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lIBRARY
Michigan State
University

This is to certify that the

dissertation entitled

"The Pathology and Transmission

of Mycobacterium bovis in Michigan Wildlife"

presented by

Kelly Lynn Butler

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Veterinary Pathology

W28“ %{/¢«/¢Z

Major progs’sgv
Date Z/L 0 /0 /

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

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PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE I DATE DUE

DATE DUE

 

f Eli-‘03. g 3005

‘U 3 1
MAY 1 0 2008

 

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6/01 c:/CIRC/DateDue.p65-p.15

 

THE PATHOLOGY AND TRANSMISSION OF M YCOBA C TERIUM BOVIS IN
MICHIGAN WILDLIFE

By

Kelly Lynn Butler

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pathobiology and Diagnostic Investigation

2001

ABSTRACT

THE PATHOLOGY AND TRANSMISSION OF MYCOBACTERIUM BOVIS IN
MICHIGAN WILDLIFE

By
Kelly Lynn Butler

Michigan currently has an endemic strain of Mycobacterium bovis tuberculosis in its
free-ranging white-tailed deer (Odocoileus virginianus) herd. The disease has been
identified in deer and a large number of small mammalian species throughout the Lower
Peninsula of the state, has spread to domestic cattle herds, and has been identified in a
captive cervid herd. Cases identified through surveillance programs in wildlife are
primarily concentrated in the northeastern portion of the Lower Peninsula thus far, but
new counties with positive cases are identified each year as the span of surveillance
widens and the numbers of animals included in the program grow.

The objectives of this body of work all sought to better define this endemic strain of
tuberculosis in Michigan wildlife. A primary objective was to evaluate several common
species for their potential in the transmission of the disease, two native passerine bird
species, the European starling (Sturnus vulgaris) and the American crow (Corvus
brachyrhynchos), and the North American opossum (Didelphis virginianus) were studied.
Although they would certainly figure prominently in transmission if they were efficient
hosts of the disease, none of these species had ever been challenged with the M. bovis
organism experimentally, and no reports describing natural infection exist, so a large part
of this thesis was to describe any pathology resultant from experimental inoculation of

these animals. Birds were challenged intraperitoneally and orally with M. bovis, but

infections were only established by the intraperitoneal exposure route. Opossums were
challenged intramuscularly and orally with M. bovis. Infection was established via both
exposure routes in Opossums.

Major concerns with tuberculosis in all species include the zoonotic potential of the
disease and the emerging threat of these microorganisms becoming increasingly resistant
to drugs used in treatment. A fundamental question that had not been examined until the
time of this work was whether or not the strain of M. bovis causing endemic tuberculosis
in Michigan’s wildlife had any evidence of drug resistance. The objective to determine
the susceptibility of the strain to common antimycobacterials was achieved successfully,
it was found that there is no evidence of resistance to date in this strain. Moreover,
comparison of the strain to that of several human cases of M. bovis identified in Michigan
showed differences in both susceptibility profiles and in genetic fingerprints when
compared to the animal case strain.

The final objective of this dissertation was to evaluate a novel cytologic technique
for its potential application in the detection of mycobacteria in clinical samples. Cells
were obtained from the tracheal, nasal, and oral mucosae of white-tailed deer
experimentally inoculated with M. bovis. These samples were examined for the presence
of intracellular mycobacteria after being prepared using the new technique, the ThinPrep
2000 automated cytology device. Results were compared to mycobacteria] cultures taken
from the same sites in these deer. The technique showed some promise in the
antemortem diagnosis of mycobacterial disease, but was less sensitive and specific than

existing veterinary diagnostic tests.

DEDICATION

For Mom, Dad, and Rob...

I suppose this would be where I say “I couldn’t have done it without you”-
I consider myself very lucky that I’ll never have to find out whether
or not I could have done so.

For being my strength, my hmnor, and my reality check

in times when all of these things are so hard to find on my own-
Thank you. I love you.

iv

ACKNOWLEDGEMENTS

The scope of the tuberculosis project in Michigan makes it impossible to
mention all of those people who have been my mentors in one way or another
over the years, but there are certainly those without whom this project could

not have been realized.

My deepest gratitude goes to my mentor, major research advisor, and
friend for the entirety of this graduate program, Dr. Scott Fitzgerald. His
continued support and superior advice have made the completion of my
graduate program possible. And his sense of humour has made it enjoyable.

Thank you, Dr. Fitzgerald.

I would like to give special thanks to Dr. Jim Sikarskie, not only for being
a helpful member of my graduate committee, but also for helping to obtain and
care for many of the experimental subjects used in both projects that are now

completed and those that are in planning.

Dr. John Kaneene has been a great help, not only as a committee member,
but also as an instructor. He continues to make less nebulous those fields that
don’t always come naturally to pathologists, statistics and epidemiology.
RoseAnn Miller also deserves special mention for her help in preparing

statistical analyses presented in this dissertation.

