I' “ | f ‘ '\ W ‘ . ' '9'. 'l'. J". ‘_ . " "'1 I """' ' ""‘”'.'. " ‘ ! ‘JI I." ‘ ‘lm tutu- “pangs
a: “ 9 I. ‘3 ‘ y 9 . I l l I ‘ 9" . '4'99 .1" ‘9 9'9“”: J 9 9““919-9’9‘99‘.L‘l'"'"" . '9‘ .t\f“'(-l r: ' :19,qu 9" "7‘ i“
,. .. ; I II ‘ II .I v .. 9H h: 9 I, 9.’ I >09 .9 9,, .. '9? H; .,_;'. 1- :‘rf~""£-r, I. -'
3" rs ‘ . '9 I . ‘ 9 w 9 R’ ' 9 ' 9 .I l,‘ I In". I. I‘ I‘9 .9 L9 l. ' . "'pv‘ - . -Y-:‘-9'.IJ1'§' :9- ‘.‘-.~*"‘
I 9.9-) I I 9 II.’ on '. .II,- I | b I Y‘v;"" “2’5 ’7‘ 5‘&
7' ‘ .-, .‘W. . . ." g , ; r , . db“ _‘." ~' 2;: _z”
| a " A. ' I u 4 I . . . f" ' — 7 . _I' _ I _-£7¢7.
t I .u‘ I v . I F 19 ' h ‘
. . .: H' ‘ - .
. ‘ ‘ I .. .2» -
. IIY \ I 9 "
' WI I I ; ' 4' a 7< V_#
. I 9 9 > . ‘ 9A. ‘ .
o I I .II
I _ 9 9 9 9 ‘ ‘ F
I ‘ \ ‘ ’ — TI I -—I .
I \ I v - ‘
‘ l I ' I_ — z ‘- - V
I I - ‘ ‘ "' ' —— ~ -
. .. . .:_ . . _ .
.9 . . . ' -, ‘ "-“ “ ‘
r. ‘ '0 -
I 9 I |: . ‘
r ‘I . _- "“ ‘ L .' '
0| - - "' b . «T; -’-
I . 'l ‘
0" . . I 9 .9 " ’9': ' A - t
I . . ‘ . —
' y I I ~ _~ - é -,
. .'. 0-1. v "' _ :‘Iz'; ‘
r . . v ' ‘
I 9 .h I i I 'l ‘ - --
l ‘ .-“. -
I - 0 O 3 ’ ~ _-
0
. , ..
. v . . . . »
9o 9 ' 9 i 9 A.
y . I , -- ‘
I . ' ~ ‘ ' ' " ' ' “
. I l . ' - .l
.. I
- . 9 n 9 _ ':c opt. - -
‘ ‘ r _ w- r
I ' ' I I‘ l h -
; 9
d t 9 .
‘ .
’ I . . ‘ 9" ..
U‘ D ‘ . v : 9-,. ,I ' ‘
I ' “9 u .. ‘ - ‘ " - :
: I! I3 I . _ 0 \"I'. ' '
-. ._.,. I . .
: . , '- rrrzn‘ ‘
z I ‘ ‘ ‘ ‘VY'. ’ _
. 'v' 'He' -. ,
‘9' y." _ , . .-.. I
.| -. "" 9"; ' :' 'Ij. .
‘ BeleE PYE DNEPHRIT 8 J-- V 'm‘;.-V .9! I . i . ‘
ll; . _ .9..-‘. 3 ' _ . _ ‘
9 | r I .‘99' . ' I . i ‘9: ‘-
. I. ‘ . ‘| I I. l . —‘ ."r‘. l'.l‘-. I a
g I . 'I‘ 4 " ‘ - I ‘ . I _
.. )I , 2‘3, ' ~ 15 . - "H "
I A I I I ,
" i. ' ' ""1 I . y.. . ~ .I'I I . ' a: . I _' \
, . D I 7..
,~: . the D we ‘ ' ' ' ‘ '
’ ‘ .
' . f as o . 1.‘ .
i \ _‘ . l. .. .
I VI ‘
I I I ‘3 4 '1 ' ._ I “.9 - "—
: II -- '. l
I -\ ., ." .
z . - ' I 9‘ " ' T
. . . I -- .,i . . - ‘ ‘A . .. ' .
:9 - .689 can 1m:- - '
1 iv. — ‘ ‘.I .. '.‘ , u ’8' ‘ -— 9' “ ' "' .-
y . . ;_ -;. l. ‘ I O. "> " . - ' ‘
\ I: - V ' -
'9 I .. .
I 9 9 9 , .. I . ‘. ,, .
I | ' 9 - 9
.- I , . ' '9 I II' x
9 . . r. 9. 3‘". . , ‘ l 4'
9' 9 '9' I l‘ . ‘ . - ‘ ‘ ‘
I ' ‘ l u I .l 9 .9
z. ' . T ‘ - . 9 ' "3' ‘ '
. I If 9 . ‘. -' . I '
34 . ‘2 . """' I '
O l I .
0‘ .9 9 I I, 9 . I h ‘
: I ‘ l ‘9 . k , . t .0. . I
Q“ i , ' . 9 9 .‘v
0 D . ' . ’ 9' . l 9 ‘
I I v u 9 -
o . 9 I I " -' I 5
‘ I ‘ l‘ I I"
:- . [I I ' " 9 "9 9 9 9
. I *‘I I . I —
,9 ‘ .1 . I” . AI
. , ' 9 -I ' , . ' ‘ ' 9. ~:I 9' 9
l .. 9 ~, ' .3 . . . I V...- 0:
I .9 . .
\ g 1 . . ~ . ~ ‘ ‘
‘ I Alva. I |
. . ;.. ‘ 1" ' ' ' 7 ‘
I 9| . I 9 I 9
. r ‘ W‘I'II " ’ .. - - '
. 'I I- l .
: :n '
I . I ' I J ,I . I
I _ . . -
L I "
’t . . . I ' ' . .
I . , ‘v f ' . 9 ~
' . 9 ‘ ‘
‘ " -‘
l ‘3 _ ' J .
V
. f! : l . . ,' -
z. o ' . ’ -
. 9 I v ‘
o~ . - 9 ‘ I,
: ’ 9
‘ )0 I
. . I |
U D.
. " . II
‘ O
I
. V t I I I h l
“ ‘ub .
I ' 9
. "0' '
. I
O
a I : 9
I
| .
: I
. .
v
s I
U
‘ _
f . ' '
..
I
I 9 .
I I .
. | I I
: ' '
. : " '
; ‘l on
7 I ‘
V
. 9 I
| " 9 . 9' I
v 'I
.0
l
. 9" '
: . " .
D . ..
.
|
, I
I 9 I

 

,‘
L:

 

I e . . .
_-_. '_—__ h-- —>..- ..

 

This is to certify that the

thesis entitled

Studies on the Etiology and Pathology
of Infectious Bovine Pyelonephritil

presented by

Ernest S. Feenstra

has been accepted towards fulfihnent
of the requirements for

Mo 8. degree in Animal P8131010”

 

 

-7§:7\ALJV§V< ‘ i\ 1%?»-
Major professor Frank 0 _, Jr.

Date La! 25. 19M

STUDIES ON m I'M OLOGY AND PATHOLOGY
or
13136110118 BOVINE MOWTIS

3!

Ernest s. genetra

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

mm or 8611!“)!
Department of Animal Pathology

19m;

 

THESIS

9-!

 

 

CONTENTS
INTRODUCTION
SOURCES OF CULTURES

BACTERIOLOGY 0F CORYNEBACTERIUM RENALE AND RELATED DIPHTHEROIDS

 

Morphological and Staining Characteristics
Cultural and Biological Characteristics
Fermentation Reactions

Immunology

Discussion of the Bacteriological Properties

Summary

EXPERIMENTAL STUDIES
Isolation of Diphtheroids from Apparently Normal Cows
.Artificially Induced Case of Pyelonephritis in a Bovine

Sulfonamide Bacteriostasis of Diphtheroids

BACTERIOPATHOLOGY OF INFECTIOUS BOVINE PYELONEPHRITIS

ACKNOWLEDGMENT

BIBLIOGRAPHY
References Available for Use
References Compiled.But Not Used

Bibliography of Ritzenthaler

33

3h
35

39
5h

59
67

65
69
72
73

-1-

INTRODUCTION

 

These studies were instituted in behalf of the livestock
industry on whose resources infectious bovine pyelonephritis is a
continual drain. i'he disease does not always attract widespread
attention because it is insidious in character and spreads slowly in
contrast to some diseases of a more acute nature. Usually the condi-
tion is not diagnosed until it is too late to salvage the cow. It is
with this in mind that these studies were made so that an earlier
diagnosis might be made possible.

Clarity and simplicity are considered to be keynotes in
scientific description. Following this premise this thesis has been
divided into its separate phases and is presented in short inter-

related parts.

~8-

SOURCIS OF CULTURES

The cultures used in these studies were obtained from the follow-
ing general sources:
1 - 20 - l'rom kidneys or urine of cases of pyelonephritis.

21 - 28 - rrom urine and vaginal swabs from apparently normal
cows.

29 - 33 .. Diphtheroids from various other sources used for
comparison.

Numerical Designation of Cultures

l. Accession 1826 Hard No. 2 Pyelonephritie
2. I 18311 I s 3 ..

3. AutOpsy 6022 I I h s

h. I 6273 I I 5 I

5. I 6830 I I 1 s

6. Accession 1888 I I 6 w

7. Autopsy 5950 I

8. I Ky. Ag. hp. Sta. I

9. 1469 a. of man. Vet. Div. I

10. 5R7 I I I I I I

11. Accession 1766 Hard No. l I

12. I 1817 I I 7 I

13. I 1827 Abattoir I

it. I 1830 I I

15. Autopsy 5350 Herd lo. 1 I

16. I 6270 I I 1 I

17. " 7155 morimental animal infected with No. 16
18. Accession 1850 Herd so. 6 Pyelonephri tie

l9. Accession 1853 Herd No. 8 Pyelonephritis

20. I 1902 I I 9 I

21. I 1867 I I l Apparently nomal cow
22. I 1879 I I 1 I I I
23. I 1880 I I l I s w
2h. I 1882 I I 1 I I I
25. I 1883 I I l I I I
26. . I 1881; I I l I I I
27. I 1871 _ I I 1 I I I
28. I . 1887 I I 2 I I I
29. Auto": 7036 Ram -' nephritis

30. Accession 1703 Abscess on cow

31. Autopsy 5328 Pneumonia - lamb

32. I 6039 I I

33. I 1990 I calf

Appreciation is expressed to Drs. r. Hauser. J. Ritchie. R. M. Orr.

and especially to Dr. C. 1'. Clark for bringing in specimens.

