynebacterium
Otu00035__Pasteurellaceae__Pasteurella
Otu00037__Clostridiales_Incertae_Sedis_XI__Tissierella
Otu00041__Neisseriaceae__Neisseria
Otu00042__Ruminococcaceae__
Otu00044__Neisseriaceae__
Otu00045__Moraxellaceae__Acinetobacter
Otu00047__Streptococcaceae__Streptococcus
Otu00051__Corynebacteriaceae__Corynebacterium
Otu00052__Lactobacillaceae__Lactobacillus
Otu00054__Clostridiales_Incertae_Sedis_XI__Sedimentibacter
Otu00056__Enterobacteriaceae__Yersinia
Otu00059__Lactobacillaceae__Lactobacillus
Otu00064__Aerococcaceae__
Otu00066__Planococcaceae__
Otu00070__Pasteurellaceae__Actinobacillus
Otu00082__Ruminococcaceae__Faecalibacterium
Otu00091__Lachnospiraceae__Roseburia
Otu00097__Alcaligenaceae__
Otu00100__Lachnospiraceae__Blautia
Otu00104__Erysipelotrichaceae__Sharpea
Otu00105__Ruminococcaceae__
Otu00109__Prevotellaceae__
Otu00114__Veillonellaceae__Megasphaera
Otu00118__Prevotellaceae__Paraprevotella
Otu00124__Prevotellaceae__Prevotella
Otu00125__Corynebacteriaceae__Turicella
Otu00130__Propionibacteriaceae__Propionibacterium
Otu00135__Veillonellaceae__
Otu00140__Planococcaceae__
Otu00174__Erysipelotrichaceae__
Otu00181__Enterococcaceae__Enterococcus
Otu00193__Porphyromonadaceae__
Otu00194__Prevotellaceae__Prevotella
Otu00200__Moraxellaceae__Moraxella
Otu00240__Acidaminococcaceae__Acidaminococcus
Otu00261__Ruminococcaceae__Butyricicoccus
Otu00262__Prevotellaceae__Prevotella
Otu00269__Ruminococcaceae__
Otu00284__Veillonellaceae__Mitsuokella
Otu00307__Prevotellaceae__Prevotella
Otu00313__Lachnospiraceae__Dorea
Otu00315__Prevotellaceae__Alloprevotella
Otu00338__Lachnospiraceae__
Otu00352__Sutterellaceae__Sutterella
Otu00367__Ruminococcaceae__
Otu00438__Streptococcaceae__Streptococcus
Otu00480__Prevotellaceae__Alloprevotella
Otu00517__Lachnospiraceae__Blautia
Otu00519__Prevotellaceae__Prevotella
Otu00543__Ruminococcaceae__Ruminococcus
Otu00609__Lachnospiraceae__Ruminococcus2
Otu00637__Ruminococcaceae__
Otu00660__Erysipelotrichaceae__
Otu00675__Erysipelotrichaceae__
Otu00694__Streptococcaceae__Streptococcus
Otu00783__Streptococcaceae__Streptococcus
Otu01058__Ruminococcaceae__Butyricicoccus
Otu01339__Alcaligenaceae__Oligella

Litter	
1700

Litter	
1631

Litter	
1445

*Litter	
1402

Litter	
1711

Average

28.8
46.3
3.0
0.5
1.2
0.1
0.1
1.9
0.0
0.0
0.3
0.1
1.1
0.3
0.0
0.1
0.3
0.0
0.0
0.2
0.1
0.0
4.8
0.3
0.1
0.8
0.1
0.1
0.3
0.3
0.2
0.1
0.2
0.2
0.0
0.1
0.1
0.0
0.2
0.1
0.0
0.0
0.0
0.0
0.1
0.0
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.1
0.1
0.0
0.0
0.0
0.1
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

23.1
51.0
0.9
0.2
0.7
0.0
0.4
2.5
0.0
0.3
0.1
0.0
0.7
0.0
0.1
0.2
0.2
0.1
0.2
0.2
0.1
0.1
0.6
0.0
0.0
0.1
0.0
0.0
0.1
0.6
0.5
0.0
0.5
0.1
0.1
0.2
0.1
0.0
0.3
0.0
0.0
0.1
0.1
0.0
0.2
0.1
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.1
0.2
0.3
0.1
0.0
0.1
0.1
0.2
0.1
0.1
0.1
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0

21.3
28.8
9.0
0.3
2.5
2.9
0.7
0.5
0.2
0.1
1.0
0.1
1.5
0.0
0.2
0.2
3.9
0.2
1.9
0.0
0.0
0.0
1.2
0.0
0.0
0.4
0.1
0.0
0.0
0.6
0.4
0.1
0.9
0.9
0.2
0.2
0.3
0.2
0.6
0.0
0.0
0.4
0.0
0.1
0.2
0.2
0.5
0.0
0.3
0.2
0.2
0.0
0.5
0.1
0.1
0.0
0.1
0.0
0.1
0.1
0.1
0.1
0.2
0.1
0.1
0.0
0.3
0.0
0.0
0.1
0.1
0.0

15.2
63.2
5.1
0.0
4.3
1.1
0.6
0.0
0.0
0.0
1.4
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.7
0.0
0.4
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.5
0.8
0.0
0.7
0.2
0.0
0.0
0.0
0.0
0.1
0.0
0.1
0.0
0.0
0.9
0.0
0.0
0.2
0.1
0.0
0.0
0.0
0.6
0.0
0.0
0.0
0.0
0.3
1.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.4
0.0
0.1
0.3
0.2
0.0
0.3

20.3
40.0
13.1
0.1
1.3
2.9
0.3
0.6
0.0
0.0
0.3
0.3
0.4
0.2
0.0
0.2
1.0
0.1
0.8
0.2
0.2
0.1
0.4
0.5
0.2
0.3
0.2
0.2
0.2
0.5
0.7
0.3
0.6
0.3
0.1
0.3
0.1
0.0
0.1
0.0
0.0
0.2
0.0
0.0
0.1
0.2
0.1
0.0
0.1
0.1
0.0
0.0
0.0
0.1
0.1
0.2
0.1
0.0
0.1
0.1
0.0
0.0
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0

21.7
45.9
6.2
0.2
2.0
1.4
0.4
1.1
0.1
0.1
0.6
0.1
0.7
0.1
0.1
0.1
1.1
0.1
0.6
0.2
0.1
0.1
1.4
0.2
0.1
0.3
0.1
0.1
0.1
0.5
0.5
0.1
0.6
0.3
0.1
0.2
0.1
0.0
0.3
0.0
0.0
0.2
0.0
0.2
0.1
0.1
0.2
0.0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.0
0.1
0.0
0.1
0.1
0.1
0.0
0.1
0.1
0.0
0.1

	
  

Numbers	
   in	
   different	
   colors	
   identify	
   core	
   OTUs	
   at	
   different	
   percentages	
   of	
   relative	
  
abundance.	
  Red	
  shows	
  core	
  OTUs	
  at	
  1%	
  relative	
  abundance.	
  Blue	
  identify	
  core	
  OTUs	
  at	
  0.1%	
  
relative	
  abundance.	
  Black	
  color	
  denotes	
  OTUs	
  that	
  were	
  not	
  core.	
  *	
  indicates	
  litters	
  with	
  less	
  
than	
  4	
  samples	
  for	
  this	
  specific	
  time.	
  The	
  absence	
  of	
  litter	
  1707	
  samples	
  means	
  that	
  there	
  
were	
  not	
  available	
  samples	
  in	
  this	
  time.	
  
	
  

70	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
REFERENCES	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

71	
  

REFERENCES	
  
	
  
	
  
	
  
1.	
  
	
  
2.	
  
	
  
3.	
  

	
  
4.	
  
	
  
5.	
  
	
  
6.	
  
	
  
7.	
  

	
  
8.	
  
	
  
9.	
  
	
  
10.	
  
	
  
11.	
  

	
  

Horter	
  DC,	
  Yoon	
  KJ,	
  Zimmerman	
  JJ:	
  A	
  review	
  of	
  porcine	
  tonsils	
  in	
  immunity	
  and	
  
disease.	
  Anim	
  Health	
  Res	
  Rev	
  2003,	
  4:143-­‐‑155.	
  
Smith	
   TC,	
   Harper	
   AL,	
   Nair	
   R,	
   Wardyn	
   SE,	
   Hanson	
   BM,	
   Ferguson	
   DD,	
   Dressler	
   AE:	
  
Emerging	
   swine	
   zoonoses.	
   Vector-­‐‑Borne	
   and	
   Zoonotic	
   Diseases	
   2011,	
   11:1225-­‐‑
1234.	
  
MacInnes	
   JI,	
   Gottschalk	
   M,	
   Lone	
   AG,	
   Metcalf	
   DS,	
   Ojha	
   S,	
   Rosendal	
   T,	
   Watson	
   SB,	
  
Friendship	
   RM:	
   Prevalence	
   of	
   Actinobacillus	
   pleuropneumoniae,	
   Actinobacillus	
  
suis,	
   Haemophilus	
   parasuis,	
   Pasteurella	
   multocida,	
   and	
   Streptococcus	
   suis	
   in	
  
representative	
   Ontario	
   swine	
   herds.	
   Canadian	
   Journal	
   of	
   Veterinary	
   Research-­‐‑
Revue	
  Canadienne	
  De	
  Recherche	
  Veterinaire	
  2008,	
  72:242-­‐‑248.	
  
Kernaghan	
  S,	
  Bujold	
  AR,	
  MacInnes	
  JI:	
  The	
  microbiome	
  of	
  the	
  soft	
  palate	
  of	
  swine.	
  
Animal	
  Health	
  Research	
  Reviews	
  2012,	
  13:110-­‐‑120.	
  
Williams	
  LP,	
  Newell	
  KW:	
  Salmonella	
  excretion	
  in	
  joy-­‐‑riding	
  pigs.	
  American	
  Journal	
  
of	
  Public	
  Health	
  and	
  the	
  Nations	
  Health	
  1970,	
  60:926-­‐‑929.	
  
Weyrich	
   LS,	
   Feaga	
   HA,	
   Park	
   J,	
   Muse	
   SJ,	
   Safi	
   CY,	
   Rolin	
   OY,	
   Young	
   SE,	
   Harvill	
   ET:	
  
Resident	
   microbiota	
   affect	
  Bordetella	
   pertussis	
  infectious	
   dose	
   and	
   host	
  
specificity.	
  Journal	
  of	
  Infectious	
  Diseases	
  2014,	
  209:913-­‐‑921.	
  
Ghartey	
   JP,	
   Smith	
   BC,	
   Chen	
   ZG,	
   Buckley	
   N,	
   Lo	
   YT,	
   Ratner	
   AJ,	
   Herold	
   BC,	
   Burk	
   RD:	
  
Lactobacillus	
   crispatus	
   dominant	
   vaginal	
   microbiome	
   is	
   associated	
   with	
  
inhibitory	
   activity	
   of	
   female	
   genital	
   tract	
   secretions	
   against	
  Escherichia	
   coli.	
  
Plos	
  One	
  2014,	
  9:8.	
  
Croswell	
   A,	
   Amir	
   E,	
   Teggatz	
   P,	
   Barman	
   M,	
   Salzman	
   NH:	
   Prolonged	
   impact	
   of	
  
antibiotics	
   on	
   intestinal	
   microbial	
   ecology	
   and	
   susceptibility	
   to	
  
enteric	
  Salmonella	
  infection.	
  Infection	
  and	
  Immunity	
  2009,	
  77:2741-­‐‑2753.	
  
Kim	
   Y-­‐‑G,	
   Sakamoto	
   K,	
   Seo	
   S-­‐‑U,	
   Pickard	
   JM,	
   Gillilland	
   MG,	
   III,	
   Pudlo	
   NA,	
   Hoostal	
   M,	
   Li	
  
X,	
  Wang	
  TD,	
  Feehley	
  T,	
  et	
  al:	
  Neonatal	
  acquisition	
  of	
  Clostridia	
  species	
  protects	
  
against	
  colonization	
  by	
  bacterial	
  pathogens.	
  Science	
  2017,	
  356:312-­‐‑315.	
  
Willing	
   BP,	
   Vacharaksa	
   A,	
   Croxen	
   M,	
   Thanachayanont	
   T,	
   Finlay	
   BB:	
   Altering	
   host	
  
resistance	
  to	
  infections	
  through	
  microbial	
  transplantation.	
  Plos	
  One	
  2011,	
  6:9.	
  
Mulder	
  IE,	
  Schmidt	
  B,	
  Stokes	
  CR,	
  Lewis	
  M,	
  Bailey	
  M,	
  Aminov	
  RI,	
  Prosser	
  JI,	
  Gill	
  BP,	
  
Pluske	
   JR,	
   Mayer	
   CD,	
   et	
   al:	
   Environmentally-­‐‑acquired	
   bacteria	
   influence	
  

72	
  

	
  
12.	
  
	
  
13.	
  
	
  
14.	
  

	
  
15.	
  
	
  
16.	
  
	
  
17.	
  
	
  
18.	
  
	
  
19.	
  
	
  
20.	
  

	
  
21.	
  

	
  
22.	
  

	
  

microbial	
   diversity	
   and	
   natural	
   innate	
   immune	
   responses	
   at	
   gut	
   surfaces.	
   Bmc	
  
Biology	
  2009,	
  7:20.	
  
Lowe	
  BA,	
  Marsh	
  TL,	
  Isaacs-­‐‑Cosgrove	
  N,	
  Kirkwood	
  RN,	
  Kiupel	
  M,	
  Mulks	
  MH:	
  Defining	
  
the	
  "core	
  microbiome"	
  of	
  the	
  microbial	
  communities	
  in	
  the	
  tonsils	
  of	
  healthy	
  
pigs.	
  Bmc	
  Microbiology	
  2012,	
  12:14.	
  
Bokulich	
   NA,	
   Chung	
   J,	
   Battaglia	
   T,	
   Henderson	
   N,	
   Jay	
   M,	
   Li	
   H,	
   Lieber	
   AD,	
   Wu	
   F,	
   Perez-­‐‑
Perez	
   GI,	
   Chen	
   Y,	
   et	
   al:	
   Antibiotics,	
   birth	
   mode,	
   and	
   diet	
   shape	
   microbiome	
  
maturation	
  during	
  early	
  life.	
  Science	
  Translational	
  Medicine	
  2016,	
  8:13.	
  
Koenig	
  JE,	
  Spor	
  A,	
  Scalfone	
  N,	
  Fricker	
  AD,	
  Stombaugh	
  J,	
  Knight	
  R,	
  Angenent	
  LT,	
  Ley	
  
RE:	
  Succession	
  of	
  microbial	
  consortia	
  in	
  the	
  developing	
  infant	
  gut	
  microbiome.	
  
Proceedings	
  of	
  the	
  National	
  Academy	
  of	
  Sciences	
  of	
  the	
  United	
  States	
  of	
  America	
  2011,	
  
108:4578-­‐‑4585.	
  
Palmer	
   C,	
   Bik	
   EM,	
   DiGiulio	
   DB,	
   Relman	
   DA,	
   Brown	
   PO:	
   Development	
   of	
   the	
   human	
  
infant	
  intestinal	
  microbiota.	
  Plos	
  Biology	
  2007,	
  5:1556-­‐‑1573.	
  
Inoue	
   R,	
   Tsukahara	
   T,	
   Nakanishi	
   N,	
   Ushida	
   K:	
   Development	
   of	
   the	
   intestinal	
  
microbiota	
  in	
  the	
  piglet.	
  Journal	
  of	
  General	
  and	
  Applied	
  Microbiology	
  2005,	
  51:257-­‐‑
265.	
  
Konstantinov	
  SR,	
  Awati	
  AA,	
  Williams	
  BA,	
  Miller	
  BG,	
  Jones	
  P,	
  Stokes	
  CR,	
  Akkermans	
  
ADL,	
   Smidt	
   H,	
   De	
   Vos	
   WM:	
   Post-­‐‑natal	
   development	
   of	
   the	
   porcine	
   microbiota	
  
composition	
  and	
  activities.	
  Environmental	
  Microbiology	
  2006,	
  8:1191-­‐‑1199.	
  
Stephens	
  WZ,	
  Burns	
  AR,	
  Stagaman	
  K,	
  Wong	
  S,	
  Rawls	
  JF,	
  Guillemin	
  K,	
  Bohannan	
  BJM:	
  
The	
  composition	
  of	
  the	
  zebrafish	
  intestinal	
  microbial	
  community	
  varies	
  across	
  
development.	
  Isme	
  Journal	
  2016,	
  10:644-­‐‑654.	
  
Frese	
  SA,	
  Parker	
  K,	
  Calvert	
  CC,	
  Mills	
  DA:	
  Diet	
  shapes	
  the	
  gut	
  microbiome	
  of	
  pigs	
  
during	
  nursing	
  and	
  weaning.	
  Microbiome	
  2015,	
  3:10.	
  
Bergstrom	
   A,	
   Skov	
   TH,	
   Bahl	
   MI,	
   Roager	
   HM,	
   Christensen	
   LB,	
   Ejlerskov	
   KT,	
   Molgaard	
  
C,	
  Michaelsen	
  KF,	
  Licht	
  TR:	
  Establishment	
  of	
  intestinal	
  microbiota	
  during	
  early	
  
life:	
  a	
  longitudinal,	
  explorative	
  study	
  of	
  a	
  large	
  cohort	
  of	
  Danish	
  infants.	
  Applied	
  
and	
  Environmental	
  Microbiology	
  2014,	
  80:2889-­‐‑2900.	
  
Konstantinov	
  SR,	
  Favier	
  CF,	
  Zhu	
  WY,	
  Williams	
  BA,	
  Kluss	
  J,	
  Souffrant	
  WB,	
  de	
  Vos	
  WM,	
  
Akkermans	
   ADL,	
   Smidt	
   H:	
   Microbial	
   diversity	
   studies	
   of	
   the	
   porcine	
  
gastrointestinal	
   ecosystem	
   during	
   weaning	
   transition.	
   Animal	
   Research	
   2004,	
  
53:317-­‐‑324.	
  
Konstantinov	
  SR,	
  Zhu	
  WY,	
  Williams	
  BA,	
  Tamminga	
  S,	
  de	
  Vos	
  WM,	
  Akkermans	
  ADL:	
  
Effect	
  of	
  fermentable	
  carbohydrates	
  on	
  piglet	
  faecal	
  bacterial	
  communities	
  as	
  
73	
  

	
  
23.	
  
	
  
24.	
  
	
  
25.	
  
	
  
26.	
  

	
  
27.	
  

	
  
28.	
  

	
  
29.	
  
	
  
30.	
  
	
  
31.	
  
	
  
32.	
  
	
  

	
  

revealed	
  by	
  denaturing	
  gradient	
  gel	
  electrophoresis	
  analysis	
  of	
  16S	
  ribosomal	
  
DNA.	
  Fems	
  Microbiology	
  Ecology	
  2003,	
  43:225-­‐‑235.	
  
Thompson	
  CL,	
  Wang	
  B,	
  Holmes	
  AJ:	
  The	
  immediate	
  environment	
  during	
  postnatal	
  
development	
   has	
   long-­‐‑term	
   impact	
   on	
   gut	
   community	
   structure	
   in	
   pigs.	
   Isme	
  
Journal	
  2008,	
  2:739-­‐‑748.	
  
Kim	
  D,	
  Hofstaedter	
  CE,	
  Zhao	
  C,	
  Mattei	
  L,	
  Tanes	
  C,	
  Clarke	
  E,	
  Lauder	
  A,	
  Sherrill-­‐‑Mix	
  S,	
  
Chehoud	
   C,	
   Kelsen	
   J,	
   et	
   al:	
   Optimizing	
   methods	
   and	
   dodging	
   pitfalls	
   in	
  
microbiome	
  research.	
  Microbiome	
  2017,	
  5:14.	
  
Liu	
  WT,	
  Marsh	
  TL,	
  Cheng	
  H,	
  Forney	
  LJ:	
  Characterization	
  of	
  microbial	
  diversity	
  by	
  
determining	
   terminal	
   restriction	
   fragment	
   length	
   polymorphisms	
   of	
   genes	
  
encoding	
  16S	
  rRNA.	
  Applied	
  and	
  Environmental	
  Microbiology	
  1997,	
  63:4516-­‐‑4522.	
  
Caporaso	
   JG,	
   Lauber	
   CL,	
   Walters	
   WA,	
   Berg-­‐‑Lyons	
   D,	
   Lozupone	
   CA,	
   Turnbaugh	
   PJ,	
  
Fierer	
  N,	
  Knight	
  R:	
  Global	
  patterns	
  of	
  16S	
  rRNA	
  diversity	
  at	
  a	
  depth	
  of	
  millions	
  of	
  
sequences	
  per	
  sample.	
  Proceedings	
  of	
  the	
  National	
  Academy	
  of	
  Sciences	
  of	
  the	
  United	
  
States	
  of	
  America	
  2011,	
  108:4516-­‐‑4522.	
  
Schloss	
  PD,	
  Westcott	
  SL,	
  Ryabin	
  T,	
  Hall	
  JR,	
  Hartmann	
  M,	
  Hollister	
  EB,	
  Lesniewski	
  RA,	
  
Oakley	
   BB,	
   Parks	
   DH,	
   Robinson	
   CJ,	
   et	
   al:	
   Introducing	
   mothur:	
   open-­‐‑source,	
  
platform-­‐‑independent,	
   community-­‐‑supported	
   software	
   for	
   describing	
   and	
  
comparing	
  microbial	
  communities.	
  Applied	
  and	
  Environmental	
  Microbiology	
  2009,	
  
75:7537-­‐‑7541.	
  
Kozich	
  JJ,	
  Westcott	
  SL,	
  Baxter	
  NT,	
  Highlander	
  SK,	
  Schloss	
  PD:	
  Development	
  of	
  a	
  dual-­‐‑
index	
   sequencing	
   strategy	
   and	
   curation	
   pipeline	
   for	
   analyzing	
   amplicon	
  
sequence	
   data	
   on	
   the	
   MiSeq	
   Illumina	
   sequencing	
   platform.	
   Applied	
   and	
  
Environmental	
  Microbiology	
  2013,	
  79:5112-­‐‑5120.	
  
Quast	
  C,	
  Pruesse	
  E,	
  Yilmaz	
  P,	
  Gerken	
  J,	
  Schweer	
  T,	
  Yarza	
  P,	
  Peplies	
  J,	
  Gloeckner	
  FO:	
  
The	
  SILVA	
  ribosomal	
  RNA	
  gene	
  database	
  project:	
  improved	
  data	
  processing	
  and	
  
web-­‐‑based	
  tools.	
  Nucleic	
  Acids	
  Research	
  2013,	
  41:D590-­‐‑D596.	
  
Hammer	
   O,	
   Harper	
   DAT,	
   Ryan	
   PD:	
   PAST:	
   paleontological	
   statistics	
   software	
  
package	
  for	
  education	
  and	
  data	
  analysis.	
  Palaeontologia	
  Electronica	
  2001,	
  4.	
  
Schneider	
  CA,	
  Rasband	
  WS,	
  Eliceiri	
  KW:	
  NIH	
  Image	
  to	
  ImageJ:	
  25	
  years	
  of	
  image	
  
analysis.	
  Nature	
  Methods	
  2012,	
  9:671-­‐‑675.	
  
Glassing	
   A,	
   Dowd	
   SE,	
   Galandiuk	
   S,	
   Davis	
   B,	
   Chiodini	
   RJ:	
   Inherent	
   bacterial	
   DNA	
  
contamination	
  of	
  extraction	
  and	
  sequencing	
  reagents	
  may	
  affect	
  interpretation	
  
of	
  microbiota	
  in	
  low	
  bacterial	
  biomass	
  samples.	
  Gut	
  Pathogens	
  2016,	
  8:12.	
  

74	
  

33.	
  

	
  
34.	
  
	
  
35.	
  
	
  
36.	
  
	
  
37.	
  
	
  
38.	
  
	
  
39.	
  
	
  
40.	
  
	
  
41.	
  
	
  
42.	
  

	
  
43.	
  

	
  

	
  

Lauder	
  AP,	
  Roche	
  AM,	
  Sherrill-­‐‑Mix	
  S,	
  Bailey	
  A,	
  Laughlin	
  AL,	
  Bittinger	
  K,	
  Leite	
  R,	
  Elovitz	
  
MA,	
  Parry	
  S,	
  Bushman	
  FD:	
  Comparison	
  of	
  placenta	
  samples	
  with	
  contamination	
  
controls	
   does	
   not	
   provide	
   evidence	
   for	
   a	
   distinct	
   placenta	
   microbiota.	
  
Microbiome	
  2016,	
  4:29.	
  
Mandar	
   R,	
   Mikelsaar	
   M:	
   Transmission	
   of	
   mother's	
   microflora	
   to	
   the	
   newborn	
   at	
  
birth.	
  Biology	
  of	
  the	
  Neonate	
  1996,	
  69:30-­‐‑35.	
  
Park	
  SJ,	
  Kim	
  J,	
  Lee	
  TS,	
  Rhee	
  SK,	
  Kim	
  H:	
  Characterization	
  of	
  the	
  fecal	
  microbiome	
  in	
  
different	
  swine	
  groups	
  by	
  high-­‐‑throughput	
  sequencing.	
  Anaerobe	
  2014,	
  28:157-­‐‑
162.	
  
Braem	
   G,	
   De	
   Vliegher	
   S,	
   Verbist	
   B,	
   Heyndrickx	
   M,	
   Leroy	
   F,	
   De	
   Vuyst	
   L:	
   Culture-­‐‑
independent	
   exploration	
   of	
   the	
   teat	
   apex	
   microbiota	
   of	
   dairy	
   cows	
   reveals	
   a	
  
wide	
  bacterial	
  species	
  diversity.	
  Veterinary	
  Microbiology	
  2012,	
  157:383-­‐‑390.	
  
Grice	
  EA,	
  Kong	
  HH,	
  Conlan	
  S,	
  Deming	
  CB,	
  Davis	
  J,	
  Young	
  AC,	
  Bouffard	
  GG,	
  Blakesley	
  
RW,	
   Murray	
   PR,	
   Green	
   ED,	
   et	
   al:	
   Topographical	
   and	
   temporal	
   diversity	
   of	
   the	
  
human	
  skin	
  microbiome.	
  Science	
  2009,	
  324:1190-­‐‑1192.	
  
Wegener	
   HC,	
   Skovjensen	
   EW:	
   A	
   longitudinal	
   study	
   of	
   Staphylococcus	
   hyicus	
  
colonization	
   of	
   vagina	
   of	
   gilts	
   and	
   transmission	
   to	
   piglets.	
   Epidemiology	
   and	
  
Infection	
  1992,	
  109:433-­‐‑444.	
  
Mach	
  N,	
  Berri	
  M,	
  Estelle	
  J,	
  Levenez	
  F,	
  Lemonnier	
  G,	
  Denis	
  C,	
  Leplat	
  JJ,	
  Chevaleyre	
  C,	
  
Billon	
  Y,	
  Dore	
  J,	
  et	
  al:	
  Early-­‐‑life	
  establishment	
  of	
  the	
  swine	
  gut	
  microbiome	
  and	
  
impact	
  on	
  host	
  phenotypes.	
  Environmental	
  Microbiology	
  Reports	
  2015,	
  7:554-­‐‑569.	
  
Alain	
  B	
  Pajarillo	
  E,	
  Chae	
  J-­‐‑P,	
  Balolong	
  MP,	
  Bum	
  Kim	
  H,	
  Kang	
  D-­‐‑K:	
  Assessment	
  of	
  fecal	
  
bacterial	
   diversity	
   among	
   healthy	
   piglets	
   during	
   the	
   weaning	
   transition.	
   The	
  
Journal	
  of	
  general	
  and	
  applied	
  microbiology	
  2014,	
  60:140-­‐‑146.	
  
Looft	
  T,	
  Allen	
  HK,	
  Casey	
  TA,	
  Alt	
  DP,	
  Stanton	
  TB:	
  Carbadox	
  has	
  both	
  temporary	
  and	
  
lasting	
  effects	
  on	
  the	
  swine	
  gut	
  microbiota.	
  Frontiers	
  in	
  Microbiology	
  2014,	
  5:9.	
  
Looft	
   T,	
   Johnson	
   TA,	
   Allen	
   HK,	
   Bayles	
   DO,	
   Alt	
   DP,	
   Stedtfeld	
   RD,	
   Sul	
   WJ,	
   Stedtfeld	
   TM,	
  
Chai	
   BL,	
   Cole	
   JR,	
   et	
   al:	
   In-­‐‑feed	
   antibiotic	
   effects	
   on	
   the	
   swine	
   intestinal	
  
microbiome.	
   Proceedings	
   of	
   the	
   National	
   Academy	
   of	
   Sciences	
   of	
   the	
   United	
   States	
   of	
  
America	
  2012,	
  109:1691-­‐‑1696.	
  
Rettedal	
  E,	
  Vilain	
  S,	
  Lindblom	
  S,	
  Lehnert	
  K,	
  Scofield	
  C,	
  George	
  S,	
  Clay	
  S,	
  Kaushik	
  RS,	
  
Rosa	
   AJM,	
   Francis	
   D,	
   Brozel	
   VS:	
   Alteration	
   of	
   the	
  ileal	
   microbiota	
   of	
   weanling	
  
piglets	
   by	
   the	
   growth-­‐‑promoting	
   antibiotic	
   chlortetracycline.	
   Applied	
   and	
  
Environmental	
  Microbiology	
  2009,	
  75:5489-­‐‑5495.	
  

75	
  

44.	
  

Sekirov	
   I,	
   Tam	
   NM,	
   Jogova	
   M,	
   Robertson	
   ML,	
   Li	
   YL,	
   Lupp	
   C,	
   Finlay	
   BB:	
   Antibiotic-­‐‑
induced	
  perturbations	
  of	
  the	
  intestinal	
  microbiota	
  alter	
  host	
  susceptibility	
  to	
  
enteric	
  infection.	
  Infection	
  and	
  Immunity	
  2008,	
  76:4726-­‐‑4736.	
  

	
  

	
  

76	
  

CHAPTER!3.!DEVELOPMENT!OF!THE!TONSILLAR!MICROBIOME!IN!PIGS!FROM!
NEWBORN!TO!MARKET!AGE!
!
!
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77!

INTRODUCTION!
!
Numerous!bacteria!and!viruses!can!access!the!host!using!the!oropharynx!and!
nasopharynx!as!portals!of!entrance.!Tonsils,!!lymphoQepithelial!tissues!located!at!their!
junction,!play!a!key!role!in!surveillance!of!pathogens!accessing!host![1].!Tonsils!play!a!
significant!role!in!the!initial!process!of!pathogenQhost!colonization!and!as!a!reservoir!of!
hostQspecific!pathogens!as!well!as!zoonotic!pathogens!highly!transmissible!to!humans!
[2].!Multiple!bacterial!pathogens!are!regularly!isolated!from!tonsils!of!asymptomatic!
animals.!Pathogens!residing!in!the!tonsils!can!spread!systemically!or!be!transmitted!to!
other!animals!including!humans,!with!such!transmission!often!triggered!by!stressful!
conditions! such! as! transport! [3].! It! has! been! suggested! that! in! the! process! of! the!
colonization!of!host!by!pathogenic!microorganisms,!the!microbiome!plays!an!important!
preventive!role![4Q7]!as!well!as!a!regulatory!role!in!resistance!to!infection![8,!9].!!
There! are! limited! numbers! of! studies! addressing! the! tonsillar! microbiome! in!
humans!or!pigs![10Q16].!Conversely,!there!is!a!growing!number!of!studies!on!intestinal!
microbiome!in!different!species.!Some!studies!suggest!a!gradual!and!sequential!process!
in!the!development!of!intestinal!microbiome![17,!18],!where!some!taxa!persisted!and!
were! stable! while! others! were! acquired! over! time.! However,! microbial! communities!
tended!to!achieve!an!adultQlike!profile!as!time!progressed![17,!19].!This!trend!was!seen!
despite!the!fact!that!during!development!there!were!significant!shifts!in!the!structure!
of!the!population![19]!as!well!as!in!the!diversity![18],!and!many!of!these!shifts!were!
associated!with!life!events,!for!example!diet!changes!and!antibiotic!treatment,!among!
others![17].!