The remaining members of my graduate committee, Dr. Tom Bell and Dr.
Willie Reed, have been supportive not only through my doctoral degree work,

but more or less since I set foot into the AHDL as a resident many years ago.

Dale Berry, Steve Church, Angie Schooley, and Sandy Arduin of the
Michigan Department of Community Health have made much of my research
possible by providing inocula and by culturing tissues of both surveillance and
research project subjects. I am particularly grateful to Dale and Steve for their
help in the delicate inoculation of what were at times some rather wild wildlife

subjects.

Many members of the Michigan Department of Natural Resources have
encouraged my work and certainly have helped in surveillance projects. I
thank Dr. Steve Schmitt, Dr. Dan O’Brien, Jean Fierke, Tim Lyons and Tom
Cooley for their hard work and for being able to maintain a sense of humor

during many mentally and physically challenging months of surveillance work.

Dr. Mitchell Palmer and Diana Whipple of the National Animal Disease
Center have both been extremely encouraging, and working in collaboration
with them has been a true pleasure. I admire them both as great scientists and

as great individuals.

vi

Special mention should be made to Drs. Bob Dunstan and Dr. Tom Bell
for their guidance in the formative stages of my graduate program. They have
allowed me to ask honest questions and have always given me honest answers,
an invaluable trait that I admire in them both. I am truly lucky to have worked

with these men.

My office mates, past and present: Dr. Mike Slanker, Dr. Kelly Credille,
Dr. Kathy-Anne Clarke, and Dr. Laura Zwick have been an unmatched source
of support, comfort, and sometimes, a much needed laugh. I will always
consider these people my extended family. I am so happy our paths have

allowed us to meet.

vii

TABLE OF CONTENTS

Page
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xiv
CHAPTER 1: Literature Review and

Surveillance Background 1
Literature Review 2
Mycobacteria: Structure and Virulence 2
Pathogenesis and the Immune Response in Mycobacteria] Disease 5
Introduction 5

Basic Pathophysiology and Immunology 6

Cell Mediated Immunity Versus Delayed-Type Hypersensitivity] 11

Pathologic Presentation of Tuberculosis in Animals 12
Species of Mycobacteria 12

Mammalian Versus Avian Presentations of Tuberculosis 14
Immunology 14

Clinical Presentation of Disease 17

Comparative Pathology 18

Tuberculosis in Cattle and Deer 22
Surveillance Program Background-M. bovis in Michigan 25
Mycobacterium bovis 25
Tuberculosis in Michigan Deer 26
Tuberculosis in Other Michigan Wildlife Species 29
Objectives for Research 32

References 51

viii

CHAPTER 2: Experimental Inoculation of European
Starlings (Stumus vulgaris) and American Crows (Corvus brachyrhynchos)

with Mycobacterium bovis 58
Abstract 59
Introduction 59
Materials and Methods 60

Birds 60
Inoculum 61
Experimental Design 61
Starling experiment 61

Crow experiment 63
Mycobacterial Isolation and Identification 63
Statistical Analysis 64
Results 65
Clinical Condition of Birds 65
Weight Change Data 65
Gross Necropsy Findings 66
Microscopic Lesions 67
Mycobacterial Isolation and Identification 69
Statistical Analysis 69
Discussion 70
References 82

CHAPTER 3: Experimental Inoculation of North
American Opossums (Didelphys virginiana) with

M ycobacterium bovis 84
Abstract 85
Introduction 85
Materials and Methods 86

Opossums 86
Inoculum 87
Experimental Design 88
Mycobacterial Isolation and Identification 90
Surveillance Data 91
Statistical Analysis 91
Results 92
Discussion 96
References 109

ix

CHAPTER 4: Antimicrobial susceptibility patterns of
human and wildlife Mycobacterium bovis isolates
from Michigan

Abstract

Introduction

Materials and Methods

Results

Discussion

References

CHAPTER 5: A Novel Cytologic Evaluation
Technique for the Detection of Mycobacteria
Abstract
Introduction
Materials and Methods
Deer Subjects
Mycobacterial Strain
Specimen Handling
Cytology Preparation and Scoring
Statistical Analysis
Results
Discussion
References

CHAPTER 6: Conclusions and Future Directions

E

111
112
113
115
117
118
123

125
126
126
131
131
132
132
133
134
135
136
147

149

LIST OF TABLES
Table 1.1. Yearly prevalence rates for M. bovis tuberculosis in deer in
Michigan, 1995-2000.

Table 1.2. Positive cases of M. bovis tuberculosis in Michigan
carnivore/ omnivore surveillance through the year 2000.

Table 2.1. Laboratory findings in starlings.

Table 2.2. Laboratory findings in crows.

Table 3.1. Laboratory results.

Table 3.2. Summary of cases with evidence of M. bovis tuberculosis.

Table 3.3. Carnivore and omnivore surveillance results for M. bovis in
Michigan (1996-2000).