Isolation of Cultures
The cultures. were isolated by streaking blood agar plates with
infected material from diseased organs or urine. Any colonies that
developed were IfishedI and stained. to identify them.- Diphtheroide thus

isolated were added to the stock collection and maintained on tryptose

agar or blood agar.

-14-

BACTIRI 012.00! 01' CORYNIBAGTIRIUII m AND RELATED DIPHTIEROIDS
Morphological and Staining Characteristics
Cultural and Biological Characteristics
Fermentation Reactions
lumunolog

Discussion of the Bacteriological Properties

Summary

-5-

Morphological and Staining Characteristics

Ritsenthaler (1910) reviewed the literature on pyelonephritis
and listed.more than twenty investigators who mentioned that bacteria
were associated.with pyelonephritis. Many of these references are
rather general but there are some studies which have contributed to the
morphological study of the specific etiological agent of pyelonephritis.
Inderlen (1891) found in.pure culture a small rod shaped organism
measuring 0.712.1-2.8 u which stained readily by Gram's method and still
better by'Weigert's. Hoflich (1891) described the organism as being
rod shaped 0.711-3 u and easily stained.by Gram's method. He called it
the Bacillus pyelonephritidis boua. lrnst (1905) placed the organism in
the group of coryne bacteria and named it Cogzgebacterium renalis 33:13
and described it as being 0.5-0.6:2-‘4 u in size.

Gair (1918) found the Corynobacillus in turbid urine of a cow.
He described the organism as gram positive. non-motile. 0.5x2-h u in
sise. aerobic. and irregular of form. length and staining. In stained
smears directly from urine the characteristic clumping of organisms was
noticed.

Jones and Little (1926) described the organisms as non-motile.
gram positive slender rods 2-3 u in length. IThose from the tissues are
not as pleomorphic as those from the earlier transfer cultures. although
many may show polar granules or swollen ends. The morphology of cultures
grown in broth is more variable; short coccoid forms, beaded rods with
swollen ends. as well as the usual slender rods are abundant. Occasionally
a stalk with rudimentary branches may be observedI.

In 1930 the same authors stated that the organism was non-motile

and stained intensely by the Gran method.

-6-

Ilerchant (1935) reported that g. renalis was the largest of the
four species included in his study. IThe organism had a distinct bacil-
lary shape in all media. The direct smear from kidneyerudate reveals
striated bacilli. When grown on serum agar the cells were generally
quite large and uniform. but segmented shapes were seen alsoI. All
strains used were gram positive. non-motile and did not produce spores
or capsules. Palisade grouping was observed. Other characteristics of
the organism were the swollen as well as pointed ends. striations.
coccoid shapes. and metachromatic granules.

In 19h} Thorp. Lengham. Clark and Doll described the organisms
as gram positive. non-motile. non-spore forming rods which exhibit beaded
staining characteristics and show palisade groupings.

Also in 19h} Coffin described C. 53931; as Ia gram positive
diphtheroid which tends to form aggregations in palisade-like clusters.
It is a medium sized rod with some evidence of pleomorphism. Thus.
sausage shaped. slightly coma shaped and coccoid forms are observed in
the same urinary sediment. Some will _show evidence of banding when stained
by methylene blueI. Cocooid forms develop on artificial media with loss
of gram positive staining.

In 19% Pass reported an organism resembling g. renalis as being
gram positive and having typical bars and metachromtic granules in
characteristically grouped rods. This organism was isolated from the
kidney of a horse.

laterial and lethods

hcept as mentioned. media were prepared and techniques followed
as outlined in the Manual of Hethods for Pure Culture Study of Bacteria
(1936). Smears were stained both by Gram's method and with 'right's

stain. liaterial for making the smear preparations was obtained from the

-7-

following sources: tissues of kidneys and urinary bladders showing
lesions of pyelonephritis and cystitis. urine sediment from infected cows
and 2h hour. R8 hour and one week old cultures grown on blood agar.
tryptose agar and in beef extract broth. Regular beef extract broth was
fortified with double amounts of peptone and beef extract. The pH of the
media was adJusted by means of Beckman pH meter to approximately 7.5
which was found to be Optimum for growth: sterilization was carried out
by autoclaving at 15 pounds pressure for 20 minutes. Measurements were
made with an eyepiece micrometer.

Bacteria from twenty cases of pyelonephritis and eight urine
samples from apparently normal cows were examined and the results
included in this study.

issue.

The diphtheroids in direct smears from affected kidneys and.
from urine specimens were always gram positive. as were bacteria from
young cultures (214 hours and less) on and in artificial media. As the
cultures grew older (more than 2“ hours) more and more gram negative
forms were observed.

The limits of sise for stained bacteria were about 0.7 u in
width to 3 u in length. Most organisms were about 0.8 u wide by 1.3 u
long. Unstained diphtheroids appeared to be 0.1 - 0.2 u larger. No
differentiation of cultures could be based on sise'of the microgrganisms.
although there was often difference in appearance.

With Gram's stain the microgrganisms almost always stained '
solidly but with Iright's stain they often appeared beaded and stained
a lighter more iridescent blue than most of the other bacteria.

The shapes varied from short thick ovals to medium length rods

with rounded ends. The diphtheroids were found singly. in straight and

-8.

curved.pairs and in palisade groups. In liquid media there was a tend-
ency toward the occurrence of coccoid forms in short chains.

Cultures l - 6 consistently had more curved pairs when grown on
blood agar than other cultures. In broth this difference was less

discernable.

Pig. 1.
I 2.
s 3.
I h.
s 5.
I 6.

Culture (1) 211 hr. blood agar culture.

I (2)
I (3)
I (I)
I (5)

I (6)

w s s
w s w
broth I

blood agar culture.

'right' s stain.

Gram stain.
s s
I! l
s w
s I

x1160.

x1160.

11160.

x1160.

x1160.

x1160.

 

 

 

 

 

 

Pig. 1

\
/
r. 8
av.
..¢
.0...
\ h I
1.1"
.s.
U. at;
.1.
... . -lv
. N.)
x 1
- r.
.. u
.IIV . -
.a.w a n
I | \ 1 ll! ,
. v
s aw
3..
la .. .—

em.

is

1

 

 

 

“Se n

'I

I
e
\’ \\
\ I
- . . r a v
\ \ \
\
a
\s

x I Ala ~
I”
I
Is I
ss\ .
«K as
I‘ Q'. \d
1*. \ “11¢
sin, I i I II I

 

.fi. ..

. Jar. \m‘
. r J

. a J

1" ..

a? I:

. K

. —

'\

Pig. 6

 

 

 

Pig. 5

fig.

7.

9.

10.

11.

12.

culture (10)

(11)
(1h)
(17)
(19)

(20)

21} hr. broth culture. Gram stain. x1160.

I I blood agar culture. I I x1160.
I I broth culture. I I x1160.
let mount of urine sediment. 1550.
an hr. broth culture. I I x1160.

Smear of urine sediment. lright's stain. 11080.

-10..

 

 

 

 

I‘ig. 10
Pig. 9

 

 

 

 

V

Fig. 13.
I it.
I 15.
I 16.
I 17.
I 18.

culture (17) 2% hr. blood agar culture.

(21)

(22)

(2h)

(27)

(28)

I broth

Gram stain.
I I
I I
I I
I! I
it I

11160.

11160.

11160.

11160.

11160.

11160.

~11-

 

 

| -/"\ 'I: ‘ o ' CI 4 k " I .
7 _. -. p ,, 1 w a a I:
’f ‘ T ~ ' ‘ ’ ‘_
1.. 'I J. .’ «N
" “‘ 3' \ ’ . vi
1 ‘ , I
- \ 1 I ‘ e I P I It 40‘ A]
i :\ )1 '\ ‘ T } a n
.1 f “ I. v m
l V, I, ‘ ~ g
‘ g. . ‘
l ’1 v. x 15' ‘. '1 :: 1
. ’ If p I
y . l I . I "
‘ m ‘ I n 0.3..) Q
7 A! is“ -
“a ”'1' I " ‘-
_ _ L. _ _ 1:. 1____. “wait: . §___L‘_____
Pig. 13 Pig. 1h

 

 

 

’ 4,,7tfltt"1"A
.9 .-' ' ‘fi fi ,. 331‘: ~. '9. ‘
j .’ s ’ .r‘z ”‘8’?" t. - .bttig‘L “a..."
’1 .‘ sp- ' i s— I t ‘ -- ’I ' O
1 O ‘ I Y”. ‘a ‘ ’
N I“ \ 4/ " 9 .5"?
\f‘ " r H" '2’ ‘él' “1%:1‘flflnt‘;
fz’ 7)” x , I ‘3‘ '74 “1&3 ’(fl. §u h‘ :d
{affirm 0' L . ,1, * $1 ,1/ ‘2‘, I‘ ‘6 ‘,\ (I)? J A»: u}
3 ‘I O _ ; 4’ r 4. .
‘ s .1 .‘ 7 ‘3” u .- ‘J‘ I .
I ' 4 ’L To '
I ilfl‘” .3". ’3 res-f.
Pig. 16
i ‘l '
\ g ‘
s' hi
3 . I.
' e, ’

 

 

 

-12.

gucltural and Biological characteristics

Ritsenthaler (1910) in his review stated that Inderlen (1891)
and lrnst (1905 ) cultivated the organism of pyelonephritis but makes
scant mention of any cultural characteristics other than luxuriant
growth in urine.

In 1926 Jones and um. stated “The bacillus is readily grown
on ordinary media. In liquids such as broth or urine. aggregates of
considerable sise are found at the botton of the tube. On the surface
of 'agar or serum the growth is raised. grayish-white and dry. On potato
the growth at first is usually grayish-white. but later it becomes
dingy yellow and the potato turns brown. Blood serum and gelatin are
not liquefied and henolysin is not prohced. In litmus milk the growth
reaction is characteristic. The lit-us at the bottom of the tube is
reduced. here the casein coagulates. but the upper stratum is deep blue
and coagulates slowly. the casein is digested slowly". In 1930 they
reiterate their description of the reaction in litmus milk.

Merchant (1935) stated '9. renalis grew in a mist cream colored
colony. l'he edges were irregular, but secondary growth was not observed.
The culture became drier as it aged but never was as dry as 9. 22332-
tuberculosis'. The action on lit-us milk was variable. 'ltine of the
twenty strains studied caused a digestion of the silk casein with a result-
ing alkaline reaction but no coagulation. The digestion continued till
the nediun was of a blue transparent nature. lo change occurred in eleven
of the cultures". The organism was non-motile. Henolysin. indol and
hydrogen sulfide were not produced. nitrates were not reduced and all
cultures were Voges-Proskauer negative. "In serum bouillon. g. renalis

produced a white powdery growth which collected along the walls and in the

-13.

bottom of the tube and in some cultures a thin pellicle was present.‘ The
liquid remained clear“.