!

78!

It!has!been!demonstrated!that!common!management!practices!such!as!the!use!
of!antibiotic!treatments!can!significantly!affect!microbial!communities!and!predispose!
the! host! to! infections! [20].! However,! the! microbiota! also! can! be! shifted! towards! a!
microbial!community!that!would!protect!the!host!from!potential!infections,!as!in!the!
case!of!altering!the!intestinal!microbiota!through!fecal!microbiota!transplantation![6].!
Notwithstanding!the!relevant!role!that!the!microbiota!can!play!in!maintaining!a!good!
health!status!in!the!host,!very!little!is!known!about!the!tonsillar!microbial!communities!
of! pigs! based! on! a! cultureQindependent! approach.! Two! studies! have! described! the!
normal!tonsillar!microbiome!in!finishing!pigs![12,!15]!and!one!study!has!described!the!
metabolically!active!microbiome!of!slaughter!pigs![10].!The!core!tonsil!microbiome!in!
18Q20! week! old! growerQfinisher! pigs! was! comprised! of! members! of! the! families!
Pasteurellaceae,! Moraxellaceae,. Streptococcaceae,. Fusobacteriaceae,! Veillonellaceae,.
Enterobacteriaceae,. Neisseriaceae,. and! Peptostreptococcaceae,. as! well! as. the! order!
Clostridiales.! Currently,! tonsils! are! an! underQexplored! habitat! of! the! mammalian!
microbiome.!How!the!microbial!community!in!the!tonsils!is!established!and!develops!
over!time!and!!what!!role!it!plays!in!the!acquisition!and!carriage!of!pathogens!!and!thus!
in! host! health! and! disease! is! not! known! at! this! time.! ! Microbiome! development! is!
suggested!to!be!based!on!specific!bacterial!interactions!and!not!on!random!assembly!of!
microorganisms! [17].! Numerous! authors! have! shown! that! human! or! animal!
microbiome!development!in!different!body!locations!frequently!follows!a!gradual!and!
successional!process![17Q19,!21].!Whether!a!similar!process!occurs!in!tonsils!remains!
to!be!established.!

!

79!

The! goal! of! the! current! study! was! to! utilize! a! cultureQindependent! approach!
using! highQthroughput! sequencing! of! 16S! rRNA! genes! to! follow! and! describe! the!
development!of!the!tonsillar!microbial!communities!in!pigs!from!birth!through!market!
age.!This!characterization!of!the!development!of!the!swine!tonsillar!microbiome!lays!a!
base!for!future!studies!to!alter!this!microbiome!to!reduce!pathogen!load!and!improve!
overall!animal!health.!
!
MATERIALS!AND!METHODS!
!
Animals.) The! Michigan! State! University! Institutional! Animal! Care! and! Use!
Committee!approved!this!study!and!the!animal!procedures.!The!pigs!used!in!this!study!
were!from!a!high!health!status!farrowQtoQfinish!herd!with!~200!sows.!!Relevant!medical!
history! for! this! herd! included! no! recent! respiratory! disease;! freedom! from!
Actinobacillus. pleuropneumoniae,. Mycoplasma. hyopneumoniae,! and! PRRSV;! a! recent!
outbreak!of!porcine!epidemic!diarrhea!virus!(PEDV),!under!control!prior!to!this!study;!
and! routine! vaccination! against! erysipelas,! atrophic! rhinitis,! and! porcine! circovirus!
type!2!(PCV2).)
Four! crossbred! sows! (Yorkshire! X! Hampshire)! of! different! parity! (number! of!
pregnancies)! were! selected! for! this! study! and! included! sow! 1700! (first! parity),! sow!
1631! (second! parity),! sow! 1445! (fifth! parity)! and! sow! 1711! (tenth! parity).! ! Four!
randomly!selected!piglets!from!each!of!the!four!sows!were!sampled!within!a!period!no!
longer! than! 8! hours! after! birth! (newborn)! and! the! same! piglets! were! sampled!
subsequently!at!1,!2,!3,!4,!6,!8,!10,!12,!16,!and!19!weeks!of!age.!!

!

80!

Newborn! piglets! received! a! single! intramuscular! injection! of! IronQDextran!
during!their!first!week!of!life.!Between!the!third!and!fourth!weeks!of!age!(21!to!24!days!
–! average! weight! 18! pounds)! piglets! were! weaned,! vaccinated,! and! moved! from! the!
farrowing! room! where! they! were! housed! with! the! sow! and! littermates! to! a! nursery!
room,!with!litters!maintained!as!pen!mates.!At!this!time!the!piglets!were!weaned!from!
milk! to! a! solid! pellet! ration! diet! (Pig! 1300®,! Akey! Nutrition,! Brookville! OH)!
supplemented!with!Carbadox®!at!a!dose!of!50!g/ton.!Two!weeks!after!being!moved!to!
the!nursery!facility!(at!5!weeks!of!age),!Carbadox®!supplementation!was!removed!from!
the!feed,!and!food!was!changed!to!a!ground!ration!supplemented!with!Tylan®!at!a!dose!
of!100!g/ton.!!At!~nine!weeks!of!age!(63!to!67!days!–!average!weight!60!pounds),!piglets!
were!moved!to!a!finishing!room!and!were!assigned!to!different!pens!based!on!criteria!
such!as!gender!and!weight;!separation!by!litter!was!no!longer!maintained.!At!this!time,!
Tylan®! supplementation! was! discontinued! and! a! ground! ration! without!
supplementation!was!provided.!Finally,!at!~eighteen!weeks!of!age,!piglets!were!moved!
again!to!another!finishing!room!(with!mixing!of!prior!penmates)!where!they!remained!
until! being! moved! to! the! slaughterhouse! (average! weight! 240! pounds).! ! These!
management!practices!are!summarized!in!Figure!3.1.!
Collection) of) microbiome) samples.! Tonsil! brushes! developed! by! our! group!
and!validated!in!previous!studies![12]!were!used!to!collect!tonsil!microbiome!samples!
from! sows! and! larger! piglets,! while! CytosoftTM! cytology! brushes! (Medical! Packaging!
Corporation,! Camarillo,! CA)! were! used! for! smaller! piglets.! Collection! and! storage! of!
samples!was!as!previously!described![12].!!

!

81!

Isolation)of)community)DNA.!Extraction!of!community!DNA!from!samples!!was!
performed! using! a! PowerSoil! DNA! Isolation! Kit! and! PowerBead! tubes! (MoBio!
Laboratories,!Carlsbad,!CA)!as!previously!described![12,!22].!
Illumina) sequencing) and) sequence) analysis.! Sequencing! was! performed! at!
the! MSU! Research! Technology! Support! Facility! (RTSF)! as! previously! described! [22].!!
Negative! controls! consisting! of! DNAQfree! water! or! MoBio! C6! reagent! were! used! as!
“blank! library! controls”! [23]! and! included! in! each! sequencing! run.! Briefly,! uniquely!
indexed!primers!were!used!to!amplify!the!V4!region!of!the!16S!rRNA!gene!from!the!
community! DNA,! as! described! by! Caporaso! [24].! A! SequalPrep! normalization! plate!
(Invitrogen)!was!used!to!normalize!the!amplification!products,!which!were!then!pooled!
and!the!reaction!cleaned!using!AMPure!XP!beads.!The!pooled!sample!was!sequenced!on!
an! Illumina! MiSeq! v2! flow! cell! using! a! 500! cycle! v2! reagent! kit! (PE250! reads).! Base!
calling!was!performed!using!Illumina!Real!Time!Analysis!Software!(RTA)!v1.18.54!and!
output! of! RTA! demultiplexed! and! converted! to! FastQ! files! using! Illumina! Bcl2fastq!
v1.8.4.!
The! openQsource,! platformQindependent,! communityQsupported! software!
program! mothur! v.1.35.0! (http://www.mothur.org)! [25]! was! used! for! amplicon!
analysis.! Raw! sequencing! data! was! processed! according! to! the! mothur! standard!
operating! procedure! (http://www.mothur.org/wiki/MiSeq_SOP)! [26]! and! aligned!
using! the! mothurQformatted! version! 123! of! Silva! 16S! ribosomal! gene! database! [27].!
After!sequences!were!classified,!all!sequences!classified!as!Chloroplast,!Mitochondria,!
unknown,!Archaea,!or!Eukaryota!were!removed!from!the!data!set.!Subsampling!at!7000!
sequences! per! sample! was! done,! followed! by! a! preclustering! of! the! sequences! and!

!

82!

removal! of! chimeric! sequences! using! a! mothur! formatted! version! of! the! Ribosomal!
Database!Project!(RDP)!training!set!version!14!and!uchime,!based!on!mothur!protocol.!
A! cutoff! of! ≥97%! sequence! identity! was! used! to! classify! sequences! into! Operational!
Taxonomic!Units!(OTUs).!Singleton!and!doubleton!reads!were!removed!before!the!final!
analysis.!For!the!final!analysis!of!the!data,!samples!were!subsampled!to!5179!reads!per!
sample.! ! The! full! data! set! analyzed! is! available! as! a! supplemental! file! at!
(https://figshare.com/s/e910143b694abb664fcc).! Samples! from! newborn! piglets!
through!weaning!were!used!in!a!smaller!study![22]!!!
Diversity)and)statistical)analysis.)A!clustering!cutoff!of!3%!for!the!processed!
sequences! was! used! in! the! statistical! analysis.! Mothur! output! files! were! used! to!
estimate! alpha! diversity! (sobs)! and! beta! diversity! indexes,! as! well! as! representative!
sequences,!all!of!which!were!calculated!in!mothur!v.1.35.0!(http://www.mothur.org)!
[25].!PAST3!(Version!3.14;!http://folk.uio.no/ohammer/past/)!was!used!for!statistical!
analysis!

of!

the!

samples.!

FigTree!

(Version!

1.4.3;!

http://tree.bio.ed.ac.uk/software/figtree/)!was!used!for!construction!of!dendrogram!
figures.!ImageJ!was!used!to!measure!the!area!of!the!ellipses!for!the!two!dimensional!
scatter! plot! [28].! RStudio! (Version! 0.99.446;! https://www.rstudio.com/)! and! library!
gplots!(https://CRAN.RQproject.org/package=gplots)!were!used!to!generate!heatmaps.!
Inkscape! 0.91! (https://inkscape.org/en/download/macQos/),! was! used! to! process!
images!and!edit!labels.!Taxonomy!tables!and!OTU!plots!were!generated!in!Microsoft !
®

Excel !2011,!where!the!analysis!of!samples!was!done!with!data!that!represented!higher!
®

than!0.1%!of!the!total!reads!for!the!samples!analyzed.!

!

83!

Availability)of)supporting)data.!Raw!sequence!data!and!metadata!is!available!
at!NCBI!database!(SRA!accession!number:!!in!submission!process).!!
!

!

!

84!

RESULTS!
!
A!total!of!128!tonsil!microbiome!samples!from!pigs!from!birth!through!market!
age! were! sequenced! and! analyzed.! Of! these,! 64! samples! were! collected! from! piglets!
before!weaning!(newborn!–!third!week),!which!included!samples!from!16!piglets!(4!per!
sow)!at!each!time!point.!!In!addition,!64!samples!were!collected!from!pigs!after!weaning,!
which! included! samples! from! 16! piglets! at! week! 4! and! 8! piglets! (2! per! sow)! at! all!
subsequent!time!points!(Table!3.1.).!!
Table!3.1.)Samples)processed)by)sampling)time)and)litter!

!

!
!
!
!
!
!
!

!

85!

Management) practices) are) related) with) changes) in) population) diversity.)
Tonsil!samples!for!microbiome!analysis!were!collected!at!eleven!time!points!during!the!
life! of! the! pigs! in! this! study.! Some! of! the! sampling! times! were! chosen! specifically! to!
represent!times!associated!with!management!practices!significant!in!the!life!of!the!pigs,!
including!immediately!prior!to!and!after!weaning,!alteration!in!feed!and!inQfeed!growth!
promoters,!and!movement!to!new!rooms!(Figure!3.1.)!!

!
!
Figure)3.1.)Significant)management)practices)at)the)swine)farm)during)the)life)of)
the)pigs)in)this)study.)General!management!features!experienced!by!the!pigs!during!
their! life! at! the! farm! are! depicted! here,! including! changes! in! feed,! use! of! in! feed!
antibiotics,!and!movement!to!new!housing.!
Analysis! of! the! alpha! diversity! of! the! tonsil! microbiome! (sobs:! refers! to! the!
number!of!observed!OTUs)!and!the!relation!with!the!different!changes!experienced!by!
the!pigs!during!their!life!showed!that!the!alpha!diversity!varied!widely!(Table!3.2).!For!
newborn!piglets,!the!average!value!of!sobs!was!110!(range!=!26Q376,!SD!=!101).!!The!
average! sobs! value! decreased! steadily! in! the! following! weeks! (first! to! third! week),!!!
!

86!

reaching!a!value!of!83!(range!=!57Q132).!This!was!accompanied!by!a!marked!decrease!
in!the!standard!deviation!to!24,!indicating!that!the!microbiome!became!very!similar!in!
all!pigs!by!3!weeks!of!age.!In!contrast,!from!week!four!to!ten!there!was!an!abundant!
increase!in!diversity!that!coincided!with!specific!challenging!events!experienced!by!the!
piglets.!Between!the!third!and!fourth!week,!the!piglets!were!weaned,!and!at!the!same!
time!they!were!moved!to!a!nursery!room,!vaccinated!and!their!diet!was!changed.!These!
changes!were!reflected!in!a!slight!increase!in!the!average!and!maximum!sobs!as!well!as!
the!standard!deviation.!However,!the!biggest!change!in!the!diversity!occurred!during!
the!period!where!Carbadox !was!removed!from!the!diet!and!Tylan !was!supplemented.!
®

®

Diversity!increased!to!over!three!times!the!previous!registered!values!for!average!sobs.!
Conversely,!the!removal!of!Tylan ,!accompanied!by!the!transfer!of!pigs!to!a!finishing!
®

room! where! they! were! no! longer! segregated! by! litter,! led! to! trend! of! decreasing!
diversity.!By!week!nineteen,!this!progressive!decrease!in!the!diversity!led!to!a!value!of!
average! sobs! of! 108! (range! =! 73Q178,! SD! =! 35).! ! Overall,! there! was! a! pattern!
demonstrating! that! extended! time! under! constant! conditions! led! to! fewer! sobs! and!
reduced!standard!deviation.!
!
)

!

)

87!

Table)3.2.)Number)of)observed)OTUs)(sobs))during)the)different)sampling)times))

!

!

sobs:!number!of!observed!OTUs!
Min!sobs:!Minimum!number!of!observed!sobs!
Max!sobs:!Maximum!number!of!observed!sobs!
!
Challenging) management) conditions) during) development) of) the) pigs)
generated)disruption)in)the)microbiome.!We!wondered!if!the!development!of!the!
tonsillar!microbiome!followed!a!succession!in!time!and!if!there!was!any!similarity!in!
the! microbiome! between! the! different! sampling! periods.! An! unrooted! dendrogram!
based!on!a!BrayQCurtis!analysis!(Figure!3.2.)!shows!the!clustering!of!the!pig!tonsillar!
microbiome! samples! from! newborn! through! the! nineteenth! week.! Samples! from!
newborn!piglets!were!mainly!distributed!in!two!groups,!one!corresponding!to!pigs!from!
a!first!parity!sow!(4/16;!25%)!and!the!other!from!pigs!of!multiparous!sows!(10/16;!
62.5%),!the!remaining!two!samples!were!clustered!with!microbiome!samples!of!older!
pigs.! At! one! week! of! age,! the! microbiome! samples! were! clustered! by! litter! in! four!
different!groups.!!!

!

88!

A
G, H, I
A, F
1
1 5 1F
A

18A
26A

22 A
24
A
23A
36A
3
4 29AA

17A
16A

E
248EE
1
17 1E
1 0E
4 5EE
113

10

42E
40 A
39E
263EE
2
16EH
1 55 F
1

E

11
1 G
1 21 H
15 G
G
23
G
2135 I
I

A, E, F, H

92

E

J
11 2JJ
1156J
1
K
11 K
12

B
E, H

2
2 6B
22243BB
1 B
122EH

75

36K
42K
22
2 3 KK
16K

K

E

80

99

78

70

I
16
2422JJI
42
23J

88

79

36I
36J

1631
170 0
1711

94

77

36E
22E

I, J, K, Sow

92

75

100
B, C, H

40C
42B
40B
39B
36 B
16 H
18C

11I
12I
22
I
H
222
3H

97

71

23F

42C
36C

B

G
16 2G
4

B

12F
16F
22G
36
42 H
22 H
36 F
42 F
36 F
14 G
45

B
11 B
102B
1 3B
1

K

156BB
1 B
18
17 B

C

15 K

16C
23C

22
3 C
11 9 C
1 778DD
2 C
23 96 D
4 04 DD
D
2234D
C
D
11 2D
131D
10
D

F
A - Newborns

1 32CC
11 1 CC
10
D
1 65 DC
11 5
D
43262DD
2 6C
2

C, D

H, I

F, G, H, Sow
A
11
A
12 A
13 A
10

A

0.1

B - First week
C - Second week
D - Third week

Weaning. Carbadox supplementation

E - Fourth week

Carbadox removed. Tylan supplementation

F - Sixth week

G - Eighth week

Tylan removed. Moved to finishing room 1

H - Tenth week

I - Twelfth week
J - Sixteenth week

Moved to finishing room 2

K - Nineteenth week
Sow tonsils

!

Figure'3.2.'Unrooted'Bray3Curtis'dendrogram'for'all'sampled'weeks.!The!dendrogram!shows!the!clustering!of!the!samples!
collected!from!pigs!from!newborn!through!nineteen!weeks,!as!well!as!sow!tonsillar!samples.!Samples!are!color!coded!by!week!
of!sampling.!Small!legends!indicate!some!of!the!challenges!that!took!place!in!specific!times.

!

89!

!
The!following!weeks!showed!that!as!pigs!aged,!their!tonsil!microbiomes!tended!
to! become! more! similar.! During! the! second! week,! the! samples! clustered! together! in!
three!related!groups.!In!the!third!week,!all!sixteen!samples!clustered!together!in!one!
group,!which!also!included!ten!of!the!week!2!samples.!The!fourth!week,!which!marked!
a!transitional!time!after!a!challenge,!i.e.,!weaning!plus!movement!to!new!housing!plus!
addition! of! Carbadox ! to! the! new! solid! feed,! showed! a! split! of! the! previously! tightly!
®

clustered!samples!into!four!separate!groups,!which!were!not!clustered!by!litter.!The!
sixth!week,!again!marked!by!a!transition!after!a!challenge,!i.e.,!removal!of!Carbadox !and!
®

addition!of!Tylan !to!the!feed,!again!showed!samples!clustered!in!four!separate!groups,!
®

which!were!neither!clustered!by!litter!nor!the!same,!with!one!exception,!as!the!groups!
for!the!fourth!week!samples.!However,!for!the!eighth!week,!samples!clustered!in!only!
two! groups.! Once! again,! in! the! tenth! week,! which! marked! the! transition! after! a!
challenging! condition,! i.e.,! removal! of! Tylan ! from! feed! and! reassignment! to! new!
®

finishing! rooms! with! litter! groups! broken! up,! showed! a! major! disruption! in! the!
clustering! pattern! with! samples! falling! into! six! different! groups.! The! sampling! times!
corresponding!to!weeks!twelve!and!sixteen,!a!time!of!stability!for!the!piglets,!once!again!
showed! coalescing! of! the! microbiota;! the! twelfth! week! samples! clustered! into! three!
groups,!while!the!sixteenth!week!samples!all!clustered!into!a!single!group.!Finally,!for!
the!last!sampling!period!corresponding!to!the!nineteenth!week,!also!a!transitional!time!
after! a! challenge,! i.e.,! movement! to! new! finishing! rooms! with! reGassortment! of! the!
groups,! the! samples! once! again! showed! a! split! into! three! different! groups.! The!
clustering! pattern! also! showed! that! as! the! pigs! aged,! most! samples! clustered! with!

!

90!

tonsillar!samples!from!sows,!despite!no!longer!having!contact!with!the!sows.!Based!on!
the! above! analysis! we! identified! three! sampling! times! (third,! eighth! and! sixteenth!
weeks),!which!were!immediately!before!a!challenging!condition,!where!the!microbiome!
tended!to!be!more!similar!between!pigs.!Statistical!support!for!this!clustering!pattern!
is!shown!in!an!unrooted!dendrogram!based!on!BrayGCurtis!analysis!(Figure!3.3.),!where!
samples! derived! from! the! third,! eighth! and! sixteenth! weeks! formed! three! distinct!

22D

D

23
40 D
D

13 D

11D
D

12
26

39
24 D
D

10 D

groups!which!were!supported!by!bootstrap!values!higher!than!70.!

D
36 D
42

18D
17 D

15D
16 D
99

70

71
85

15 G
78

12G

11

98

22J
42J

97

G

81

G
11
J

J
12
J
15
16J

16 G

42G

36J

22

G

36

G

23

23
J

D - Third week
G - Eighth week
J - Sixteenth week
Litter 1700
Litter 1631
Litter 1445
Litter 1711

0.09

!

Figure'3.3.'Unrooted'Bray2Curtis'dendrogram'for'three'pre2transition'times'.!The!
dendrogram! shows! the! clustering! of! tonsil! microbiome! samples! from! pigs! at! three!
times!immediately!before!challenging!events:!week!3,!week!8,!and!week!16.!Samples!
are!color!coded!by!week!of!sampling.!Bootstrap!values!higher!than!70!are!shown.!
!

91!

!
We!also!analyzed!the!clustering!shown!in!Figure!3.2.!to!determine!whether!there!
were!effects!of!litter!or!of!pen!on!the!clustering.!!Samples!from!newborn!and!1!week!old!
pigs! clustered! by! litter,! but! older! animals! did! not.! ! We! saw! no! correlation! of! the!
clustering! with! groups! in! the! same! pens! except! as! related! to! the! litter! effect! seen! in!
newborn!and!1!week!old!animals!
Tonsil'microbiome'membership'throughout'the'life'of'the'pigs.'To!visualize!
how!the!membership!of!the!tonsillar!microbiome!changes!through!the!life!of!the!pigs,!
we! plotted! the! proportion! of! the! 20! most! commonly! identified! bacterial! families! in!
piglets!at!each!sampling!time,!as!well!as!in!sows!(Figure!3.4.).!!Members!of!the!phyla!
Actinobacteria! (Family! Micrococcaceae),! Bacteroidetes! (Families! Bacteroidaceae,!
Porphyromonadaceae,! Prevotellaceae,! Flavobacteriaceae),! Firmicutes! (Families!
Bacillaceae9 1,! Staphylococcaceae,! Streptococcaceae,! Clostridiaceae9 1,! Clostridiales9
Incertae9 Sedis9 XI,! Lachnospiraceae,! Peptostreptococcaceae,! Ruminococcaceae,!
Erysipelotrichaceae,! Veillonellaceae),! Fusobacteria! (Family! Fusobacteriaceae),! and!
Proteobacteria9 (Families! Burkholderiaceae,! Neisseriaceae,! Pasteurellaceae! and!
Moraxellaceae)9were!identified!as!the!most!abundant!bacterial!phyla!and!families!in!pig!
tonsils.!!The!distribution!and!proportions!of!these!bacterial!families!fluctuated!through!
the! sampling! period! (Table! 3.3.),! with! the! largest! shifts! related! with! challenging!
conditions! experienced! by! the! pigs.! Three! families! that! consistently! represented! a!
major! portion! of! the! tonsil! microbiome! across! all! time! points! were! the!
Streptococcaceae,9Pasteurellaceae,9and!Moraxellaceae.9

!

92!

!
Figure!3.4.!Twenty!most!abundant!families!identified!in!the!tonsillar!microbiome!of!pigs!from!newborn!to!market!age!
and!in!the!sows.!The!chart!shows!the!twenty!most!abundant!families!identified!in!the!tonsillar!microbiome!of!the!sampled!!!

!

93!

Figure!3.4!(cont´d).!!piglets!from!newborn!through!market!age!as!well!as!the!sows.!Text!boxes!in!the!plot!indicate!times!when!
a!challenging!event!occurred.!“Others”!represent!members!of!bacterial!families!different!from!the!20!most!abundant!families!
identified.!!
!
!

!

94!

The! microbiome! of! newborns! was! characterized! by! the! abundant! presence! of!
the! families! Streptococcaceae,! Moraxellaceae,! Staphylococcaceae. and! Micrococcaceae,!
each! representing! 10! to! 23%! of! the! total;! members! of! families! Pasteurellaceae,!
Burkholderiaceae!and!Bacillaceae!as!well!as!members!of!the!order!Clostridiales!were!
identified! in! smaller! proportions.! In! the! first! week,! Pasteurellaceae! and!
Porphyromonadaceae! increased! dramatically,! to! 25%! and! 8.1%,! respectively.!
Moraxellaceae! also! increased! slightly,! while! there! was! a! slight! decrease! in!
Streptococacceae..A!more!dramatic!decrease!was!evident!for!Staphylococcaceae,!which!
almost! disappeared,! and! Micrococcaceae.! Over! the! next! two! weeks,! members! of! the!
Streptococcaceae!continued!to!decrease,!and!Micrococcaceae!virtually!disappeared.!In!
contrast,!members!of!Moraxellaceae!continued!to!increase.!!Members!of!Pasteurellaceae.
remained! constant.! Fusobacteriaceae. appeared! in! week! 2! and! remained! present! in!
week! 3.! Multiple! members! of! the! order! Clostridiales! (Clostridiaceae. 1,! Clostridiales!
Incertae!Sedis!XI,!Lachnospiraceae,!Peptostreptococcaceae!and!Ruminococcaceae)!were!
present!in!proportions!lower!than!one!percent,!each,!throughout!the!first!3!weeks!of!
life!in!these!piglets.!!!
The! transition! between! the! third! and! fourth! weeks,! when! the! piglets! were!
weaned!and!shifted!to!solid!food!containing!Carbadox ,!was!marked!by!drastic!shifts!in!
®

the!tonsil!microbiome.!!Moraxellaceae!decreased!dramatically!from!31.2!%!in!week!3!to!
7.9!%!in!week!4,!Streptococcaceae!bloomed!from!7.4!%!to!41.6!%,!while!Pasteurellaceae.
and!

Clostridiales!

remained!

steady.!

Members!

Porphyromonadaceae!almost!disappeared.!

!

95!

of!

Fusobacteriaceae!

and!

Week!6,!after!another!major!transition!when!Carbadox !was!removed!from!feed!
®

and!Tylan !added,!was!again!marked!by!drastic!shifts!in!the!tonsil!microbiome.!!Overall!
®

sobs,!as!described!above,!increased!from!111!to!368,!indicating!a!massive!increase!in!
diversity.! Members! of! the! Streptococcaceae! and! Pasteurellaceae! both! decreased!
dramatically,!from!41.6%!to!11.6%!and!23.2!%!to!10%,!respectively,!and!Moraxellaceae!
decreased! and! almost! disappeared.! However,! members! of! Bacillaceae. 1! and! some!
members!of!the!order!Clostridiales!(Clostridiales.Incertae.Sedis.XI,!Lachnospiraceae.and!
Ruminococcaceae)! began! to! flourish! and! increased! substantially,! particularly!
Bacillaceae. 1! which! increased! from! 0.9! to! 13.1%.! Interestingly,! almost! 44%! of! the!
members! of! the! tonsillar! microbiome! did! not! fit! into! these! twenty! most! abundant!
bacterial!families!for!this!time!point.!!!
In! the! eighth! week,! the! decreasing! trend! for! Streptococcaceae! and!
Pasteurellaceae! continued! and! each! family! dropped! to! a! relative! abundance! of! 7%.!
Moraxellaceae! remained! in! very! low! abundance.! However,! Clostridiales,. particularly!
Clostridiaceae.1,!increased,!!!as!did!Bacteroidales..The!proportion!of!identified!bacterial!
families! that! were! not! included! in! the! twenty! most! abundant! was! still! close! to! forty!
percent.!!
The!tenth!week,!which!corresponded!to!another!significant!transition!period!for!
the!pigs,!i.e.,!removal!of!Tylan !from!feed!as!well!as!movement!to!finishing!rooms!and!
®

reassortment!of!litter!members,!was!again!marked!by!a!major!shift!in!the!microbiome.!
The!three!predominant!families,!Pasteurellaceae,.Streptococcaceae,.and!Moraxellaceae,.
all! increased,! particularly! the! Pasteurellaceae! that! increased! from! 7%! to! 30.7%.! In!
contrast,!members!of!the!Clostridiales!(Clostridiaceae.1,!Clostridiales.Incertae.Sedis.XI,!

!

96!

Lachnospiraceae! and. Ruminococcaceae)! and! Prevotellaceae. decreased,! as! did! the!
proportion!of!the!microbiome!classified!as!“Others”.!!
Over!the!next!6!weeks,!represented!by!sampling!times!at!12!weeks!and!16!weeks,!
the! tonsil! microbiomes! in! all! of! the! pigs! coalesced! to! a! common! core! (Figure! 3.2.).!
Overall,!there!was!a!massive!increase!in!the!Clostridiales,!particularly.Clostridiaceae.1!
and! Peptostreptococcaceae,! from! 2.2%! and! 0.6%! in! week! 10! to! 44.1%! and! 7%,!
respectively!in!week!16.!Over!the!same!period,!Pasteurellaceae.decreased!from!30.7%!
to! 7.1%,! and. Bacillaceae. 1. decreased! from! 10.1%! to! 0.6%.! Streptococcaceae,.!
Moraxellaceae,. and. Bacteroidales. ! remained! relatively! stable.! The! proportion! of!
identified! bacterial! families! that! were! not! included! into! the! twenty! most! abundant!
families! decreased! to! ~10%.! ! By! week! 16,! Fusobacteriaceae! and! Neisseriaceae!
reappeared!in!low!proportions.!
!Finally,! the! nineteenth! week,! which! coincided! with! a! transitional! period! in!
which!penmates!were!reassorted!into!new!rooms,!was!marked!by!another!significant!
disruption! of! the! microbiome.! Overall,! an! increase! in! Pasteurellaceae,. Moraxellaceae,.
Neisseriaceae! and! Fusobacteriaceae! was! paired! with! a! dramatic! decrease! in!
Clostridiales,!particularly!!Clostriaceae.1.and.Peptostreptococacceae.!!
It!should!be!noted!that,!after!the!first!3!weeks!and!weaning,!these!shifts!in!the!
microbiome!were!not!synchronous!in!all!piglets.!Figure!3.2.!shows!several!clusters!that!
contain!samples!from!sequential!weeks,!e.g.,!weeks!6,!8!and!10;!!weeks!8,!10,!and!12;!
and!weeks!10,!12,!and!16,!indicating!that!common!microbiomes,!represented!by!the!
clusters,!!were!reached!at!different!times!in!different!pigs.!This!is!further!illustrated!in!
Figure!3.6.!which!shows!the!most!abundant!microbial!families!over!time!in!4!different!

!