Table 4.1. Wildlife-source M. bovis cases/ isolates from Michigan
showing growth on proportion plate drug susceptibility testing.

Table 4.2. Human-source M. bovis cases/ isolates from Michigan
showing growth on proportion plate drug susceptibility testing.

Table 5.1. Deer numbers at post—inoculation (PI) sampling dates.
Table 5.2. Deer which cultured positive for M. bovis.

Table 5.3. Deer with positive cytology readings.

Table 5.4. Cytology scores for those samples with positive readings.

Table 5.5. Results of descriptive statistical analysis of Thinprep
cytoprep technique.

xi

50

77

77

104

104

106

122

122

142

142

142

144

144

LIST OF FIGURES

Figure 1.1. Tuberculosis in the lung of a Michigan white-tailed
deer (Odocoileus virginianus).

Figure 1.2. Tuberculous granuloma on the pleural surface of the
lungs of a Michigan white-tailed deer.

Figure 1.3. Purulent tuberculous abscesses in the lymph nodes of a
Michigan white-tailed deer.

Figure 1.4. Caseous tuberculous abscess in a lymph node of a
Michigan white—tailed deer, cut section.

Figure 1.5. Geographic location of initial M. bovis case in
Michigan, 1975.

Figure 1.6. Geographic location of current M. bovis endemic
white-tailed deer propositus, 1994.

Figure 1.7. White-tailed deer M. bovis cases identified in first
year of surveillance, 1995.

Figure 1.8. White-tailed deer M. bovis cases identified in 1996.
Figure 1.9. White-tailed deer M. bovis cases identified in 1997.
Figure 1.10. White-tailed deer M. bovis cases identified in 1998.
Figure 1.11. White-tailed deer M. bovis cases identified in 1999.
Figure 1.12. White-tailed deer M. bovis cases identified in 2000.

Figure 1.13. Geographic location of index case of M. bovis identified
in carnivore surveillance, a coyote from DMU 452, 1996.

Figure 1.14. Camivore/ omnivore surveillance data
(positive M. bovis cases) from the most current year of
surveillance, 2000.

Figure 2.1. Weight data for starlings.

Figure 2.2. Weight data for crows.

xii

37

39

39

41

41

42

42

43

43

46

46

79

79

Figure 2.3. Photomicrograph of liver from a starling orally
inoculated with M. bovis.

Figure 2.4. Photomicrograph of liver from a starling orally
inoculated with M. bovis.

Figure 2.5. Photomicrograph of liver from a crow intraperitoneally
inoculated with M. bovis.

Figure 3.1. Section of skeletal muscle from an opossum inoculated
intramuscularly with M. bovis.

Figure 3.2. Section of lung from an opossum intramuscularly
inoculated M. bovis.

Figure 3.3. Section of lung from an opossum intramuscularly
inoculated with M. bovis.

Figure 5.1. Positive (2+) cytology obtained using ThinPrep
technique, tonsilar sample.

Figure 5.2. Positive cytology (2+) obtained using ThinPrep
technique, nasal sample.

Figure 5.3. Cytology example with large intracellular cocci and
no acid-fast bacilli noted, ThinPrep technique.

xiii

81

81

108

108

108

146

146

146

AHDL

aB-T
BALT

BCG
BTB
CD
CI
CIP
CFU
CM
CMI
CS
DMU
DTH
EA
ELISA

EMB
GALT

GSF

H&E
HEPA

MCP I

MDR

LIST OF ABBREVIATIONS

xiv

Animal Health Diagnostic
Laboratory

alpha-beta T cell
bronchial-associated
Lymphoid tissue

bacillus Calmette-Guerin
blood tuberculosis test
cluster of differentiation
confidence interval
ciproflozacin

colony forming unit
capreomycon

cell mediated immunity
cycloserine

deer management unit
delayed type hypersensitivity
ethionamide
enzyme-linked
immunosorbent assay
ethambutol

gut-associated lymphoid
tissue

gamma-delta T cell
growth index

granulocyte stimulating
factor

hematoxylin and eosin
high efficiency particulate air
high power field/ 40X
gamma interferon
interleukin

intramuscular

isoniazid

intraperitoneal
kanamycin
lipoarabinomanan

M. avium complex
monocyte chemoattractant
protein I
multidrug-resistant

MHC

MOTT
MDCH
MDNR

MSU
NADC

NK
PBS
PI
PPD
PZA
RA
RFLP

SM
TAD/ TDM
TCH

Thl/ 2
TGFB
TNF-a

major histocompatibility
complex

mycobacteria other than
tuberculosis

Michigan Department of
Community Health
Michigan Department of
Natural Resources
Michigan State University
National Animal Disease
Center

natural killer T cell
phosphate buffered saline
post inoculation

purifed protein derivative
pyrazinamide

rifampin

restriction fragment length
polymorphism
streptomycin
trehalose-6,6’ dimycolate
thiophen-2-carboxylic acid
Hydrazide