In 19h} Thorp. Langhan. Clark and Doll reported that none of six
cultures of diphtheroids fora indol or hydrogen sulfide. neither did they
reduce nitrates.

Coffin (19143) nsntioned that on agar. small beaded. non-hemolytic
colonies forn which become yellow-tinged and dry. In bouillon a granular
precipitate is forled attended by clearing of the medium. A pellicle may
or say not fora. Litmus milk usually became alkaline in two to three
days.

loss in 19th reported that 'prinary isolation on serun agar pro-
duced small maline colonies which enlarged slowly. In veal infusion.
slush agar and in veal broth. the growth was very granular. Indol was

not produced“. This organisa cane from the kidney of a horse.

genial and lethods

Ihe media were prepared and the tests conducted very much as
described in the Ianual for Pure Culture Study of Bacteria (1936) except
for these variations which are mentioned. All cultures were incubated at
37°C. i'he pH of all media was adjusted to approximately 7.5 by means of
a Beck-an pH meter. Iryptose agar (Difco) and tryptose agar plus 10 per
cent defibrimted sheep blood were used to observe colony characteristics.
Regular beef extract broth was not suited to the requirements of all the
cultures so the amounts of peptone and beef extract were doubled. All
media except litmus milk were sterilised by autoclaving at 15 pounds
pressure for 20 minutes. Duplicate tests were nade in each case. Motility
in broth cultures was checked after 12 hours incubation at 37's. Production

of hemolysin was checked by streaking the cultures on thin blood agar plates.

-114.-

Hydrogen sulfide production was determined by observing the blackening of
lead acetate strips suspended over a medium containing litte's peptone and
beef heart infusion as the sources of sulfur bearing amino acids. Indole
production and nitrate reduction were checked after four days incubation
in suitable media. 'l'he ability of 10 per cent gelatin to solidify after
inoculation was checked at regular intervals for two weeks by placing
the tubes in the refrigerator for an hour. Starch hydrolysis was checked
by growing the cultures on starch agar plates for one week and then
flooding them with a 50 per cent alcoholic solution saturated with
iodine. Litmus milk was prepared by adding powdered litmus to skim milk
till a satisfactory color was obtained. Sterilization was performed by
placing the tubed medium in flowing steam 30 minutes daily for 1t days.
Observations of the inoculated medium were made at intervals for two
weeks.

Results

flaw cultural and biological properties my be found tabulated
in chart form. The diphtheroids under study were non-motile and non-
spore forming.

In general the cultures were separated into two groups by their
growth requirements - one group growing well on tryptose agar and the
other group requiring the addition of blood for optimum growth. Iithin
these groups there were many dissimilarities.

i'he rapidly growing. easily cultivated group say be separated
into so-called “smooth. and “rough. types. The "smooth" type fits the
requirements for g. Egg-is; very well. On tryptose agar incubated at 37.0.
for an hours there developed moist. punctiform. grayish to creamy white
colored colonies about 1 mm in diameter. which appeared. microscopically.

granular with slightly lacy edges. With age the colonies became much

-15.

drier. The “rough“ type produced colonies of similar size but of a much
different consistency - dry and brittle rather than soft and moist. The
‘rough' type produced a flat. slightly spreading secondary growth while
the “smooth“ type of colony slowly enlarged. no true rough types were
isolated from actual cases of pyelonephritis but from urine of cows with
apparently normal kidneys. The fine growing. more exacting. group of
diphtheroids produced on blood agar in an hours at 37°C. dewdrOp or
streptococcus like colonies which.were about 0.3-0.5 mm in diameter.
This group was isolated only from cases of pyelonephritis but differed
from typical g.‘gggglg cultures in several ways other than growth
characteristics as will be noted under various headings.

In nutrient broth the same two “or groups were apparent. The
fine growing cultures grow slowly and produced a rather granular sedi-
ment with not much clouding of the liquid and no pellicle. The other
more rapidly growing cultures produced.mnch granular sediment with.vary-
ing degrees of clouding of the liquid. Often the growth seemed to be
around the side of the tube. Most of these cultures formed pellicles on
the surfaces which varied from light.to very heavy. There were my
minor dissimilarities between cultures.

Very few of the cultures caused.hemolysis of erythrocytes. The
zones of hemolysis around the colonies were not always as clearly defined
as those usually observed around the extremely hemolytic streptococci
colonies but there was a definite clear zone around the colonies on the
blood agar plates.

The production of hydrogen sulfide seemed to be confined mainly
to the fine growing group of cultures although a few of the other cultures
also prochiced hydrogen sulfide.

None of the cultures produced indol. nor did any hydrolyze starch.

-16-

Culture 7 liquefied gelatin and three other cultures reduced
nitrates.

most of the cultures turned litmus milk alkaline and slowly
digested the casein with a resulting clear blue medium. The cultures
which were able to ferment lactose or galactose turned the litmus milk
acid indicating primarily that the lactose was fermented by the micro-
drganisms and also that some of the lactose is hydrolyzed to galactose in

the process of sterilization.

 

 

Culture Type of Growth Hemolysin Production H25 Production Nitrate Reduction Litmus Milk
+ od. + N reduction auacid
S-smooth. typical (+) hemolysis ( ) Eés pr ( ) 08 bubasic

 

 

 

colonies
' ” t r d. a no N " c-coagulation
R—Zggfgiegry flaky (1) Slight hem°1y315 (+> sligh Has p o ( ) 03 r-reduction
S 8
F-fine, slow. grow» (+) very slight (+) very slight td~tota1 digegtion
ing colonies ' pd-partial
P—pellicle formed on (a) no Has prod.
broth
End-secondary colony
growth
1 F + + a b. td
S
2 F + 3 a b, to
z A
3 F * + a b, td a,
- T‘
h F + + a b, td
5 F + i r b. pd
6 r u 2 ~ b, td
7 F a a r a. c
S S. P - g a b, pd
9 s. r 1 , - b. pd
10 S. P - a a b, pd
11 s. P - 1 - b, pd
12 So P + I ~ ‘b
13 s 2 - - b
1h 5. P - _ _ b, pd
15 s. P + g .. b, pd
16 s, P - - _ b, pd
17 s. P am _ u b, pd
19 S. P : - _ b. Pd
20 S. P - - _ b, pd
21 S, P - 1 - b, pd
22 S. P - a a b, td
23 s. P - - + b
2h S. P - _ - b, td
25 s. r - : _ b, pd
26 -
Rs P, and. - + - b. pd
27 Rs P+s 2nd. _ - +
a

28 R. P+. 2nd - _ + a c

 

 

 

Fig. 19. Cultures 6. 20 and 26. Blood agar. an hours.
Fig. 20. " 6. 20 and 26. 0 " RS '

-18-

 

19

fig.

 

 

 

Fig. 20

Fig. 21. Cultures 3. l6 and 27. Blood agar. 2h hours.

fig. 22. ' 3. 16 and 27. Tryptose agar. 2h hours.

-19..

 

Fig. 23. Cultures 6. 20 and 26. Tryptose agar. 2% hours.

Pig. 2“. " 18 and 19. Blood agar. 2h '

-20.

 

Pi g. 23

fig. 25.
Pi g. 26o
Fig. 27.

“so ago

culture 3. Colonies on blood agar.

17, s w w s
13 , s w s w
20. w w w a

(All 2’4 hour cultures)

-

x7.
x6.

17.

~21-

 

Fig. 25 Fig. 26

 

Fig. 27 Fig. 28

Culture 26. Colonies on blood agar. 17.

s 27. s I I w x7.
I 20. " " tryptose agar. x9.
I 16s ' I I . x9.

(all 2h hour cultures)

~22-

 

Fig. 29
Fig. 30

 

Fig. 31 Fig 32

Fig. 33. Culture 3. Colonies on tryptose agar. 1130.
Pig. 3h. ' l6. ' ' I ' x108.

(Both 2h hour cultures)

-23.

(1

.t‘ .

 

 

 

 

Pig. 3"

rig. 35. Culture 17. Colonies on tryptose agar. X68.
Hg. 36. I! 26. e I I w 1130.

(Both 2’4 hour cultures)

-21.

 

lie. 35

 

-25..
Fermentation Reactions

Jones and Little (1926) reported that only dextrose was fermented
and that lactose. saccharose. maltose and mannitol were not attacked.by
the diphtheroids isolated from cases of pyelonephritis. In 1930 the same
workers showed in a chart that some other diphtheroids produced acid in
lactose and/or maltose as well as in dextrose. Merchant (1935) reported
that‘g. renalis fermented glucose and that some strains also fermented
levulose and mannose while one strain was unique in that it fermented
only dextrin. He used arabinose. xylose. glucose. levulose, galactose.
mannose. sucrose. lactose. maltose. dextrin. inulin. salicin. dulcitol.
mannitol and glycerol.

In l9h2 Morgan mentioned an atypical.g. renalis which was unable
to ferment glucose.

Thorp. Langham. Clark and.Doll reported (l9h3) that of six
cultures from cases of pyelonephritis. all fermented glucose and fructose
while one of the cultures also fermented arabinose. dextrin. galactose.
lactose. maltose and xylose. Another of the cultures also fermented
xylose. None of the organisms attacked dulcitol. glycerol. inulin.
inositol. mannitol. raffinose. salicin. sorbitol. sucrose or trehalose.

Coffin (l9h3) mentioned that the formation of acid in dextrose
was a diagnostic feature of'g.‘£gg§lg.

loss (l9hh) isolated from a horse kidney an organism resembling

‘Q. renalis which fermented only dextrose.

‘gaterials and.Methodg
A,broth medium was used in the fermentation studies. TwO'variae
tions in constituents of the broth were tried. The first medium consisted

of Bacto-Peptone l per cent. Bacto Beef lxtract 0.3 per cent. nncl 0.5 per

-26-

cent at a.pH of approximately 7.0 using brom cresol purple as an indi-
cator. The second medium contained.Bacto-Tryptose 2 per cent. Bacto Beef
Extract 0.7 per cent. laC1 0.5 per cent at the same pH and with the same
indicator as the first medium.

The fermentable substances were added in amounts of 0.5 per cent
except in cases of the more rare ones in which case 0.1 per cent was
used. Sterilization of media was performed by autoclaving after adding
the substances except in the cases of those complex compounds which may
be broken down to simpler forms by heat. These were autoclaved in 2 per
cent solution and added to the sterile tubed medium.

Inoculations were made directly from agar slant cultures and
tubes were incubated at 37‘c. for two weeks. Observations were made at
2h hour intervals for the first three days and at MS hour intervals for
the remaining time. Doubtful reactions were checked on the Beckman.pfl
meter.

12.82122.