97!

pigs.!!As!examples,!a!microbiome!with!a!preponderance!of!Pasteurellaceae!is!seen!in!
week!10!in!pig!23,!week!12!in!pig!11,!both!weeks!10!and!12!in!pig!22,!and!not!at!all!in!
pig!36.!!A!microbiome!with!a!preponderance!of!Clostridiaceae,!mainly!Clostridiaceae.1!
and!Peptostreptococcaceae,!is!seen!in!weeks!12!and!16!in!pig!36,!week!16!in!pigs!22!and!
23,!and!weeks!16!and!19!in!pig!11.!
The!tonsillar!microbiome!of!sows!was!dominated!by!members!of!Clostridiaceae.
1! (~23%)! and! Peptostreptococcaceae! (~12%).! Other! families! present! in! proportions!
between! 1! and! 8%! included! Erysipelotrichaceae,! Pasteurellaceae,! Bacillaceae. 1,!
Streptococcaceae,!Burkholderiaceae,!Moraxellaceae,!Neisseriaceae.!Micrococcaceae!and!
other!members!of!the!Clostridiales!(Clostridiales.Incertae.Sedis.XI,!Lachnospiraceae!and!
Ruminococcaceae).!
Distribution* of* specific* OTUs* throughout* the* life* of* the* pigs.* While!
presentation! of! the! microbiome! data! at! the! taxonomic! level! of! family! gives! the! best!
overview! of! the! data! over! time,! we! also! examined! the! presence! and! abundance! of!
specific! OTUs! over! time! (Figure! 3.5.).! At! the! family! level,! Pasteurellaceae,!
Streptococcaceae,. and. Moraxellaceae! predominate! throughout! the! life! of! the! pigs.!
However,! within! the! top! 40! OTUs! there! were! three! OTUs! of! Pasteurellaceae. seen,!
including! OTU0001,! which! was! present! in! high! concentration! during! weeks! 1Z4! but!
never! lost,! OTU0016! which! appeared! in! weeks! 6,! 10Z12;! and! OTU0031,! which! was!
mainly! seen! in! one! week! old! piglets.! Similarly,! there! were! three! OTUs! of!
Streptococcaceae,!including!OTU002!which!was!seen!throughout!the!lives!of!the!pigs,!
OTU009!which!was!seen!in!newborns!and!weeks!1Z4,!and!OTU0024,!which!was!seen!
mainly! in! older! piglets.! Finally,! there! were! three! OTUs! of! Moraxellaceae,! including!

!

98!

OTU003,!which!was!a!major!component!of!the!microbiome!in!newborns!through!week!
4!and!then!disappeared!to!return!in!the!week!19!samples;!OTU0006,!which!was!present!
in!lower!amounts!than!OTU0003!in!weeks!1Z4!but!in!much!higher!amounts!in!weeks!16!
and!19;!and!OTU0046,!which!was!a!minor!component!of!the!microbiome.!!!

Nineteenth week

Sixteeenth week

Twelfth week

Tenth week

Eighth week

Sixth week

Fourth week

Third week

Second week

First week

Newborn

Otu00001__Pasteurellaceae__
Otu00002__Streptococcaceae__Streptococcus
Otu00003__Moraxellaceae__Moraxella
Otu00004__Bacillaceae_1__
Otu00005__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00006__Moraxellaceae__
Otu00007__Micrococcaceae__Rothia
Otu00008__Porphyromonadaceae__Porphyromonas
Otu00009__Streptococcaceae__Streptococcus
Otu00010__Bacillaceae_1__
Otu00011__Staphylococcaceae__Staphylococcus
Otu00012__Bacillaceae_1__Bacillus
Otu00013__Fusobacteriaceae__Fusobacterium
Otu00014__Clostridiales_Incertae_Sedis_XI__Tissierella
Otu00015__Burkholderiaceae__Ralstonia
Otu00016__Pasteurellaceae__Actinobacillus
Otu00017__Neisseriaceae__
Otu00020__Ruminococcaceae__
Otu00021__Peptostreptococcaceae__Clostridium_XI
Otu00022__Planococcaceae__Lysinibacillus
Otu00023__Prevotellaceae__Prevotella
Otu00024__Streptococcaceae__Streptococcus
Otu00027__Veillonellaceae__Veillonella
Otu00029__Flavobacteriaceae__
Otu00031__Pasteurellaceae__Pasteurella
Otu00032__Bacillales_Incertae_Sedis_XI__Gemella
Otu00033__Prevotellaceae__
Otu00035__Leptotrichiaceae__
Otu00036__Enterobacteriaceae__Escherichia.Shigella
Otu00037__Bacteroidaceae__Bacteroides
Otu00038__Xanthomonadaceae__Ignatzschineria
Otu00039__Fusobacteriaceae__Fusobacterium
Otu00040__Bacteroidaceae__Bacteroides
Otu00041__Erysipelotrichaceae__Turicibacter
Otu00046__Moraxellaceae__Acinetobacter
Otu00051__Lachnospiraceae__Clostridium_XlVb
Otu00053__Peptostreptococcaceae__Clostridium_XI
Otu00055__Flavobacteriaceae__
Otu00056__Clostridiaceae_1__Clostridium_sensu_stricto
Otu00059__Burkholderiaceae__Burkholderia

0

10

20

30

40

% OTU relative abundance

!
Figure* 3.5.* Forty* most* abundant* Operational* Taxonomic* Units* (OTUs)* for* pigs*
through*different*sampling*times.!HeatZmap!showing!the!relative!abundance!of!the!
top!40!OTUs!identified!per!sampling!time!through!the!life!of!these!pigs.!Note!that!any!
OTUs!identified!as!“unclassified”!at!the!family!level!were!not!included!in!this!list.!
!
Aerobic,* anaerobic,* and* facultatively* anaerobic* organisms* in* the* tonsils.!
An!analysis!of!the!distribution!of!the!bacterial!families!identified!in!the!tonsils!based!on!

!

99!

their! classification! by! use! of! oxygen! as! aerobes,! anaerobes! or! facultative! anaerobes!
(Figure!3.7)!showed!that!in!piglets!aged!newborn!to!4!weeks!the!microbial!population!
was! comprised! of! ~70%! aerobes! and! facultative! anaerobes.! ! The! abundance! of!
facultative! anaerobes! decreased! from! birth! through! week! 3,! but! increased! after!
weaning,! most! likely! due! to! the! bloom! in. Streptococcaceae.! ! The! proportion! of!
anaerobes! increased! after! the! weaning! period,! with! a! concomitant! decrease! in!
facultative!anaerobes!and!aerobes,!and!reached!~65%!of!the!total!microbiome!in!week!
16.!!!
!
!

!

!

100!

DISCUSSION!
!
We!have!previously!characterized!the!tonsil!microbiome!in!healthy!18Z20!week!
old!growerZfinisher!pigs![12]!and!sought!in!this!study!to!characterize!how!that!tonsil!
microbial!community!develops!during!the!life!of!pigs!from!newborn!to!market!age.!!In!
particular,! we! wished! to! determine! when! specific! members! of! the! tonsil! microbial!
community! appeared,! and! whether! there! was! a! temporal! succession! in! the!
development!of!the!community.!
There! are! strong! parallels! between! our! current! data! and! that! from! the! prior!
study! [12].! In! both! studies,! members! of! the! tonsil! microbiome! were! found! to!
predominantly! belong! to! 5! phyla:! Proteobacteria,. Firmicutes,. Bacteroidetes,.
Fusobacteria,. and! Actinobacteria,! with! Proteobacteria. ! and! Firmicutes! together!
representing! ~85Z90%! of! the! tonsil! microbiome.! In! both! studies,! Pasteurellaceae,.
Moraxellaceae,.Neisseriaceae,.Streptococcaceae,.Peptostreptococcaceae,.Veillonallaceae,.
and!Fusobacteriaceae.!were!identified!as!among!the!most!abundant!bacterial!families!
found.!!In!the!current!study,!many!families!in!the!orders!Clostridiales!and!Bacteroidetes!
were!also!found!to!be!among!the!most!abundant!taxa!seen.!!Improvements!in!both!the!
sequencing! technology! and! the! databases! that! facilitate! identification! of! bacteria! via!
16s!rRNA!gene!sequencing!likely!account!for!these!differences.!!In!the!earlier!study,!it!
was! not! possible! to! identify! most! Clostridiales! below! the! order! level,! which! is! now!
possible.! ! Further,! in! the! earlier! study! it! was! recognized! that! Bacteroidetes! were!
underrepresented!in!the!final!data,!possibly!due!to!amplification!bias!with!the!primers!
used!in!that!study![12].!

!

101!

We!collected!samples!from!eleven!different!sampling!periods!from!newborn!to!
19!weeks!of!age!(Figure!3.1.)!as!well!as!the!tonsillar!microbiome!of!the!sows.!In!our!
analysis!of!the!taxa!(at!the!family!level!and!the!OTU!level)!in!these!samples,!we!observed!
that!the!development!of!tonsillar!communities!in!pigs!followed!a!successional!process.!!
Some!members!of!the!community!were!acquired!very!early!in!life,!from!the!sow!vaginal!
tract!and!the!teat!skin!or!milk![22],!while!others!were!acquired!later.!!Some!members!
of! the! community,! particularly! Streptococcus,. Pasteurellaceae,! and! Moraxella,! were!
present! throughout! the! life! of! the! pigs,! while! others! such! as! Staphylococcus. and!
Fusobacterium.seemed!to!be!transient!(Figure!3.4.).!!Further,!specific!OTUs!of!some!of!
these!major!taxa!also!appeared!to!be!either!permanent!or!transient!(Figure!3.5.).!The!
relative!proportions!of!the!major!members!of!the!microbiome!did!change!through!time,!
however,! as! pigs! aged,! their! microbiome! seemed! to! become! more! similar! to! the!
microbiome!of!older!pigs!(Table!3.3.).!This!process!was!not!always!synchronous,!but!
the!overall!progression!was!very!similar!in!most!pigs.!
As!we!examined!the!changes!in!the!microbiome!over!time,!it!became!clear!that!
at!certain!time!points,!e.g.,!at!3,!8,!and!16!weeks,!the!microbiomes!of!all!of!the!animals!
became! very! similar,! or! coalesced! (Figure! 3.2.! and! 3.3.! and! Table! 3.2.).! When! we!
analyzed!this!in!comparison!to!management!of!the!pigs!(Figure!3.1.),!we!concluded!that!
stretches!of!time!with!constant!conditions,!such!as!newborn!through!week!3,!led!to!this!
coalescing!of!the!microbiome.!This!coalescing!occurred!regardless!of!litter!source!for!
the! pigs! or! room! in! which! they! were! housed.! In! contrast,! times! where! there! were!
changes! in! management! conditions,! such! as! addition! or! removal! of! in! feed! growth!
promoter!antibiotics!or!movement!of!pigs!to!new!housing,!and!especially!weaning,!led!

!

102!

to!perturbations!in!the!microbiome!(e.g.,!weeks!4,!6!,10!and!19.!The!taxonomic!data!was!
supported!by!an!analysis!of!the!alpha!diversity!(sobs:!number!of!observed!OTUs,!Table!
3.2.).! Whether! these! perturbations! occurred! in! response! to! specific! stresses,! such! as!
presence!of!antibiotics,!or!were!adaptations!of!the!microbiome!to!new!conditions,!such!
as!new!feed,!remains!unclear.!!
There! are! no! studies! available! that! describe! the! development! of! the! tonsillar!
microbiome!of!pigs!or!other!mammals,!except!this!study!and!one!recently!submitted!by!
our! lab! that! followed! the! development! of! the! tonsillar! microbiome! of! piglets! from!
newborn!to!weaning,!focusing!on!the!source!of!members!of!the!microbiome!and!the!
litter!effect!as!well!as!the!overall!development!and!the!effect!of!weaning![22].!!Most!of!
the! available! data! following! the! development! of! microbial! communities! in! mammals!
has! been! focused! on! the! gastrointestinal! tract.! Pajarillo! et! al! [29]! assessed! the! fecal!
bacterial!diversity!of!healthy!piglets!during!the!weaning!transition,!and!suggested!that!
this!period!was!related!to!a!trend!of!increasing!bacterial!diversity,!which!may!be!related!
with!the!changes!in!diet.!However,!they!did!not!discard!a!possible!additional!influence!
of!stress!or!disruption!associated!with!the!weaning!period.!Another!study!describing!
the! bacterial! diversity! of! pig! feces! over! time! followed! the! development! of! fecal!
microbiome! from! pigs,! between! 10! to! 22! weeks! old,! ! and! identified! that! calculated!
diversity!indices!suggested!similar!diversity!profiles!for!all!the!samples![30].!Although!
these! prior! studies! examined! the! fecal! microbiome,! they! support! our! results! of!!
increased!bacterial!diversity!when!the!piglets!were!weaned,!which!decreased!after!10Z
12!weeks,!following!challenges!or!disruptions,!such!as!addition!or!removal!of!in!feed!
antibiotics.!!

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103!

It!should!be!noted!that!the!shifts!in!the!membership!and!diversity!of!the!tonsil!
microbiota! that! we! observed! were! often! associated! with! challenges! that! occurred! in!
several! combinations! and! rarely! were! associated! with! a! singular! change! in! the!
management! of! the! pigs.! Changes! in! diet,! the! administration! of! antibiotics,! and! the!
environment! frequently! occurred! simultaneously,! making! it! difficult! to! determine!
which! challenges! were! associated! with! the! observed! microbiota! shifts.! However,!
absence!of!challenges!or!disruptions!led!to!stabilization!of!the!microbiome,!with!most!
pigs!developing!similar!microbiomes!over!times!with!constant!conditions.!
There! is! extensive! research! data! showing! that! that! the! balance! of! microbial!
communities!is!altered!by!the!use!of!antibiotic!treatments![20,!31].!Rettedal!et!al![32]!
studied!the!effect!of!the!growth!promoter!chlortetracycline!on!the!ileal!microbiota!of!
pigs!and!found!that!use!of!this!antibiotic!was!associated!with!a!significant!shift!in!the!
gut!!microbiota,!including!a!replacement!of!the!dominant!species!of!Lactobacillus!and!
decrease!in!relative!abundance!of!Turicibacter!.!However,!Poole!et!al![33]!did!not!find!
changes! in! diversity! in! feces! associated! with! a! similar! dose! of! chlortetracycline!
supplementation! .! The! inZfeed! supplementation! of! pigs! with! a! mixture! of! antibiotics!
known! as! ASP250,! containing! chlortetracycline,! sulfamethazine! and! penicillin,! was!
correlated! with! a! shift! in! the! bacterial! phylotypes! present! in! the! intestine,! where!
microbial!community!membership!changed!over!time!associated!with!administration!
of! the! product.! The! changes! were! related! mainly! with! a! decrease! in! Bacteroidetes.
abundance! and! an! increase! in! Proteobacteria,! among! other! changes! [34].! Carbadox !
®

supplementation! inZfeed! was! associated! with! significant! changes! in! community!
structure!and!bacterial!membership!in!the!intestinal!microbiota!of!pigs.!An!immediate!

!

104!

effect! was! noticed! in! the! bacterial! community! after! the! administration! of! this! drug,!
although! the! microbiome! structure! recovered! later! despite! the! continued! use! of! the!
medication.! The! authors! reported! a! relative! increase! in! Prevotella! associated! with!
Carbadox !administration,!while!Carbadox !withdrawal!was!associated!with!an!increase!
®

®

in!the!E..coli!population![35].!The!use!of!the!growth!promoter!Tylosin!(also!known!as!
Tylan )! was! associated! with! a! pronounced! shift! in! the! intestinal! microbiome!
®

distribution! and! quantity,! altering! the! abundance! of! specific! genera! such! as!
Lactobacillus.among!others.!These!changes!occurred!at!specific!times!in!the!growing!pig!
as!the!pigs!aged![36].!!!These!prior!studies!have!tried!to!identify!the!effects!of!medicated!
food!on!the!gut/feces!microbiome,!but!there!are!no!studies!that!characterize!the!effect!
of!ingested!medications!on!the!tonsillar!microbiome.!However,!it!can!be!concluded!that!
regardless!of!the!medication,!the!administration!of!antibiotics!or!growth!promoters!in!
food! exerts! an! effect! on! the! bacterial! communities.! In! this! study,! we! observed! large!
shifts!in!the!tonsil!microbiome!related!to!specific!periods!where!medicated!food!was!
added,!changed!or!removed.!However,!because!in!feed!medication!was!not!an!isolated!
factor! but! supplementary! to! other! changes! at! the! same! time,! we! cannot! make! a!
definitive!conclusion!about!the!specific!effect!of!the!administration!of!this!medication.!
We!do!consider!the!microbiome!shifts!seen!to!be!relevant!!and!the!potential!subject!of!
further!research.!!
We!identified!the!first!major!shift!associated!with!supplementation!of!Carbadox !!
®

coinciding! with! a! huge! bloom! in! members! of! Streptococcaceae! and! a! decrease! in!
Moraxellaceae,.Fusobacteriaceae!and!Porphyromonadaceae,!reported!previously!by!our!
group! [22].! Another! shift! was! associated! with! the! removal! of! Carbadox ! and!
®

!

105!

supplementation!with!Tylan ,!with!a!major!decrease!in!members!of!Streptococcaceae,!
®

Moraxellaceae! and! Pasteurellaceae! with! a! concurrent! increase! in! members! of!
Clostridiales!and!Bacillaceae.1.!The!removal!of!Tylan !from!the!diet!was!associated!with!
®

a!slight!increase!in!members!of!Streptococcaceae!and!Moraxellaceae,!parallel!to!a!higher!
increase!in!members!of!Pasteurellaceae.!We!believe!it!is!relevant!to!highlight!that!the!
presence!of!Tylan !in!the!diet!is!associated!with!an!increase!in!the!bacterial!diversity!
®

(Table!3.2.!and!Figure!3.4.).!We!emphasize!that!our!goal!was!not!directed!towards!the!
identification!of!specific!effects!of!antibiotics!or!growth!promoters!in!the!development!
of!the!tonsil!microbiome,!but!instead!towards!characterization!of!the!development!of!
tonsillar!microbiome!of!pigs!from!a!healthy!farm.!Our!results!open!an!avenue!for!future!
research! on! the! specific! effect! of! these! medications! on! the! tonsillar! microbiome! and!
how!they!can!potentially!influence!the!acquisition!of!pathogenic!flora.!!
Another!big!change!experienced!by!the!pigs!was!dietary,!particularly!at!weaning.!
In! human! infants,! the! introduction! to! a! new! diet! associated! with! cessation! of! breast!
feeding!has!been!shown!to!be!associated!with!profound!changes!in!the!composition!of!
the! intestinal! microbiome! [37].! It! has! been! suggested! that! the! diet! to! which! an!
individual! has! been! exposed! rapidly! alters! the! structure! of! the! intestinal! microbial!
communities![38].!Similarly,!in!pigs!it!has!been!shown!that!the!diet!can!have!an!effect!
in!the!intestinal!microbiome![39],!and!in!particular!that!the!diet!supplemented!after!
weaning! in! piglets! can! alter! the! fecal! microbiota! considerably.! A! diet! supplemented!
with! fermentable! carbohydrates! was! related! with! greater! bacterial! diversity! when!
compared!to!control!diets![39].!Diet!changes!during!weaning!transition!can!exert!an!

!

106!

effect! on! the! composition! of! the! intestinal! microbiota! [40,! 41]! where! bacterial!
community!structure!can!change!as!the!diet!changes![42].!
Finally,!the!environmental!changes!experienced!by!the!pigs!could!play!a!role!in!
the!development!of!the!tonsillar!microbiome.!!These!can!include!both!changes!in!the!
physical!environment!and!exposure!to!new!penmates!after!reassortment!of!pigs!into!
new!housing.!The!immediate!environment!in!which!pigs!grow!has!been!suggested!to!
have! a! profound! influence! on! the! initial! acquisition! and! development! of! fecal! and!
colonic!microbiota![43].!!A!recent!study!following!the!development!of!gut!microbiota!
and! the! effect! of! early! changes! in! the! environment! demonstrated! that! microbial!
diversity!was!disturbed!by!changes!in!environmental!hygiene,!and!that!the!effect!of!the!
generated!changes!remained!for!a!long!time!in!the!affected!animals![8].!!In!our!study,!
we!saw!increased!fecal!anaerobes,!such!as!Clostridiales,!in!the!tonsils!(Figure!3.4.!and!
Figure!3.7.)!after!weaning!and!especially!after!Tylan !was!removed!from!feed.!!Pigs!are!
®

coprophagic,!and!it!is!likely!that!these!anaerobes!were!acquired!from!ingestion!of!feces!
from!the!pen!floors.!!In!the!older!pigs,!crypt!abscesses!in!the!deeper!areas!of!elongating!
crypts!might!provide!a!niche!for!colonization!by!the!acquired!anaerobes,!or!conversely!
these!anaerobes!may!cause!the!formation!of!the!crypt!abscesses.!We!have!previously!
observed!that!pigs!housed!in!a!very!clean!high!biosecurity!environment!had!almost!no!
Clostridiales!in!the!tonsils!(unpublished!data).!
The! sixteenth! week! constituted! the! highest! threshold! for! members! of!
Clostridiales!with!Clostridiaceae.1.and!Peptostreptococcaceae!comprising!~51%!of!the!
identified! members! of! the! microbiome! for! this! period.! Our! results! are! supported! by!
Bokulich!et!al![31],!who!studied!the!development!of!fecal!microbiota!in!children!during!

!

107!

early! life! and! associated! the! administration! of! antibiotics! in! children! during! first!
months!of!life!with!deficit!in!members!of!Clostridiales.!Further,!the!authors!associated!
a!gradual!increase!in!members!of!this!order!with!the!introduction!to!solid!food.!!Our!
findings!become!especially!relevant!when!compared!with!recent!findings!reported!by!
Kim!et!al![7],!which!found!that!the!presence!of!members!of!Clostridiales!in!the!enteric!
microbiota!of!mice!is!critical!to!prevent!the!growth!of!enteric!pathogens!in!the!intestine.!
We!do!not!know!how!this!finding!can!be!translated!to!the!tonsillar!microbiome!of!pigs,!
but!it!is!interesting!to!see!that!one!of!the!most!vulnerable!periods!for!pigs!to!acquire!
diseases!(weaning!through!eighth!week)!was!marked!by!a!low!abundance!of!members!
of!the!Clostridiales.!!
We! need! to! emphasize! that! the! goal! of! this! research! was! not! to! examine! the!
individual! effect! of! each! one! of! the! possible! factors! associated! with! the! challenges!
experienced!by!the!pigs!during!their!lives!but!on!the!contrary,!the!purpose!of!this!work!
was!to!characterize!the!development!of!porcine!tonsillar!microbiome!of!healthy!pigs!
from!newborn!through!market!age!under!normal!management!conditions!and!thus!to!
create!a!base!knowledge!in!a!topic!so!far!little!studied.!!!
In!this!study,!we!found!that!there!were!some!bacterial!families!that!dominated!
the!tonsillar!microbiome!throughout!the!life!of!pigs;!however,!their!relative!abundance!
often! changed! significantly! after! the! challenging! events.! Similarly,! other! bacterial!
families!appeared!and/or!disappeared!at!specific!ages.!We!identified!that!members!of!
Pasteurellaceae,!Streptococcaceae!and!Moraxellaceae!were!the!most!abundant!families!
through! the! life! of! pigs! despite! their! fluctuation! at! certain! ages.! Nevertheless,!
Pasteurellaceae!was!the!most!abundant!family!throughout!the!study!period.!The!greater!

!

108!

presence! of! members! of! Clostridiales! particularly! Clostridiaceae. 1! and!
Peptostreptococcaceae! in! older! pigs! needs! to! be! highlighted,! as! well! as! the! sudden!
increase! in! members! of! Fusobacteriaceae! at! certain! ages.! A! longitudinal! study! of!
bacterial!diversity!in!feces!of!commercial!pigs!found!that!some!phyla!dominated!the!
microbiome!regardless!of!the!age!of!the!animals,!supporting!our!findings!in!the!tonsils.!
Further,! it! was! observed! that! a! small! group! of! organisms! were! the! most! prevalent!
microbes!as!pigs!aged,!and!their!microbiome!converged!with!the!time!when!they!were!
maintained!under!similar!conditions![30].!Although!this!study!was!focused!on!the!fecal!
microbiome,!it!supports!our!results!in!the!development!of!tonsillar!microbiome,!where!
we!identified!some!bacterial!families!that!dominated!and!were!present!throughout!the!
study!period,!as!well!as!other!bacterial!families!that!were!transient!and!appeared!at!
different!times,!and!further!saw!a!convergence!of!the!tonsil!microbiome!in!all!the!pigs!
when!they!were!maintained!under!constant!conditions.!
Jensen! et! al! [11]! characterized! the! microbiome! of! tonsillar! crypts! of! human!
patients! either! with! chronic! tonsillitis! or! tonsils! from! healthy! patients! which! were!
removed! because! of! hyperplasia.! The! authors! could! identify! a! core! microbiome!
population! at! the! species! level! in! the! crypts! of! humans! independent! of! their! health!
status! and! age,! which! involved! the! genus! Streptococcus,! Prevotella,! Fusobacterium,!
Porphyromonas,!

Neisseria,!

Parvimonas,!

Haemophilus,!

Actinomyces,!

Rothia,!

Granulicatella!and!Gemella.!The!above!identified!genera!are!members!of!the!families!
Streptococcaceae,!

Prevotellaceae,!

Fusobacteriaceae,!

Porphyromonadaceae,!

Neisseriaceae,! Clostridiales! Incertae! Sedis. XI,! Pasteurellaceae,! Actinomycetaceae,!
Micrococcaceae,! Carnobacteriaceae! and! Bacillales! Incertae! Sedis. XI,! respectively.!

!

109!

Similarly,! other! studies! identifying! the! human! microbiome,! recognize! members! of!
families! Streptococcaceae,! Prevotellaceae! and! Fusobacteriaceae! as! abundant! in! the!
tonsillar!microbiome!of!healthy!humans![13,!16,!44].!!!Although!we!did!not!characterize!
specifically!the!microbiome!of!tonsillar!crypts!and!we!were!not!able!to!characterize!the!
members!of!the!community!further!than!family!or!genus!level!for!some!taxa,!our!results!
also!show!that!members!of!the!above!mentioned!families!except!Actinomycetaceae.and!
Carnobacteriaceae,. comprised! some! of! the! most! abundant! families! identified! in! pig!
tonsils.!It!was!found!that!members!of!the!genus!Staphylococcus.were!present!only!in!
low! proportions! in! human! tonsils! [11].! Similarly,! we! identified! that! members! of! the!
family! Staphylococcaceae! were! abundant! only! in! the! newborns! and! decreased!
noticeably!and!almost!disappeared!on!the!following!weeks.!
A! BrayZCurtis! analysis! of! the! development! of! pigs! microbiome! from! birth! to!
market!age!(Figure!3.2.)!showed!us!that!as!the!pigs!were!getting!older,!the!acquired!
microbial!population!tended!to!be!more!similar!to!the!microbiome!present!in!adult!pigs,!
i.e.,!the!tonsillar!microbiome!of!sows!(Figure!3.4).!We!identified!that!between!the!sixth!
to!tenth!week,!some!samples!clustered!with!a!sample!from!the!tonsillar!microbiome!of!
sows.! However! a! higher! percentage! of! samples! from! older! pigs,! especially! between!
twelfth! to! nineteenth! weeks,! were! clustered! together! with! samples! from! tonsillar!
microbiome!of!sows.!These!findings!demonstrate!both!that!there!is!a!succession!in!the!
development!of!tonsillar!microbiome!in!pigs!and!that!the!final!status!of!the!microbiome!
in! grower/finisher! pigs! develops! to! resemble! that! of! adult! animals.! Similar! findings!
were! reported! by! other! authors! studying! the! development! of! the! human! intestinal!
microbiota![19,!31],!which!found!that!as!infants!aged,!their!gut!microbiome!began!to!

!

110!

look!like!the!adult!microbiome,!although!it!did!not!reach!a!mature!stage!found!in!adults.!
Our!results!shows!that!although!the!microbiome!of!older!pigs!was!more!similar!to!the!
microbiome! of! the! sows,! there! are! still! observable! differences! in! the! abundance! of!
certain! families,! as! the! case! of! members! of! families! Peptostreptococacceae,!
Erysipelotrichaceae! and! Burkholderiales! which! were! more! prominent! in! sow!
microbiome.! Many! other! studies! have! also! shown! that! there! is! a!
succession/sequentiality!in!the!development!of!microbial!communities!in!mammalian!
tissue![17,!18,!30,!45,!46]!
!
CONCLUSIONS!
!
This! study! provides! baseline! information! on! the! development! of! tonsillar!
microbiome!of!piglets!from!newborn!to!market!age,!as!well!as!the!tonsillar!microbiome!
of!sows.!We!demonstrate!that!there!was!a!succession!in!the!development!of!the!tonsillar!
microbiome!of!piglets!as!they!age,!which!was!not!synchronous!on!all!pigs!but!was!highly!
similar.! ! The! tonsil! microbiome! tended! to! stabilize! and! become! very! similar! in! all!
animals! over! times! where! management! conditions! are! constant.! ! However,! the!
challenges!associated!with!management!procedures!typical!in!a!swine!farm!generated!
prominent! changes! in! the! microbiome! composition! and! the! abundance! of! diverse!
bacterial!families.!Nonetheless,!over!time!the!microbiome!of!these!young!pigs!tended!
to!be!more!similar!to!the!microbiome!of!older.!We!do!not!know!if!the!observed!patterns!
would!be!similar!for!all!pigs!from!this!farm,!or!if!the!same!pattern!would!be!observed!
independent!of!the!breed!or!the!specific!farm.!!This!study!lays!the!baseline!for!future!

!

111!

research!to!examine!the!effect!of!specific!conditions,!such!as!use!of!antibiotics,!on!the!
development!of!the!tonsil!microbiome!and!of!acquisition!of!specific!pathogens!on!the!
tonsil!microbiome!and!conversely!of!the!effect!of!the!composition!and!structure!of!the!
tonsil!microbiome!on!acquisition!of!pathogens.!!Manipulation!of!the!tonsil!microbiome!
to!provide!enhanced!resistance!to!acquisition!and!carriage!of!pathogens!is!a!potential!
outcome!of!these!studies.!
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112!

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APPENDIX!
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113!

!
Table!3.3.!Top!20!most!abundant!families!per!sampling!time!
!
Row	Labels
Corynebacteriaceae
Micrococcaceae
Bacteroidaceae
Porphyromonadaceae
Prevotellaceae
Rikenellaceae
Flavobacteriaceae
Chitinophagaceae
Bacillaceae_1
Bacillales_Incertae_Sedis_XI
Planococcaceae
Staphylococcaceae
Aerococcaceae
Lactobacillaceae
Streptococcaceae
Clostridiaceae_1
Clostridiales_Incertae_Sedis_XI
Lachnospiraceae
Peptostreptococcaceae
Ruminococcaceae
Erysipelotrichaceae
Veillonellaceae
Fusobacteriaceae
Leptotrichiaceae
Caulobacteraceae
Sphingomonadaceae
Burkholderiaceae
Comamonadaceae
Neisseriaceae
Succinivibrionaceae
Enterobacteriaceae
Pasteurellaceae
Moraxellaceae
Pseudomonadaceae
Xanthomonadaceae
Spirochaetaceae
Others

Newborns
2.02
10.25
0.30
0.85
0.51
0.24
0.59
0.17
2.40
0.19
0.15
11.60
0.36
0.16
22.77
1.30
2.25
0.78
0.24
1.21
0.69
0.59
0.09
0.00
2.20
0.31
3.73
0.75
0.17
0.04
0.93
7.68
13.19
0.12
1.15
0.23
9.80

First	
week
0.14
2.63
1.14
8.11
1.79
0.07
2.06
0.01
2.10
1.32
0.16
0.07
0.57
0.60
16.71
0.67
0.49
0.87
0.45
0.67
0.47
1.28
0.23
0.00
0.05
0.10
0.38
0.17
0.43
0.01
0.49
24.90
17.67
0.06
0.16
0.22
12.76

Second
week
0.07
0.74
0.74
6.11
1.58
0.02
2.38
0.00
1.29
1.93
0.10
0.06
0.18
0.31
10.23
1.21
0.73
0.85
0.67
1.41
0.41
0.61
4.11
2.58
0.02
0.01
0.13
0.11
1.87
0.02
0.25
24.93
24.72
0.01
0.05
0.47
9.07

Third
week
0.07
0.42
1.33
5.69
2.97
0.00
4.22
0.01
0.13
1.22
0.03
0.03
0.07
0.03
7.45
0.49
0.59
0.49
0.50
1.24
0.32
0.28
4.29
3.55
0.07
0.03
0.08
0.02
1.07
0.00
0.04
23.40
31.22
0.01
0.01
0.11
8.51

Fourth
week
0.22
1.68
0.29
0.21
2.05
0.04
0.18
0.04
0.94
0.51
0.10
0.08
0.09
2.25
41.64
0.58
0.59
2.06
0.11
1.64
0.54
1.57
0.12
0.00
0.26
0.31
1.68
0.27
2.17
0.05
0.49
23.25
7.85
0.09
0.23
0.08
5.71

Sixth
week
0.68
0.43
0.93
1.34
1.90
1.16
0.25
0.15
13.13
0.06
0.82
0.34
0.00
0.55
11.63
1.35
3.82
3.21
0.08
4.18
0.06
0.27
0.01
0.06
0.06
0.52
1.54
0.97
0.36
0.05
2.32
10.00
0.87
0.33
1.47
0.56
34.54

Eighth
week
0.68
0.38
2.94
0.78
5.40
0.54
0.11
0.10
14.60
0.14
0.88
0.30
0.00
0.21
7.09
6.90
3.29
4.92
0.65
4.19
0.29
0.93
0.13
0.01
0.07
0.51
1.73
1.11
0.48
1.45
1.10
7.02
1.15
0.71
0.95
1.39
26.88

Tenth
week
0.28
0.29
1.07
0.81
3.75
0.70
0.17
0.14
10.07
0.09
0.65
0.25
0.04
0.19
11.86
2.21
2.48
2.18
0.63
2.52
0.25
1.05
0.34
0.00
0.14
0.33
0.62
0.73
1.01
0.03
0.86
30.70
3.68
0.45
0.67
0.57
18.22

Twelfth
week
0.10
0.30
1.60
3.54
5.52
0.04
0.10
0.56
1.83
0.04
0.08
0.19
0.17
0.23
4.90
24.10
0.32
2.30
4.75
2.17
0.53
1.20
0.54
0.01
0.04
0.19
1.39
0.40
0.40
0.06
0.37
31.87
1.16
0.91
0.34
0.33
7.43

Sixteenth
week
0.25
0.23
2.33
3.34
2.92
0.02
0.08
0.28
0.65
0.44
0.24
0.08
0.33
0.22
7.83
44.14
0.56
0.77
7.09
1.21
1.33
0.39
2.28
0.16
0.00
0.03
0.69
0.18
2.39
0.02
0.43
7.16
5.28
0.03
0.11
0.35
6.15

Nineteenth
week
0.30
0.26
3.78
5.11
2.21
0.11
1.67
0.04
1.37
0.82
0.11
0.36
1.23
0.06
7.58
10.19
0.43
0.72
1.97
0.53
0.39
0.95
4.96
1.88
0.00
0.20
0.35
0.04
8.90
0.00
0.27
15.47
18.87
0.00
0.04
1.50
7.33

Sow	
tonsils
1.50
1.11
0.93
0.59
0.55
0.00
0.18
0.11
6.83
0.76
1.25
0.35
0.15
0.08
4.56
22.48
2.03
1.17
12.31
1.00
7.77
0.39
0.49
0.49
0.48
0.98
3.32
0.98
1.08
0.14
0.78
7.17
2.61
0.38
0.36
0.81
13.84

!
!!
The!!!table!shows!the!twenty!most!abundant!families!identified!in!the!tonsillar!microbiome!of!the!sampled!!!piglets!from!newborn!
through!market!age!as!well!as!the!sows.!The!color!code!!shows!in!red,!the!families!that!!represents!the!top!twenty!most!abundant!
families!for!a!specific!period!or!sample.!The!blue!color!represents!!families!that!were!not!part!of!the!top!20!families!for!that!
period.!In!black!color!are!other!members!of!microbiome!not!shown!in!the!table.!
!