helper T cell subset 1/ 2
transforming grth factor B
tumor necrosis factor alpha

Chapter One

Literature Review and Surveillance Background

Literature Review

Mycobacteria: Structure and Virulence

Organisms classified as Mycobacterium spp. are numerous, and include those
ranging from nonpathogenic or opportunistic saprophytes, to highly pathogenic,
zoonotic disease agents. A large part of the virulence of these organisms is
determined by certain structural components and biochemical or enzymatic functions
inherent to each species. In general, mycobacteria are non-spore forming, rod-shaped
(bacillar), slow-growing, variably obligate aerobes. Unique to mycobacteria (and to a
lesser extent, corynebacteria and nocardiae) is their characteristic acid-fastness. This
term describes the fact that once stained, mycobacteria will retain stain after treatment
with acid or alcohol (Brooks et a1 1991). Both the difficulty in initial staining and the
resistance to decolorization that mycobacteria exhibit are due to the composition of
their cell walls. The largest part of the cell wall of mycobacteria is an amalgamation
of lipids and lipid-containing compounds, including waxes, which contribute to most
of their acid-fastness. Proteins and polysaccharides make up the remaining
constituents. Together, these components elicit both delayed type hypersensitivity and
cell mediated immunity to varying degrees. Humoral immunity is not protective in
mycobacteria] disease, and does not play a significant role in lesion development
(Thoen and Bloom 1995; Dungworth 1993). The components of the specific chemical
makeup of cell walls differ proportionately between species of mycobacteria and are

intimately linked to the virulence of the strain in question. Generally speaking, those

bacteria that have the “biochemical ingredients” which allow them to most efficiently
multiply and survive within host cells while eliciting the least significant inflammatory
response in the host will prove most virulent (Lagrange et a1 1999).

Among the defenses of the mycobacteria leading to disease are the variably
effective, species-specific abilities to survive and grow in number both intracellularly,
within unactivated host macrophages, and extracellularly, within caseous centers of
lesions resulting from infection (Dannenberg 1999; Thoen and Bloom 1995). The
ability of mycobacteria to prevent fusions of phagosomes and lysosomes within
macrophages is one of its most effective defense mechanisms. Acidic lipids known as
sulfatides were thought to be primarily responsible for this property (Thoen and
Bloom 1995), but data does exist that raises questions toward the validity of this
finding (Goren 1988). It is known that these sulfolipids do contribute to the
inflammatory response, however, possibly by inducing prostaglandin synthesis and
altering superoxide production in inflammatory cells (Moulding 1999).

Regardless of the yet to be determined mechanism, it is known that virulence does
typically coincide with sulfolipid content of cell walls in mycobacteria (Goren et a1
1974). Another primary virulence factor is cord factor, trehalose-6,6’ dimycolate
(TAD;TDM) (Glickman 2001; Dungworth 1993; Brooks et a1 1991). The specific
effects of this cell wall glycolipid include it’s leukotoxic abilities (partially due to its
stimulation of cachectin) and inhibition of chemotaxis of leukocytes (Silva and
Faccioli 1988; N011 et al 1956). It also has been noted as toxic to mitochondria, is

linked to ribosome detachment fi'om the rough endoplasmic reticulum, and contributes

to the inhibition of phagolysosome membrane fusion (Glickman 2001; Spargo et al
1991; Thoen and Himes 1986). Interestingly, bacilli will morphologically form
ropelike cords due to the presence of this factor, and their ability to do so can be
positively correlated with their species-specific virulence (Dungworth 1993).

Also important to mycobacterial virulence is lipoarabinomanan (LAM), a
glycolipid thought to be responsible for not only deterring adherence of the bacteria by
macrophages, but also having unique free radical scavenging capabilites (Moulding
1999; Strohmeir and Fenton 1999; Thoen and Bloom 1995). Adherence and uptake of
bacilli into macrophages is due to the binding of LAM to the CD14 and macrophage
mannose receptors. This nonspecific binding then allows mycobacteria to enter the
cytoplasm of the macrophage to begin replication. Interesting effects of LAM also
include its abilities to not only attract lymphocytes to the site of infection by
stimulating the production of chemokines, but also its ability to suppress the function
of these same lymphocytes once attracted to the site through promotion of enhanced
transforming growth factor B (TGFB) production (see immunology review below) and
reduced gamma interferon (IFN-y) production (Glickman 2001). Such duality in
effects may lead to enhanced mycobacterial proliferation within cells while at the
same time, the overall immune response to infection is thought to diminish. Poorly
understood mechanisms leading to suppressed lymphocyte activation have also been
attributed to LAM (Strohmeir and Fenton 1999).