The fermentation reactions are recorded in chart form. No gas
was formed.by any of the cultures. The great maJority of the cultures
fermented fructose and glucose and only those two substances. One
culture failed to ferment fructose. Two "rough” cultures also fer-
mented galactose. one of them also fermenting lactose and the other
fermenting maltose. Culture 7 which was isolated from a case of
pyelonephritis in a calf fermented almost all of the substances to some
degree. Seven of the cultures fermented xylose and are thereby .

separated from the rest of the cultures.

 

Fermentation Reactions *

 

omoahx

encamnmns

economm

Headphom

nwowfimm

mmommmmmm

Hovwcnwfi

omcuamz

emooomg

fldefiH

HoawmonH

Hoaoohac

omooeac

cmouomamw

omouosah

Hoewoflfin

m«aaxon

$03924

moanuaco

 

a...

~+

.4.

2 3h.

Q"!

P.)

um?

=+ _ a a

(«a

complete change to yellow color (acid)

(H
(1)
(~)

 

u

i!

partial

no change

 

 

 

.28...

mm

In 1925 Jones and Little reported attempts to recognize early
cases of pyelonephritis by use of serological and biological tests. In
agglutination tests the titers were about the same for both the infected
and uninfected animals. Also. no recognisable reactions resulted from
introduction of culture filtrate into and beneath the skin and on the
cornea. In 1926 these same workers reported that in the main the cultures
they isolated from scattered sources reacted to a greater or lesser
degree with three agglutinating sera.prepared by immunizing rabbits with
single strains. In 1930 they reported on the agglutination relationships
of strains of organisms isolated from apparently normal calves to strains
obtained from cases of pyelonephritis. The calf group of organisms had
agglutination affinities similar to the other organisms mentioned and
was capable of absorbing agglutinin from specific antiserum. In 1935
lerchant reported a close serological relationship between‘g. renalis
and‘g.jppeudotuberculosis. In a few cases there seemed to be a relation-
ship between C. renalis and g. pzoggnes and also 9. diphtheriae.

7 In 192.1 Bruner and ldwards reported in their work on the
classification of‘g. 2321 that one culture of‘g. renalis caused slight
fixation in the complement fixation test using‘g.‘gggi immune serum.

By treating‘g.12ggi with.heat and.acid they were able to remove some of
the type specific antigens. Then by the complement fixation test they

were able to demonstrate species specific antigens.

Materials and.uethng
Rabbits were inoculated intravenously with twelve cultures of
diphtheroids to producejpotent immune sera. Six of these cultures were

from cases of pyelonephritis. four were cultures similar in many ways to

-29-

those from actual cases but were from normal cows. and the last two were
isolated.from a.pneumonia case and an abscess and resembled‘g. pzogene .
Antigens for the plate agglutination test were prepared by
suspending the organisms from a 2h-hour tryptose agar slant in phenolated
(0.5 per cent) physiological saline to make a dense suspension. The test
was carried out by adding .05 ml of antigen to .05 ml of antiserum and
noting the reaction. The results were recorded after five minutes in
order to eliminate some of the non-specific reactions occurring later.
Antigens for the complement fixation tests were prepared.by
suspending the organisms from 2h hour tryptose agar slant cultures in
physiological saline. adding one-tenth volume of 0.5 N HCl and.placing
the tubes in a boiling water bath for ho minutes. The bacteria were then
centrifuged and washed.repeatedly with sterile saline till the reaction

was approximately neutral.

Results

The agglutination reactions are recorded in the chart. The
organisms seem to be grouped into serological types which are fairly
distinct except that there are cultures outside of the groups which have
agglutinative relationships not shared.by those of any group. In some
cases the reaction was rather indefinite or late and in other cases
spontaneous agglutination interfered.with observations. In one instance
an antigen was not agglutinated by its homologous antiserum.

The results of the complement fixation tests showed the organisms
to be type specific even after the treatment with acid and.heat and not
species specific as was hoped. Therefore. the reactions are not tabulated

here.

 

 

 

 

 

 

 

 

 

 

Group gglutination affinities
Antisera 3 8 12 15 16 18 21 25 26 27 3o 31
Antigens ___
11 a + + + a + + + + a a -
19 u + + + a + + + + a a -
20 - + + + + + + + + - - a
21 - + + + + + + g + a g -
22 - + + + + + + + + a a g 6L
‘ 5’
2h 3 + + + + + + + + a + +
no 5
26 - + + + + + + + + a + +
9 a + + + + + ' + + + + - a
16 - + + + + + + + + + a ,
17 - + + + + + : + + + a a
28 I' + + z + + + z 3: f a a.
10 Auto agglutinating
8 + + + + + + + + + + a a
1% 2 + + + + + + + + 3 a g
B H - me cm .0 mm
27 t + + + + f + + + ’ “ +
13 2 . 1 1 + + r; a; _, .. 3: 3
18 ~ I - + 1‘ ~ f 1' - - 1' :c
12 en as + + + a. .. u + : ~ “
23 mm as em on a — - mm a. - r em
1 : In em as am am a. a. u a 5 sm
3 + a - - 1 - - z _ a + +
n O - c- - ms - ., me a u a. a.
5 3 3 ' r + + ; 2 a + a 3
6 H em a u a e- um cm . as me me
7 me ~ - a u c- - 1‘ b 9’ mm H
+
a - Q ~ - a a as . a - M
0 - - - - _ - _ u a
3 _, _, g + +
31 a - _ g u _ _ _ _ - a +
32 a - - - _ _ n - - - _
” +

(+) agglutination
(1) partial agglutination
(i) very slight agglutination

(-) no agglutination

 

 

-31-
Discussion of thegpacteriological Properties

To one studying diphtheroids the variations met with are amazing
and interesting. It might facetiously be stated that variation is their
only constant feature. This does not refer to the properties of any one
diphtheroid species as much.as to the many diphtheroids which have never
been described. There are also some intra species variations such as
type of colony. chromogenesis and agglutination affinities.

Culture 7 has many characteristics which seem to indicate
relationship to‘g. pzoggnes. The case from which it was isolated ran an
acute course rather than the chronic one usually associated with
g. m infections.

Consideration of the prOperties of cultures 1-6 would place them
in a group by themselves. They do not fit any description of diphtheroids
given‘by'nergey (1939). These cultures were isolated from cases of
pyelonephritis. yet they are otherwise very different from typical
‘9. Eggglg'which has been incriminated as the causative agent in bovine
pyelonephritis. The similarities of these cultures to 3. Legal: are
habitat. staining characteristics. formation of acid in fructose and
glucose (with minor exception). no indol production. no starch hydrolysis.
no gelatin liquefaction and the fact that litmus milk becomes alkaline.
The differences are that cultures l-G produce acid also in xylose.
peptonise litmus milk more vigorously. show a much greater tendency to
produce hemolysin and hydrogen sulfide. show little agglutination affinity
for typical gy‘gggglg, have more curved.pairs in smears from blood agar
cultures and grow more slowly and produce much smaller colonies on arti-
ficial media. In view of all these differences it is not possible to
classify these cultures as'g.‘ggnalg,or even as a strain of this species.

The possibility that cultures 1-6 might be related to Q, Exogenes could

-32.

be brought out but not substantiated.because they also ferment xylose
but do not ferment lactose and sucrose as does 9, pzoggnes. Also
cultures 1.6 do not form acid in litmus milk and do grow fairly well on
plain media. The results of the agglutination tests showed the possi-
bility of a slight affinity between cultures 1-5 andwg. pyogenes but not
to any marked degree. There were some minor differences within the
group.

The rest of the cultures (8-20) which were isolated from cases of
pyelonephritis are relatively similar and fit very well the description
given for‘g. Eggglg in Bergey's Manual (1939). The variation within this
group is likely due to the fact that different strains each having its
own characteristics make up the group. There are slight differences in
growth and.pigment formation. The colony color ranges from grayishpwhite
to a creamy white. There are no differences in the fermentation reactions.
all cultures forming acid in only fructose and glucose. In the aggluti-
nation reactions differences appear and the cultures are classified into
different subgroups by slight variation in agglutination affinities.

The group of cultures (21-28) isolated from apparently normal
cows present somewhat more of a problem in classification. Several of
the cultures appeared rather similar in all characteristics to the
diphtheroids from cases of pyelonephritis and which were tentatively
classified as‘g.'gggalg. The fact that their habitat is the urine and
vaginas of cows does throw a ray of suspicion upon them even though their
pathogenicity has not been established. Several of the cultures show
properties such as nitrate reduction and fermentation of lactose.
galactose. sucrose and maltose which would tend to separate them from
.Q.‘£gg§lg. .Llso. the colony characteristics were grossly different from
those resembling‘g.‘£gg§lg even though the stained organisms showed only

sinor differences.

~33-

Sumaiz

Some of the morphological. staining. cultural. biological.
fermentation and immunological preperties of 28 diphtheroids have been
given and an attempt was made to classify them especially as they
seemed related to _c_. m. From the data presented it seems likely
that there are two distinct species of Corynebacteria involved as
etiological agents in infectious bovine pyelonephritis although hereto-

fore only one has been so described.

-3h-

IIPIRIMENEAL STUDIES

Isolation of Diphtheroids from Apparently
normal Cows

‘Artificially Induced Case of Pyelonephritis
in a Bovine

Sulfonamide Bacteriostasis of Diphtheroids

~35-
‘ggg Isolation of Diphtheroids from_Apparently Nbrmal Cows

Jones and Little (1930) isolated 128 strains of diphtheroids from
the external genitalia of 3h male calves. Twenty-five strains from 12 of
the 3k calves were similar in morphology. cultural characteristics and
certain immunological preperties to Corynebacterium renale. They were
able to produce the disease in one cow by inJecting intra-urethrally one
of the strains of organisms isolated from the sheath of one of the
calves. They pointed out the great difficulty in tracing the infection
from animal to animal in the herd.

The insidious character and long incubation period of infectious
pyelonephritis is well known. Jones and Little (1926) mention the
possibility of the organisms being disseminated from cow to cow by‘brushp
ing over the vulva of an infected cow and then brushing over the vulva
of another cow.

This study was made to determine the occurrence of diphtheroids.
especially‘g.‘gggalg. in the apparently normal cow. Host of the cows
chosen were those stabled next to or near cows that had died of pyelo-
nephritis.

Materials and Methods

Urine samples and.vaginal swabs from 13 cows were collected.by
Dr. C. r. Clark and brought to the laboratory for examination.

Each urine sample was centrifuged. 4A loOpful of sediment was
streaked on a blood agar plate. Another Ioepful was placed on a glass
slide. a cover slip added. and thus examined under the microscope using
the high.power dry objective. Two more loopfuls were smeared out on two
glass slides. one to be stained by Gram's method and the other with
Iright' s stain.

The vaginal swabs were streaked on blood agar plates and also on

-36-

glass slides which were stained. one by Gram's method and the other with
Iright's stain.