114!

100%

100%

Others

90%

90%

Others

Xanthomonadaceae

Moraxellaceae

Moraxellaceae

80%

Pasteurellaceae

80%

Pasteurellaceae

Enterobacteriaceae

Neisseriaceae
70%

Neisseriaceae
70%

Burkholderiaceae

Burkholderiaceae

Caulobacteraceae

Leptotrichiaceae

Fusobacteriaceae

60%

Fusobacteriaceae

60%

Ruminococcaceae

Veillonellaceae

Peptostreptococcaceae
50%

Ruminococcaceae
50%

Lachnospiraceae

Peptostreptococcaceae
Lachnospiraceae

Clostridiales_Incertae_Sedis_XI
Clostridiaceae_1

40%

Clostridiales_Incertae_Sedis_XI

40%

Clostridiaceae_1

Streptococcaceae

Streptococcaceae

Lactobacillaceae

30%

Bacillales_Incertae_Sedis_XI

30%

Staphylococcaceae
Flavobacteriaceae

Bacillaceae_1
20%

Prevotellaceae

20%

Flavobacteriaceae

Porphyromonadaceae

Prevotellaceae

Bacteroidaceae

Porphyromonadaceae

10%

10%

Micrococcaceae

Corynebacteriaceae

A

0%
11A

11B

11C

11D

11E

11F

11G

11H

11I

11J

11K

100%

B

0%
22A

22B

22C

22D

22E

22F

22G

22H

22I

22J

22K

100%

90%

90%

Others

Others

Moraxellaceae

Spirochaetaceae

Pasteurellaceae

80%

Moraxellaceae

80%

Neisseriaceae

Pasteurellaceae

Burkholderiaceae
70%

Neisseriaceae
70%

Leptotrichiaceae

Leptotrichiaceae

Fusobacteriaceae

Fusobacteriaceae

Veillonellaceae

60%

Veillonellaceae

60%

Ruminococcaceae

Ruminococcaceae

Peptostreptococcaceae
50%

Peptostreptococcaceae
50%

Lachnospiraceae

Lachnospiraceae

Clostridiales_Incertae_Sedis_XI
Clostridiaceae_1

40%

Clostridiales_Incertae_Sedis_XI
Clostridiaceae_1

40%

Streptococcaceae

Streptococcaceae

Staphylococcaceae
30%

Staphylococcaceae
30%

Bacillaceae_1

Bacillaceae_1
Flavobacteriaceae

Flavobacteriaceae
Prevotellaceae

20%

Prevotellaceae

20%

Porphyromonadaceae

Porphyromonadaceae

Bacteroidaceae

Bacteroidaceae
10%

10%

Micrococcaceae

0%
23A

23B

23C

23D

23E

23F

23G

23H

23I

23J

23K

C

Micrococcaceae

0%
36A

36B

36C

36D

36E

36F

36G

36H

36I

36J

36K

D

!

Figure!3.6.!Top!20!most!abundant!families!per!sampling!time!for!4!selected!pigs.!Top!20!families!through!the!time!for!pig!!
!
11!(A),!pig!22!(B),!pig!23!(C),!pig!36!(D)!

!

115!

!
100%
90%
80%
70%
60%
Not	included
50%

Anaerobes
Facultative

40%

Aerobes

30%
20%
10%
0%

Newborns

First	
week

Second	
week

Third	
week

Fourth	
week

Sixth	
week

Eighth	
week

Tenth	
week

Twelfth	 Sixteenth	 Nineteenth	 Sow	
week
week
week
tonsils

!

Figure!3.7.!Proportions!of!aerobes,!anaerobes!and!facultative!bacteria.!The!plot!chart!shows!the!twenty!most!abundant!
families!identified!in!the!tonsillar!microbiome!of!the!sampled!pigs!from!newborn!through!market!age!as!well!as!sows!and!their!
proportion!once!classified!as!aerobes,!anaerobes!or!facultative!organisms!based!on!Bergey´s!Manual!of!Systematic!
Bacteriology![47]!
!

116!

!

!
!
!
!
!

!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
REFERENCES!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!

117!

REFERENCES!
!
!
!
1.!
!
2.!
!
3.!
!
4.!
!
5.!

!
6.!
!
7.!

!
8.!

!
9.!
!
10.!
!

!

Horter!DC,!Yoon!KJ,!Zimmerman!JJ:!A"review"of"porcine"tonsils"in"immunity"
and"disease.!Anim%Health%Res%Rev%2003,!4:143@155.!
Smith!TC,!Harper!AL,!Nair!R,!Wardyn!SE,!Hanson!BM,!Ferguson!DD,!Dressler!AE:!
Emerging"swine"zoonoses.!Vector3Borne%and%Zoonotic%Diseases%2011,!11:1225@
1234.!
Williams! LP,! Newell! KW:! Salmonella" excretion" in" joy>riding" pigs.! American%
Journal%of%Public%Health%and%the%Nations%Health%1970,!60:926@929.!
Weyrich!LS,!Feaga!HA,!Park!J,!Muse!SJ,!Safi!CY,!Rolin!OY,!Young!SE,!Harvill!ET:!
Resident" microbiota" affect"Bordetella, pertussis"infectious" dose" and" host"
specificity.!Journal%of%Infectious%Diseases%2014,!209:913@921.!
Ghartey!JP,!Smith!BC,!Chen!ZG,!Buckley!N,!Lo!YT,!Ratner!AJ,!Herold!BC,!Burk!RD:!
Lactobacillus, crispatus" dominant" vaginal" microbiome" is" associated" with"
inhibitory" activity" of" female" genital" tract" secretions" against"Escherichia,
coli.!Plos%One%2014,!9:8.!
Willing! BP,! Vacharaksa! A,! Croxen! M,! Thanachayanont! T,! Finlay! BB:! Altering"
host" resistance" to" infections" through" microbial" transplantation.! Plos% One%
2011,!6:9.!
Kim!Y@G,!Sakamoto!K,!Seo!S@U,!Pickard!JM,!Gillilland!MG,!III,!Pudlo!NA,!Hoostal!
M,!Li!X,!Wang!TD,!Feehley!T,!et!al:!Neonatal"acquisition"of"Clostridia"species"
protects" against" colonization" by" bacterial" pathogens.! Science% 2017,!
356:312@315.!
Mulder!IE,!Schmidt!B,!Stokes!CR,!Lewis!M,!Bailey!M,!Aminov!RI,!Prosser!JI,!Gill!
BP,!Pluske!JR,!Mayer!CD,!et!al:!Environmentally>acquired"bacteria"influence"
microbial"diversity"and"natural"innate"immune"responses"at"gut"surfaces.!
Bmc%Biology%2009,!7:20.!
Croswell!A,!Amir!E,!Teggatz!P,!Barman!M,!Salzman!NH:!Prolonged"impact"of"
antibiotics" on" intestinal" microbial" ecology" and" susceptibility" to"
enteric"Salmonella,infection.!Infection%and%Immunity%2009,!77:2741@2753.!
Mann! E,! Pinior! B,! Wetzels! SU,! Metzler@Zebeli! BU,! Wagner! M,! Schmitz@Esser! S:!
The"metabolically"active"bacterial"microbiome"of"tonsils"and"mandibular"
lymph"nodes"of"slaughter"pigs.!Frontiers%in%Microbiology%2015,!6:10.!

118!

11.!
!
12.!
!
13.!

!
14.!

!
15.!
!
16.!
!
17.!

!
18.!
!
19.!
!
20.!
!
21.!

!

Jensen!A,!Fago@Olsen!H,!Sorensen!CH,!Kilian!M:!Molecular"mapping"to"species"
level"of"the"tonsillar"crypt"microbiota"associated"with"health"and"recurrent"
tonsillitis.!Plos%One%2013,!8:15.!
Lowe! BA,! Marsh! TL,! Isaacs@Cosgrove! N,! Kirkwood! RN,! Kiupel! M,! Mulks! MH:!
Defining" the" "core" microbiome"" of" the" microbial" communities" in" the"
tonsils"of"healthy"pigs.!Bmc%Microbiology%2012,!12:14.!
Segata!N,!Haake!SK,!Mannon!P,!Lemon!KP,!Waldron!L,!Gevers!D,!Huttenhower!C,!
Izard! J:! Composition" of" the" adult" digestive" tract" bacterial" microbiome"
based"on"seven"mouth"surfaces,"tonsils,"throat"and"stool"samples.!Genome%
Biology%2012,!13:18.!
Liu!CM,!Cosetti!MK,!Aziz!M,!Buchhagen!JL,!Contente@Cuomo!TL,!Price!LB,!Keim!
PS,! Lalwani! AK:! The" otologic" microbiome" >" a" study" of" the" bacterial"
microbiota" in" a" pediatric" patient" with" chronic" serous" otitis" media" using"
16S" rRNA" gene>based" pyrosequencing.! Archives% of% Otolaryngology3Head%
&%Neck%Surgery%2011,!137:664@668.!
Lowe! BA,! Marsh! TL,! Isaacs@Cosgrove! N,! Kirkwood! RN,! Kiupel! M,! Mulks! MH:!
Microbial"communities"in"the"tonsils"of"healthy"pigs.!Veterinary%Microbiology%
2011,!147:346@357.!
Aas! JA,! Paster! BJ,! Stokes! LN,! Olsen! I,! Dewhirst! FE:! Defining" the" normal"
bacterial" flora" of" the" oral" cavity.! Journal% of% Clinical% Microbiology% 2005,!
43:5721@5732.!
Koenig!JE,!Spor!A,!Scalfone!N,!Fricker!AD,!Stombaugh!J,!Knight!R,!Angenent!LT,!
Ley! RE:! Succession" of" microbial" consortia" in" the" developing" infant" gut"
microbiome.! Proceedings% of% the% National% Academy% of% Sciences% of% the% United%
States%of%America%2011,!108:4578@4585.!
Inoue!R,!Tsukahara!T,!Nakanishi!N,!Ushida!K:!Development"of"the"intestinal"
microbiota" in" the" piglet.! Journal% of% General% and% Applied% Microbiology% 2005,!
51:257@265.!
Palmer! C,! Bik! EM,! DiGiulio! DB,! Relman! DA,! Brown! PO:! Development" of" the"
human"infant"intestinal"microbiota.!Plos%Biology%2007,!5:1556@1573.!
Sekirov!I,!Tam!NM,!Jogova!M,!Robertson!ML,!Li!YL,!Lupp!C,!Finlay!BB:!Antibiotic>
induced"perturbations"of"the"intestinal"microbiota"alter"host"susceptibility"
to"enteric"infection.!Infection%and%Immunity%2008,!76:4726@4736.!
Yin! Y,! Lei! F,! Zhu! L,! Li! S,! Wu! Z,! Zhang! R,! Gao! GF,! Zhu! B,! Wang! X:!Exposure"of"
different" bacterial" inocula" to" newborn" chicken" affects" gut" microbiota"
development"and"ileum"gene"expression.!Isme%Journal%2010,!4:367@376.!
119!

!
22.!
!
23.!
!
24.!

!
25.!

!
26.!

!
27.!
!
28.!
!
29.!
!
30.!
!
31.!

!

!

Peña@Cortes!LC,!LeVeque!RM,!Funk!J,!Marsh!TL,!Mulks!MH:!Development"of"the"
tonsillar"microbiome"in"pigs"from"newborn"through"weaning.!!(University!
MS!ed.;!2017.!
Kim!D,!Hofstaedter!CE,!Zhao!C,!Mattei!L,!Tanes!C,!Clarke!E,!Lauder!A,!Sherrill@Mix!
S,! Chehoud! C,! Kelsen! J,! et! al:! Optimizing" methods" and" dodging" pitfalls" in"
microbiome"research.!Microbiome%2017,!5:14.!
Caporaso!JG,!Lauber!CL,!Walters!WA,!Berg@Lyons!D,!Lozupone!CA,!Turnbaugh!PJ,!
Fierer! N,! Knight! R:! Global" patterns" of" 16S" rRNA" diversity" at" a" depth" of"
millions" of" sequences" per" sample.! Proceedings% of% the% National% Academy% of%
Sciences%of%the%United%States%of%America%2011,!108:4516@4522.!
Schloss!PD,!Westcott!SL,!Ryabin!T,!Hall!JR,!Hartmann!M,!Hollister!EB,!Lesniewski!
RA,!Oakley!BB,!Parks!DH,!Robinson!CJ,!et!al:!Introducing"mothur:"open>source,"
platform>independent,"community>supported"software"for"describing"and"
comparing"microbial"communities.!Applied%and%Environmental%Microbiology%
2009,!75:7537@7541.!
Kozich!JJ,!Westcott!SL,!Baxter!NT,!Highlander!SK,!Schloss!PD:!Development"of"a"
dual>index" sequencing" strategy" and" curation" pipeline" for" analyzing"
amplicon" sequence" data" on" the" MiSeq" Illumina" sequencing" platform.!
Applied%and%Environmental%Microbiology%2013,!79:5112@5120.!
Quast!C,!Pruesse!E,!Yilmaz!P,!Gerken!J,!Schweer!T,!Yarza!P,!Peplies!J,!Gloeckner!
FO:! The" SILVA" ribosomal" RNA" gene" database" project:" improved" data"
processing"and"web>based"tools.!Nucleic%Acids%Research%2013,!41:D590@D596.!
Schneider! CA,! Rasband! WS,! Eliceiri! KW:! NIH" Image" to" ImageJ:" 25" years" of"
image"analysis.!Nature%Methods%2012,!9:671@675.!
Alain!B!Pajarillo!E,!Chae!J@P,!Balolong!MP,!Bum!Kim!H,!Kang!D@K:!Assessment"of"
fecal" bacterial" diversity" among" healthy" piglets" during" the" weaning"
transition.!The%Journal%of%general%and%applied%microbiology%2014,!60:140@146.!
Kim! HB,! Borewicz! K,! White! BA,! Singer! RS,! Sreevatsan! S,! Tu! ZJ,! Isaacson! RE:!
Longitudinal" investigation" of" the" age>related" bacterial" diversity" in" the"
feces"of"commercial"pigs.!Veterinary%Microbiology%2011,!153:124@133.!
Bokulich!NA,!Chung!J,!Battaglia!T,!Henderson!N,!Jay!M,!Li!H,!Lieber!AD,!Wu!F,!
Perez@Perez! GI,! Chen! Y,! et! al:! Antibiotics," birth" mode," and" diet" shape"
microbiome" maturation" during" early" life.! Science% Translational% Medicine%
2016,!8:13.!

120!

32.!

!
33.!
!
34.!

!
35.!
!
36.!

!
37.!

!
38.!
!
39.!

!
40.!
!
41.!

Rettedal!E,!Vilain!S,!Lindblom!S,!Lehnert!K,!Scofield!C,!George!S,!Clay!S,!Kaushik!
RS,! Rosa! AJM,! Francis! D,! Brozel! VS:! Alteration" of" the"ileal" microbiota" of"
weanling" piglets" by" the" growth>promoting" antibiotic" chlortetracycline.!
Applied%and%Environmental%Microbiology%2009,!75:5489@5495.!
Poole!TL,!Suchodolski!JS,!Callaway!TR,!Farrow!RL,!Loneragan!GH,!Nisbet!DJ:!The"
effect" of" chlortetracycline" on" faecal" microbial" populations" in" growing"
swine.!Journal%of%Global%Antimicrobial%Resistance%2013,!1:171@174.!
Looft!T,!Johnson!TA,!Allen!HK,!Bayles!DO,!Alt!DP,!Stedtfeld!RD,!Sul!WJ,!Stedtfeld!
TM,!Chai!BL,!Cole!JR,!et!al:!In>feed"antibiotic"effects"on"the"swine"intestinal"
microbiome.! Proceedings% of% the% National% Academy% of% Sciences% of% the% United%
States%of%America%2012,!109:1691@1696.!
Looft!T,!Allen!HK,!Casey!TA,!Alt!DP,!Stanton!TB:!Carbadox"has"both"temporary"
and" lasting" effects" on" the" swine" gut" microbiota.! Frontiers% in% Microbiology%
2014,!5:9.!
Kim! HB,! Borewicz! K,! White! BA,! Singer! RS,! Sreevatsan! S,! Tu! ZJ,! Isaacson! RE:!
Microbial"shifts"in"the"swine"distal"gut"in"response"to"the"treatment"with"
antimicrobial"growth"promoter,"tylosin.!Proceedings%of%the%National%Academy%
of%Sciences%of%the%United%States%of%America%2012,!109:15485@15490.!
Bergstrom! A,! Skov! TH,! Bahl! MI,! Roager! HM,! Christensen! LB,! Ejlerskov! KT,!
Molgaard!C,!Michaelsen!KF,!Licht!TR:!Establishment"of"intestinal"microbiota"
during" early" life:" a" longitudinal," explorative" study" of" a" large" cohort"
of"Danish" infants.! Applied% and% Environmental% Microbiology% 2014,! 80:2889@
2900.!
David!LA,!Maurice!CF,!Carmody!RN,!Gootenberg!DB,!Button!JE,!Wolfe!BE,!Ling!
AV,! Devlin! AS,! Varma! Y,! Fischbach! MA,! et! al:! Diet" rapidly" and" reproducibly"
alters"the"human"gut"microbiome.!Nature%2014,!505:559@563.!
Konstantinov!SR,!Zhu!WY,!Williams!BA,!Tamminga!S,!de!Vos!WM,!Akkermans!
ADL:! Effect" of" fermentable" carbohydrates" on" piglet" faecal" bacterial"
communities" as" revealed" by" denaturing" gradient" gel" electrophoresis"
analysis"of"16S"ribosomal"DNA.!Fems%Microbiology%Ecology%2003,!43:225@235.!
Frese!SA,!Parker!K,!Calvert!CC,!Mills!DA:!Diet"shapes"the"gut"microbiome"of"
pigs"during"nursing"and"weaning.!Microbiome%2015,!3:10.!
Konstantinov!SR,!Favier!CF,!Zhu!WY,!Williams!BA,!Kluss!J,!Souffrant!WB,!de!Vos!
WM,! Akkermans! ADL,! Smidt! H:! Microbial" diversity" studies" of" the" porcine"
gastrointestinal" ecosystem" during" weaning" transition.! Animal% Research%
2004,!53:317@324.!

!
!

121!

42.!

!
43.!
!
44.!
!
45.!
!
46.!
!
47.!
!

!

Leser!TD,!Lindecrona!RH,!Jensen!TK,!Jensen!BB,!Moller!K:!Changes"in"bacterial"
community"structure"in"the"colon"of"pigs"fed"different"experimental"diets"
and" after" infection" with" Brachyspira, hyodysenteriae.! Applied% and%
Environmental%Microbiology%2000,!66:3290@3296.!
Thompson! CL,! Wang! B,! Holmes! AJ:! The" immediate" environment" during"
postnatal"development"has"long>term"impact"on"gut"community"structure"
in"pigs.!Isme%Journal%2008,!2:739@748.!
Huse! SM,! Ye! YZ,! Zhou! YJ,! Fodor! AA:! A" core" human" microbiome" as" viewed"
through"16s"rRNA"sequence"clusters.!Plos%One%2012,!7:12.!
Mortensen!MS,!Brejnrod!AD,!Roggenbuck!M,!Abu!Al@Soud!W,!Balle!C,!Krogfelt!KA,!
Stokholm! J,! Thorsen! J,! Waage! J,! Rasmussen! MA,! et! al:! The" developing"
hypopharyngeal"microbiota"in"early"life.!Microbiome%2016,!4.!
Borewicz!KA,!Kim!HB,!Singer!RS,!Gebhart!CJ,!Sreevatsan!S,!Johnson!T,!Isaacson!
RE:!Changes"in"the"porcine"intestinal"microbiome"in"response"to"infection"
with"Salmonella,enterica"and"Lawsonia,intracellularis.!Plos%One%2015,!10.!
Krieg! NR,! Staley! JT,! Brown! DR,! Hedlund! BP,! Paster! BJ,! Ward! NL,! Ludwig! W,!
Whitman! W:! Bergey's" Manual" of" Systematic" Bacteriology,% Vol% 4,% Second%
Edition.!Springer;!2011.!

122!

CHAPTER	
  4.	
  SPATIOTEMPORAL	
  DEVELOPMENT	
  OF	
  THE	
  TONSIL	
  MICROBIOME	
  IN	
  
PIGS	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

123	
  

INTRODUCTION	
  
	
  
	
  
Tonsils	
   are	
   lympho-­‐‑epithelial	
   tissues	
   strategically	
   located	
   at	
   the	
   junction	
   of	
  
oropharynx	
   and	
   nasopharynx	
   where	
   they	
   fulfill	
   an	
   important	
   role	
   in	
   immune	
  
surveillance	
   and	
   protection	
   of	
   the	
   host	
   from	
   pathogens	
   gaining	
   access	
   via	
   oral	
   or	
  
respiratory	
   routes	
   [1].	
   Numerous	
   host-­‐‑specific	
   and	
   zoonotic	
   pathogens	
   such	
   as	
  
bacteria	
   and	
   viruses	
   can	
   colonize	
   tonsils	
   and	
   use	
   them	
   as	
   a	
   reservoir,	
   generating	
   a	
  
source	
   that	
   has	
   high	
   potential	
   for	
   transmission	
   to	
   other	
   animals	
   and	
   to	
   humans	
   [2].	
  
There	
  is	
  a	
  paucity	
  of	
  data	
  pertaining	
  to	
  the	
  tonsillar	
  microbiome	
  in	
  mammals.	
  Further,	
  
there	
  are	
  no	
  published	
  studies	
  about	
  the	
  development	
  of	
  the	
  spatiotemporal	
  structure	
  
of	
  microbial	
  communities	
  of	
  pig	
  tonsils	
  or	
  tonsils	
  of	
  any	
  other	
  mammal.	
  	
  
Studies	
   on	
   the	
   development	
   of	
   the	
   spatiotemporal	
   structure	
   of	
   microbial	
  
communities	
  of	
  other	
  regions	
  of	
  the	
  oral	
  cavity,	
  such	
  as	
  dental	
  plaque	
  in	
  humans,	
  have	
  
shown	
  that	
  a	
  key	
  characteristic	
  of	
  oral	
  microbiome	
  communities	
  is	
  the	
  presence	
  of	
  a	
  
high	
   diversity	
   and	
   temporal	
   succession	
   in	
   their	
   population	
   [3].	
   	
   In	
   dental	
   plaque,	
  
members	
  of	
  the	
  genus	
  Streptococcus	
  function	
  as	
  early	
  colonizers	
  opening	
  the	
  way	
  for	
  
attachment	
   of	
   other	
   bacteria,	
   creating	
   the	
   conditions	
   suitable	
   for	
   the	
   arrival	
   of	
   late	
  
colonizers	
   which	
   are	
   frequently	
   pathogenic	
   bacteria	
   [4].	
   Bacteria	
  in	
   the	
   oral	
   cavity,	
   as	
  
well	
  as	
  in	
  other	
  tissues,	
  have	
  specific	
  adherence	
  molecules	
  on	
  their	
  surface	
  which	
  are	
  
necessary	
  for	
  initiating	
  colonization	
  [4].	
  In	
  the	
  development	
  of	
  microbial	
  communities	
  
of	
   oral	
   plaque,	
   the	
   community	
   is	
   structured	
   spatially	
   by	
   the	
   temporal	
   succession	
   of	
  
colonizing	
   bacteria,	
   where	
   members	
   of	
   the	
   genus	
   Streptococcus	
   are	
   early	
   colonizers	
  
and	
  have	
  receptors	
  for	
  co-­‐‑aggregation	
  of	
  other	
  early	
  colonizers,	
  such	
  as	
  members	
  of	
  

	
  

124	
  

the	
   genera	
   Actinomyces	
   and	
   Veillonella.	
   After	
   the	
   initial	
   development	
   of	
   the	
  
microbiome	
   of	
   the	
   human	
   dental	
   plaque,	
   interbacterial	
   cell-­‐‑cell	
   recognition	
   plays	
   a	
  
significant	
  role	
  in	
  the	
  spatiotemporal	
  development	
  of	
  these	
  communities	
  [5].	
  	
  
After	
   attachment	
   of	
   early	
   colonizers	
   to	
   the	
   dental	
   surface,	
   members	
   of	
   the	
  
genus	
   Fusobacterium	
   arrive	
   and	
   act	
   as	
   a	
   bridge	
   between	
   early	
   and	
   late	
   colonizers,	
  
which	
   are	
   frequently	
   known	
   pathogenic	
   bacteria	
   such	
   as	
   Porphyromonas	
   and	
  
Treponema,	
  among	
  others	
  [6].	
  An	
  important	
  characteristic	
  of	
  these	
  associations	
  is	
  that	
  
there	
   is	
   no	
   coadhesion	
   or	
   coaggregation	
   between	
   early	
   and	
   late	
   colonizers;	
   only	
  
Fusobacterium	
  can	
  coaggregate	
  first	
  with	
  early	
  and	
  then	
  with	
  late	
  colonizers	
  [6].	
  	
  
Coaggregation	
   is	
   the	
   cell-­‐‑cell	
   recognition	
   between	
   different	
   types	
   of	
   bacteria	
  
[7].	
  It	
  has	
  been	
  suggested	
  that	
  relationships	
  between	
  coaggregation	
  partners	
  lead	
  to	
  a	
  
temporal	
  relationship	
  with	
  respect	
  to	
  colonization	
  of	
  surfaces.	
  Studies	
  done	
  by	
  Palmer	
  
et	
   al.	
   [5],	
   using	
   fluorescent	
   labeled	
   antibodies	
   and	
   confocal	
   laser	
   scanning	
   microscopy	
  
(CLSM),	
   	
   for	
   the	
   first	
   time	
   showed	
   the	
   significance	
   of	
   coaggregation	
   in	
   the	
  
development	
   of	
   microbial	
   communities	
   and	
   emphasized	
   its	
   importance	
   in	
   the	
  
spatiotemporal	
  development	
  of	
  microbial	
  communities.	
  
The	
   spatial	
   distribution/localization	
   of	
   diverse	
   bacteria	
   has	
   been	
   studied	
   in	
  
different	
  tissues	
  [8-­‐‑13],	
  	
  using	
  methods	
  such	
  as	
  Scanning	
  Electron	
  Microscopy	
  (SEM)	
  
[13],	
   	
   immunohistochemistry	
   [9],	
   and	
   FISH	
   (Fluorescence	
   In	
   Situ	
   Hybridization)	
   and	
  
detection	
  of	
  bacteria	
  using	
  epifluorescence	
  and	
  CLSM	
  [8,	
  11,	
  13-­‐‑16].	
  These	
  techniques	
  
have	
   helped	
   understand	
   the	
   interplay	
   of	
   microbes	
   in	
   different	
   tissues.	
   However,	
  
limited	
  knowledge	
  is	
  available	
  about	
  the	
  spatial	
  distribution,	
  assembly	
  and	
  structure	
  
of	
  tonsillar	
  microbial	
  communities	
  in	
  any	
  mammal,	
  including	
  pigs.	
  

	
  

125	
  

Tonsils	
   are	
   colonized	
   by	
   bacteria	
   from	
   the	
   genus	
   Streptococcus,	
   specifically	
  
Streptococcus	
  suis,	
  early	
  in	
  the	
  lives	
  of	
  pigs,	
  as	
  early	
  as	
  during	
  the	
  birth	
  process	
  [17-­‐‑
19].	
   Studies	
   using	
   segregated	
   early	
   weaned	
   pigs	
   have	
   suggested	
   a	
   temporality	
   in	
  
colonization	
  by	
  other	
  bacteria,	
  such	
  as	
  Haemophilus	
  parasuis,	
  which	
  were	
  detected	
  in	
  
the	
  first	
  day	
  of	
  life,	
  followed	
  by	
  other	
  bacterial	
  species,	
  such	
  as	
  Pasteurella	
  multocida	
  
and	
  Bordetella	
  bronchiseptica,	
  which	
  arrive	
  after	
  the	
  second	
  week	
  of	
  life	
  or	
  later	
  [17,	
  
20].	
   Although	
   there	
   has	
   been	
   no	
   comprehensive	
   study	
   of	
   the	
   patterns	
   of	
   tonsillar	
  
colonization	
   by	
   different	
   bacteria	
   or	
   spatiotemporal	
   structure	
   of	
   the	
   bacterial	
  
communities	
  in	
  tonsils,	
  the	
  aforementioned	
  studies	
  suggest	
  that	
  there	
  is	
  a	
  pattern	
  of	
  
temporality	
  in	
  the	
  building	
  of	
  tonsillar	
  communities	
  of	
  pigs,	
  at	
  least	
  in	
  early	
  life.	
  	
  
The	
   goal	
   of	
   this	
   study	
   was	
   to	
   characterize	
   how	
   the	
   three	
   most	
   abundant	
  
members	
   of	
   the	
   pig	
   tonsillar	
   microbiome,	
   Pasteurellaceae,	
   Streptococcaceaae	
   and	
  
Moraxellaceae	
  [21,	
  22],	
  are	
  spatially	
  localized	
  at	
  different	
  times	
  in	
  the	
  life	
  of	
  pigs.	
  The	
  
use	
   of	
   FISH	
   probes	
   targeting	
   bacterial	
   16S	
   rRNA	
   genes	
   and	
   CLSM	
   allowed	
   us	
   to	
  
identify	
   the	
   presence	
   of	
   these	
   different	
   bacterial	
   taxa	
   in	
   tonsils.	
   Scanning	
   Electron	
  
Microscopy	
   (SEM)	
   gave	
   us	
   a	
   detailed	
   view	
   of	
   the	
   tonsils,	
   their	
   general	
   appearance	
  
over	
  time,	
  and	
  an	
  overview	
  of	
  the	
  organization	
  of	
  the	
  microbial	
  communities	
  and	
  how	
  
they	
  developed	
  over	
  time.	
  