Activation of monocytes may also be impaired by the mycobacterial protein

antigen 85A. In fact, of the specific mycobacterial proteins studied in detail, antigen

85A is among the most important of those that directly enhance virulence by binding
fibronectin, preventing it from adhering to these bacilli (Thoen and Bloom 1995).
Another protein from this complex, antigen 853, seems to have a role in mycolic acid
production (Cooper and Flynn 1995). Proteins in the antigen 85 complex, along with
ESAT-6, another mycobacterial protein, have the ability to potently stimulate the
proinflammatory cytokine IFN-y (Cooper et al 2001; Weinrich et al 2001; Cooper
1995). Most of the remaining mycobacterial structural proteins, and indeed many of
the lipids as well, have immunomodulation of the host as their primary fimction
(Brooks et a1 1991). These antigens are among the important contributors to those
soluble fractions harvested as purified protein derivatives (PPD) from M. bovis or M.
avium cultures for use in the tuberculin skin test (Adams 2001).
Pathogenesis and the Immune Response in Mycobacterial Disease:

Introduction

As mentioned in the previously section discussing virulence, the main host
defenses against mycobacterial infection in all species in which they are of importance
are primarily the cell mediated immune responses (CMI). CMI and delayed type
hypersensitivity (DTH) are both major (separate) factors in tuberculosis, with humoral
immunity playing a much less significant role that does not contribute to memory
immunity (Dungworth 1993). Humoral responses are, when detected, most often
present in chronic and/ or disseminated infections (Reddy and Andersen 1998). This
is not to say that antibody opsonization of organisms and resultant enhanced

phagocytosis does not occur in all stages of this disease, but it is primarily the T cell

generated response to the bacteria that is responsible for resolution of infection and
generation of memory immunity in mycobacterial infections (Thoen and Himes,
1986). Interestingly, the host cellular response to mycobacterial invasion is of great
importance, not only because it determines the course of the disease, but also because
it is largely involved in the development of the unique pathologic features of
tuberculosis that ensue as clinical disease develops. In fact, it is the host’s own
immune response, and the balance between protective acquired immunity and delayed
type hypersensitivity that determine much of the course of the disease (Dannenberg
1999)

Basic Pathophysiology and Immunology

Mycobacteria enter, most typically, as inhaled droplets (less than 20 pm in
diameter and containing one to three bacilli) into pulmonary alveoli or, less
commonly, are ingested and invade the gastrointestinal system mucosa (Dannenberg
1999; Thoen and Himes 1986). As organisms are engulfed by local tissue
polymorphonuclear cells and non-activated monocytes, their intracellular existence
begins. It is at this stage that infection may be initially limited as the host’s immune
system clears itself of infectious bacilli (Dannenberg 1999). But unique properties
that allow mycobacteria to escape degradation by this, the body’s first defense against
them, immediately become important to the survival of the bacteria within host cells.
These defenses include an overall impaired difficulty by the host in the destruction of
the relatively rigid cell walls containing mycolic acids, waxes, cord factor, and other

lipids or protein complexes which enhance virulence and prevent phagolysosome

formation; survival of mycobacteria within phagolysosomes by microorganism-
generated pH elevations, or by structural defenses; and phenolic glycolipids which
directly deter superoxide radical degradation of bacteria, even allowing
intracytoplasmic replication of the organisms (Rhoades and Ullrich 2000; Brooks et al
1991). These specific virulence factors were discussed in the first section of this
chapter and will not be reviewed in detail.

Products resulting from the degradation of bacilli and inflammatory cells cause an
initial release of tumor necrosis factor alpha (TNF-or), various interleukins
(interleukins one, six, and ten most importantly; IL-1, IL—6, and IL-10) and other
chemokines, particularly monocyte chemoattractant protein I (MCP I) and IL-8, which
modulate the activation of further inflammatory cells and the chemotaxis of these cells
to the site of initial infection (Skinner et al 2001). An important subset of cells
involved in the production of MCP I in infection are the gamma-delta receptor-bearing
subset (78) of T cells, which have some evidence of participation in driving the
formation of tuberculosis lesions towards more purely monocyte-type reactions than
suppurative, neutrophilic ones (Orrne 1999). Dendritic cells travel from the forming
“primary complex” to regional lymph nodes in response to antigen presentation from
initially insulted macrophages. Antigen presentation to CD (cluster of differentiation)
4+ T cells allows for expansion of subsets of these cells specific to mycobacterial
antigens. The involved subsets of CD 4+ cells are primarily class one helper T cells,
Th1 cells, which are recruited in tuberculosis infections, until lesion resolution and

chronic immune responses begin to be developed by type 2, Th2, subsets and Th1

involvement subsides (Ortana and Cauda 1998; Griffin et al 1995). Interleukin two is
important in initial signals to activate macrophages and recruit new circulating
monocytes towards the now-forming granuloma in this cascade. Activated
macrophages in turn produce IL-12, furthering recruitment of CD 4+ helper cells.
Natural killer (NK) T cells are activated by IL-12 and produce IFN-y, along with 75 T
cell subsets (Orme and Cooper 1999).