Ihen bacteria were stained with Wright's stain using the same
procedure as for blood cells. the bacteria were rather lightly stained.
They may be stained darker by allowing one application of Wright's stain
to dry or almost dry. then adding more Iright's stain which is diluted
with distilled water after one minute. After several minutes the excess
stain is washed.off. Inhyl alcohol (95%) is used for 10 seconds to clear
the smear. The slide is then rinsed in distilled water for several
seconds and allowed to dry. d

The inoculated blood agar plates were incubated for 2h hours and
then examined. If there were no colonies present which resembled
diphtheroid colonies the plates were incubated longer and examined daily
until it was determined.that no diphtheroid colonies were going to
develop.

All suspicious appearing colonies were "fished" and the organisms
stained and examineda

The pH of the centrifuged.urine was determined.by means of the
BeckIan pH meter.

was

The results of this work are summarised in the table. The colony
growth of the diphtheroids on tryptose agar varied from a moist. raised
grayish or creamy white type to a dry flatter whitish type. All cultures
grew luxuriantly in nutrient broth and on tryptose agar. The reactions
of these organisms are tabulated elsewhere in this thesis but may be
reviewed here. The fermentation reactions of these organisms were similar
to those of typical C. 5232}: in that acid was produced in fructose and

glucose except that one culture produced.acid in galactose. lactose and

-37"

sucrose. and another produced acid in galactose. maltose and sucrose.
Three of the cultures reduced nitrates. lith the exception of cultures
23 and 27 agglutination reactions of these diphtheroids were similar to
those of diphtheroids isolated from cases of pyelonephritis although

most of the cultures were grouped together by the agglutination test.

Discussion

These data are not intended to show the incidence of Q'.£22§l2 in
a dairy herd.becanse relatively few cows were included in the observa-
tions. Rather. it is to show that in apparently normal cows diphtheroids
are present which in many cases are indistinguishable from typical
‘§.‘£gg§lg. These facts are easily correlated.with the general history
and course of the disease. The organism seems to be practically
ubiquitous and also is an Opportunist. It is conceivable that a cow may
carry these diphtheroids all her life with no harm to herself. The chain
of circumstances initiating pyelonephritis is not clear. It seems likely
that any injury or break in the continuity of the mucous membrane of the
urinary system would.give the organism an advantage which might lead to
the initiation of disease.

One observation worthy of note is that not once was there found
an organism similar to the slow growing 'xylose positive” type which is

found in many cases of pyelonephritis.

Md Conclusion
Diphtheroids. some of which were similar to those isolated from

 

cases of pyelonephritis in cows. have been isolated from urine specimens
and vaginas of apparently normal cows. The pathogenicity of these

cultures has not been established.

-38-

lgplation of Diphtheroids from Apparently Normal Cows

 

 

 

Culture Age of Cow pH of Diphtherom;aginal
lo. (yrs.) Urine Urine Swab
2l 6 7.67 + -
22 6 7.9 - +
-- 9 8.00 - -
.. s 8.23 I - -
.. 9 8.1% - -
~- 8 8.21 - ..
23 8 7.65 + -
-- 6 7.5 - -
2h 6 8.03 ~ +
25 6 7.88 - +
26 - 8.2 + +
27 9 3-05 + +
28 - 8.25 + -
(+) diphtheroids present 3 (+)

(-) no diphtheroids found 5 (-)

-39-

[Agtificially Induced Case of gyelonephritis in a Bovine

The epidemiology of specific infectious pyelonephritis of cows
remained somewhat controversial until the work of Jones and Little. The
common laboratory animals seem to be refractory to the disease except
under one condition - that of ligation of ureter or ureters of the rabbit

at the time of intravenous injection of a suspension of Corynebacterium

 

‘gggalg. Inderlen in 1891 did this experiment which.was repeated.by Doll
in l9h2. Typical pyelonephritis lesions appear in the affected.kidney
under these conditions.

In 1905 Ernst induced cystitis in a cow by introducing 50 gm of
sterile sand into the bladder and following this in 2h hours with a sus-
pension of 'Corynebacillus renalis bovigfi. The cystitis subsided after
9 days--

In 1926 Jones and Little reported the results of several attempts
to produce the disease in rabbits. guinea pigs. white mice and cows. The
laboratory animals were refractory as was reported above. The intravenous
injection of the organismsinto cows failed to infect as did the introduc-
tion of organisms into the vagina. One cow of two fed the organisms
developed a typical cystitis: these investigators were unable to repeat
this work and get positive results. InJection of a suspension of
organisms into the bladder resulted in a cystitis.

In 1930 Jones and.Little reported inoculating 5 cows intra-
urethrally with broth cultures of diphtheroids. One of the h cows
developed a typical cystitis and pyelonephritis.

An analysis of the transmission work done thus far would seem to
indicate that the infection gains entrance to the body by a urogenous route.

The diphtheroid organisms have been found in the genito-urinary

tracts of supposedly normal cows. Seven of 12 cows examined in one herd

~ho-

had diphtheroids in either urine or vagina. Jones and Little (1930)
report the isolation of many varieties of diphtheroids from calves. They
also suggest that the microgrganisms are possibly spread from cow to cow
by brushing over the vulvas of cows with a brush contaminated with
material from the vulva of a cow harboring the infectious agent.

The object of this part of the thesis is to report an artifi~
cially induced case of cystitis and pyelonephritis in a Jersey steer.
This animal exhibited the typical symptoms and an autopsy 9 months after
intra-urethral inoculation showed typical lesions of specific infectious
cystitis and pyelonephritis. One criticism which may be leveled at the
experiment is that of inadequate control. The urine was not examined
culturally for diphtheroids prior to inoculation with 9. 53592:. It say
be stated. however. that the steer seemed in the best of health and was
physically negative at the beginning of the experiment and no infected
animal had been housed in this barn previously. Also. the organism
cultivated from the kidney was similar morphologically. culturally.
biologically and serologically to the original culture used to inoculate
the animal. In the Opinion of the writer there is little doubt but that

the injected organism caused the disease.

Clinical History Lrectal palpation _performed by Dr. C. 1'. Clark)
h/l‘j/h}. Suspension of g. g_gn_a_l_e_ from autOpsy No. 6270 (Culture 16)
was injected intra-urethrally into the bladder of an experimental steer.
(Culture from autopsy No. 6270 was used since it seemed to satisfy all
the requirements for g. _r_e_n_a_l_e).
5/6/14}. Both ureters seem slightly enlarged.
5/lh/h3. Left ureter seemed enlarged but right one of normal size.
5/2h/h3. Both ureters definitely enlarged.

6/11/14}. Ureters enlarged all the way from bladder to kidneys.

Chlo-

Bight ureter about 1/2I in diameter and left ureter slightly smaller.
Bladder feels contracted and doughy. Herdsman reports that steer kicks
at abdomen as if in pain and also grunts and strains a little during
micturition.

6/7/h3. Condition of urinary tract about the same as on 6/h/h3.
Herdsman reports that steer has not eaten for several days. reces appear
slightly dehydrated.

Glee/I3. Bight ureter still enlarged but left ureter appears less
so. Bladder still feels contracted and less than 50 ml of urine voided
upon stimulation. Animal appears to be regaining flesh and is active.

7/lh/h3. Ureters seem smaller. Bladder is still contracted.
Small amount of urine voided.

7/28/h3. no noticeable change.

10/11/u3. I I I

1/5/hh. Bladder wall definitely thickened. ureter and kidneys
seem to be slightly enlarged. Steer in good flesh and excellent spirits.

l/lS/hh. Condition is about the same as on l/5/hh.

Killed for autOpsy.

Urine Imamination

Sggple Taken p;g Sediment
VEGCB 8.7 erythrocytes fibrin _C_. renale pus
5
5/1 / 3 9.2 I I I
5/2h h} 9.0 I I I
6/h/ 3 8.85 I I I
smug 3.7 . .. . ..
6/28/ 8.7 I I I I
7/1I/I3 I
7/28/“3 8.78 I I
10/11/ 3 8.68 I I I
11/12 ‘43 w
1/5/ 8.7 I I
ills/uh 8.8 I I I I

Urine samples were taken by manual pressure on the bladder after

scrubbing the sheath.

-ll2.

.Rma 522.8 88833.3.” 8385 .o .3825 e5 .m .m .ursm A2
.33 $5388 1338 3333323 .1». .e 5338 Se

aneo men .53 e

 

 

 

 

 

 

93 3.9. NA 3.0 8.8 8.3. an. *2 Rm 885 08.9.3.2 was 81:}
on: new. «A m; 8...: 8.8 an. awn me. can.» 80.288 9.: mini:
om: 88H we." .. 9.2 $8 mm «8 En 08.9 828:5 m8” 9.2m?
.Am .883?82388 .3 .88 a demos—«Sn 3 «mafia 6.9- .3 .mm swan

men oo
N...“ « Hendc Hm

-113.

After a nine months' period of observation the affected animal

was autopsied and examined to determine the extent of infection.

Bacteriology
The kidneys and.bladder were cultured and smears of these organs
and.wet mount preparations of urine sediment were made. Diphtheroids
were present in great numbers in all preparations and typical diphtheroid

colonies grew on the blood agar plates.

Tissue Technic
Blocks of tissue were taken from the kidneys. ureters. bladder.
urethra. heart. liver and.spleen and fixed in Zenker's fluid. The tissues

were stained with eosin and.hematoxylin.

Gross Patholggz

The urinary bladder contained about 100 ml of bloodptinged.
mucus thickened fluid with some necrotic debris. The walls of the urinary
bladder appeared thickened. The mucosa was edematoue and hemorrhagic.

One area of the mucosa. roughly 1' x 2'. was necrotic. being a dull grayish
brown color. lxtending from the bladder for about hI the urethra was
inflamed. Ureters were thickened very slightly. The mucosa of the left
ureter was slightly congested. The right ureter exhibited a diverticulum
about 1-1/2' long situated.approximately 8I from the'bladder.

The left kidney was apparently normal with possibly a mild con-
gestion in the areas of the renal pyramids. The right kidney was very
adherent to the surrounding fat. then removed. it was found to have a
grayish mottled appearance and was very firm in consistency. When out it
seemed slightly gritty and.had more resistance to the knife than normally.
On longitudinal section the renal pyramids were necrotic and cupped.

Several of the calyces contained concretions of varying sise. The mucosa

—hh-

of the calyces was slightly edematous and hemorrhagic. The color of the
cortex varied from a reddish brown to grayish brown to almost white in
some areas. The capsule was thickened and adherent to the parenchyma.
The remainder of the body seemed normal with the exception of a small

(3 mm diam.) cyst on one leaf of the right atrioventricular valve.

lgggroscopic Patholggy
M-

Capsul . The capsule was greatly thickened.with connective and
vascular tissue.