	
  
MATERIALS	
  AND	
  METHODS	
  
	
  
Animals.	
   Approval	
   for	
   all	
   experimental	
   procedures	
   was	
   obtained	
   from	
   The	
  
Michigan	
   State	
   University	
   Institutional	
   Animal	
   Care	
   and	
   Use	
   Committee	
   (IACUC).	
  

	
  

126	
  

Piglets	
   derived	
   from	
   three	
   crossbred	
   sows	
   (Yorkshire	
   x	
   Hampshire)	
   from	
   a	
   high	
  
health	
   status	
   herd	
   were	
   used	
   in	
   this	
   study.	
   	
   A	
   detailed	
   description	
   of	
   this	
   herd	
   has	
  
been	
  published	
  previously	
  elsewhere	
  [21].	
  One	
  piglet	
  from	
  each	
  of	
  the	
  three	
  sows	
  was	
  
selected	
  randomly	
  from	
  the	
  litter	
  at	
  each	
  sampling	
  time:	
  1,	
  3,	
  4,	
  8,	
  10	
  and	
  17	
  weeks.	
  
Pigs	
  were	
  initially	
  anesthetized	
  with	
  a	
  combination	
  of	
  Tiletamine-­‐‑Zolazepam	
  (Telazol ,	
  
®

Zoetis,	
   USA)	
   and	
   Ketamine	
   (Kethatesia,	
   Henry	
   Schein	
   Animal	
   Health,	
   Dublin,	
   OH),	
  
administered	
  intramuscularly.	
  	
  Once	
  anesthetized,	
  they	
  were	
  euthanized	
  by	
  overdose	
  
of	
   a	
   pentobarbital	
   solution	
   (Fatal-­‐‑Plus,	
   Vortech	
   Pharmaceuticals	
   -­‐‑	
   Dearborn,	
   MI),	
  
delivered	
  intravenously	
  into	
  the	
  vena	
  cava,	
  following	
  standard	
  procedures	
  approved	
  
by	
  IACUC.	
  
Tissue	
   collection.	
   	
   	
   The	
   entire	
   right	
   soft	
   palate	
   tonsil	
   from	
   each	
   piglet	
   was	
  
collected	
  and	
  the	
  excess	
  connective	
  tissue	
  removed	
  using	
  sterile	
  scalpels	
  and	
  forceps.	
  
Each	
  right	
  tonsil	
  was	
  placed	
  in	
  a	
  sterile	
  glass	
  Petri	
  dish	
  and	
  divided	
  into	
  quarters.	
  A	
  
random	
   quarter	
   of	
   the	
   tissue	
   was	
   carefully	
   placed	
   in	
   a	
   labeled	
   disposable	
   tissue	
  
cassette	
   previously	
   covered	
   with	
   a	
   thin	
   film	
   of	
   HistoPrep	
   embedding	
   medium	
   (Fisher,	
  
Fair	
   Lawn,	
   NJ).	
   Samples	
   were	
   oriented	
   carefully	
   to	
   allow	
   access	
   to	
   tonsillar	
   crypts	
  
when	
  trimming.	
  Following	
  this,	
  the	
  cassette	
  was	
  fully	
  filled	
  with	
  the	
  embedding	
  media	
  
and	
  immediately	
  frozen	
  at	
  -­‐‑80°C.	
  After	
  solidification,	
  the	
  sample	
  blocks	
  were	
  removed	
  
from	
   the	
   cassettes,	
   wrapped	
   in	
   aluminum	
   foil,	
   labeled	
   and	
   stored	
   at	
   -­‐‑80°C	
   until	
  
processing.	
   Blocks	
   were	
   sectioned	
   on	
   a	
   Leica	
   Cryostat	
   (Leica	
   Microsystems,	
   Vienna,	
  
Austria).	
   Tissue	
   sections	
   (5	
   µm)	
   were	
   collected	
   on	
   positively	
   charged	
   microscope	
  
slides,	
  Leica	
  BONDTM	
  Plus	
  Slide	
  (Leica	
  Biosystems	
  Richmond,	
  Illinois,	
  USA),	
  and	
  stored	
  
at	
  -­‐‑20°C	
  until	
  processing	
  for	
  FISH.	
  Another	
  random	
  tonsil	
  quarter	
  was	
  placed	
  into	
  a	
  15	
  

	
  

127	
  

ml	
   sterile	
   test	
   tube	
   containing	
   10	
   ml	
   of	
   4%	
   paraformaldehyde	
   (PFA)	
   for	
   4	
   -­‐‑	
   6	
   h	
   and	
  
then	
  removed	
  and	
  stored	
  in	
  50%	
  ethanol	
  until	
  processed.	
  	
  
Bacterial	
  strains	
  and	
  smears	
  on	
  slides.	
   To	
   validate	
   the	
   FISH	
   probes,	
   we	
   used	
  
the	
   following	
   bacterial	
   isolates:	
   Streptococcus	
   suis,	
   Staphylococcus	
   aureus,	
   Pasteurella	
  
multocida,	
  Escherichia	
  coli	
  (all	
  from	
  the	
  laboratory	
  collection	
  of	
  MHM)	
  and	
  Moraxella	
  
bovis	
   (kindly	
   provided	
   by	
   Dr.	
   Rinosh	
   Mani).	
   Overnight	
   cultures	
   of	
   BHI	
   agar	
   (BBLTM	
  
Brain	
  Heart	
  Infusion,	
  Becton	
  Dickinson	
  and	
  Company,	
  Sparks,	
  MD)	
  streaked	
  with	
  each	
  
individual	
  isolate	
  were	
  used	
  to	
  inoculate	
  5	
  ml	
  of	
  BHI	
  broth	
  to	
  an	
  initial	
  optical	
  density	
  
at	
   520nm	
   (OD520)	
   of	
   0.15.	
   Inoculated	
   broths	
   were	
   incubated	
   at	
   35°C	
   shaking	
   at	
   160	
  
rpm	
   until	
   an	
   OD520	
   of	
   approximately	
   0.5	
   was	
   reached.	
   Bacterial	
   suspensions	
   were	
  
mixed	
  with	
  equal	
  amounts	
  of	
  4%	
  paraformaldehyde	
  (4%	
  PFA)	
  and	
  fixed	
  for	
  1.5	
  hours,	
  
followed	
  by	
  three	
  washes	
  in	
  PBS,	
  pH	
  7.4.	
  Pellets	
  were	
  suspended	
  in	
  equal	
  amounts	
  of	
  
PBS	
  and	
  absolute	
  ethanol	
  and	
  stored	
  at	
  -­‐‑20°C	
  in	
  aliquots	
  until	
  used	
  for	
  validation	
  of	
  
the	
  probes	
  in	
  slide	
  smears	
  and	
  muscle	
  tissue.	
  
Bacterial	
   strains	
   and	
   muscle	
   slides.	
   To	
   validate	
   the	
   FISH	
   probes	
   against	
  
bacterial	
   cells	
   present	
   in	
   mammalian	
   tissue,	
   50	
   ml	
   of	
   overnight	
   cultures	
   of	
  
Streptococcus	
   suis,	
   Pasteurella	
   multocida,	
   Escherichia	
   coli	
   and	
   Moraxella	
   bovis	
   were	
  
pelleted	
  by	
  centrifugation	
  and	
  suspended	
  in	
  5	
  ml	
  of	
  sterile	
  broth.	
  	
  Equal	
  proportions	
  
of	
   all	
   the	
   bacterial	
   isolates	
   were	
   mixed	
   in	
   a	
   sterile	
   10	
   ml	
   tube	
   and	
   inoculated	
   onto	
  
samples	
   of	
   muscle	
   of	
   approximately	
   3	
   x	
   3	
   x	
   10mm,	
   which	
   were	
   collected	
   aseptically	
  
and	
   randomly	
   from	
   a	
   euthanized	
   pig	
   used	
   in	
   the	
   study.	
   Inoculated	
   muscle	
   was	
  
processed	
  in	
  the	
  same	
  way	
  as	
  tonsil	
  tissue.	
  	
  	
  

	
  

128	
  

Oligonucleotide	
  probes.	
   FISH	
   experiments	
   were	
   carried	
   out	
   using	
   the	
   probe	
  
EUB338	
  [23]	
  which	
  targets	
  a	
  region	
  of	
  the	
  bacterial	
  16S	
  rRNA	
  genes,	
  present	
  in	
  most	
  
bacteria,	
   and	
   the	
   nonsense	
   probe	
   NON338	
   [24],	
   which	
   is	
   the	
   antisense	
   probe	
   of	
  
EUB338	
   and	
   was	
   used	
   to	
   exclude	
   non-­‐‑specific	
   probe	
   binding.	
   Specific	
   nucleotide	
  
probes	
   for	
   the	
   family	
   Pasteurellaceae	
   PAS111	
   [16],	
   the	
   genus	
   Streptococcus	
   STR405	
  
[25]	
   and	
   the	
   genus	
   Moraxella	
   MOR575	
   (designed	
   in	
   this	
   study),	
   were	
   selected	
   to	
  
target	
   the	
   three	
   most	
   abundant	
   taxa	
   identified	
   in	
   the	
   tonsillar	
   microbiome	
   of	
   pigs	
   [21,	
  
22]	
  (Table	
  4.1.).	
  The	
  sequences	
  for	
  these	
  FISH	
  probes	
  were	
  obtained	
  after	
  a	
  search	
  of	
  
available	
  journal	
  publications	
  or	
  in	
  probeBase	
   [26]	
  or	
  using	
  the	
  probe	
  design	
  function	
  
of	
   ARB	
   [27],	
   and	
   compared	
   with	
   representative	
   sequences	
   for	
   the	
   respective	
   OTUs,	
  
using	
  the	
  ribosomal	
  database	
  project	
  (RDP)	
  sequence	
  analysis	
  tools	
  [28].	
  	
  Probes	
  were	
  
custom	
  synthesized	
  by	
  IDT 	
  (Integrated	
  DNA	
  Technologies,	
  Coralville,	
  IA).	
  
®

Table	
  4.1.	
  Oligonucleotide	
  probes	
  

	
  
	
  
Fluorescent	
   In	
   Situ	
   Hybridization	
   (FISH).	
   For	
   this	
   procedure	
   the	
   probes	
  
were	
   first	
   validated	
   against	
   PFA-­‐‑fixed	
   pure	
   cultures	
   of	
   the	
   bacterial	
   strains	
   and	
  
mixtures	
   of	
   the	
   strains	
   spread	
   on	
   positively	
   charged	
   slides,	
   Leica	
   BONDTM	
   Plus	
   Slide	
  
(Leica	
   Biosystems	
   Richmond,	
   Illinois,	
   USA),	
   and	
   against	
   samples	
   of	
   muscle	
   tissue	
  
inoculated	
   with	
   a	
   mix	
   of	
   bacterial	
   cells	
   as	
   explained	
   above.	
   These	
   slides	
   were	
   also	
  

	
  

129	
  

used	
   as	
   positive	
   controls	
   for	
   probe	
   hybridization	
   and	
   were	
   included	
   in	
   all	
  
hybridization	
   experiments.	
   From	
   every	
   tonsil	
   sample,	
   four	
   sequential	
   sections	
   were	
  
hybridized,	
   each	
   with	
   a	
   different	
   set	
   of	
   probes.	
   Section	
   one	
   was	
   hybridized	
   with	
   the	
  
EUB338	
  	
  and	
  NON338	
  	
  probes;	
  section	
  two	
  was	
  hybridized	
  with	
  the	
  EUB338,	
  MOR575	
  
and	
   PAS111	
   	
   probes;	
   section	
   three	
   was	
   hybridized	
   with	
   the	
   EUB338,	
   STR405	
   and	
  
PAS111	
  probes;	
  and	
  finally	
  a	
  fourth	
  section	
  was	
  hybridized	
  with	
  the	
  MOR575,	
  STR405	
  
and	
   PAS111	
   probes.	
   Tissues	
   were	
   initially	
   immersed	
   in	
   a	
   PBS-­‐‑Tween	
   20,	
   pH	
   7.4	
  
buffer,	
  then	
  incubated	
  with	
  a	
  lysis	
  buffer	
  (100	
  µg/ml	
  lysozyme	
  in	
  0.1	
  mM	
  Tris-­‐‑HCl,	
  pH	
  
8.0)	
  at	
  37°C	
  for	
  10	
  min,	
  serially	
  washed	
  with	
  PBS-­‐‑Tween	
  buffer	
  and	
  then	
  submitted	
  to	
  
the	
   hybridization	
   protocol.	
   A	
   pre-­‐‑warmed	
   (46°C)	
   solution	
   containing	
   4	
   µl	
   of	
   each	
  
selected	
   oligonucleotide	
   probe	
   (50	
   ng/µl)	
   was	
   mixed	
   with	
   hybridization	
   buffer	
  
containing	
  0.9	
  M	
  NaCl,	
  20	
  mM	
  Tris-­‐‑HCl,	
  pH	
  8,	
  20%	
  formamide	
  (Fisher,	
  Fair	
  Lawn,	
  NJ)	
  
and	
   0.01%	
   SDS,	
   to	
   make	
   up	
   a	
   volume	
   of	
   100	
   µl	
   per	
   hybridized	
   slide.	
   The	
   solution	
   was	
  
applied	
  on	
  the	
  tissue	
  sections	
  and	
  incubated	
  in	
  a	
  dark-­‐‑humid	
  histochemistry	
  staining	
  
tray	
   (IBI	
   Scientific,	
   Kapp	
   Court	
   Peosta,	
   IA)	
   for	
   3h	
   at	
   46°C.	
   Immediately	
   following	
  
hybridization,	
   slides	
   were	
   washed	
   twice	
   for	
   25	
   min	
   at	
   48°C	
   each,	
   with	
   pre-­‐‑warmed	
  
(48°C)	
  washing	
  buffer	
  containing	
  0.9	
  M	
  NaCl,	
  20	
  mM	
  Tris-­‐‑HCl,	
  pH	
  8,	
  and	
  0.01%	
  SDS.	
  
Finally,	
  the	
  slides	
  were	
  rinsed	
  with	
  ice-­‐‑cold	
  ultrapure	
  water,	
  gently	
  dried	
  with	
  a	
  paper	
  
towel	
  and	
  mounted	
  with	
  ProLong 	
  Diamond	
  antifade	
  mountant	
  with	
  DAPI	
  (Molecular	
  
®

Probes,	
  USA).	
  The	
  mounting	
  media	
  was	
  cured	
  for	
  at	
  least	
  24	
  h	
  at	
  room	
  temperature	
  
before	
  analysis	
  of	
  the	
  slides.	
  	
  
Confocal	
  Laser	
  Scan	
  Microscopy	
  (CLSM).	
  A	
  Nikon	
  C2	
  Laser	
  Scanning	
  Confocal	
  
Microscope	
   (Nikon	
   Instruments	
   Inc.,	
   Melville,	
   NY)	
   equipped	
   with	
   405nm,	
   488nm,	
  
	
  

130	
  

561nm	
   and	
   633	
   nm	
   lasers	
   was	
   used	
   to	
   acquire	
   the	
   images.	
   Image	
   processing	
   was	
  
performed	
  with	
  NIS	
  Element	
  viewer	
  software	
  	
  (Nikon,	
  version	
  4.1.11).	
  
Scanning	
  Electron	
  Microscopy	
  (SEM).	
  Tissue	
  samples	
  stored	
  in	
  50%	
  ethanol	
  
were	
  dehydrated	
  in	
  a	
  series	
  of	
  graded	
  ethanol	
  solutions	
  (50%,	
  75%,	
  95%)	
  for	
  20	
  min	
  
at	
  each	
  gradation	
  followed	
  by	
  three	
  10	
  min	
  changes	
  in	
  100%	
  ethanol,	
   dried	
   by	
   critical	
  
point	
   drying	
   with	
   a	
   Leica	
   Microsystems	
   model	
   EM	
   CPD	
   300	
   (Leica	
   Microsystems,	
  
Vienna,	
   Austria),	
   mounted	
   on	
   aluminum	
   stubs	
   using	
   carbon	
   suspension	
   cement	
   (SPI	
  
Supplies,	
   West	
   Chester,	
   PA)	
   and	
   adhesive	
   tabs	
   (M.E.	
   Taylor	
   Engineering,	
   Brookville,	
  
MD),	
   coated	
  with	
  iridium	
   for	
  30	
  sec	
   in	
  a	
  Quorum	
  Technologies/Electron	
  Microscopy	
  
sciences	
  Q150T	
  turbo	
  pumped	
  sputter	
  coater	
  (Quorum	
  Technologies,	
  Laughton,	
  East	
  
Sussex,	
  England	
  BN8	
  6BN),	
  and	
  observed	
  with	
  a	
  JEOL	
  7500F	
  (field	
  emission	
  emitter)	
  
scanning	
  electron	
  microscope	
  (JEOL	
  LTd.,	
  Tokyo,	
  Japan).	
  
	
  
RESULTS	
  
	
  
Previous	
  research	
  by	
  our	
  group	
  analyzing	
  the	
  composition	
  and	
  development	
  of	
  
microbial	
   communities	
   present	
   in	
   the	
   tonsillar	
   tissue	
   of	
   pigs	
   using	
   culture-­‐‑
independent	
   sequencing	
   of	
   bacterial	
   16s	
   rRNA	
   genes	
   demonstrated	
   both	
   the	
  
successional	
  development	
  of	
  the	
  tonsil	
  microbiome	
  and	
  that	
  the	
  three	
  most	
  common	
  
taxa	
   seen	
   throughout	
   the	
   lives	
   of	
   pigs	
   were	
   Streptococcus,	
   Pasteurellaceae,	
   and	
  
Moraxella	
  	
  [21,	
  22,	
  29,	
  30].	
  Here	
  we	
  used	
  both	
  FISH	
  to	
  localize	
  these	
  three	
  major	
  taxa	
  
in	
  tonsil	
  tissue	
  and	
  SEM	
  to	
  characterize	
  the	
  overall	
  structure	
  of	
  the	
  tonsil	
  tissue	
  over	
  
the	
  lives	
  of	
  pigs.	
  

	
  

131	
  

Validation	
   of	
   the	
   probes.	
   We	
   validated	
   our	
   specific	
   probes	
   using	
   fixed	
   cells	
  
from	
   pure	
   cultures	
   of	
   the	
   bacteria	
   Streptococcus	
   suis,	
   Staphylococcus	
   aureus,	
  
Pasteurella	
  multocida,	
  Escherichia	
  coli	
  and	
  Moraxella	
  bovis.	
  Smears	
  of	
  mixed	
  bacterial	
  
cells	
   and	
   the	
   above	
   mentioned	
   combinations	
   of	
   probes	
   were	
   used	
   to	
   identify	
   the	
  
positive	
   labeling	
   of	
   cells	
   with	
   the	
   morphological	
   features	
   of	
   members	
   of	
   the	
   genera	
  
Streptococcus,	
   Pasteurella	
   and	
   Moraxella,	
   when	
   hybridized	
   with	
   their	
   specific	
   probe	
  
and	
  when	
  stained	
  with	
  DAPI,	
  which	
  stains	
  nuclear	
  DNA	
  (Figure	
  4.1.A.).	
  Bacterial	
  cells	
  
inoculated	
  onto	
  muscle	
  tissue	
  showed	
  similar	
  results	
  to	
  those	
  obtained	
  with	
  smears	
  of	
  
mixed	
  bacterial	
  cells	
  (Figure	
  4.1.B).	
  	
  
Spatial	
  structure	
  of	
  communities	
  on	
  the	
  tonsils	
  using	
  FISH.	
   After	
   validating	
  
the	
   probes,	
   tonsil	
   samples	
   were	
   processed	
   from	
   all	
   the	
   1,	
   3,	
   4	
   and	
   17	
   week	
   piglets.	
  
FISH	
   was	
   performed	
   with	
   combinations	
   of	
   probes	
   EUB338-­‐‑NON338,	
   EUB338-­‐‑
STR405-­‐‑PAS11,	
   MOR575-­‐‑EUB338-­‐‑PAS11	
   or	
   MOR575-­‐‑STR405-­‐‑PAS11,	
   all	
   mounted	
  
with	
  ProLong 	
  Diamond	
  antifade	
  mountant	
  with	
  DAPI.	
  There	
  was	
  positive	
  labeling	
  of	
  
®

Streptococcus	
  cells	
  with	
  EUB338	
  or	
  STR405	
  probes,	
  and	
  positive	
  staining	
  with	
  DAPI	
  in	
  
pig	
  tonsil	
  tissue;	
  however	
  there	
  was	
  no	
  consistent	
  specific	
  labeling	
  of	
  tonsil	
  specimens	
  
with	
   either	
   the	
   PAS111	
   or	
   MOR575	
   probes	
   at	
   any	
   time	
   point	
   tested	
   (Figure	
   4.1.C	
   to	
  
4.1.F).	
  There	
  was	
  extensive	
  non-­‐‑specific	
  labeling	
  of	
  granular	
  structures	
  disseminated	
  
on	
   the	
   epithelial	
   surface	
   (Figure	
   4.1.F	
   and	
   Figure	
   4.2.A.)	
   as	
   well	
   as	
   in	
   other	
   tissue	
  
locations	
   (intraepithelial	
   and	
   in	
   submucosa).	
   The	
   non-­‐‑specific	
   labeling	
   was	
   evident	
  
with	
   all	
   combinations	
   of	
   probes	
   and	
   made	
   the	
   definitive	
   identification	
   of	
   positively	
  
labeled	
  bacterial	
  cells	
  extremely	
  difficult.	
  An	
  SEM	
  image	
  of	
  the	
  tonsillar	
  surface	
  from	
  
an	
   eight	
   week	
   old	
   pig	
   showed	
   some	
   elements	
   that	
   could	
   have	
   been	
   part	
   of	
   the	
  

	
  

132	
  

unspecific	
  labeled	
  material	
  observed	
  in	
  FISH	
  (Figure	
  4.2.B),	
  and	
  demonstrated	
  all	
  the	
  
material	
   observed	
   on	
   the	
   tonsillar	
   surface	
   that	
   made	
   the	
   use	
   of	
   FISH	
   difficult	
   and	
  
problem	
  prone.	
  	
  
Although	
   previous	
   research	
   by	
   our	
   group	
   [21,	
   22,	
   29,	
   30]	
   using	
   culture-­‐‑
independent	
   sequencing	
   of	
   bacterial	
   16s	
   rRNA	
   genes	
   showed	
   that	
   Streptococcus,	
  
Pasteurellaceae,	
   and	
   Moraxellaceae	
   as	
   well	
   as	
   many	
   other	
   taxa	
   are	
   present	
   in	
   pig	
  
tonsils,	
   we	
   were	
   not	
   able	
   to	
   reliably	
   identify	
   bacterial	
   taxa	
   other	
   than	
   members	
   of	
  
genus	
  Streptococcus	
  or	
  bacterial	
  cells	
  stained	
  with	
  the	
  generic	
  eubacterial	
  probe	
  using	
  
FISH.	
   Therefore,	
   we	
   decided	
   to	
   use	
   SEM	
   to	
   identify	
   the	
   presence	
   and	
   appearance	
   of	
  
microbial	
  communities	
  in	
  the	
  tonsillar	
  tissue	
  of	
  the	
  sampled	
  pigs.	
  
	
  

	
  

133	
  

	
  
Figure	
   4.1.	
   Representative	
   images	
   for	
   the	
   validation	
   of	
   FISH	
   probes	
   and	
  
identification	
   of	
   Streptococcus	
   cells	
   on	
   the	
   tonsillar	
   surface.	
   A,	
   Oligonucleotide	
  
probes	
   were	
   validated	
   with	
   bacterial	
   cell	
   smears	
   using	
   DAPI	
   staining	
   (blue)	
   and	
  
probes	
   specific	
   for	
   Moraxella	
   (green),	
   Streptococcus	
   (orange)	
   and	
   Pasteurellaceae	
  
(purple).	
   B,	
   Validated	
   oligonucleotide	
   probes	
   using	
   aseptically	
   collected	
   muscle	
  
inoculated	
   	
   with	
   a	
   mixed	
   bacterial	
   population.	
   Nuclear	
   DNA	
   of	
   myocytes	
   (blue),	
  
	
  

134	
  

Figure	
   4.1.	
   (cont´d).	
  	
  Moraxella	
  (green),	
  Streptococcus	
  (orange)	
  	
  and	
  	
  Pasteurellaceae	
  	
  
(purple).	
  C,	
  	
  	
  Tonsil	
  tissue	
  from	
  a	
  one	
  week	
  old	
  pig.	
  Streptococcus	
  cells	
  are	
  visualized	
  in	
  
orange	
  (short	
  arrow),	
  lying	
  on	
  the	
  surface	
  of	
  the	
  nonkeratinized	
  stratified	
  squamous	
  
epithelia	
  of	
  tonsils	
  (*).	
  D,	
  Tonsil	
  tissue	
  from	
  a	
  three	
  week	
  old	
  pig.	
  Streptococcus	
  cells	
  
barely	
  visualized	
  in	
  orange	
  (short	
  arrow),	
  on	
  the	
  surface	
  of	
  the	
  epithelium	
  (*),	
  covered	
  
by	
   a	
   layer	
   of	
   cellular	
   debris	
   (long	
   arrow).	
   E,	
   Tonsil	
   tissue	
   from	
   a	
   four	
   week	
   old	
   pig.	
  
Chain	
   of	
   Streptococcus	
   (orange	
   –	
   short	
   arrow)	
   lying	
   on	
   the	
   epithelial	
   surface	
   (*).	
  
Cellular	
   debris	
   (long	
   arrow).	
   F,	
   Tonsil	
   tissue	
   from	
   a	
   ten	
   week	
   old	
   pig.	
   Long	
   chain	
   of	
  
Streptococcus	
   (orange	
   -­‐‑	
   short	
   arrow)	
   and	
   cellular	
   debris	
   (long	
   arrow),	
   lying	
   on	
   the	
  
epithelial	
  surface	
  (*).	
  Multiple	
  structures	
  with	
  granular	
  appearance	
  and	
  different	
  sizes	
  
(arrow	
  heads)	
  are	
  evident	
  as	
  well	
  as	
  cellular	
  debris	
  (long	
  arrow)	
  staining	
  with	
  DAPI	
  
(blue).	
  
	
  

	
  
Figure	
  4.2.	
  Representative	
  images	
  of	
  bacterial	
  cells	
  and	
  cellular	
  debris	
  in	
  tonsils	
  
using	
   FISH	
   and	
   SEM.	
   A,	
   Confocal	
   image	
   of	
   tonsil	
   tissue	
   from	
   a	
   one	
   week	
   old	
   pig	
  
showing	
   positively	
   hybridized	
   Streptococus	
   (orange	
   -­‐‑	
   short	
   arrow)	
   on	
   the	
   epithelial	
  
surface	
   (*)	
   and	
   structures	
   of	
   diverse	
   sizes	
   displaying	
   non-­‐‑specific	
   labeling	
   (orange	
   –	
  	
  
	
  

135	
  

Figure	
   4.2.	
   (cont´d).	
   	
   arrow	
   heads)	
   and	
   	
   	
   cellular	
   	
   	
   debris	
   (long	
   arrows).	
   	
   B,	
   	
   SEM	
  	
  
image	
   	
   from	
   	
   an	
   	
   eight	
   	
   week	
   	
   old	
   pig	
   showing	
   bacterial	
   cells	
   (short	
   arrows)	
   on	
   the	
  
tonsillar	
  epithelial	
  surface	
  (*),	
  surrounded	
  by	
  cellular	
  debris	
  (long	
  arrows)	
  and	
  a	
  layer	
  
of	
  mucous/exopolysaccharide	
  material	
  (+).	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

136	
  

Spatial	
   structure	
   of	
   communities	
   on	
   the	
   tonsils	
   using	
   SEM.	
   Using	
   SEM,	
  	
  
tonsillar	
  tissues	
  of	
  one,	
  three,	
  four,	
  eight,	
  ten	
  and	
  seventeen	
  week	
  old	
  piglets	
  showed	
  a	
  
pattern	
   of	
   succession	
   in	
   the	
   development	
   of	
   microbial	
   communities	
   in	
   the	
   tonsillar	
  
tissue	
   of	
   pigs.	
   The	
   characteristics	
   of	
   tonsils	
   tended	
   to	
   vary	
   between	
   animals	
   of	
   the	
  
same	
   age,	
   with	
   respect	
   to	
   the	
   number	
   of	
   bacterial	
   cells	
   and	
   amount	
   of	
   detritus	
  
identified.	
   	
   However,	
   there	
   were	
   general	
   features	
   displayed	
   by	
   the	
   pigs	
   at	
   each	
   time	
  
point.	
   The	
   tonsillar	
   surface	
   of	
   one	
   week	
   old	
   pigs	
   showed	
   a	
   smooth,	
   clean	
   surface	
   with	
  
little	
   debris	
   (Figure	
   4.3.A),	
   with	
   crypts	
   relatively	
   clean	
   and	
   devoid	
   of	
   debris	
   (Figure	
  
4.3.B-­‐‑4.3.C).	
  There	
  were	
  scattered	
  small	
  communities	
  of	
  bacterial	
  cells	
  (Figure	
  4.3.D-­‐‑
4.3.E)	
   composed	
   mainly	
   of	
   lancet-­‐‑shaped	
   diplococci,	
   long	
   rods,	
   short	
   rods	
   and	
  
diplobacilli.	
   Also,	
   two	
   types	
   of	
   epithelial	
   cell	
   surfaces	
   were	
   evident:	
   those	
   with	
   loosely	
  
arranged	
   microplicae	
   in	
   microridges,	
   and	
   those	
   with	
   densely	
   packed	
   microplicae	
  
appearing	
   as	
   very	
   short	
   microvilli	
   [31].	
   The	
   initial	
   attachment	
   of	
   bacterial	
   cells	
   to	
  
surface	
   microplicae	
   of	
   the	
   epithelial	
   cells	
   (Figure	
   4.3.F),	
   using	
   their	
   pili,	
   was	
   clearly	
  
seen.	
  	
  	
  
In	
  three	
  week	
  old	
  piglets,	
  the	
  tonsils	
  were	
  characterized	
  by	
  a	
  tonsillar	
  surface	
  
with	
   a	
   more	
   rugose	
   appearance	
   (Figure	
   4.4.A)	
   and	
   wider	
   crypt	
   entrances.	
   Increased	
  
debris	
   was	
   evident	
   in	
   the	
   lumen	
   of	
   crypts	
   (Figure	
   4.4.B	
   to	
   4.4.D)	
   and	
   larger	
   micro-­‐‑
communities	
   of	
   diverse	
   bacteria	
   were	
   seen	
   (Figure	
   4.4.C	
   to	
   4.4.E).	
   The	
   random	
  
appearance	
   of	
   an	
   echynocite	
   was	
   also	
   observed	
   (Figure	
   4.4.D).	
   Bacterial	
   micro-­‐‑
communities	
   composed	
   mainly	
   of	
   short	
   rods,	
   diplococci,	
   diplobacilli	
   and	
   cocci	
   were	
  
observed	
  (Figure	
  4.4.E	
  –	
  4.4.F).	
  	
  

	
  

137	
  

	
  
Figure	
  4.3.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
   one	
   week	
   old	
   pigs.	
   A,	
   tonsillar	
   tissue	
   of	
   one	
   week	
   old	
   pigs	
   showing	
   shallow	
  
crypts	
  (c)	
  with	
  little	
  debris	
  (long	
  arrow)	
  and	
  a	
  very	
  clean	
  and	
  smooth	
  tonsil	
  surface.	
  B,	
  
Crypt	
   (c)	
   showing	
   presence	
   of	
   a	
   small	
   amount	
   of	
   debris	
   (long	
   arrow)	
   and	
  
desquamating	
  epithelial	
  	
  cells	
  	
  (d).	
  	