CD 8+ cytotoxic T cells are also important in the elimination of mycobacteria
from the site of initial infection, and as they spread throughout the body. Cytotoxic T
cells are primarily involved in destruction of those phagocytes with major
histocompatibility complex (MHC) class I expression, which induce immediate
cytolysis by CD 8+ cells, whereas CD 4+ cells produce lymphokines in response to
recognition of MHC 11 associated presentation (Reddy and Andersen 1998). Both CD
4+ and CD 8+ T cells secrete IFN-y, which enhances overall inflammatory cell
activity in the body by recruiting both specific and non-specific phagosomes.
Although both cell types are implicated in this defense, CD 4+ cells apparently are the
more important contributors (Dannenberg 1999). Both CD 8+ and CD 4+ Th1 cell
types are specific to the mycobacterial antigens produced by the infection and
presented to them on associated MHC receptors (Reddy and Andersen 1998). Certain
non-peptide components of the cell wall are presented by macrophages to lymphocytes
on non-MHC receptors. The lymphocytes that recognize these antigens are sometimes
referred to as CD1 lymphocytes or CD 4-8- lymphocytes, based on the molecular

structure of the receptor. Such cells are important in the cytotoxic killing of microbe-

laden cells and in producing IFN-y, not only in tuberculosis infections, but in other
bacterial diseases as well (Dannenberg 1999; Cooper and Flynn 1995).

Tumor necrosis factor alpha (TNF-a) is secreted by both CD 4+ and CD 8+ T cells
as well. This cytokine is quite paroxysmal in its mechanisms, in that it not only helps
to enhance bactericidal activity of immune cells, but also leads to host tissue
destruction that results in the formation of caseous granulomas typical of tuberculosis
(Cooper and Flynn 1995). A balance is thought to exist between TNF—or and
transforming growth factor beta (TGF-B) in tuberculous lesions, with cells located
centrally in granulomas producing more of the former, and cells peripherally (within
the fibro-epilthelioid “capsule”, see basic pathology section below), producing more
of the latter (Aung et al 2000; Vanham et al, 1997). Interestingly, TGF-B may even
play a role in the regression of tubercles (Maeda 1993).

TGF-B expression typically increases in the typical tuberculosis immunologic
cascade of events as infection subsides, while TNF-or expression is most extreme
during the initial stages of infection and during primary tissue destructive phases
(Hernandez-Pando 1997). Interleukin ten and IL-4 are also associated with lesion
resolution phases in tuberculosis, and are secreted by type 2 CD 4+ lymphocytes,
which dominate the immune response at these late stages of infection (Ortona and
Cauda 1998). Along with IL-13, these cytokines downregulate type 1 CD 4+
lymphocyte activity, and promote the limited humoral immune response typical of

mycobacterial disease (Dannenberg 1999).

The majority of the T cell response to mycobacterial infection involves alpha-beta
(a0) receptor type, CD 4+ and CD 8+ lymphocytes. Recently, interest has formed
regarding the potentially intriguing role of )6 receptor type T cell subsets as
modulating mycobacterial infections in very specific ways (Delves 2000a). Current
research indicates that these cells are important in establishing secretory IgA in
response to repeat intracellular bacterial infections (Delves 2000b). They may play a
role in chronic, granulomatous stages of disease or in infectious recrudescence (Delves
2000b; Orrne and Cooper 1999), but do have a significant part in nonspecific killing of
macrophages laden with organisms along with NK cells very early in infection
(Dannenberg 1999). Their diagnostic value (the ability to identify them
immunohistochemically in tissue sections) for the investigation of responses to PPD
and other antigen-derived injection responses plays a potentially beneficial role in
vaccine technologies. They may form part of the link between cell mediated
immunity and delayed-type hypersensitivity in tuberculosis, a line which is currently
relatively still blurred pathophysiologically (Cooper and Flynn 1995).

The inflammatory events described lead to the formation of the primary and
secondary, or post-primary, complexes of infection. These lesions may bring
resolution to infection and a level of memory immunity in the host to the
mycobacterial agent involved. Conversely, they may lead to devastating and
widespread tissue destruction and death; or they may lead to temporary cessation of
inflammation, as viable, yet dormant, mycobacteria become essentially walled-off to

further body inflammatory defenses by the host’s own defenses. Immunologic

10

compromise of any sort in the host with such surviving mycobacteria-laden
granulomas can lead to reinstatement of the entire disease process.

Cell Mediated Immunity Versus Delayed-Type Hypersensitivity

Perhaps the most unique feature of the pathogenesis of mycobacterial disease are
the intimately related cellular events and antigenic responses that lead to both
protective immunity and lesion formation, often simultaneously. A fine line exists
between the immunologic mechanisms by which a beneficial host response is created
and those mechanisms which lead to tissue destruction.

In a beneficial host response (CMI), memory T cell immunity develops and
activated macrophages are signaled to sites of bacterial invasion in order to kill
multiplying bacilli. In delayed-type hypersensitivity (DTH), tubercle bacilli are
contained by non-activated macrophages which eventually rupture, inciting caseous
inflammation, but at the same time limiting the more favorable environment for
mycobacterial growth, that within inflammatory cells. Each response can be regarded
as protective in its own right, yet it is that of DTH which leads to host tissue damage
and the hallmark caseous granulomas of tuberculosis (Dannenberg 1999).