‘gggggg. There were many subcapsular foci of lymphocytes with a
few mononuclear phagocytes. In some cases these foci seemed to extend
into the cortex a short distance along the medullary rays. Other similar
foci were scattered throughout the cortex. In some of these foci there
was indication of fibroblastic activity. The renal corpuscles and tubules
in these areas were in various stages of atrOphy. In a few cases the
parietal epithelium of the renal corpuscles appeared thickened and there
was a pericapsular increase in connective tissue. Adhesions of the
glomerular tuft to Bowman‘s capsule were not rare.

Medulla. In the affected lobes the pyramidal shape of the medulla
was lost and a crater took the place of the papilla. Lymphocytes were
present diffusely throughout with the greater number being present in the
inner medulla next to the necrotic areas. Activity of the interstitial
connective tissue was marked. The epithelium and medullar tissue in many
areas were necrotic and.had lost all form; all that remained was cellular
debris. Many of the papillary ducts contained cellular debris. Capillary
congestion was also consistently present.

Calyces and Pericglygal Connective Tissue. Lymphocytes and

mononuclear phagocytes were present diffusely and in foci. The connective

-145.

tissue elements showed increased activity. The epithelium lining the
calyces was destroyed in some areas and.a cellular debris was accumu-
lating. Hyperemia was also present.

35ft Kidney.

The left kidney was essentially normal except for a very few small
foci of lymphocytes.

Calycal epithelium was desquamated in some areas and in the
surrounding connective tissue foci of lymphocytes were found. The
capillaries in this area were congested.
ureters.

The epithelium was denuded from the mucosa in many places. The
lamina propria was thickened due to an infiltration of lymphocytes. plasma
cells and edema. These cells were present diffusely and in scattered foci.
the greater number being found adJacent to the lumen.

Bladder.

A severe cystitis was present in some areas characterized by deep
necrosis of the mucosa. capillary congestion. edema and infiltration by
lymphocytes and plasma cells. Again these cells were present diffusely

and in foci. These phenomena account for the great thickening of the

1M1” propria.

W

The foregoing protocol is one typical of many field cases in which
the onset is usually unnoticed and the course chronic with symptoms appearb
ing intermittently during the early part of the disease. ‘When this case
was terminated after nine months. one kidney was found.badly damaged.
The animal might have lived for some time as the renal reserve capacity
had not been exhausted as was shown by the results of blood analyses.
The course in field cases may last for several years but unless the

process is stopped. and that fairly early. death is almost inevitable.

4+6-

The path of the organisms to the kidney is still a matter of
conjecture. We may feel fairly certain that the bacteria enter the body
and gain access to the urinary bladder by a urogenous route but the path
from there to the kidney may be one of several ways. Continuity of infec-
tion from a primary cystitis up the ureter would be one way. Another
would be inflammation of the bladder terminus of the ureter: there might
be a stasis and a baclcing up of contaminated urine into the kidney. Still
other possibilities would be the spread from the bladder by way of the
lymphatic vessels or even the blood stream. The blood stream idea is not
corroborated very well by the fact that the lesion seems to begin in the

medulla and calyces rather than in the glomeruli.

W
The course of an artificially induced case of pyelonephritis in

a bovine has been followed and reported.

Pig. 37. Cross specimen from case of pyelonephritis.

J47-

 

 

 

1'16- 37

Fig. 38. Ureter. Cross section showing thickened mucosa with
foci of lymphocytes and plasma cells. 15.

Fig. 39. Ureter. Same as Fig. 38. 182.

J48-

 

 

 

l'ig. 38

  
   
    
  

  
 

1"...049 . 4 5‘0 , '; 9 . "
- .3: Al’s -
ffl'f'ih'ut-I'L" c" " ‘ “.... .
A,“ ‘ «1'3 '1': '

' e.‘ .
.’.'-:: -."d-‘:" ..
V "'

   
 
  

. .l . I ' '
u ' > I .
' f t
' ' \.
£37.22. "3’
? "%V‘ "we.

.“ - ’-:'_ L,

h

fight”? ‘ ' ¢~".~' . ‘1‘ ' r
P.‘ 5‘" T a . E‘;..::A. :fixne .Q."\ 2

Q
;_e'..~’ e‘, n if- I" I
re- . . i 7'..- a.“ .- e i
" "L"??? ‘1’ $9.9.- . "5. £3.25, ..
3-“: - *~.-.-~4-.~.~.s-31»’~, ta, .
i . _ . . ;. ~ , ' ‘

 
   
 

0 . .65 .
flu,“ whirl} . .
h ‘ - o .'- .3 _
3" 3.2-?“ '3‘ ‘34"? 3‘,"
‘3’ ' ’

b
v
I

    

" ' ‘L;.V‘*- . .l" v -
@3133??? ’5'” 7' 3:” J, a".
‘1 . 'r’?‘ 'lfu' e if. ‘

3 V. -' .2. ”I .- , .
‘15-. ‘. Jr
1‘ ‘ \Il ‘

       
  

‘..

   

L.

(-
~:£"t:

‘ a
‘
€

rig. no. Bladder. Cross section showing necrotic area with
tissue debris and inflammatory zone. 26.

Fig. kl. Bladder. Same as Fig. ho. X36.

-hg-

1. .

“a. .. ,
.3. 2.“...
refine.“
,1...

a...

. i .
TX!
\

A .

 

l'i g. 141

Fig. “2. Kidney. Cross section showing general design and
location of inflammatory areas. 16.5.

fig. “3. Kidney cortex and capsule showing inflammatory sons
and thickening of capsule. xhz.

-50..

 

rig. he

 

d o. . . 0* "JIU. §
\“W . w r .l ,cr .
. .. . . _ wax.

.0

spam .5

. p a;

. \;»~w.e.spe,

we

’

We? '7‘? n Penman?»

“, we

rig. R3

mg. nu. Kidney cortex and capsule. 139.

Fig. ”5. Kidney medulla showing necrosis of the surface tissue
and increase in the interstitial connective tissue.
11:2.

 

. [.5

1’

-5].

' 4.3.M_5.’ :‘9..."‘P£ ‘. .1.
4.? ’_l'x‘,-“’rfi

e ‘ I ‘.

.s

3.311;.

Pig. ‘6

  
  
 
 

. a. ' -
e- e"
e"

"I .' .
. .‘ .
.I -' '
.‘ i
-. . '5" . . .
‘ - ! .Q’f "‘1‘." ‘
~w' n I ‘ « ‘- .
‘ e . _.
‘. ,‘.. . ‘ 7»
. a ‘ ‘
.

as ,a

  

   
  

 

Fig. ’46. Kidney. Deep cortical area showing foci of lymphocyte
around blood vessels. 135.

Fig. ’47. Kidney. Pericalycal connective tissue showing foci
of lymphocytes. x32.

-52.

v

. a
Li so.

a... .2.
unsung)...
l U

..r

C u.
I. I. :-
. . .. , : . ...
. .. . .. p.
A.... .. .. ., . 4
. ,. v p. .
. l. I . v
s. . . . 4 . .. ... .. l.
w s. I , u
I D . .1. \
. v i . . A .u u A, I
l _ 1 . a
l o a, .aa s
. e u . . .I p: , .. .2
. .... o
I I, I . s.I , .1 a
\ e w . . )1 V I
. e — s ‘
II . l.‘ - !

si.
..

.3‘ '

.A.‘

Fig. ‘16

vg'

(. ..
. '3';
‘ Q
..m

I
s

n .-
....~.-a 1
.(‘up u!\(\.
1 n.

y b. .
. IJ...

 

Pig. W

Fig. 1#8. Kidney. Glomerulus with thickened capsule of Bownan.
atr0phied tubules. Increased interstitial connective
tissue and lymphocytes. xhl'j.

Fig. 19. Kidney. Phenomena 31mm to those shown in Fig. us
are demonstrated. 11:15.

 

-514.
Sulfonamide Bacteriostasis of Diphtheroids

As an indicator of possible therapeutic action of any drug in
case of a disease caused.by a specific microgrganism the ”in vitro'
method may be used. Many strains of the type species of the genus
Corynebacterium have been used by Ouyang (lShl) to ascertain the action
of sulfonamides. but as far as is known no reports on the bacteriostasis
of the diphtheroids of pyelonephritis have been published.

The sulfonamide bacteriostasis of any organism seems to depend
on the essentiality of p-aminObenzoic acid for the growth of that
organism. The more the organism depends on this nutritional factor. the
more susceptible it is to the sulfonamide substitution in its metabolism.
Woods reported in l9hO on the relation of p—aminobenzoic acid to the
mechanism of action of sulfanilamide. Since then it has been reported
that only the ionized.part of the drug is active and that the difference
between the various members of the sulfonamide group is due to the
difference in degree of ionization which characterizes each member at any
one pH level (lyss. l9hl; Fox and Rose. l9h2).

This study was instituted as preliminary work in the search for
an effective therapeutic agent to be used in the treatment of pyelo-
nephritis of cattle.

Materials and Methods

 

The cultures used were from cases of pyelonephritis in cows as
may be noted in the section devoted to sources of cultures in the begin-
ning part of the thesis. The medium used was the regular beef extract
broth containing sodium chloride 0.5 per cent. Bacto beef extract 0.3 per
cent. and Bacto peptone 0.5 per cent in distilled water and was adjusted
to a pH of approximately 7.3 with sodium hydroxide by means of a Bookman
pH meter. Sulfanilamide.*sulfapyridine’and sulfathiazole‘ were dissorved

* Kindly supplied by Merck and Company. Rahway. N.J.

~55~

in the broth in the amounts mentioned in the chart. tubed and then auto.
claved at 15 lbs. for 20 minutes. The inoculum was one lOOpful of a
2“ hour broth culture. The amount of growth in all tubes was estimated
and noted every 2h hours for h days though only the final reading is
recorded here.

Results

The chart contains the essential data. There were differences
between the individual sulfonamides as well as between the diphtheroids.
Some cultures were able to grow in broth solutions of sulfanilamide in
concentrations up to 200 mgms per cent.in sulfapyridine up to 25 mgms
per cent which was the highest used and in sulfathiazole up to 20 mgms
per cent. is may be noted, some cultures did not grow at all in
sulfonamide solutions. These were mainly of the fine growing type which
require blood for Optimum growth. Sulfathiazole inhibited the growth.of
almost all of the cultures.

ggpcussion and Conclusions

The difference in degrees of action of the three sulfonamides was
expected due to their differences in degree of ionization. Also. in view
of the various growth characteristics of the diphtheroids the individual
differences were expected.

To evaluate the "in vitro' action of sulfonamides as an indica-
tion of their ”in vivo' or therapeutic action many factors are to be con»
sidered. The nutritional needs of the organism in artificial media. the
tolerance of the animal body for the drugs. the effective concentration
or drug level in body fluids. the degrees of solubility and ionization of
the drugs in body fluids. the minimal therapeutic period. economy of
effective treatment and the presence of irreversible pathological changes

are some of the factors to be considered.