  C,	
  	
  Demonstration	
  	
  of	
  	
  small	
  	
  sparse	
  	
  micro-­‐‑colonies	
  	
  	
  
	
  

138	
  

Figure	
   4.3.	
   (cont´d).	
   (short	
   arrow)	
   around	
   a	
   crypt	
   (c)	
   entrance.	
   D,	
   Small	
   micro-­‐‑
colonies	
   of	
   bacterial	
   cells	
   (short	
   arrows)	
   on	
   the	
   epithelial	
   surface	
   as	
   well	
   as	
   few	
  
dispersed	
   bacterial	
   cells.	
   Two	
   types	
   of	
   epithelial	
   cell	
   surfaces	
   with	
   loosely	
   arranged	
  
(e1)	
  or	
  densely	
  packed	
  (e2)	
  microplicae	
  are	
  shown.	
  E,	
  Detailed	
  view	
  of	
  a	
  small	
  micro-­‐‑
colony	
  (short	
  arrow)	
  on	
  epithelial	
  cells	
  showing	
  the	
  two	
  types	
  of	
  epithelial	
  cells	
  with	
  
surface	
   microplicae.	
   Diverse	
   bacterial	
   morphologies:	
   lancet-­‐‑shaped	
   diplococci	
   (dc),	
  
long	
  rods	
  (lr),	
  short	
  rods	
  (sr),	
  diplobacillus	
  (db),	
  were	
  present.	
  F,	
  Diplococci	
  (dc)	
  using	
  
pili	
  (arrow	
  head)	
  to	
  attach	
  to	
  the	
  crests	
  of	
  microplicae	
  (long	
  arrow).	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

139	
  

	
  
Figure	
  4.4.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
   three	
   week	
   old	
   pigs.	
   A,	
   tonsillar	
   tissue	
   showing	
   the	
   edges	
   of	
   crypts	
   (c)	
  
becoming	
   more	
   rugose,	
   and	
   crypt	
   entrance	
   wider	
   and	
   with	
   debris	
   (long	
   arrow).	
   B,	
  
Lots	
  of	
  debris	
  (long	
  arrow)	
  within	
  crypts	
  and	
  some	
  desquamating	
  epithelial	
  cells	
  (d).	
  	
  
C,	
  Small	
  clusters	
  of	
  	
  bacteria	
  	
  	
  micro-­‐‑colonies	
  	
  (short	
  arrow)	
  	
  scattered	
  	
  around	
  a	
  crypt	
  
	
  

140	
  

	
  Figure	
  4.4.	
   (cont´d).	
  	
   entrance.	
  	
  D,	
  An	
  echynocite	
  (ec)	
  lying	
  beside	
  a	
  bacterial	
  micro-­‐‑
colony	
  on	
  the	
  epithelial	
  surface.	
  Bacterial	
  cells	
  are	
  densely	
  packed.	
  	
  E,	
  Detailed	
  view	
  of	
  
a	
  small	
  micro-­‐‑colony	
  (short	
  arrow)	
  on	
  an	
  epithelial	
  cell	
  showing	
  a	
  population	
  formed	
  
mainly	
   by	
   small	
   rods	
   (sr).	
   F,	
   Diverse	
   bacterial	
   morphologies:	
   diplococci	
   (dc),	
  
diplobacilli	
   (db),	
   cocci	
   (co)	
   and	
   small	
   rods	
   (sr)	
   interacting	
   using	
   their	
   pili	
   (arrow	
  
head).	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

141	
  

The	
   fourth	
   week,	
   which	
   is	
   marked	
   as	
   a	
   stressful	
   post	
   weaning	
   time	
   with	
  
movement	
   of	
   the	
   animals	
   to	
   new	
   rooms,	
   as	
   well	
   as	
   switching	
   to	
   solid	
   feed	
  	
  
supplemented	
  	
  with	
  	
  an	
  	
  antibiotic	
  	
  growth	
  	
  promoter	
  	
  in	
  	
  the	
  	
  diet,	
  	
  showed	
  a	
  different	
  
view.	
   Tonsillar	
   crypt	
   width	
   continued	
   to	
   increase	
   (Figure	
   4.5.A)	
   and	
   parts	
   of	
   the	
  
tonsillar	
   surface	
   were	
   covered	
   by	
   patches	
   and	
   strands	
   of	
   debris	
   and/or	
  
mucous/exopolysaccharide	
   material	
   (Figure	
   4.5.A	
   to	
   4.5.E).	
   The	
   lumen	
   of	
   crypts	
  
appeared	
   particularly	
   empty.	
   However,	
   small	
   micro-­‐‑communities,	
   mainly	
   composed	
  
of	
   diplococci	
   and	
   short	
   rods	
   and	
   few	
   large	
   rods	
   were	
   identified	
   around	
   crypts	
   (Figure	
  
4.5.C	
  to	
  4.5.F).	
  	
  
By	
   the	
   eighth	
   week,	
   after	
   the	
   piglets	
   were	
   on	
   a	
   solid	
   diet	
   supplemented	
   with	
  
two	
  different	
  antibiotics	
  used	
  as	
  growth	
  promoters	
  in	
  feed	
  for	
  4-­‐‑5	
  weeks,	
  the	
  rugose	
  
appearance	
   of	
   the	
   tonsillar	
   surface	
   returned	
   and	
   there	
   was	
   a	
   noticeable	
   amount	
   of	
  
debris	
  and	
  mucous/exopolysaccharide	
  material	
  covering	
  the	
  lumen	
  of	
  crypts	
  and	
  the	
  
tonsillar	
   surface	
   (Figure	
   4.6.A	
   –	
   4.6.B).	
   Small	
   clusters	
   of	
   micro-­‐‑communities	
   were	
  
evident	
   in	
   the	
   periphery	
   of	
   the	
   crypts	
   (Figure	
   4.6.C).	
   Long	
   rods	
   displaying	
   a	
  
characteristic	
   boxcar	
   shaped	
   morphology	
   were	
   widely	
   seen	
   (Figure	
   4.6.D	
   –	
   4.6.E)	
   as	
  
well	
  as	
  lower	
  proportions	
  of	
  cocci,	
  short	
  rods	
  and	
  diplococci	
  interacting	
  and	
  attaching	
  
to	
  the	
  cell	
  surface	
  and	
  to	
  the	
  neighboring	
  bacterial	
  cells	
  (Figure	
  4.6.E	
  –	
  4.6.F).	
  	
  

	
  

142	
  

	
  
Figure	
  4.5.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
   four	
   week	
   old	
   pigs.	
   A,	
   tonsillar	
   tissue	
   of	
   four	
   week	
   old	
   pigs	
   showing	
   few	
  
patches	
   of	
   debris	
   (long	
   arrow)	
   in	
   the	
   crypts	
   (c),	
   which	
   had	
   wider	
   entrances,	
   and	
  
covering	
  the	
  surface	
  of	
  tonsils.	
  B,	
  	
  	
  Crypt	
  	
  	
  (c)	
  	
  	
  almost	
  	
  	
  empty,	
  	
  	
  with	
  	
  	
  very	
  little	
  debris	
  
(long	
  arrow).	
  	
  C,	
  	
  Small	
  	
  	
  clusters	
  	
  	
  of	
  	
  bacterial	
  	
  micro-­‐‑colonies	
  	
  (short	
  arrow)	
  	
  scattered	
  	
  
	
  

143	
  

Figure	
  4.5.	
  (cont´d).	
   	
   around	
   a	
   crypt	
   entrance	
   (c),	
   surrounded	
   and/or	
   covered	
   with	
  
strands	
   of	
   mucous/exopolysaccharide	
   material	
   (*).	
   	
   D,	
   Bacterial	
   micro-­‐‑colony	
   (short	
  
arrow)	
  on	
  epithelial	
  surface.	
  Bacterial	
  cells	
  are	
  densely	
  packed	
  and	
  covered	
  by	
  strands	
  
and	
   patches	
   of	
   mucous/exopolysaccharide	
   material	
   (*).	
   E,	
   Detailed	
   view	
   of	
   a	
   small	
  
micro-­‐‑colony	
   (short	
   arrow)	
   on	
   an	
   epithelial	
   cell	
   (e1)	
   showing	
   a	
   relatively	
   clear	
  
epithelial	
   surface	
   with	
   presence	
   of	
   strands	
   of	
   mucous/exopolysaccharide	
   material	
   (*).	
  
Bacterial	
  cells	
  of	
  the	
  type	
  diplococci	
  (dc)	
  as	
  well	
  as	
  small	
  rods	
  (sr)	
  and	
  a	
  large	
  curved	
  
rod	
   (lr)	
   were	
   evident.	
   F,	
   Lancet-­‐‑shaped	
   diplococci	
   (dc)	
   and	
   long	
   rods	
   (lr)	
   interacting	
  
and	
  covered	
  by	
  strands	
  of	
  mucous/exopolysaccharide	
  material	
  (*).	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

144	
  

	
  
Figure	
  4.6.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
  eight	
  week	
  old	
  pigs.	
   A,	
   Tonsillar	
   tissue	
   showing	
   rugose	
   crypts	
   (c)	
   and	
   a	
   rugose	
  
appearance	
   of	
   the	
   surface,	
   with	
   patches	
   of	
   scattered	
   mucous/exopolysaccharide	
  
material	
  (*)	
  covering	
  the	
  surface	
  of	
  tonsils.	
  B,	
  Crypt	
  (c)	
  packed	
  with	
  abundant	
  debris	
  
and	
  	
  	
  	
  some	
  	
  	
  	
  desquamating	
  	
  	
  	
  epithelial	
  	
  	
  	
  cells	
  	
  	
  	
  (d).	
  	
  	
  C,	
  	
  	
  	
  Small	
  	
  	
  	
  clusters	
  	
  	
  	
  of	
  	
  	
  bacterial	
  
	
  

145	
  

Figure	
   4.6.	
   (cont´d).	
   	
   micro-­‐‑colonies	
   (arrow)	
   scattered	
   around	
   a	
   crypt,	
   with	
   some	
  
desquamating	
   cells	
   (d)	
   and	
   debris	
   (long	
   arrow).	
   D,	
   Bacterial	
   micro-­‐‑colony	
   (short	
  
arrow)	
   on	
   the	
   epithelial	
   surface	
   and	
   on	
   an	
   apparently	
   desquamating	
   cell	
   (d).	
   Bacterial	
  
cell	
  morphology	
  was	
  dominated	
  by	
  large	
  boxcar-­‐‑shaped	
  rods	
  (lr).	
  E,	
  Detailed	
  view	
  of	
  a	
  
small	
   micro-­‐‑colony	
   (short	
   arrow)	
   on	
   an	
   epithelial	
   cell	
   showing	
   large	
   boxcar-­‐‑shaped	
  
rods	
  (lr)	
  intermingled	
  with	
  some	
  cocci	
  (co)	
  and	
  small	
  rods	
  (sr).	
  F,	
  Chain	
  of	
  diplococci	
  
(dc)	
   and	
   small	
   rods	
   (sr),	
   using	
   their	
   pili	
   (arrow	
   head)	
   to	
   attach	
   to	
   the	
   crests	
   of	
  
microplicae	
  and	
  to	
  the	
  neighboring	
  bacterial	
  cells.	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

146	
  

The	
   panorama	
   for	
   the	
   tenth	
   week	
   looked	
   similar	
   to	
   week	
   eight,	
   where	
   the	
  
tonsillar	
  surface	
  and	
  crypts	
  had	
  a	
  rugose	
  appearance,	
  crypts	
  appeared	
  wider,	
  and	
  the	
  
presence	
   of	
   debris	
   and	
   mucous/exopolysaccharide	
   material	
   was	
   evident	
   (Figure	
   4.7.A	
  
–	
  4.7.B).	
  Broad	
  micro-­‐‑communities	
  were	
  observed	
  in	
  the	
  periphery	
  of	
  the	
  crypt	
  lumen	
  
(Figure	
   4.7.C	
   –	
   4.7.D),	
   and	
   were	
   characterized	
   by	
   the	
   presence	
   of	
   multiple	
   cell	
  
morphologies:	
   cocci,	
   diplococci,	
   and	
   large	
   and	
   small	
   rods,	
   which	
   were	
   interacting	
  
closely	
  via	
  pili	
  with	
  each	
  other	
  and	
  with	
  the	
  epithelial	
  surface	
  (Figures	
  4.7.E	
  –	
  4.7.F).	
  	
  
Finally,	
   tonsils	
   in	
   the	
   seventeenth	
   week	
   displayed	
   a	
   very	
   rugose	
   tonsillar	
  
surface,	
   extensively	
   covered	
   by	
   a	
   layer	
   of	
   mucous/exopolysaccharide	
   material,	
   with	
  
crypts	
   that	
   were	
   very	
   wide	
   and	
   full	
   of	
   debris	
   (Figure	
   4.8.A	
   –	
   4.8.B).	
   There	
   was	
  
considerable	
  debris	
  covering	
  the	
  epithelial	
  surface	
  (Figure	
  4.8.C),	
  as	
  	
  well	
  	
  as	
  extensive	
  	
  
bacterial	
  	
  micro-­‐‑communities	
  	
  (Figure	
  4.8.D),	
  	
  some	
  	
  of	
  	
  which	
  were	
  covered	
  by	
  a	
  layer	
  
of	
   mucous/exopolysaccharide	
   material	
   (Figure	
   4.8.E).	
   Many	
   bacterial	
   cells	
   appeared	
  
almost	
   embedded	
   in	
   the	
   surface	
   of	
   the	
   tonsil	
   epithelium.	
   The	
   bacterial	
   cells	
   were	
  
interconnecting	
   extensively	
   with	
   the	
   neighbor	
   cells	
   as	
   depicted	
   by	
   the	
   presence	
   of	
  
multiple	
  pili	
  intermingling	
  (Figure	
  4.8.F).	
  
Similarly	
  to	
  the	
  changes	
  observed	
  in	
  the	
  tonsillar	
  surface	
  of	
  tonsils	
  throughout	
  
the	
   different	
   samples,	
   multiple	
   bacterial	
   members	
   of	
   micro-­‐‑communities	
   were	
  
identified	
  through	
  the	
  different	
  sampling	
  times.	
  These	
  bacteria	
  were	
  characterized	
  by	
  
diverse	
   cellular	
   morphologies,	
   varying	
   from	
   lancet-­‐‑shaped	
   diplococci	
   and	
   small	
   thin	
  
rods	
  in	
  the	
  first	
  week	
  (Figure	
  4.9.A);	
  large	
  chains	
  of	
  palisade	
  shaped	
  Alyssiella,	
  chains	
  
of	
   cocci,	
   short	
   rods	
   and	
   long	
   rods,	
   in	
   the	
   third	
   week	
   	
   (Figure	
   4.9.B);	
   diplococci,	
   long	
  
rods	
   and	
   short	
   rods,	
   in	
   the	
   fourth	
   week	
   (Figure	
   4.9.C);	
   box-­‐‑car	
   shaped	
   rods,	
   lancet-­‐‑

	
  

147	
  

shaped	
   diplococci,	
   cocci	
   and	
   long	
   rods,	
   in	
   the	
   eighth	
   week	
   (Figure	
   4.9.D);	
   long	
   rods,	
  
cocci,	
   lancet-­‐‑shaped	
   diplococci	
   and	
   small	
   rods,	
   in	
   the	
   tenth	
   week	
   (Figure	
   4.9.E);	
   and	
  
finally,	
  cocci	
  and	
  small	
  rods	
  in	
  the	
  seventeenth	
  week	
  (Figure	
  4.9.F).	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

148	
  

	
  
Figure	
  4.7.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
  ten	
  week	
  old	
  pigs.	
   A,	
   Tonsillar	
   tissue	
   showing	
   rugose	
   crypts	
   (c)	
   and	
   a	
   rugose	
  
appearance	
   of	
   the	
   surface.	
   Abundant	
   debris	
   (long	
   arrow)	
   in	
   the	
   crypt	
   entrance	
   and	
  
mucous/exopolysaccharide	
   material	
   (*)	
   covering	
   patches	
   of	
   the	
   tonsil	
   surface.	
   B,	
  	
  
Crypt	
  	
  (c)	
  	
  with	
  	
  abundant	
  	
  debris	
  	
  (long	
  arrow)	
  	
  in	
  	
  the	
  	
  lumen	
  	
  and	
  	
  covering	
  	
  the	
  walls.	
  	
  
	
  

149	
  

Figure	
   4.7.	
   (cont´d).	
  C,	
  Large	
  bacterial	
  micro-­‐‑colony	
  (short	
  arrow)	
  in	
  the	
  entrance	
  of	
  
a	
   crypt	
   (c),	
   surrounded	
   by	
   scattered	
   debris	
   (long	
   arrow).	
   D,	
   Bacterial	
   micro-­‐‑colonies	
  
(short	
   arrows)	
   with	
   diverse	
   clustered	
   cells	
   covering	
   large	
   areas	
   of	
   the	
   epithelial	
  
surface.	
   E,	
   Detailed	
   view	
   of	
   a	
   bacterial	
   micro-­‐‑colony	
   showing	
   an	
   intermingled	
  
population	
  of	
  cocci	
  (co),	
  large	
  rods	
  (lr),	
  small	
  rods	
  (sr)	
  and	
  diplococci	
  (dc).	
  F,	
  A	
  diverse	
  
bacterial	
   community	
   showing	
   interactions	
   between	
   small	
   rods	
   (sr),	
   cocci	
   (co),	
  
diplococci	
  (dc)	
  extending	
  their	
  pili	
  (arrow	
  head)	
  to	
  the	
  neighboring	
  cells.	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

150	
  

	
  
Figure	
  4.8.	
  Representative	
  scanning	
  electron	
  microscopy	
  images	
  of	
  tonsil	
  tissue	
  
from	
  seventeen	
  week	
  old	
  pigs.	
   A,	
   Tonsillar	
   surface	
   extensively	
   covered	
   by	
   a	
   layer	
   of	
  
mucous/exopolysaccharide	
   material	
   (*)	
   and	
   crypts	
   (c)	
   full	
   of	
   debris	
   (long	
   arrow).	
   The	
  
epithelial	
   surface	
   has	
   a	
   very	
   rugose	
   appearance.	
   B,	
   Crypt	
   fully	
   clogged	
   with	
   debris	
  
(long	
  arrow).	
  Clear	
  	
  	
  presence	
  	
  	
  of	
  	
  	
  round	
  	
  	
  structures	
  	
  	
  with	
  	
  smooth	
  	
  surface,	
  	
  likely	
  	
  	
  
	
  

151	
  

Figure	
   4.8.	
   (cont´d).	
   lymphocytes	
   (l).	
   C,	
   Epithelial	
   surface	
   extensively	
   covered	
   with	
  
debris	
   (long	
   arrow).	
   A	
   bacterial	
   micro-­‐‑colony	
   (short	
   arrow)	
   and	
   multiple	
   round	
   and	
  
smooth	
   structures,	
   likely	
   lymphocytes	
   (l)	
   are	
   present.	
   D,	
   Epithelial	
   surface	
   extensively	
  
covered	
  by	
  a	
  bacterial	
  micro-­‐‑colony	
  (short	
  arrow).	
  E,	
  Detailed	
  view	
  of	
  diplococci	
  (dc)	
  
and	
   small	
   rods	
   (sr),	
   appearing	
   almost	
   embedded	
   in	
   the	
   tonsil	
   epithelial	
   surface	
   and	
  
covered	
   by	
   a	
   layer	
   of	
   mucous/exopolysaccharide	
   material	
   (*).	
   F,	
   Diplococci	
   (dc)	
   and	
  
cocci	
  (co)	
  interacting	
  extensively,	
  	
  with	
  pili	
  (arrow	
  head)	
  extended	
  to	
  the	
  neighboring	
  
cells.	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

152	
  

	
  
Figure	
   4.9.	
   Representative	
   bacterial	
   micro-­‐‑colonies	
   identified	
   through	
   the	
  
different	
   sampling	
   times.	
  Scanning	
  electron	
  microscopy	
  images	
  from	
  tonsil	
  samples	
  
of	
   pigs	
   in	
   different	
   times	
   of	
   their	
   life.	
   A,	
   Micro-­‐‑colony	
   formed	
   by	
   lancet-­‐‑shaped	
  
diplococci	
   (dc)	
   and	
   small	
   thin	
   rods	
   (sr),	
   identified	
   in	
   one	
   week	
   old	
   pigs.	
   Bacterial	
   cells	
  
are	
  	
  	
  loosely	
  	
  	
  disseminated	
  	
  on	
  	
  	
  the	
  	
  epithelial	
  	
  surface.	
  	
  B,	
  	
  Micro-­‐‑colony	
  	
  composed	
  	
  of	
  
	
  

153	
  

Figure	
   4.9.	
   (cont´d).	
   	
   several	
   large	
   palisade-­‐‑shaped	
   chains	
   of	
   actively	
   dividing	
  
bacteria	
   with	
   pili	
   along	
   one	
   short	
   end	
   of	
   each	
   cell,	
   which	
   are	
   likely	
   Alyssiella	
  
[32](arrow	
  head),	
  interacting	
  with	
  a	
  chain	
  of	
  cocci	
  (short	
  arrow),	
  short	
  rods	
  (sr)	
  and	
  
long	
  rods	
  (large	
  arrow),	
  identified	
  in	
  a	
  three	
  week	
  old	
  pig.	
  C,	
  Micro-­‐‑colony	
  composed	
  
of	
  diplococci	
  (dc),	
  long	
  rods	
  (lr),	
  and	
  short	
  rods	
  (sr),	
  identified	
  in	
  a	
  four	
  week	
  old	
  pig.	
  
D,	
   Micro-­‐‑colony	
   composed	
   of	
   box-­‐‑car	
   shaped	
   rods	
   (arrow	
   head),	
   lancet-­‐‑shaped	
  
diplococci	
  (short	
  arrow),	
  cocci	
  (long	
  arrow)	
  and	
  long	
  rods	
  (lr),	
  identified	
  in	
  an	
  eight	
  
week	
  old	
  pig.	
  	
  E.	
  Micro-­‐‑colony	
  composed	
  of	
  long	
  rods	
  (lr),	
  cocci	
  (arrow	
  head),	
  lancet-­‐‑
shaped	
  diplococci	
  (short	
  arrow),	
  small	
  rods	
  (long	
  arrow),	
  identified	
  in	
  a	
  ten	
  week	
  old	
  
pig.	
   Extensive	
   interactions	
   between	
   bacteria.	
   F.	
   Micro-­‐‑colony	
   composed	
   of	
   cocci	
   (short	
  
arrow)	
   and	
   small	
   rods	
   (large	
   arrow),	
   closely	
   interacting.	
   Bacterial	
   cells	
   seem	
  
embedded	
  in	
  the	
  surface	
  of	
  the	
  epithelial	
  cells.	
  
	
  
	
  
	
  
	
  
	
  
	
  	
  	
  	
  
	
  
	
  
	
  
	
  
	
  	
  	
  	
  	
  	
  

	
  

154	
  

DISCUSSION	
  
	
  
The	
   present	
   study	
   allowed	
   us	
   to	
   investigate	
   the	
   temporal	
   succession	
   of	
  
tonsillar	
   bacterial	
   community	
   structures	
   in	
   pigs,	
   from	
   one	
   week	
   through	
   seventeen	
  
weeks	
  old.	
  The	
  validation	
  of	
  the	
  oligonucleotide	
  probes	
  with	
  smears	
  of	
  bacterial	
  cells	
  
as	
   wells	
   as	
   with	
   muscle	
   inoculated	
   with	
   bacterial	
   cells	
   was	
   successful.	
   However,	
   when	
  
the	
   FISH	
   probes	
   were	
   used	
   on	
   tissues	
   derived	
   from	
   pig	
   tonsils,	
   only	
   bacteria	
   from	
  
genus	
   Streptococcus	
   were	
   reliably	
   identified	
   on	
   the	
   epithelial	
   surface	
   of	
   tonsils	
   from	
  
the	
  sampled	
  pigs.	
  While	
  Streptococcus	
  was	
  not	
  present	
  in	
  every	
  slide	
  for	
  each	
  animal,	
  	
  
there	
  was	
  positive	
  identification	
  of	
  Streptococcus	
  in	
  at	
  least	
  one	
  sample	
  examined	
  for	
  
each	
  sampling	
  time.	
  The	
  initial	
  aim	
  of	
  this	
  study	
  was	
  to	
  characterize	
  the	
  assembly	
  of	
  
the	
  three	
  most	
  abundant	
  taxa	
  identified	
  -­‐‑	
  Pasteurellaceae,	
  Streptococcus	
  and	
  Moraxella	
  
-­‐‑	
   through	
   the	
   development	
   of	
   the	
   tonsillar	
   microbiome	
   of	
   pigs,	
   which	
   have	
   been	
  
reported	
   as	
   being	
   present	
   throughout	
   the	
   lives	
   of	
   the	
   pigs	
   from	
   this	
   farm	
   [21,	
   22].	
  
Nevertheless,	
   the	
   presence	
   of	
   marked	
   nonspecific	
   staining	
   of	
   multiple	
   structures	
  
(Figure	
   4.1.F	
   and	
   4.2.A)	
   in	
   most	
   of	
   the	
   samples	
   with	
   all	
   the	
   fluorophores	
   used,	
  
especially	
   Cy3	
   and	
   Cy5,	
   as	
   well	
   as	
   the	
   marked	
   autofluorescence	
   of	
   the	
   tonsil	
   tissue,	
  
made	
   this	
   aim	
   unattainable	
   in	
   the	
   given	
   circumstances.	
   The	
   issues	
   of	
   non-­‐‑specific	
  
background	
  and	
  the	
  possible	
  biases	
  when	
  analyzing	
  eukaryotic	
  tissues	
  processed	
  	
  by	
  	
  
FISH	
  	
  have	
  	
  been	
  reported	
  previously	
  [33].	
  Further,	
  tissue	
  autofluorescence	
  has	
  been	
  
associated	
   with	
   a	
   decrease	
   in	
   the	
   signal-­‐‑to-­‐‑noise	
   ratio	
   and	
   with	
   masking	
   the	
   true	
  
fluorescent	
   signal,	
   when	
   working	
   with	
   FISH	
   [34].	
   We	
   did	
   not	
   quantify	
   these	
  
phenomena	
   in	
   our	
   study,	
   but	
   certainly	
   the	
   high	
   presence	
   of	
   non-­‐‑specific	
   background	
  

	
  

155	
  

and	
  autofluorescence	
  negatively	
  influenced	
  our	
  ability	
  to	
  detect	
  reliably	
  the	
  signal	
  of	
  
the	
  hybridized	
  probes.	
  	
  
It	
   is	
   difficult	
   to	
   detect	
   bacteria	
   when	
   using	
   FISH	
   probes	
   in	
   eukaryotic	
   tissue	
  
when	
   the	
   bacterial	
   concentration	
   is	
   lower	
   than	
   105/ml	
   [35].	
   Hogart	
   et	
   al	
   [36]	
  
demonstrated	
   that	
   FISH	
   is	
   a	
   highly	
   specific	
   technique,	
   albeit	
   with	
   a	
   moderate	
  
sensitivity	
  when	
  dealing	
  with	
  bacteria	
  with	
  concentrations	
  lower	
  than	
  4	
  x	
  105	
  CFU/ml	
  
in	
  sputum	
  samples.	
  Stepinska	
  et	
  al	
  [37]	
  compared	
  the	
  use	
  of	
  culture	
  isolation	
  vs	
  FISH	
  
techniques	
   for	
   the	
   identification	
   of	
   bacterial	
   cells	
   from	
   lysates	
   of	
   CD14	
   cells	
   from	
  
adenoids	
   and	
   tonsil	
   tissue,	
   and	
   although	
   they	
   stated	
   that	
   FISH	
   was	
   twice	
   more	
  
effective	
  than	
  cultivation	
  in	
  detecting	
  bacteria,	
  they	
  also	
  could	
  not	
  obtain	
  positive	
  FISH	
  
results	
   in	
   some	
   of	
   the	
   samples,	
   in	
   spite	
   of	
   being	
   able	
   to	
   isolate	
   bacterial	
   cells	
   from	
  
them.	
  We	
  do	
  not	
  know	
  the	
  exact	
  concentration	
  of	
  bacterial	
  cells	
  in	
  each	
  of	
  our	
  samples,	
  
although	
  we	
  have	
  previously	
  found	
  1	
  X	
  106	
  –	
  1.5	
  X	
  107	
  cfu	
  per	
  gram	
  of	
  tonsil	
  tissue	
  in	
  
healthy	
   grower-­‐‑finisher	
   pigs.	
   Certainly,	
   using	
   a	
   culture-­‐‑independent	
   approach	
   by	
  
sequencing	
  the	
  16s	
  rRNA	
  genes	
  of	
  tonsillar	
  samples	
  collected	
  from	
  pigs	
  from	
  the	
  same	
  
farm,	
  

we	
  

characterized	
  

these	
  

three	
  

bacterial	
  

families	
  

(Pasteurellaceae,	
  

Streptococcaceae	
  and	
  Moraxellaceae)	
  as	
  present	
  in	
  a	
  high	
  relative	
  abundance	
  through	
  
the	
  pigs’	
  life	
  [21,	
  22].	
  
Moter	
   and	
   Göbel	
   [34]	
   have	
   described	
   the	
   possible	
   problems	
   and	
   pitfalls	
   in	
  
using	
   FISH	
   for	
   visualization	
   of	
   microorganisms.	
   The	
   authors	
   mentioned	
  
autofluorescence	
  and	
  lack	
  of	
  specificity	
  of	
  nucleotide	
  probes	
  as	
  possible	
  causes	
  of	
  false	
  
positive	
   results	
   in	
   FISH	
   procedures.	
   Additionally,	
   they	
   mentioned	
   the	
   insufficient	
  
penetration	
  of	
  the	
  probes,	
  higher	
  order	
  structure	
  of	
  target/probe,	
  low	
  RNA	
  content,	
  as	
  

	
  

156	
  

well	
   as	
   photobleaching	
   and	
   failure	
   to	
   use	
   a	
   probe	
   targeting	
   bacteria	
   in	
   general,	
   as	
   a	
  
cause	
   of	
   false	
   negative	
   results.	
   In	
   the	
   present	
   study,	
   it	
   is	
   possible	
   that	
   the	
   non-­‐‑
detection	
   of	
   the	
   expected	
   microorganisms	
   in	
   the	
   samples	
   may	
   be	
   due	
   to	
   our	
   strict	
  
quality	
   control,	
   where	
   we	
   did	
   not	
   accept	
   what	
   we	
   considered	
   to	
   be	
   possible	
   false	
  
positive	
   results	
   where	
   we	
   saw	
   tissue	
   autofluorescence	
   or	
   non-­‐‑specific	
   labeling	
   of	
  
eukaryotic	
   cells	
   or	
   granules	
   with	
   our	
   probes.	
   Since	
   the	
   oligonucleotide	
   probes	
   were	
  
validated	
   with	
   bacterial	
   isolates	
   from	
   the	
   desired	
   taxa,	
   we	
   considered	
   that	
   we	
   were	
  
targeting	
   the	
   specific	
   desired	
   microorganisms.	
   Probes	
   STR405,	
   PAS111	
   and	
   EUB338	
  
were	
  validated	
  under	
  our	
  experimental	
  conditions.	
  These	
  probes	
  have	
  been	
  validated	
  
in	
  previous	
  studies	
  of	
  microbial	
  communities	
  in	
  the	
  oral	
  cavity	
  of	
  humans	
  [16].	
  	