Essentially these responses are indivisible, but the course of disease depends on
which predominates. A dose-dependent response of mononuclear phagocytes entering
the site of the developing lesion and the amount of mycobacterial antigen available for
destruction is seen (Reddy and Andersen 1998). As antigen load increases, the
number of macrophages, activated and non-activated, will increase, and a DTH type

response will predominate. As the growth of tubercle bacilli is held in check, CMI

11

will predominate and healing will begin as bacteria are eliminated (Dungworth 1993).
Interestingly, studies indicate that most species will “obey the laws of immunity” to
mycobacteria in that those with numerous bacilli present in tuberculosis lesions have
poor in vitro lymphocyte transformation readings, indicating a poorly mounted host
immune response (Thorns et al 1982).

The tuberculin skin test is the classic example of the cellular response elicited by
memory T cells, and relies heavily on the concepts of CMI and DTH. These
immunologic consequences are also important considerations in vaccine development,
as antigen sets most like those present in natural infection clearly provide the best
protection because they include both mycobacterial structural components and
secreted proteins (Collins 1994; Fifis et a1 1994). Because beneficial cellular
responses in the host can be characterized and measured, studying specific antigen
subsets leading to these responses may lead to more efficacious vaccine strategies in
the fiiture, for both humans and animals.

Pathologic Presentation of Tuberculosis in Animals

Species of Mycobacteria

The primary mycobacteria implicated as causing disease in animals in the United
States are Mycobacterium bovis, and those within the M. avium complex (MAC;
Mycobacterium avium, intracellulare, lepraemurium and paratuberculosis). The
human tuberculosis agent, Mycobacterium tuberculosis, has been known to cause
disease in both domestic and captive wild species, but is typically due to transmission

from human caretakers (Tirnoney et al 1988). It follows logically then that most

12

human cases of M. bovis tuberculosis are due to contact with infected cattle. Many
human M. bovis cases diagnosed in recent decades represent recrudescence of
extrapulmonary infections established prior to milk pasteurization and tuberculosis
eradication programs being established (Wilkins et a1 1986). Numerous additional
species of mycobacteria are reported in both animals and man, but are of much lesser
epidemiologic significance, are less virulent save for cases involving
immunocompromised hosts, and are much easier to manage clinically. Cases
involving mycobacteria other than tuberculosis group (MOTT) organisms and MAC
organisms in animals usually involve saprophytes (M. scrofulaceum, kansasii,
marinum, fortuitum, etc.), and are typically associated with individual, localized
infections.

The species Mycobacterium africanum and Mycobacterium microti, the remaining
organisms in the M. tuberculosis group along with M. tuberculosis and M. bovis, are
of medical concern in their regions of greatest import, Africa and Europe (the United
Kingdom, the Netherlands, and Germany) respectively (Rastogi et al 2001; Niemann
et al 2000; Kremer et a1 1998). Disease caused by these species can affect both
animals and man in these areas, but is not present in the United States at this time.
Cases of tuberculosis are much rarer for these mycobacteria, and are of much less
importance than those previously mentioned, even in their countries of origin
(Horskotte et al 2001; Skinner et a1 2001; Collins 2000; Alfredsen and Saxegaard
1992). All disease-causing mycobacteria can be considered zoonoses, but the

transmission of some of the less virulent, more opportunistic pathogens in the group is

13

typically of much less concern either between animals and man or within groups
(Rastogi et a1 2001). For the purpose of this discussion, “tuberculosis” in animals will
be that due to M. bovis, except where described in birds, where the described lesions
are those caused by M. avium infection.

Mammalian Versus Avian Presentations of Tuberculosis

Immunology

The hallmark lesion of tuberculosis in any of the various susceptible species is the
granuloma (Dungworth 1993). The characterization of this granuloma, and the events
leading to its formation do differ in very specific ways in the response to tuberculosis
infection in mammalian and avian (or reptilian) hosts (Montali 1988).

Perhaps most important and most basic are the distinct differences that exist
between avian and mammalian hosts in the organs responsible for the generation of
inflammatory and immune-mediated responses. In particular, their lymphoid systems
share very few similarities. The generation and maturation of B lymphocytes in birds
takes place primarily in the bursa of Fabricius, whereas in mammals, generation of
these cells is in the bone marrow, Peyer’s patches, spleen, and lymph nodes, and in the
thymus to some extent (Sharma 1991). That is not to say that the bone marrow and
spleen of birds do not contribute to B lymphocyte development as well, more so that
the task does have a more dedicated organ than it does in mammals. Both mammals
and birds have a thymus as their primary T lymphocyte source, both during

development and throughout life (Pope 1991).