-56-

On the basis of the data presented sulfathiazole seems worthy of

further study and clinical trial.

957e-

 

 

 

 

 

 

.ucmm H.603. co coasvHH—opmc some 03.3 on. o». demon m
some H602. no wousaHsopsm some 0.3.3: on. ca demon H.
Queen» acmHuchH .. a: mason» mo ecoawoc : 0.... H
363328 .3 0093 .3
museum on o
m H en» as es» s s a m a n H as ea» c
H as» a» no es» 2 a n H s m m H an» eye
H6 0 o o o co 0 o o eo o o o o o
Hso o o o o co . o o o eo o o o o 0
ac o o o 0 U0 0 o o a m es» 0 o o
m H can a» a» a a m m a a a m use as»
so 0 o o co m a» H do a so 0 o o o
Hco o o 0 eo : H H «a» a n H as» ea» ecu
eo o o o no a m H as a m H H use can
a» o o o o a» o o o a» o o o o o
co 0 o o o o o o o o o o co o o
eo o o o 0 e0 0 o 0 we 0 o o o o
co 0 o o 0 ac o o o so 0 o o o 0
0H om omul 0: am . oH mH om mm on. 00H omH com cam. can

 

 

Winema cacao anaemia We name on 3 «.HNanMHH—m

 

 

 

eaaeaoeauoaeem sandman—um

as evuemnwnemasm

MMMHHJ‘Ja-‘ffldfifizffl

 

 

 

 

 

 

BACTEBI GPA THOLOGY

-59-
Bacteriopatholggy_of Infectious Bovine Ryelonephritis

The location of the causative agent in relation to the lesion it
causes is an interesting and important factor in the study of the disease
and an aid in understanding the nature of the lesion.

Dammann (1877) found bacteria in the urinary tubules. Guillebeau
(1888) found slender. nonnmotile bacilli in renal abscesses. Hgflich
(1891) mentioned. "On one occasion only. in some sections stained with
hematoxylin. the organisms were seen in the glomeruli while felted masses
were constantly met with in the terminal portions of the straight tubes
and in the epithelium of the pelvis and ureter". Ernst (1905) describes
the condition in the early stage of the disease. "Gram's stain reveals
masses of bacteria scattered irregularly through the abscesses in the
medulla around blood.vessels: these masses surround the malpighian
corpuscles like a cap. The bacteria are collected into clumps and
cylinders in the convoluted tubes and in the loops of Henle. becoming
less frequent in the sub-cortical zone. Groups of bacteria are rare in
the thrombosed vessels. The straight tubes. which are packed with
bacteria eliminated from the labyrinth. give the medullary zone a
striated appearance. The papillary tubes appear free. but their mucous
membrane is infiltrated with leucocytes". Describing a later stage.
that of papillary necrosis. he stated. ”Disintegration of the collecting
tubes of the papilla. which are crammed with bacteria. debris and
leucocytes. occurs '.

Jones and Little (1925) stated that the organisms were present
in the infected kidney pelves. .Also. bacteria were found in large numbers
in the necrotic debris on the mucosa of the bladder. from these data and
the fact that the bacteria were never found in the deeper structures they

deduce that the organism is not very invasive. In 1930 the same authors

-60—

noted densely packed masses of diphtheroids in the large tubules lying
close to the papilla in the kidney.
Eaterials and.Methods

Sections of kidney. ureter and bladder were stained by the
Gram-Weigert method for staining bacteria in sections and examined to
determine the location of the bacteria and their relationship to the
lesion produced.

new

There was much variation between cases. In some instances the
bacteria seemed to be only superficially located in the cellular debris
of the kidney calyces and in similar material adherent to the mucosa of
the ureters and bladder. In these cases there was local necrosis and
areal increase in the number of lymphocytes and also in the activity of
the connective tissue elements. There was no direct invasion of the
living tissues as far as could be determined in these cases. In other
instances the bacteria were located superficially as those cases
mentioned.above but also were found in scattered foci throughout the
kidney cortex and in the thickened capsule. Their presence was always
attended by necrosis and inflammatory reactions of a chronic type -~
infiltration of tissues with lymphocytes and a few mononuclear phagocytes
plus an increase of the interstitial connective tissue. These foci
varied from very small ones in which only a few tubules were found.to
contain bacteria which.were causing necrosis of the epithelial cells to
fairly large cortical abscesses which contained much dead tissue and
large numbers of bacteria and were surrounded by the chronic inflammatory
reaction already mentioned. Local necrosis surrounding each clump of
bacteria was a constant feature. The clumps of bacteria varied in size

from several short rods together to large amorphous darkestaining masses

-61.

along the edges of which a few bacteria were visible, enabling recogni-
tion of the material.
Discussion

The seeming difference in virulence or invasiveness of the
diphtheroids in the various cases studied is in keeping with the variap
tion.between diphtheroids which seems to be one of their characteristics.
The difference in susceptibility of the infected animals should also be
considered.

Possibly the location of the lesions and the etiological agent
is a factor in explaining the course of the disease in different cases.
Some cases are of a chronic nature while others are fulminating and
terminate soon. The more mild and drawn out type of case may be
associated with the superficial slowly advancing type of lesion in which
the bacteria are in the calycal cellular debris and seem to cause the
medulla to slowly be necrosed and to recede. In later stages lesions may
be found throughout the kidney. The fulminating type of case may be
associated.with the more severe lesions in the kidney with scattered
abscesses throughout.

One of the more striking observations is that outside of the
lesions in the kidney tubule the bacteria seem rarely to be in contact
with living tissue. This is especially apparent in the kidney calyces
and the ureter and bladder. The bacteria are found in the tissue debris
next to a necrotic area in the mucosa which is bordered on the apposite
side by a chronic inflammatory zone. Observation of this type of lesion
and the fact that the type species of the same genus produces a powerful
toxin has stimulated the search for a toxin produced.by the diphtheroids
isolated from cases of pyelonephritis. So far no one has reported demon-

strating any such toxin. One may only conjecture that the necrosis is the

-62..

result of an endotoxin which is not apparent in culture filtrates.

The inherent difficulties in finding single or a few bacteria in
tissue sections are realized. It is possible that careful culturing
technics would.aid in further establishing the location of bacteria at
least in relation to the medulla and cortex of the kidney.

Summagy

The location of the bacteria varies in different cases. In the
milder cases the microgrganisms are present seemingly in only the
cellular debris of the kidney calyces and the ureter and.bladder mucosa.
In the more severe cases the microgrganisms may also be found in foci
scattered throughout the kidney. Bacteria may be found in the tubules
and necrotic tubular epithelium. In the strikingly characteristic
lesion bacteria are in cellular debris which is flanked.by a zone of

necrosis and outside of that a zone showing chronic inflammatory changes.

Fig. 50. Bladder mucosa shows general architecture of tissue
alterati on. Cellular debris. necrotic tissue and
zone of chronic inflammation. Gram-leigert stain.

X95-

rig. 51. Hiaer magnification of bacteria in cellular debris in
fig. 50. Gram-[eigert stain. 1770.

.63.

 

 

Fig. 50

 

 

 

 

fig. 51

Fig. 52. Ureter. General architecture of lesion.
Gram-Weigert stain. 195.

Fig. 53. Ureter. Bacteria in cellular debris.
Gram-leigert stain. x550.

 

‘

~61I~

9."
,

.fir"

.‘ - u - '
.s v i ‘, .-
. .1 z. . ‘
. f .
O
5

s I. .a‘. ‘-
”919.4
'10, ‘;V

”- .‘. '.“,.'. .c,
a -. \‘l .
My? “6%; «7.3”.-
d’f'. ' ”“5 '\.-. 3. x'r

S

‘ A.

. ““*(m.

'n

 

Fig. 5h. Kidney. Inner medulla. General design of lesion.
Gram-Weigert stain. 182.

rig. 55. Bacteria in cellular debris in lesion similar to that
in 113. She Gram-Weigert stain. 1770.

 

  

-55..

    
  
  

-. i (4'
. 7, r, ‘;
Jiggtryfl. -. A
1‘ "2‘ ’ . -7,_

Hg. 5”

 

 

Fig. 56. Cortex of kidney. Tubular lesions. Masses of bacteria
in tubules. Necrosis of epithelium. roci of lympho-
cytes. Gram-Weigert stain. 195.

Fig. 57. Bacteria in kidney tubule. Gram-Weigert stain. x770.

 

”66“

 

 

-67-

ACKNOWLEDGMENT

The guidance and imparting of knowledge by
Dr. E. T. Hellman was very much appreciated.

The patient pedagogy and driving personality
of Dr. Frank Tharp. Jr.. was a constant asset and
inspiration in conducting the work and the preparation
of this thesis.

Also very helpful was the assistance and advice
of Dre. C. 1. Clark. B. J. Killham. R. r. Langham.

H. R. Ruhland and L. B. Shall.

-68..

BIBLIOGRAPHY
1. References Available For Use.
2. References Compiled But Not Used.

3. Bibliography of Ritzenthaler.

. ..‘

.ifl'flhe- '

“.3: -7-

W315} - -

1910.

1918.

1918.

1918.

1925-

1926.

1926.

1927.

1930.

1931.

1935-

1936.

1937.

-69-
1. References Ayailable For Use

Ritsenthaler. l.
The Pathology of Bacillary Pyelonephritis in Cattle.
Jour. Comp. Path. and Therap. 23:33-60.

Boyd. I. L.
Pyelonephritis in Cattle.
Corn. Vet. 8:120-121.

Gair. G.
Bacterial Pyelonephritis in a Cow.
Vet. Journal. 7hz5é~58. 5

L“;
\J

BOYd. '0 Les FltCh. Os Po and. Billings, We A. i.
Ovine PYelonephritis.
Corn. Vet. 8:2hl.

'—-mfln1'

Jones. I. s. and.Litt1e. R. B.
Specific Infectious Cystitis and.Pyelonephritis of Cows.
Jour. Exp Med. ha: 593-607.

s-i- .s.

1*

Jones. r. 8. and Little. R. B.

The Organism Associated with Specific Infectious Cystitis and
Pyelonephritis of Cows.

Jour. Exp. Med. uh:1l-20.

361111! ’ He ‘0
Nephrectomy in Bovine Practice.
Vet. Med. 21: .161—162.

Boyd. l. L.
Pyelonephritis of Cattle.
Corn. Vet. 17: h5-6o.

Jones. r. 8. and.Little. R. B.

A Contribution to the Epidemiology of Specific Infectious
Cystitis and Pyelonephritis of Cows.

Jour. Exp. lied. 513909.920.

Palmer. c. Cs
Pyelonephritis in Cattle. ,
Vet. Med. 26:168-177. .