  The	
  
aforementioned	
  probes	
  targeted	
  a	
  very	
  broad	
  spectrum	
  of	
  microorganisms	
  in	
  each	
  of	
  
the	
  desired	
  taxa.	
  We	
  also	
  validated	
  them	
  in	
  silico	
  against	
  representative	
  sequences	
  for	
  
the	
   taxa	
   identified	
   as	
   more	
   abundant	
   in	
   samples	
   from	
   the	
   sampled	
   farm	
   (data	
   not	
  
shown).	
  We	
  were	
  expecting	
  positive	
  identification	
  of	
  members	
  of	
  bacterial	
  taxa	
  related	
  
with	
  the	
  probes.	
  On	
  the	
  other	
  hand,	
  we	
  designed	
  a	
  probe	
  targeting	
  a	
  broad	
  spectrum	
  
of	
   microorganisms	
   of	
   the	
   genus	
   Moraxella	
   (MOR831),	
   that	
   was	
   tested	
   in	
   silico	
   as	
   a	
  
good	
   probe	
   however,	
   we	
   were	
   not	
   able	
   to	
   validate	
   the	
   probe	
   against	
   multiple	
  
Moraxella	
   isolates	
   (data	
   not	
   shown).	
   After	
   analyzing	
   our	
   designed	
   probe,	
   we	
   found	
  
that	
   it	
   was	
   targeting	
   a	
   16s	
   rRNA	
   location	
   characterized	
   by	
   difficult	
   accessibility	
   and	
  
giving	
  a	
  very	
  poor	
  fluorescence	
  [38].	
  We	
  designed	
  a	
  second	
  probe	
  (MOR575)	
  targeting	
  
members	
   of	
   genus	
   Moraxella.	
   Unfortunately,	
   the	
   designed	
   probe	
   only	
   targeted	
   a	
  
narrow	
   group	
   of	
   microorganisms	
   in	
   this	
   genus;	
   however	
   we	
   validated	
   the	
   probe	
   in	
  
silico	
   against	
   representative	
   sequences	
   of	
   this	
   taxa	
   identified	
   in	
   our	
   previous	
   studies	
  

	
  

157	
  

[21,	
   22],	
   as	
   well	
   as	
   against	
   diverse	
   Moraxella	
   isolates	
   stored	
   in	
   our	
   lab	
   collection	
   of	
  
microorganisms.	
  We	
  did	
  not	
  culture	
  Moraxella	
  isolates	
  from	
  the	
  sampled	
  pigs,	
  which	
  
would	
   have	
   been	
   the	
   ideal	
   to	
   validate	
   our	
   probe	
   with	
   even	
   more	
   specificity.	
   The	
  
designed	
   probe	
   when	
   examined,	
   still	
   provided	
   a	
   relative	
   low	
   fluorescence	
   intensity	
  
(40%)	
  as	
  mentioned	
   by	
   Fush	
   et	
   al	
  [38].	
  It	
  may	
  be	
  possible	
  that	
  the	
  inability	
  to	
  identify	
  
microorganisms	
   from	
   the	
   genus	
   Moraxella	
   in	
   the	
   samples,	
   was	
   due	
   to	
   the	
   excessive	
  
autofluorescence	
  of	
  the	
  tissue	
  samples	
  and	
  the	
  low	
  signal-­‐‑to-­‐‑noise	
  ratio	
  of	
  the	
  probe.	
  
It	
  also	
  may	
  be	
  due	
  to	
  the	
  fact	
  that	
  the	
  microorganisms	
  from	
  this	
  genus	
  present	
  in	
  the	
  	
  
samples	
  were	
  not	
  targeted	
  specifically	
  by	
  the	
  probe.	
  	
  
Swidsinski	
   and	
   Loening-­‐‑Baucke	
   [39],	
   reported	
   the	
   almost	
   total	
   absence	
   of	
  
bacteria	
   in	
   the	
   epithelial	
   surface	
   of	
   human	
   tonsils,	
   even	
   in	
   samples	
   derived	
   from	
  
tonsillectomy	
   of	
   patients	
   with	
   chronic	
   tonsillitis.	
   However,	
   the	
   positive	
   identification	
  
of	
  bacteria	
  by	
  FISH,	
  in	
  tonsils	
  derived	
  from	
  patients	
  with	
  chronic	
  tonsillitis	
  has	
  been	
  
reported	
   [35,	
   39].	
   The	
   authors	
   identified	
   high	
   concentrations	
   of	
   bacteria	
   attached	
   to	
  
tonsils/adenoids	
   epithelia,	
   bacterial	
   infiltrations	
   of	
   superficial	
   epithelia,	
   bacteria	
   in	
  
tissue	
   fistulas	
   and/or	
   fissures,	
   as	
   well	
   as	
   bacteria	
   contained	
   in	
   macrophages,	
   in	
  
regions	
   of	
   tissue	
   associated	
   with	
   an	
   inflammatory	
   response.	
   In	
   the	
   present	
   study,	
   it	
  
may	
   be	
   possible	
   that	
   what	
   we	
   sometimes	
   identified	
   as	
   non-­‐‑specific	
   labeling	
   of	
  
granular	
  structures	
  in	
  the	
  submucosa	
  may	
  in	
  fact	
  be	
  positive	
  hybridization	
  to	
  bacterial	
  
cells	
  inside	
  or	
  attached	
  to	
  phagocytic	
  cells,	
  masked	
  by	
  the	
  autofluorescence	
  and	
  non-­‐‑
specific	
   labeling	
   of	
   other	
   tissue	
   components	
   that	
   we	
   observed.	
   	
   	
   Further,	
   the	
   large	
  
amount	
  of	
  mucous/exopolysaccharide	
  often	
  seen	
  on	
  the	
  tonsil	
  surface	
  and	
  within	
  the	
  

	
  

158	
  

crypts,	
  as	
  well	
  as	
  the	
  layers	
  of	
  bacteria	
  seen	
  in	
  colonies	
  in	
  the	
  older	
  piglets,	
  may	
  have	
  
blocked	
  access	
  of	
  the	
  probes	
  to	
  many	
  bacteria.	
  
Despite	
   the	
   negative	
   results	
   attained	
   in	
   this	
   study	
   to	
   investigate	
   the	
   temporal	
  
succession	
  of	
  tonsillar	
  bacterial	
  community	
  structures	
  in	
  pigs	
  by	
  using	
  FISH,	
  when	
  we	
  
used	
   SEM,	
   we	
   were	
   able	
   to	
   identify	
   a	
   pattern	
   of	
   succession	
   in	
   the	
   appearance	
   of	
   the	
  	
  
tonsillar	
   surface	
   as	
   well	
   as	
   in	
   the	
   width	
   of	
   the	
   crypts	
   and	
   more	
   importantly,	
   in	
   the	
  
structure	
  of	
  the	
  tonsillar	
  micro-­‐‑communities	
  observed.	
  Initially	
  for	
  newborn	
  pigs,	
  the	
  
tonsillar	
   surface	
   had	
   a	
   smooth	
   and	
   relatively	
   clean	
   surface	
   but	
   as	
   pigs	
   aged,	
   the	
  
tonsillar	
  surface	
  acquired	
  a	
  more	
  rugose	
  texture.	
  The	
  crypt	
  entrances	
  also	
  changed	
  as	
  
pigs	
   aged,	
   with	
   the	
   width	
   of	
   the	
   crypts	
   on	
   average	
   increasing	
   and	
   the	
   crypts	
   being	
  
filled	
   with	
   debris	
   including	
   mucous/exopolysaccharide	
   material,	
   lymphocytes	
   and	
  
cellular	
  detritus,	
  among	
  other	
  material.	
  The	
  presence	
  of	
  leucocytes,	
  Periodic	
  acid-­‐‑Shiff	
  
(PAS)	
  positive	
  content	
  and	
  bacteria	
  in	
  the	
  lumen	
  of	
  crypts	
  has	
  been	
  reported	
  [40].	
  
Samples	
   from	
   one	
   week	
   old	
   pigs	
   were	
   characterized	
   by	
   small	
   micro-­‐‑
communities	
   composed	
   mainly	
   of	
   lancet-­‐‑shaped	
   diplococci	
   and	
   small	
   rods.	
   We	
  
identified	
  the	
  clear	
  attachment	
  of	
  many	
  of	
  these	
  diplococci	
  to	
  the	
  crest	
  of	
  microplicae	
  
in	
   the	
   epithelial	
   surface	
   of	
   tonsils.	
   It	
   has	
   been	
   suggested	
   that	
   the	
   crest	
   of	
   epithelial	
  
microplicae	
  are	
  used	
  for	
  initial	
  attachment	
  of	
  bacteria	
  [41]	
  and	
  this	
  was	
  demonstrated	
  
for	
   Streptococcus	
  pyogenes	
   [42].	
   	
   Certainly,	
   the	
   number	
   of	
   members	
   of	
   the	
   identified	
  
micro-­‐‑communities	
   increased	
   with	
   the	
   time	
   as	
   well	
   as	
   the	
   interactions	
   between	
  
individual	
  cells	
  with	
  the	
  neighboring	
  cells.	
  We	
  observed	
  micro-­‐‑colonies	
  with	
  bacterial	
  
cells	
   apparently	
   embedded	
   in	
   the	
   epithelial	
   surface.	
   Similar	
   findings	
   of	
   bacteria	
  
forming	
  apparent	
  depressions	
  in	
  the	
  cell	
  surface	
  of	
  epithelial	
  cells	
  in	
  tonsils	
  have	
  been	
  

	
  

159	
  

reported	
  [41].	
  	
  We	
  saw	
  a	
  greater	
  number	
  of	
  cells	
  connected	
  via	
  pili	
  to	
  neighboring	
  cells	
  
as	
  the	
   pigs	
  aged.	
  The	
  microbial	
  communities	
  identified	
  for	
  seventeen	
  weeks	
  old	
  pigs	
  
seemed	
  to	
  be	
  covered	
  by	
  a	
  layer	
  of	
  mucous/exopolysaccharide	
  material.	
  It	
  was	
  not	
  an	
  
aim	
  of	
  this	
  study	
  to	
  classify	
  the	
  chemical	
  nature	
  of	
  this	
  material.	
  However,	
  Kania	
  et	
  al	
  
[43],	
   with	
   the	
   use	
   of	
   SEM,	
   FISH	
   and	
   special	
   stains,	
   demonstrated	
   the	
   presence	
   of	
  
bacterial	
   micro-­‐‑colonies	
   immersed	
   in	
   a	
   matrix	
   of	
   glycocalix	
   in	
   tonsils	
   from	
   children	
  
with	
   chronic	
   or	
   recurrent	
   tonsilitis,	
   which	
   they	
   called	
   biofilms.	
   We	
   only	
   can	
   say	
   that	
  
the	
  structure	
  of	
  the	
  micro-­‐‑communities	
  or	
  micro-­‐‑colonies	
  changed	
  with	
  time	
  as	
  more	
  
cells	
   were	
   included	
   in	
   the	
   micro-­‐‑community.	
   Similarly,	
   the	
   interactions	
   of	
   the	
  
members	
  of	
  those	
  micro-­‐‑communities	
  seemed	
  to	
  increase	
  with	
  the	
  age	
  of	
  the	
  pigs,	
  as	
  
we	
   observed	
   more	
   cells	
   interacting	
   via	
   pili	
   with	
   the	
   neighboring	
   cells	
   in	
   the	
   micro-­‐‑
community.	
  	
  We	
  also	
  saw	
  large	
  numbers	
  of	
  lymphocytes	
  and	
  red	
  blood	
  cells,	
  as	
  well	
  as	
  
echynocytes	
  (deformed	
  red	
  blood	
  cells)	
  in	
  many	
  of	
  the	
  samples.	
  
Although	
   unfortunately,	
   we	
   cannot	
   classify	
   the	
   bacterial	
   cells	
   observed	
   with	
  
SEM	
   taxonomically	
   beyond	
   the	
   cell	
   morphology,	
   we	
   certainly	
   saw	
   different	
   bacterial	
  
shapes	
  forming	
  the	
  micro-­‐‑communities	
  throughout	
  the	
  life	
  of	
  the	
  pigs.	
  We	
  have	
  been	
  
able	
  to	
  isolate	
  Streptococcus	
  suis	
  in	
  many	
  piglets	
  from	
  this	
  farm	
  at	
  birth,	
  and	
  from	
  all	
  
the	
  piglets	
  from	
  the	
  same	
  litter	
  by	
  one	
  day	
  of	
  age	
  (unpublished	
  data).	
  We	
  studied	
  the	
  
development	
   of	
   the	
   tonsillar	
   microbiome	
   in	
   pigs	
   from	
   the	
   farm	
   and	
   we	
   identified	
  
different	
   OTUs	
   assigned	
   to	
   Streptococcus,	
   but	
   particularly	
   Streptococcus	
  suis	
   in	
   at	
   birth	
  
piglets	
   as	
   well	
   as	
   constantly	
   throughout	
   the	
   life	
   of	
   pigs	
   [21,	
   22].	
   It	
   has	
   been	
  
demonstrated	
  that	
  in	
  the	
  formation	
  of	
  dental	
  plaque	
  in	
  humans,	
  Streptococcus	
  acts	
  as	
  a	
  
first	
   colonizer	
   and	
   facilitates	
   the	
   attachment/aggregation	
   of	
   other	
   members	
   of	
   the	
  

	
  

160	
  

bacterial	
   community	
   [7].	
   This	
   pattern	
   of	
   adhesions	
   and	
   interactions	
   between	
   mixed	
  
species	
   forms	
   the	
   basis	
   of	
   spatiotemporal	
   development	
   of	
   the	
   dental	
   plaque	
   [5].	
   We	
  
can	
  only	
  speculate	
  that	
  the	
  diplococcus-­‐‑shape	
  cells	
  that	
  we	
  saw	
  almost	
  constantly	
  in	
  
all	
  the	
  samples	
  may	
  be	
  a	
  member	
  of	
  genus	
  Streptococcus	
  acting	
  as	
  first	
  colonizer	
  and	
  
facilitating	
   the	
   building	
   up	
   of	
   the	
   bacterial	
   micro-­‐‑communities	
   observed	
   in	
   all	
   our	
  
samples.	
   Members	
   of	
   genus	
   Streptococcus	
   were	
   positively	
   identified	
   in	
   samples	
   from	
  
these	
   pigs,	
   using	
   FISH	
   probes	
   targeting	
   members	
   of	
   this	
   genus.	
   It	
   is	
   possible	
   that	
  
bacteria	
   from	
   genus	
   Streptococcus	
   are	
   facilitating	
   the	
   attachment	
   of	
   bacterial	
   cells	
  
from	
   other	
   taxa	
   (different	
   bacterial	
   shapes	
   observed)	
   and	
   building	
   up	
   the	
  
communities	
  identified.	
  	
  	
  
In	
  conclusion,	
  we	
  used	
  FISH	
  and	
  SEM	
  to	
  identify	
  the	
  structure	
  and	
  presence	
  of	
  
diverse	
  bacterial	
  communities	
  on	
  the	
  surface	
  of	
  pig	
  tonsils	
  through	
  different	
  times	
  in	
  
their	
  life.	
  We	
  identified	
  morphological	
  changes	
  in	
  the	
  appearance	
  of	
  pig	
  tonsils	
  and	
  the	
  
bacterial	
  members	
  of	
  the	
  communities	
  on	
  tonsils	
  from	
  one,	
  three,	
  four,	
  eight,	
  ten	
  and	
  
seventeen	
   weeks	
   old.	
   To	
   the	
   best	
   of	
   our	
   knowledge,	
   this	
   is	
   the	
   first	
   published	
   study	
  
describing	
  the	
  morphological	
  changes	
  of	
  the	
  tonsillar	
  surface	
  in	
  pigs	
  associated	
  with	
  	
  
changes	
  in	
  the	
  microbial	
  communities	
  observed	
  through	
  different	
  times	
  in	
  their	
  life.	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

161	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
REFERENCES	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

162	
  

1.	
  
	
  
2.	
  
	
  
3.	
  
	
  
4.	
  
	
  
5.	
  
	
  
6.	
  
	
  
7.	
  
	
  
8.	
  

	
  
9.	
  
	
  
10.	
  
	
  
11.	
  

	
  

REFERENCES	
  
	
  
	
  
	
  
Horter	
  DC,	
  Yoon	
  KJ,	
  Zimmerman	
  JJ:	
  A	
  review	
  of	
  porcine	
  tonsils	
  in	
  immunity	
  
and	
  disease.	
  Anim	
  Health	
  Res	
  Rev	
  2003,	
  4:143-­‐‑155.	
  
Smith	
   TC,	
   Harper	
   AL,	
   Nair	
   R,	
   Wardyn	
   SE,	
   Hanson	
   BM,	
   Ferguson	
   DD,	
   Dressler	
  
AE:	
   Emerging	
   swine	
   zoonoses.	
   Vector-­‐‑Borne	
   and	
   Zoonotic	
   Diseases	
   2011,	
  
11:1225-­‐‑1234.	
  
Kolenbrander	
   PE,	
   Palmer	
   RJ,	
   Periasamy	
   S,	
   Jakubovics	
   NS:	
   Oral	
   multispecies	
  
biofilm	
  development	
  and	
  the	
  key	
  role	
  of	
  cell-­‐‑cell	
  distance.	
  Nature	
  Reviews	
  
Microbiology	
  2010,	
  8:471-­‐‑480.	
  
Kolenbrander	
   PE,	
   London	
   J:	
   Adhere	
   today,	
   here	
   tomorrow:	
   oral	
   bacterial	
  
adherence.	
  Journal	
  of	
  Bacteriology	
  1993,	
  175:3247-­‐‑3252.	
  
Palmer	
   RJ,	
   Gordon	
   SM,	
   Cisar	
   JO,	
   Kolenbrander	
   PE:	
   Coaggregation-­‐‑mediated	
  
interactions	
   of	
   Streptococci	
   and	
   Actinomyces	
   detected	
   in	
   initial	
   human	
  
dental	
  plaque.	
  Journal	
  of	
  Bacteriology	
  2003,	
  185:3400-­‐‑3409.	
  
Kolenbrander	
   PE,	
   Andersen	
   RN,	
   Blehert	
   DS,	
   Egland	
   PG,	
   Foster	
   JS,	
   Palmer	
   RJ:	
  
Communication	
   among	
   oral	
   bacteria.	
   Microbiology	
   and	
   Molecular	
   Biology	
  
Reviews	
  2002,	
  66:486-­‐‑505.	
  
Kolenbrander	
   PE,	
   Ganeshkumar	
   N,	
   Cassels	
   FJ,	
   Hughes	
   CV:	
   Coaggregation-­‐‑
specific	
   adherence	
   among	
   human	
   oral	
   plaque	
   bacteria.	
  Faseb	
  Journal	
  1993,	
  
7:406-­‐‑413.	
  
Moter	
   A,	
   Leist	
   G,	
   Rudolph	
   R,	
   Schrank	
   K,	
   Choi	
   BK,	
   Wagner	
   M,	
   Gobel	
   UB:	
  
Fluorescence	
   in	
   situ	
   hybridization	
   shows	
   spatial	
   distribution	
   of	
   as	
   yet	
  
uncultured	
   treponemes	
   in	
   biopsies	
   from	
   digital	
   dermatitis	
   lesions.	
  
Microbiology-­‐‑Uk	
  1998,	
  144:2459-­‐‑2467.	
  
Noiri	
   Y,	
   Li	
   L,	
   Ebisu	
   S:	
   The	
   localization	
   of	
   periodontal-­‐‑disease-­‐‑associated	
  
bacteria	
   in	
   human	
   periodontal	
   pockets.	
   Journal	
   of	
   Dental	
   Research	
   2001,	
  
80:1930-­‐‑1934.	
  
Swidsinski	
   A,	
   Loening-­‐‑Baucke	
   V,	
   Verstraelen	
   H,	
   Osowska	
   S,	
   Doerffel	
   Y:	
  
Biostructure	
   of	
   fecal	
   microbiota	
   in	
   healthy	
   subjects	
   and	
   patients	
   with	
  
chronic	
  idiopathic	
  diarrhea.	
  Gastroenterology	
  2008,	
  135:568-­‐‑579.	
  
Zijnge	
   V,	
   van	
   Leeuwen	
   MBM,	
   Degener	
   JE,	
   Abbas	
   F,	
   Thurnheer	
   T,	
   Gmur	
   R,	
  
Harmsen	
   HJM:	
   Oral	
   biofilm	
   architecture	
   on	
   natural	
   teeth.	
   Plos	
   One	
   2010,	
  
5:9.	
  
163	
  

	
  
12.	
  

	
  
13.	
  

	
  
14.	
  

	
  
15.	
  

	
  
16.	
  

	
  
17.	
  

	
  
18.	
  

	
  
19.	
  

Drescher	
  J,	
  Schlafer	
  S,	
  Schaudinn	
  C,	
  Riep	
  B,	
  Neumann	
  K,	
  Friedmann	
  A,	
  Petrich	
  
A,	
   Gobel	
   UB,	
   Moter	
   A:	
   Molecular	
   epidemiology	
   and	
   spatial	
   distribution	
   of	
  
Selenomonas	
  spp.	
  in	
  subgingival	
  biofilms.	
   European	
   Journal	
   of	
   Oral	
   Sciences	
  
2010,	
  118:466-­‐‑474.	
  
Da	
   Silva	
   S,	
   Robbe-­‐‑Masselot	
   C,	
   Raymond	
   A,	
   Mercade-­‐‑Loubiere	
   M,	
   Salvador-­‐‑
Cartier	
  C,	
  Ringot	
  B,	
  Leonard	
  R,	
  Fourquaux	
  I,	
  Ait-­‐‑Belgnaoui	
  A,	
  Loubiere	
  P,	
  et	
  al:	
  
Spatial	
   localization	
   and	
   binding	
   of	
   the	
   probiotic	
   Lactobacillus	
   farciminis	
  
to	
   the	
   rat	
   intestinal	
   mucosa:	
   influence	
   of	
   chronic	
   stress.	
   Plos	
   One	
   2015,	
  
10:16.	
  
Al-­‐‑Ahmad	
   A,	
   Wunder	
   A,	
   Auschill	
   TM,	
   Follo	
   M,	
   Braun	
   G,	
   Hellwig	
   E,	
   Arweiler	
   NB:	
  
The	
   in	
   vivo	
   dynamics	
   of	
   Streptococcus	
   spp.,	
   Actinomyces	
   naeslundii,	
  
Fusobacterium	
  nucleatum	
  and	
  Veillonella	
  spp.	
  in	
  dental	
  plaque	
  biofilm	
  as	
  
analysed	
   by	
   five-­‐‑colour	
   multiplex	
   fluorescence	
   in	
   situ	
   hybridization.	
  
Journal	
  of	
  Medical	
  Microbiology	
  2007,	
  56:681-­‐‑687.	
  
Dige	
  I,	
  Nilsson	
  H,	
  Kilian	
  M,	
  Nyvad	
  B:	
  In	
  situ	
  identification	
  of	
  Streptococci	
  and	
  
other	
   bacteria	
   in	
   initial	
   dental	
   biofilm	
   by	
   confocal	
   laser	
   scanning	
  
microscopy	
  and	
  fluorescence	
  in	
  situ	
  hybridization.	
   European	
  Journal	
  of	
  Oral	
  
Sciences	
  2007,	
  115:459-­‐‑467.	
  
Valm	
   AM,	
   Welch	
   JLM,	
   Rieken	
   CW,	
   Hasegawa	
   Y,	
   Sogin	
   ML,	
   Oldenbourg	
   R,	
  
Dewhirst	
   FE,	
   Borisy	
   GG:	
   Systems-­‐‑level	
   analysis	
   of	
   microbial	
   community	
  
organization	
   through	
   combinatorial	
   labeling	
   and	
   spectral	
   imaging.	
  
Proceedings	
  of	
  the	
  National	
  Academy	
  of	
  Sciences	
  of	
  the	
  United	
  States	
  of	
  America	
  
2011,	
  108:4152-­‐‑4157.	
  
Dritz	
   SS,	
   Changappa	
   MM,	
   Nelssen	
   JL,	
   Tokach	
   MD,	
   Goodband	
   RD,	
   Nietfeld	
   JC,	
  
Staats	
   JJ:	
   Growth	
   and	
   microbial	
   flora	
   of	
   nonmedicated,	
   segregated,	
   early	
  
weaned	
   pigs	
   from	
   a	
   commercial	
   swine	
   operation.	
   Journal	
  of	
  the	
  American	
  
Veterinary	
  Medical	
  Association	
  1996,	
  208:711-­‐‑715.	
  
Torremorell	
   M,	
   Calsamiglia	
   M,	
   Pijoan	
   C:	
   Colonization	
   of	
   suckling	
   pigs	
   by	
  
Streptococcus	
   suis	
   with	
   particular	
   reference	
   to	
   pathogenic	
   serotype	
   2	
  
strains.	
   Canadian	
   Journal	
   of	
   Veterinary	
   Research-­‐‑Revue	
   Canadienne	
   De	
  
Recherche	
  Veterinaire	
  1998,	
  62:21-­‐‑26.	
  
Swildens	
   B,	
   Nielen	
   M,	
   Wisselink	
   HJ,	
   Verheijden	
   JHM,	
   Stegeman	
   JA:	
   Elimination	
  
of	
  strains	
  of	
  Streptococcus	
  suis	
  serotype	
  2	
  from	
  the	
  tonsils	
  of	
  carrier	
  sows	
  
by	
  combined	
  medication	
  and	
  vaccination.	
   Veterinary	
  Record	
  2007,	
   160:619-­‐‑
621.	
  

	
  

	
  

164	
  

20.	
  
21.	
  
	
  
22.	
  
	
  
23.	
  

	
  
24.	
  
	
  
25.	
  
	
  
26.	
  
	
  
27.	
  
	
  
28.	
  

	
  
29.	
  
	
  
30.	
  
	
  

	
  

Alexander	
   TJL,	
   Thornton	
   K,	
   Boon	
   G,	
   Gush	
   AF,	
   Lysons	
   RJ:	
   Medicated	
  early	
  
weaning	
  to	
  obtain	
  pigs	
  free	
  from	
  pathogens	
  endemic	
  in	
  the	
  herd	
  of	
  origin.	
  
Veterinary	
  Record	
  1980,	
  106:114-­‐‑119.	
  
Peña-­‐‑Cortes	
   LC,	
   LeVeque	
   RM,	
   Funk	
   J,	
   Marsh	
   TL,	
   Mulks	
   MH:	
   Development	
   of	
  
the	
   tonsillar	
   microbiome	
   in	
   pigs	
   from	
   newborn	
   through	
   weaning.	
  	
  
(University	
  MS	
  ed.;	
  2017.	
  
Peña-­‐‑Cortes	
   LC,	
   LeVeque	
   RM,	
   Funk	
   J,	
   Marsh	
   TL,	
   Mulks	
   MH:	
   Development	
   of	
  
tonsillar	
   microbiome	
   in	
   pigs	
   from	
   newborn	
   to	
   market	
   age.	
  	
  (University	
  MS	
  
ed.;	
  2017.	
  
Amann	
   RI,	
   Binder	
   BJ,	
   Olson	
   RJ,	
   Chisholm	
   SW,	
   Devereux	
   R,	
   Stahl	
   DA:	
  
Combination	
   of	
   16S	
   rRNA-­‐‑targeted	
   oligonucleotide	
   probes	
   with	
   flow	
  
cytometry	
   for	
   analyzing	
   mixed	
   microbial	
   populations.	
   Applied	
   and	
  
Environmental	
  Microbiology	
  1990,	
  56:1919-­‐‑1925.	
  
Wallner	
   G,	
   Amann	
   R,	
   Beisker	
   W:	
   Optimizing	
   fluorescent	
   in	
   situ	
  
hybridization	
   with	
   rRNA-­‐‑targeted	
   oligonucleotide	
   probes	
   for	
   flow	
  
cytometric	
  identification	
  of	
  microorganisms.	
  Cytometry	
  1993,	
  14:136-­‐‑143.	
  
Paster	
   BJ,	
   Bartoszyk	
   IM,	
   Dewhirst	
   FE:	
   Identification	
   of	
   oral	
   streptococci	
  
using	
   PCR-­‐‑based,	
   reverse-­‐‑capture,	
   checkerboard	
   hybridization.	
   Methods	
  
in	
  Cell	
  Science	
  1998,	
  20:223-­‐‑231.	
  
Loy	
   A,	
   Maixner	
   F,	
   Wagner	
   M,	
   Horn	
   M:	
   probeBase	
   -­‐‑	
   an	
   online	
   resource	
   for	
  
rRNA-­‐‑targeted	
   oligonucleotide	
   probes:	
   new	
   features	
   2007.	
   Nucleic	
   Acids	
  
Research	
  2007,	
  35:D800-­‐‑D804.	
  
Ludwig	
  W,	
  Strunk	
  O,	
  Westram	
  R,	
  Richter	
  L,	
  Meier	
  H,	
  Yadhukumar,	
  Buchner	
  A,	
  
Lai	
   T,	
   Steppi	
   S,	
   Jobb	
   G,	
   et	
   al:	
   ARB:	
   a	
   software	
   environment	
   for	
   sequence	
  
data.	
  Nucleic	
  Acids	
  Research	
  2004,	
  32:1363-­‐‑1371.	
  
Cole	
   JR,	
   Wang	
   Q,	
   Fish	
   JA,	
   Chai	
   BL,	
   McGarrell	
   DM,	
   Sun	
   YN,	
   Brown	
   CT,	
   Porras-­‐‑
Alfaro	
   A,	
   Kuske	
   CR,	
   Tiedje	
   JM:	
   Ribosomal	
  Database	
  Project:	
  data	
  and	
  tools	
  
for	
   high	
   throughput	
   rRNA	
   analysis.	
   Nucleic	
  Acids	
  Research	
  2014,	
   42:D633-­‐‑
D642.	
  
Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Defining	
   the	
   "core	
   microbiome"	
   of	
   the	
   microbial	
   communities	
   in	
   the	
  
tonsils	
  of	
  healthy	
  pigs.	
  Bmc	
  Microbiology	
  2012,	
  12:14.	
  
Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Microbial	
   communities	
   in	
   the	
   tonsils	
   of	
   healthy	
   pigs.	
   Veterinary	
  
Microbiology	
  2011,	
  147:346-­‐‑357.	
  

165	
  

31.	
  
32.	
  
	
  
33.	
  
	
  
34.	
  
	
  
35.	
  

	
  
36.	
  

	
  
37.	
  

	
  
38.	
  

	
  
39.	
  
	
  
40.	
  

Casteleyn	
  C,	
  Cornelissen	
  M,	
  Simoens	
  P,	
  Van	
  Den	
  Broeck	
  W:	
  Ultramicroscopic	
  
examination	
  of	
  the	
  ovine	
  tonsillar	
  epithelia.	
  Anatomical	
  Record-­‐‑Advances	
  in	
  
Integrative	
  Anatomy	
  and	
  Evolutionary	
  Biology	
  2010,	
  293:879-­‐‑889.	
  
McCowan	
   RP,	
   Cheng	
   KJ,	
   Costerton	
   JW:	
   Colonization	
   of	
   a	
   portion	
   of	
   the	
  
bovine	
  tongue	
  by	
  unusual	
  filamentous	
  bacteria.	
  Applied	
  and	
  Environmental	
  
Microbiology	
  1979,	
  37:1224-­‐‑1229.	
  
Swidsinski	
  A:	
  Standards	
   for	
   bacterial	
   identification	
   by	
   fluorescence	
   in	
   situ	
  
hybridization	
   within	
   eukaryotic	
   tissue	
   using	
   ribosornal	
   rRNA-­‐‑based	
  
probes.	
  Inflammatory	
  Bowel	
  Diseases	
  2006,	
  12:824-­‐‑826.	
  
Moter	
   A,	
   Gobel	
   UB:	
   Fluorescence	
   in	
   situ	
   hybridization	
   (FISH)	
   for	
   direct	
  
visualization	
   of	
   microorganisms.	
   Journal	
   of	
   Microbiological	
   Methods	
   2000,	
  
41:85-­‐‑112.	
  
Swidsinski	
   A,	
   Goektas	
   O,	
   Bessler	
   C,	
   Loening-­‐‑Baucke	
   V,	
   Hale	
   LP,	
   Andree	
   H,	
  
Weizenegger	
   M,	
   Hoelzl	
   M,	
   Scherer	
   H,	
   Lochs	
   H:	
   Spatial	
   organisation	
   of	
  
microbiota	
   in	
   quiescent	
   adenoiditis	
   and	
   tonsillitis.	
   Journal	
   of	
   Clinical	
  
Pathology	
  2007,	
  60:253-­‐‑260.	
  