“orChant s I e ‘0
Corynebacteria.Associated with Diseases of Domestic Animals.
J. Bact. 30:95-118.

Manual of Methods for Pure Culture Study of Bacteria.
Society of American Bacteriologists.
Biotech.Publications, Geneva. N.Y.

(Current revisions used)

Boyd. I. L. and Bishop. Lucille M.
Pyelonephritis of Cattle and Horses.
J. 1.7.5.1. 90,n.s.. h3;15h.159.

1937-

1938.

1938.

1939.

19ho.

19ho.

19h1.

19hl.

l9h1.

19h1.

19h2.

19h2.

l9h2.

19n2.

~70—

Hawk. P. B. and Bergeim. 0.
Practical Physiological Chemistry. 11th Ed.
P. Blakiston's Son and Co.. Inc.. Philadelphia.

McIntosh. R. A.
Pyelonephritis.
Vet. Med. 33:h10-h11.

Palmer. C. C.
Pyelonephritis in Cattle.
J. A.V.M.A. 93:2hluah3.

Bergey. D. 8.. Breed. R. 5.. Murray. B. G. D. and Kitchens, 5. P.
Bergey's Manual of Determinative Bacteriology. 5th Ed.
The Williams and Wilkins 00.. Baltimore.

The Merck Index. 5th Ed.
Merck and Co.. Inc.. Rahway. N.J.

Woods. D. D.

The Relation of puaminobenzoic Acid to Mechanism of the Action
of Sulfanilamide.

Brit. J. Exp. Path. 21:7u-9o.

Bruner. D. I. and.Edwards. P. R.
Classification of Corynebacterium 393;,
Ky. Agr. Exp. Sta. Bul. hlhg9l~lo7.

Hoffman. I. s.
Photolometric Clinical Chemistry.
William Morrow and Co.. N.Y.

Ouyang. George.

”In vitro" Effect of Sulfanilamide. Sulfapyridine and Sulfathiazole
on‘gprynebacterium_giphtheriae.

Proc. Soc. Exp. Biol. and Med. h8:hluh3.

IVES. Os
The Nature of Sulfanilamide Inhibition.
Proc. Soc. Exp. Biol. and Med. h83122-126.

Doll. 2. a.
Unpublished data.

 

10!. Ce Ls and 3030. Ho Ho
Ionization of Sulfonamides.
Proc. Soc. EXp. 3101. and Med. 50:1u2.1h5.

Morgan. B. B. and lisnicky. W.

A Further Note on the Incidence of Trichomonas Fetus in
Slaughtered Cattle from a Wisconsin abattoir.

J. 1mm.» 100:14714472. -

Morgan. B. B.
Comparison of pH and Papulation of Trichomonas Foetus.
Proc. Soc. Exp. Biol. and Med. 51:380n382.

vac: as“.
“- 1. ..

1.3-...‘1‘.’ I.

h.

 

19h}.

19h3.

19h}.

19h}.

19hh.

-71-

Andberg. W. G. and Ieirether. F. J.

The Effect of Chemotherapeutic Agents on the Normal Bovine
Mammary Gland. I. The Effect of Novoxil.

Am. J. Vet. Res. h:l3h~lh2.

COffin. Do In

The Diagnosis of Specific Cystitis and Pyelonephritis by
Practical Office-Laboratory Procedure.

0. of Penn. Bul. nu: No. 3. pp.3—io.

Olney. R.
Pyelonephritis in a Holstein Priesian Cow.
Vet. Med. 38:397-398.

Tharp. 1.. Jr.. Langham. R. 1.. Clark. C. r. and Doll. B. R.
The Pathology of Bovine elonephritis.
Am. J. Vet. Res. h:2ho~2 9.

loss. J. 0.

Identification of Corynsbacterium renalis from the Kidney and
Bladder of a Horse.
J. A.V.u.a. ion-27.

P‘.“’ :1 . .

W)?“

 

 

 

 

1923.

1926.

1927.

1928

1928.

1929.

1930.

1931.

193k.

1936.

-72.

2. References Compiled But Not Used

 

Palmer. C. C.
Ohio State Vet. Alumni Quart. XI:22.
from Palmer. 0. 0.. Vet. Med. 26:168nl77.

Frisch. B.

Cystopyelitis from Pseudodiphtheria Bacilli.
w€}n. klin. Wchnschr. 39:11ho.

Quart. Cummulative Indicus Medicus. Vol. 1, 1927.

Declich. M.

Bacilli of Infectious Pyelonephritis in Animals.
Am. d'ig 37:683.

Q.C.I.M. Vol. 3. 1928.

Chiaudano. c. a

Pyelonephritis Due to Pseudodiphtheria Bacilli.
Gior. di batteriol. e immunol. 3:6h3.

QsCeIeMO V01. u so 19280

Hester

Pyelonephritis in Cows.

Nederl. Tijdschr. V. Geneesk. 2:M732.
Q.C.I.M. Vol. 5. 1929.

Ubertlni 9 Be
Pyelonephritis in Cattle.
Clin. Vet. 52.211.
Q.C.I.M. Vol. 6. 1929.

Borghesaleo. A.

Bacillary Pyelonephritis in Cattle; Cases.
Clin. Vet. 53. 1h}.

Q.C.I.M. V01. 7. 1930.

Ventura. L.

Pyelonephritis of Bacillary Type in Cattle.
Poll. I. Soc. eustachiana 29:229.

Q.C.I.M. Vol. 11. 1932.

Engels, C. 1‘.

Diphtheroid Infection of Bladder.
Urol. and Cutan. Rev. 38:5h3.
Q.c.1.u. Vol. 16. 193k.

Korsenssky. I. .

Properties of Corynebacterium renale.
Thesis. Budapest.

Indicus Veterinarius - Vol. 6. No. 2. 1938.

.I ~'
§~s ’w

 

1869.

1875.

1876.

1877.
1887.

1888.

1888.

1889.

1889.

1890.

1890.

1891.

1891.

1891.

1891.

~73-
3. Bibliography of Ritzenthaler

Bruckmuller
"Lohrbuch der pathologischen Zootechnie"
Vienna. p. 657

Siedamgratzky

"Ber. u. d. Veterinarw. i. Kcnigr. Sachs)!
p. 38

Pflug
"Die Krankh. d. UrOpoetischen Systems uns. Haust"
Vienna. p. 38 and 13h

Dammann
"Zeitschr. f. Tiermedizin"

Roder .
”Ber. u. d. Veterinarw. i. Konigr. vet. patholog."

Gillot
"Rec. Med. Vet."
r. va. p.159

Guillebeau and Hess
“Schweiz. Arch. f. Tierheilk
T. xxx. p. 269

Bang
"Tidskr. fur. Vet."

p. 369

Cadiot
WBull. Soc. Centr. Med. Vet."
r. x1111. p.h58

Guillebeau and Hess
”Schweiz. Arch. f. Tierheilk"
T. XXIII. p.22“

Schmidt
"Mashedschr. f. Dyrl.”
p.1h9

Bollinger. D.
"Zeitschr. f. Tiermedizin".
r. XVII. p. 3u6

Enderlen. D.
I'Zeitschr. f. Tiermedizin".

p~ 325

Guillebeau and Hess
'Schweiz. Arch. f. Tierheilk".
T. XXXIII. p. 157

Hoflich
"Monatsh. f. prakt. Tierheilk"

T. II. p. 337

.‘4.

.‘—m “A .?""~'._‘1 11 .

er;
0‘).

\

 

1892.

1892.

1893.

1893.

1893.

189h.

1895.

1895.

1896.

1897.

1897.

1897.

1898.

1899.

1900.

-7h-

Guillebeau and Hess
"Schweiz. Arch. f. Tierheilk".
To XXXIV. p. 70

Lucet
"Journ. de Med. Vet. et. de Zootechnie"
T. XLIIL p. 220

Kitt
"Monatsh. f. prakt. Tierheilk",
T. IV. pp. h33 and M81

Lucet
”Rec. Med. Vet."
T. LXX. p. 273

Rasberger
"Woohenschr. f. Tierheilk. u. Viehz"
p. 31h

Knoll
"Berlin. t. Wochenschr".

p. 197

Freytag
”Ber. u. d. Veterinarw. i. Konigr. Sachs"
p. 100

Mollereau and Porcher
”Bull. Soc. Centr. Med. Vet."
T. XLIX

Nocard
”Ann. Inst. Pasteur"

p. 609

Bartels
"Deutsche t. Vochenschr.‘

p- 303

Cadeac and Morot
”Journ. de Med. Vet. et de Zootechnie.”
T. XLVIII. p. 65

Nocard
"Rec. Med. Vet."

pp- 1 and 99

Bunge
I'Zeitschr. f. F1. u. Milchhygieine."
p. 168

Sivori
HAnn. de Med. Vet.”

p- 657

Albrecht
”Wochenschr. f. Tierheilk. u. Viehz".

ps 1‘09

mini. ' -‘ .-
i.“ ”F

\r um'l‘2‘
. e .

c.

1900.

1901.

1902.

1903.

1903.

1903-

1908.

1905.

1906.

1906.

1907-

1907.

1909.

-75-

Tauschner u
"Zeitschr. f. F1. u. Milchhygieine.

Do 131

Zimmerer
"Wochenschr. f. Tierheilk. u. Viehz.”

p- 1‘95

Lienaux and Zuaenepoel
"Ann. de Med. Vet."
p. 500

Baillet and Seres
"Rev. Gen. Med. Vet."
T. I.. p. 50h

Kunnemann

"Arch. f. Wiesenschaftl. u. prakt. Tierheilk”.

p. 128

Nocard and Leclainche
”Les Maladies Microbienne des Animaux".
T. II. p. 393. Paris

Scherzer
WBerlin.t. Wochenschr"

p- uhS

Ernst

"Centralb. f. Bakt."
r. xxx1x. pp.5h9-66o
To XL. '90 79

Friedberger
”Klin. Diagn."

p. 568

Sommer
”Berlin t. Wochenshr."
p. 900

Bergeron
TRev. Yeter".

p. 797

Lehmann and Neumann
"Atlas u. Grundriss der Bakteriologie"
Fourt Edition. Munich. pp. 151. 501 and 670

Eutyra and Marek
"Spez. Patholog. u. Therapie der Haust"
Second Edition. T. I. Jena. p. 959

 

_.
l , IIII..Lrt I- IIII..

l!- . .-_ ..t|l: I: ..-l ls . IIII‘Il.

 

 

 

 

 

 

 

 

|l|l
‘
‘1‘
\||
‘||‘
‘
‘
I||||
‘Ilu
‘||‘|l
“I‘l|
‘
I‘l‘l
‘1
\I‘
\
‘I‘II
|
"I‘ul
|I‘|I
l
|l‘|

9
2
1