Hogardt	
   M,	
   Trebesius	
   K,	
   Geiger	
   AM,	
   Hornef	
   M,	
   Rosenecker	
   J,	
   Heesemann	
   J:	
  
Specific	
   and	
   rapid	
   detection	
   by	
   fluorescent	
   in	
   situ	
   hybridization	
   of	
  
bacteria	
  in	
  clinical	
  samples	
  obtained	
  from	
  cystic	
  fibrosis	
  patients.	
  Journal	
  
of	
  Clinical	
  Microbiology	
  2000,	
  38:818-­‐‑825.	
  
Stepinska	
   M,	
   Olszewska-­‐‑Sosinska	
   O,	
   Lau-­‐‑Dworak	
   M,	
   Zielnik-­‐‑Jurkiewicz	
   B,	
  
Trafny	
   EA:	
   Identification	
   of	
   intracellular	
   bacteria	
   in	
   adenoid	
   and	
   tonsil	
  
tissue	
   specimens:	
   the	
   efficiency	
   of	
   culture	
   versus	
   fluorescent	
   in	
   situ	
  
hybridization	
  (FISH).	
  Current	
  Microbiology	
  2014,	
  68:21-­‐‑29.	
  
Fuchs	
   BM,	
   Wallner	
   G,	
   Beisker	
   W,	
   Schwippl	
   I,	
   Ludwig	
   W,	
   Amann	
   R:	
   Flow	
  
cytometric	
  analysis	
  of	
  the	
  in	
  situ	
  accessibility	
  of	
  Escherichia	
  coli	
  16S	
  rRNA	
  
for	
   fluorescently	
   labeled	
   oligonucleotide	
   probes.	
   Applied	
   and	
  
Environmental	
  Microbiology	
  1998,	
  64:4973-­‐‑4982.	
  
Swidsinski	
   A,	
   Loening-­‐‑Baucke	
   V:	
   Functional	
   structure	
   of	
   intestinal	
  
microbiota	
   in	
   health	
   and	
   disease.	
   Human	
   Microbiota:	
   How	
   Microbial	
  
Communities	
  Affect	
  Health	
  and	
  Disease	
  2013:211-­‐‑253.	
  
Baykan	
   M,	
   Celik	
   I,	
   Gezici	
   M,	
   Donmez	
   HH,	
   Eken	
   E,	
   Sur	
   E,	
   Ozkan	
   Y:	
   A	
   light	
  
microscopic	
   study	
   on	
   the	
   uptake	
   and	
   transportation	
   route	
   of	
   carbon	
  
particles	
   in	
   the	
   canine	
   palatine	
   tonsil.	
   Revue	
  De	
  Medecine	
  Veterinaire	
  2001,	
  
152:709-­‐‑715.	
  

	
  

	
  

166	
  

41.	
  
	
  
42.	
  
	
  
43.	
  

	
  

Fredriksen	
   F,	
   Raisanen	
   S,	
   Myklebust	
   R,	
   Stenfors	
   LE:	
   Bacterial	
   adherence	
   to	
  
the	
   surface	
   and	
   isolated	
   cell	
   epithelium	
   of	
   the	
   palatine	
   tonsils.	
   Acta	
  Oto-­‐‑
Laryngologica	
  1996,	
  116:620-­‐‑626.	
  
Lilja	
   M,	
   Silvola	
   J,	
   Raisanen	
   S,	
   Stenfors	
   LE:	
   Where	
   are	
   the	
   receptors	
   for	
  
Streptococcus	
   pyogenes	
   located	
   on	
   the	
   tonsillar	
   surface	
   epithelium?	
  
International	
  Journal	
  of	
  Pediatric	
  Otorhinolaryngology	
  1999,	
  50:37-­‐‑43.	
  
Kania	
  RE,	
  Lamers	
  GEM,	
  Vonk	
  MJ,	
  Huy	
  PTB,	
  Hiemstra	
  PS,	
  Bloemberg	
  GV,	
  Grote	
  
JJ:	
   Demonstration	
   of	
   bacterial	
   cells	
   and	
   glycocalyx	
   in	
   biofilms	
   on	
   human	
  
tonsils.	
  Archives	
  of	
  Otolaryngology-­‐‑Head	
  &	
  Neck	
  Surgery	
  2007,	
  133:115-­‐‑121.	
  
	
  

167	
  

CHAPTER	
  5.	
  SUMMARY	
  AND	
  FUTURE	
  DIRECTIONS	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

168	
  

SUMMARY	
  
	
  
Tonsils	
   are	
   recognized	
   as	
   one	
   of	
   the	
   first	
   tissues	
   that	
   can	
   be	
   colonized	
   by	
   pathogens	
  
entering	
   the	
   respiratory	
   or	
   oral	
   pathway.	
   Tonsils	
   can	
   be	
   used	
   as	
   a	
   primary	
   site	
   of	
  
replication	
  for	
  some	
  bacterial	
  and	
  viral	
  agents	
  that	
  can	
  be	
  either	
  host-­‐‑specific	
  or	
  zoonotic	
  
organisms.	
   Some	
   of	
   these	
   bacterial	
   agents,	
   such	
   as	
   Streptococcus	
   suis,	
   are	
   isolated	
  
continuously	
   from	
   pig	
   tonsils	
   and	
   deserve	
   special	
   attention	
   especially	
   because	
   of	
   its	
  
zoonotic	
  	
  potential	
  and	
  economic	
  impact.	
  Further,	
  it	
  is	
  recognized	
  that	
  the	
  tissue-­‐‑resident	
  
bacterial	
  microbiome	
  may	
  play	
  an	
  important	
  role	
  in	
  host	
  protection	
  against	
  colonization	
  by	
  
pathogenic	
  microorganisms.	
  The	
  goal	
  of	
  this	
  study	
  was	
  to	
  characterize	
  the	
  development	
  of	
  
the	
   composition	
   and	
   structure	
   of	
   the	
   tonsillar	
   microbiome	
   in	
   pigs	
   from	
   birth	
   through	
  
market	
   age,	
   thus	
   laying	
   the	
   foundation	
   for	
   future	
   studies	
   concerning	
   the	
   interaction	
   of	
  
pathogens	
  with	
  resident	
  microbial	
  communities	
  in	
  the	
  tonsils.	
  
Studies	
   of	
   the	
   tonsillar	
   microbiome	
   of	
   pigs,	
   using	
   a	
   culture-­‐‑independent	
   approach	
  
based	
   on	
   the	
   use	
   of	
   cloning	
   libraries,	
   have	
   identified	
   that	
   in	
   18	
   to	
   20	
   week	
   old	
   pigs	
   from	
  
two	
   different	
   healthy	
   herds,	
   ~74%	
   of	
   the	
   microbiome	
   	
   was	
   composed	
   of	
   members	
   of	
   the	
  
families	
   Pasteurellaceae,	
   Porphyromonadaceae,	
   Bacteroidaceae	
   and	
   Prevotellaceae	
   [1].	
  
However,	
   using	
   a	
   454-­‐‑pyrosequencing	
   approach	
   to	
   study	
   the	
   same	
   samples	
   and	
   new	
  
samples	
  from	
  the	
  same	
  farm,	
  it	
  was	
  found	
  that	
  ~	
  90.4%	
  of	
  the	
  microbiome	
  was	
  composed	
  
by	
   members	
   of	
   families	
   Pasteurellaceae,	
   Moraxellaceae,	
   Fusobacteriaceae,	
   Veillonellaceae,	
  
Neisseriaceae,	
   Peptostreptococcaceae,	
   Enterobacteriaceae	
   and	
   Streptococcaceae	
   [2].	
  
Notwhitstanding	
  these	
  previous	
  reports	
  studying	
  the	
  microbiome	
  in	
  pig	
  tonsils,	
  there	
  are	
  
no	
  studies	
  focused	
  on	
  characterizing	
  the	
  development	
  of	
  the	
  tonsil	
  microbiome	
  in	
  pigs.	
  

	
  

169	
  

Based	
  on	
  studies	
  conducted	
  in	
  pigs	
  and	
  humans,	
  it	
  has	
  been	
  suggested	
  that	
  there	
  is	
  a	
  	
  
gradual	
   and	
   successional	
   process	
   in	
   the	
   development	
   of	
   the	
   intestinal	
   microbiome	
   [3-­‐‑6].	
  
We	
   hypothesized	
   that	
   the	
   development	
   of	
   tonsillar	
   microbial	
   communities	
   in	
   pigs	
   also	
  
follows	
   a	
   temporal	
   succession	
   process.	
   Since	
   there	
   are	
   no	
   published	
   reports	
   characterizing	
  
the	
   development	
   of	
   microbial	
   communities	
   in	
   pig	
   tonsils,	
   we	
   decided	
   to	
   study	
   and	
  
understand	
   the	
   development	
   of	
   the	
   microbiome	
   in	
   pig	
   tonsils,	
   in	
   continuance	
   of	
   the	
  
previous	
  work	
  done	
  in	
  this	
  field	
  by	
  our	
  laboratory.	
  
Our	
   lab	
   has	
   previously	
   developed	
   and	
   validated	
   a	
   non-­‐‑invasive	
   method	
   to	
   collect	
  
tonsil	
   samples	
   and	
   used	
   this	
   method	
   to	
   describe	
   the	
   core	
   tonsil	
   microbiome	
   in	
   grower-­‐‑
finisher	
  pigs.	
  The	
  use	
  of	
  this	
  validated	
  methodology	
  allowed	
  us	
  to	
  follow	
  the	
  microbiome	
  
development	
  of	
  each	
  animal	
  continuously	
  from	
  birth	
  (day	
  0)	
  up	
  to	
  market	
  age	
  (17	
  weeks).	
  
Our	
  initial	
  approach	
  was	
  to	
  analyze	
  the	
  development	
  of	
  tonsillar	
  microbiome	
  in	
  pigs	
  during	
  
the	
  first	
  few	
  weeks	
  of	
  age,	
  giving	
  special	
  attention	
  to	
  the	
  possible	
  sources	
  of	
  the	
  members	
  
of	
   the	
   tonsil	
   community	
   and	
   to	
   the	
   challenging	
   time	
   of	
   weaning	
   and	
   the	
   effects	
   of	
   post	
  
weaning	
   in	
   the	
   tonsillar	
   microbiome	
   communities.	
   We	
   found	
   that	
   the	
   microbiome	
   in	
  
piglets,	
   collected	
   a	
   few	
   hour	
   post	
   birth	
   (PB),	
   showed	
   organisms	
   including	
   Streptococcus,	
  
Staphylococcus,	
  Moraxella,	
  Rothia,	
  and	
  Pasteurellaceae	
  (OTUs	
  002,	
  009,	
  003,	
  007,	
  and	
  001	
  
respectively)	
  as	
  the	
  most	
  abundant	
  in	
  tonsils	
  at	
  that	
  time.	
  We	
  compared	
  these	
  with	
  OTUs	
  
found	
   in	
   maternal	
   vaginal,	
   teat	
   skin,	
   tonsil	
   and	
   fecal	
   samples	
   and	
   traced	
   that,	
   piglets	
  
acquired	
   Pasteurellaceae	
   and	
   Streptococcus	
   from	
   the	
   sow	
   vaginal	
   tract	
   most	
   likely	
   during	
  
parturition,	
  while	
  	
  sow	
  teat	
  skin	
  (or	
  milk,	
  which	
  we	
  did	
  not	
  sample)	
  was	
  the	
  likely	
  source	
  
for	
   Moraxella,	
   Staphylococcus	
   and	
   Rothia.	
   	
   These	
   results	
   are	
   in	
   agreement	
   with	
   studies	
   of	
  
Mandar	
   and	
   Mikelsaar	
   [5],	
   which	
   compared	
   vaginal	
   flora	
   of	
   human	
   mothers	
   with	
   the	
  

	
  

170	
  

microorganisms	
   that	
   initially	
   colonize	
   the	
   external	
   ear	
   canal	
   in	
   their	
   newborns	
   and	
  
concluded	
   that	
   there	
   is	
   a	
   significant	
   influence	
   of	
   the	
   vaginal	
   microflora	
   in	
   the	
   initial	
  
microbial	
   population	
   found	
   in	
   the	
   newborns.	
   Streptococcus	
   and	
   Staphylococcus	
   as	
   well	
   as	
  
other	
  aerobes	
  have	
  been	
  commonly	
  reported	
  as	
  first	
  colonizers	
  in	
  infants,	
  possibly	
  derived	
  
from	
  maternal	
  sources,	
  such	
  as	
  breast	
  milk	
  and	
  vagina.	
  	
  
Our	
   results	
   also	
   demonstrated	
   a	
   strong	
   litter	
   effect	
   in	
   the	
   initial	
   development	
   of	
  
tonsillar	
   microbiome	
   in	
   pigs,	
   followed	
   by	
   a	
   gradual	
   successional	
   development,	
   where,	
   by	
  
the	
   third	
   week,	
   the	
   microbiomes	
   of	
   all	
   piglets	
   was	
   highly	
   similar.	
   The	
   piglet	
   tonsil	
  
microbiomes	
   were	
   clustered	
   tightly	
   by	
   litter	
   at	
   birth	
   and	
   at	
   week	
   1.	
   However,	
   this	
  
clustering	
   was	
   no	
   longer	
   apparent	
   by	
   3	
   weeks	
   of	
   age.	
   In	
   the	
   successional	
   development,	
  
some	
   organisms	
   found	
   in	
   high	
   proportions	
   initially	
   in	
   piglets	
   PB,	
   such	
   as	
   Staphylococcus	
  
and	
  Micrococcaceae,	
  decreased	
  dramatically	
  within	
  the	
  next	
  2	
  weeks.	
  However,	
  members	
  of	
  
the	
  families	
  Pasteurellaceae	
  and	
  Moraxellaceae,	
  increased	
  their	
  levels	
  in	
  parallel,	
  while	
  the	
  
levels	
  of	
  others	
  decreased	
  mild	
  to	
  moderate,	
  as	
  the	
  case	
  of	
  Streptococcaceae.	
  Furthermore,	
  
there	
   were	
   also	
   several	
   transitory	
   OTUs	
   or	
   families	
   that	
   appeared	
   and	
   disappeared	
   at	
  
specific	
   time	
   points	
   such	
   as	
   Porphyromonadaceae,	
   Prevotellaceae,	
   and	
   Flavobacteriaceae	
  
that	
   appeared	
   at	
   week	
   1	
   and	
   increased	
   slightly	
   over	
   the	
   next	
   2	
   weeks	
   or	
   such	
   as	
  
Fusobacteriaceae	
   and	
   Leptotrichiaceae,	
   that	
   appeared	
   for	
   two	
   weeks	
   and	
   then	
   almost	
  
disappeared.	
   	
   Similar	
   results	
   in	
   the	
   succession	
   and	
   shifts	
   in	
   the	
   population	
   assembly	
   which	
  
seemed	
  to	
  stabilize	
  over	
  time,	
  were	
  reported	
  in	
  the	
  development	
  of	
  intestinal	
  microbiome	
  
in	
  humans	
  and	
  pigs	
  [5,	
  6].	
  
Our	
   work	
   has	
   demonstrated	
   that	
   weaning	
   and	
   the	
   significant	
   stress	
   events	
  
associated	
  with	
  it,	
  such	
  as	
  environmental,	
  social	
  and	
  feed	
  changes,	
  were	
  associated	
  with	
  a	
  

	
  

171	
  

major	
   shift	
   in	
   the	
   microbiome.	
   These	
   results	
   are	
   in	
   concordance	
   with	
   studies	
   of	
  
development	
   of	
   intestinal	
   microbiome	
   which	
   demonstrated	
   that	
   transition	
   from	
   nursery	
   to	
  
weaning	
  is	
  associated	
  with	
  a	
  significant	
  change	
  in	
  the	
  intestinal	
  microbiota	
  [8,	
  9].	
  
After	
   having	
   identified	
   that	
   there	
   is	
   a	
   succession	
   in	
   the	
   microbiome	
   in	
   the	
   first	
  
weeks	
  of	
  life	
  and	
  that	
  weaning	
  generated	
  significant	
  shifts	
  in	
  the	
  microbiome,	
  we	
  followed	
  
how	
   the	
   microbiome	
   develops	
   further	
   during	
   the	
   life	
   of	
   pigs,	
   particularly	
   where	
   they	
   are	
  
exposed	
   to	
   different	
   challenges	
   associated	
   with	
   normal	
   management	
   practices	
   in	
   a	
   swine	
  
farm.	
   We	
   identified	
   four	
   major	
   challenging	
   conditions	
   that	
   were	
   related	
   with	
   perceptible	
  
changes	
   in	
   the	
   relative	
   abundance	
   of	
   the	
   tonsillar	
   microbiome	
   populations.	
   The	
   first	
  
challenge	
   was	
   previously	
   described	
   as	
   weaning,	
   which	
   occurred	
   in	
   parallel	
   with	
   the	
  
vaccination	
   of	
   piglets,	
   movement	
   to	
   a	
   nursery	
   room,	
   	
   and	
   introduction	
   to	
   a	
   solid	
   diet	
   based	
  
on	
  pellet	
  ration	
  supplemented	
  with	
  Carbadox®.	
  The	
  second	
  challenge	
  was	
  associated	
  with	
  
withdrawal	
   of	
   Carbadox®	
   supplementation	
   and	
   a	
   shift	
   to	
   feeding	
   with	
   a	
   ground	
   ration	
  
supplemented	
  with	
  Tylan®.	
  The	
  third	
  challenge	
  was	
  associated	
  with	
  the	
  move	
  to	
  a	
  finishing	
  
room,	
   as	
   well	
   as	
   the	
   separation	
   from	
   litter	
   mates	
   and	
   discontinuation	
   of	
   Tylan®	
  
supplementation	
  from	
  the	
  ground	
  ration.	
  The	
  final	
  identified	
  challenge	
  was	
  associated	
  with	
  
another	
  move	
  to	
  the	
  	
  final	
  finishing	
  room.	
  	
  
The	
  identified	
  challenges	
  were	
  associated	
  with	
  shifts	
  in	
  the	
  composition	
  and	
  relative	
  
percentages	
   of	
   families	
   of	
   the	
   microbiota.	
   These	
   challenges	
   frequently	
   occurred	
   as	
  
combinations	
   of	
   several	
   stress	
   and	
   rarely	
   involved	
   a	
   singular	
   change	
   in	
   the	
   management	
   of	
  
the	
   pigs.	
   Nevertheless,	
   supplementation	
   with	
   Carbadox®	
   or	
   Tylan®	
   have	
   been	
   associated	
  
with	
  shifts	
  in	
  the	
  distribution	
  of	
  microbial	
  communities	
  in	
  the	
  intestine	
  [10,	
  11].	
  Similarly,	
  
changes	
   in	
   the	
   environment	
   where	
   pigs	
   grow	
   [12,	
   13],	
   and	
   changes	
   in	
   their	
   diet	
   [8,	
   14,	
   15],	
  

	
  

172	
  

have	
  been	
  associated	
  also	
  with	
  shifts	
  in	
  the	
  microbial	
  communities	
  in	
  intestine.	
  However,	
  
there	
  are	
  not	
  specific	
  studies	
  that	
  refer	
  to	
  changes	
  experienced	
  by	
  microbial	
  communities	
  
of	
  pig	
  tonsils	
  when	
  the	
  aforementioned	
  conditions	
  are	
  present.	
  	
  
The	
   study	
   of	
   microbial	
   communities	
   in	
   tonsils	
   from	
   newborn	
   to	
   market	
   age	
  
confirmed	
   that	
   there	
   is	
   a	
   succession	
   in	
   the	
   development	
   of	
   the	
   communities	
   through	
  
different	
  ages	
  of	
  the	
  pigs.	
  However,	
  there	
  were	
  some	
  bacterial	
  families	
  that	
  dominated	
  the	
  
tonsillar	
   microbiome	
   through-­‐‑out	
   the	
   life	
   of	
   pigs	
   such	
   as	
   members	
   of	
   Pasteurellaceae,	
  
Streptococcaceae	
   and	
   Moraxellaceae,	
   although	
   their	
   relative	
   abundance	
   experienced	
  
changes	
   after	
   the	
   challenging	
   events.	
   These	
   findings	
   are	
   supported	
   by	
   studies	
   done	
   in	
   fecal	
  
microbiome	
  in	
  pigs,	
  reporting	
  that	
  some	
  phyla	
  dominate	
  the	
  microbiome	
  regardless	
  of	
  the	
  
age	
   [16].	
   On	
   other	
   hand,	
   we	
   found	
   that	
   older	
   pigs	
   (16	
   weeks)	
   tended	
   to	
   have	
   a	
   microbiome	
  
more	
   similar	
   to	
   the	
   microbiome	
   of	
   older	
   animals,	
   for	
   example	
   sows.	
   Similarly,	
   studies	
   in	
  
human	
   intestinal	
   microbiota	
   have	
   shown	
   that	
   as	
   infants	
   age,	
   their	
   microbiota	
   was	
   more	
  
similar	
  to	
  adults	
  microbiota	
  [3,	
  5].	
  
Finally,	
   using	
   FISH	
   and	
   SEM,	
   we	
   confirmed	
   the	
   presence	
   of	
   diverse	
   bacterial	
  
communities	
   on	
   the	
   surface	
   of	
   pig	
   tonsils	
   and	
   how	
   these	
   changed	
   through	
   their	
   life.	
  
Further,	
  we	
  confirmed	
  the	
  presence	
  of	
  members	
  of	
  genus	
  Streptococcus	
  on	
  tonsillar	
  surface	
  
of	
   pigs.	
   Finally,	
   	
   we	
   have	
   also	
   identified	
   morphological	
   changes	
   in	
   the	
   appearance	
   of	
   pig	
  
tonsils	
  and	
  the	
  bacterial	
  members	
  of	
  the	
  tonsil	
  communities	
  throughout	
  the	
  life	
  of	
  pigs.	
  	
  
	
  
	
  
	
  
	
  

	
  

173	
  

FUTURE	
  DIRECTIONS	
  
	
  
The	
  results	
  of	
  this	
  study	
  demonstrated	
  that	
  there	
  is	
  a	
  succession	
  in	
  the	
  development	
  
of	
   tonsillar	
   communities	
   in	
   pigs.	
   We	
   identified	
   the	
   presence	
   of	
   some	
   bacterial	
   families	
   all	
  
through	
   the	
   life	
   of	
   pigs	
   while	
   others	
   fluctuate	
   or	
   appeared	
   and	
   disappeared	
   sporadically.	
  	
  
We	
   can	
   only	
   speculate	
   on	
   the	
   initial	
   sources	
   of	
   tonsillar	
   microbiome.	
   We	
   don´t	
   know	
   the	
  
effect	
   of	
   the	
   use	
   of	
   	
   Carbadox 	
   and	
   Tylan 	
   or	
   other	
   antibiotics	
   in	
   the	
   development	
   and	
  
®

®

succession	
  of	
  the	
  tonsillar	
  communities.	
  More	
  importantly,	
  we	
  don´t	
  know	
  the	
  role	
  played	
  
by	
  normal	
  microbiota	
  in	
  the	
  acquisition	
  of	
  a	
  pathogenic	
  microorganism.	
  We	
  may	
  have	
  also	
  
ignored	
   the	
   exact	
   role	
   played	
   by	
   some	
   members	
   of	
   the	
   microbial	
   communities	
   in	
   the	
  
development	
  of	
  microbiome	
  structure	
  and	
  in	
  the	
  acquisition	
  of	
  potential	
  pathogens.	
  Finally,	
  
we	
  	
  do	
  not	
  know	
  if	
  the	
  arrival	
  of	
  these	
  pathogens	
  can	
  be	
  prevented	
  by	
  the	
  use	
  of	
  probiotics	
  
or	
  specific	
  changes	
  in	
  the	
  management	
  practices.	
  All	
  these	
  questions	
  will	
  be	
  addressed	
  in	
  
future	
  works	
  however;	
  initially	
  we	
  want	
  to	
  address	
  the	
  following	
  topics:	
  
Future	
  direction	
  1.	
   Follow	
  the	
  development	
  of	
  the	
  tonsil	
  microbiome	
  in	
  a	
  second	
  
herd,	
  possibly	
  one	
  with	
  a	
  chronic	
  problem	
  such	
  as	
  Salmonella.	
  
Future	
  direction	
  2.	
  Examine	
  effect	
  of	
  acquisition	
  of	
  a	
  pathogen	
  known	
  to	
  colonize	
  
the	
   tonsils,	
   such	
   as	
   Actinobacilllus	
   pleuropneumoniae	
   or	
   Salmonella	
   enterica,	
   on	
   the	
  
composition	
  of	
  the	
  tonsil	
  microbiome.	
  
	
  
	
  
	
  
	
  

	
  

174	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
REFERENCES	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

175	
  

1.	
  
	
  
2.	
  
	
  
3.	
  
	
  
4.	
  

	
  
5.	
  
	
  
6.	
  
	
  
7.	
  
	
  
8.	
  
	
  
9.	
  
	
  
10.	
  
	
  
11.	
  

REFERENCES	
  
	
  
	
  
	
  
Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Microbial	
   communities	
   in	
   the	
   tonsils	
   of	
   healthy	
   pigs.	
   Veterinary	
   Microbiology	
  
2011,	
  147:346-­‐‑357.	
  
Lowe	
   BA,	
   Marsh	
   TL,	
   Isaacs-­‐‑Cosgrove	
   N,	
   Kirkwood	
   RN,	
   Kiupel	
   M,	
   Mulks	
   MH:	
  
Defining	
   the	
   "core	
   microbiome"	
   of	
   the	
   microbial	
   communities	
   in	
   the	
   tonsils	
   of	
  
healthy	
  pigs.	
  Bmc	
  Microbiology	
  2012,	
  12:14.	
  
Bokulich	
   NA,	
   Chung	
   J,	
   Battaglia	
   T,	
   Henderson	
   N,	
   Jay	
   M,	
   Li	
   H,	
   Lieber	
   AD,	
   Wu	
   F,	
   Perez-­‐‑
Perez	
   GI,	
   Chen	
   Y,	
   et	
   al:	
   Antibiotics,	
   birth	
   mode,	
   and	
   diet	
   shape	
   microbiome	
  
maturation	
  during	
  early	
  life.	
  Science	
  Translational	
  Medicine	
  2016,	
  8:13.	
  
Koenig	
  JE,	
  Spor	
  A,	
  Scalfone	
  N,	
  Fricker	
  AD,	
  Stombaugh	
  J,	
  Knight	
  R,	
  Angenent	
  LT,	
  Ley	
  
RE:	
  Succession	
  of	
  microbial	
  consortia	
  in	
  the	
  developing	
  infant	
  gut	
  microbiome.	
  
Proceedings	
  of	
  the	
  National	
  Academy	
  of	
  Sciences	
  of	
  the	
  United	
  States	
  of	
  America	
  2011,	
  
108:4578-­‐‑4585.	
  
Palmer	
   C,	
   Bik	
   EM,	
   DiGiulio	
   DB,	
   Relman	
   DA,	
   Brown	
   PO:	
   Development	
   of	
   the	
   human	
  
infant	
  intestinal	
  microbiota.	
  Plos	
  Biology	
  2007,	
  5:1556-­‐‑1573.	
  
Inoue	
   R,	
   Tsukahara	
   T,	
   Nakanishi	
   N,	
   Ushida	
   K:	
   Development	
   of	
   the	
   intestinal	
  
microbiota	
  in	
  the	
  piglet.	
  Journal	
  of	
  General	
  and	
  Applied	
  Microbiology	
  2005,	
  51:257-­‐‑
265.	
  
Mandar	
   R,	
   Mikelsaar	
   M:	
   Transmission	
   of	
   mother's	
   microflora	
   to	
   the	
   newborn	
   at	
  
birth.	
  Biology	
  of	
  the	
  Neonate	
  1996,	
  69:30-­‐‑35.	
  
Frese	
  SA,	
  Parker	
  K,	
  Calvert	
  CC,	
  Mills	
  DA:	
  Diet	
  shapes	
  the	
  gut	
  microbiome	
  of	
  pigs	
  
during	
  nursing	
  and	
  weaning.	
  Microbiome	
  2015,	
  3:10.	
  
Alain	
   B	
   Pajarillo	
   E,	
   Chae	
   J-­‐‑P,	
   Balolong	
   MP,	
   Bum	
   Kim	
   H,	
   Kang	
   D-­‐‑K:	
   Assessment	
   of	
  
fecal	
  bacterial	
  diversity	
  among	
  healthy	
  piglets	
  during	
  the	
  weaning	
  transition.	
  
The	
  Journal	
  of	
  general	
  and	
  applied	
  microbiology	
  2014,	
  60:140-­‐‑146.	
  
Looft	
  T,	
  Allen	
  HK,	
  Casey	
  TA,	
  Alt	
  DP,	
  Stanton	
  TB:	
  Carbadox	
  has	
  both	
  temporary	
  and	
  
lasting	
  effects	
  on	
  the	
  swine	
  gut	
  microbiota.	
  Frontiers	
  in	
  Microbiology	
  2014,	
  5:9.	
  
Kim	
   HB,	
   Borewicz	
   K,	
   White	
   BA,	
   Singer	
   RS,	
   Sreevatsan	
   S,	
   Tu	
   ZJ,	
   Isaacson	
   RE:	
  
Microbial	
   shifts	
   in	
   the	
   swine	
   distal	
   gut	
   in	
   response	
   to	
   the	
   treatment	
   with	
  
antimicrobial	
   growth	
   promoter,	
   tylosin.	
   Proceedings	
   of	
   the	
   National	
   Academy	
   of	
  
Sciences	
  of	
  the	
  United	
  States	
  of	
  America	
  2012,	
  109:15485-­‐‑15490.	
  

	
  
	
  

176	
  

12.	
  

	
  
13.	
  
	
  
14.	
  

	
  
15.	
  

	
  
16.	
  

	
  

Mulder	
  IE,	
  Schmidt	
  B,	
  Stokes	
  CR,	
  Lewis	
  M,	
  Bailey	
  M,	
  Aminov	
  RI,	
  Prosser	
  JI,	
  Gill	
  BP,	
  
Pluske	
   JR,	
   Mayer	
   CD,	
   et	
   al:	
   Environmentally-­‐‑acquired	
   bacteria	
   influence	
  
microbial	
   diversity	
   and	
   natural	
   innate	
   immune	
   responses	
   at	
   gut	
   surfaces.	
   Bmc	
  
Biology	
  2009,	
  7:20.	
  
Thompson	
  CL,	
  Wang	
  B,	
  Holmes	
  AJ:	
  The	
  immediate	
  environment	
  during	
  postnatal	
  
development	
   has	
   long-­‐‑term	
   impact	
   on	
   gut	
   community	
   structure	
   in	
   pigs.	
   Isme	
  
Journal	
  2008,	
  2:739-­‐‑748.	
  
Konstantinov	
  SR,	
  Zhu	
  WY,	
  Williams	
  BA,	
  Tamminga	
  S,	
  de	
  Vos	
  WM,	
  Akkermans	
  ADL:	
  
Effect	
  of	
  fermentable	
  carbohydrates	
  on	
  piglet	
  faecal	
  bacterial	
  communities	
  as	
  
revealed	
  by	
  denaturing	
  gradient	
  gel	
  electrophoresis	
  analysis	
  of	
  16S	
  ribosomal	
  
DNA.	
  Fems	
  Microbiology	
  Ecology	
  2003,	
  43:225-­‐‑235.	
  
Konstantinov	
  SR,	
  Favier	
  CF,	
  Zhu	
  WY,	
  Williams	
  BA,	
  Kluss	
  J,	
  Souffrant	
  WB,	
  de	
  Vos	
  WM,	
  
Akkermans	
   ADL,	
   Smidt	
   H:	
   Microbial	
   diversity	
   studies	
   of	
   the	
   porcine	
  
gastrointestinal	
   ecosystem	
   during	
   weaning	
   transition.	
   Animal	
   Research	
   2004,	
  
53:317-­‐‑324.	
  
Kim	
   HB,	
   Borewicz	
   K,	
   White	
   BA,	
   Singer	
   RS,	
   Sreevatsan	
   S,	
   Tu	
   ZJ,	
   Isaacson	
   RE:	
  
Longitudinal	
   investigation	
   of	
   the	
   age-­‐‑related	
   bacterial	
   diversity	
   in	
   the	
   feces	
   of	
  
commercial	
  pigs.	
  Veterinary	
  Microbiology	
  2011,	
  153:124-­‐‑133.	
  
	
  

177