14

The lack of a well-developed lymphatic system is notable when comparing avian
anatomy to that of the mammal. Although some species (waterfowl primarily) do
have rudimentary lymph nodes, when present, these structures do not serve in
filtration as they do in mammals. Consequently, no need for well-developed
lymphatics is present in birds, and as such, they have comparatively few of these
vessels, and they figure much less prominently in immunogenesis than do those of
mammals (Pope 1996). Because birds lack organized lymph nodes, the occurrence of
secondary lymphoid tissues associated intimately with various organ parenchyma is
much more prominent in these species. Gut—associated and bronchial-associated
lymphoid tissues (GALT and BALT), cecal tonsils, hepatic, and even renal lymphoid
reserves serve to protect organs in birds where lymph nodes defenses may be absent,
along with disseminated lymphoid aggregates present in nearly all parenchymatous
tissues (King and McLelland 1984). Also noteworthy is the avian spleen being a more
central feature in many inflammatory processes than it is in mammals. The avian
spleen is best seen as a defensive spleen, with nearly equal red and white pulp
distribution, particularly when compared to that of mammals, which have spleens
featuring a larger proportion of red pulp (King and McLelland 1984; Hodges 1974).
Germinal centers occurring in the lymphoid tissues of birds are not as highly
developed as are those of mammals- with well-mapped maturation sequences present.
But these areas do often feature a reticular cell envelope that is not necessarily

prominent in mammalian germinal centers (Pope 1996).

15

Although relatively unimportant in the defense against mycobacteria, some
mention towards humoral immunity is necessary. Irnmunoglobulins shared by birds
and mammals are IgG (also called IgY in birds), IgM, and IgA (Sharma, 1997). IgD
and IgE, present in mammals, are absent in birds. Also relatively unimportant in the
discussion of tuberculosis, but nonetheless absent in birds are lymphocytes having
MHC class III antigen presentation expression. Mammals and birds both have MHC
class I and II expression, but birds lack a true counterpart to mammalian MHC HI, and
mammals lack the exclusively avian MHC class IV (Lamont 1998). In both species,
most immune responses, including that directed towards tuberculosis infection, do
primarily involve lymphocytes with expression of MHC classes I and II (Lamont
1998; Sharma 1997). Lymphocytes bearing these class markers are similar in birds
and mammals. Cytokine and chemokine cascades and the involved cellular responses
in inflammation are quite comparable as well, particularly the initial monokine
activities of IL-1, IL-2, TNF-or, and GSF (granulocyte stimulating factor)(Klasing,
1991). It should be noted that research to specifically define avian lymphokines has
not been performed nearly as extensively as it has been in mammals. (Shanna 1991;
Montali 1988). Many of those that have been researched and compared to date do,
however, seem to be functionally similar but with limited cross-reactivity (Klasing

1998)

16

Clinical Presentation of Disease

When discussing clinical cases of tuberculosis, it is important to note differences
in exposure routes typical of avian and bovine TB that lead to organ infection and
resultant clinical signs. In avian tuberculosis, ingestion of infected feed, water, or
litter is the primary route of exposure with the aerogenous route being second most
important (Gerlach 1994). As such, dissemination of infection throughout the
abdominal viscera is typical in birds (Goodwin 1996). Vertical and vector
transmissions occur, but are less common. In cattle the primary route of exposure is
aerogenous, from infective droplets produced by other infected animals (Neill et a1
1994; Thoen and Himes 1986). The caudal lung lobes and draining lymph nodes may
therefore be the only affected organs. Ingestion of infected sputum secondary to
respiratory infection in individuals may lead to pharyngeal, intestinal, hepatic, and
mesenteric lesions, as may ingestion of contaminated feeds or water, the second most
common transmission mode. Congenital, cutaneous, and genital transmission of
tuberculosis occur less frequently (Neill et a1 1994). Exposure routes are also related
to the environmental stability of the involved organisms. All mycobacteria are known
to exist for long periods environmentally in organic substrates. Yet M. avium may
persist in contaminated soil for up to two years, whereas M. bovis may lose infectivity
in a few days when exposed to sunlight (it can persist for much longer in the absence
of heat and sun)(Timoney et al 1988). Both agents (particularly M. avium) survive for

very long periods in water supplies, soil, and dust. Disinfection of the environment

17

via prolonged contact with phenols is the method of choice for either agent
(Falkinham 1998; Greene and Gunn-Moore 1998).

Clinically, tuberculous birds may or may not show evidence of disease.
Inapparent infections may exist, particularly in young birds. As infections advance,
weight loss in the presence of a hearty appetite and unthriftiness ensue. Weight loss
may or may not be associated with gastrointestinal distress such as diarrhea and
bleeding. As the disease disseminates, lameness due to bone and joint involvement
and eventual death are typical (Gerlach 1994). Cattle infected with tuberculosis may
harbor infections for very long periods without clinical signs (Smith 1990; Timoney et
al 1988). Both mammals and birds may form primary complexes of mycobacterial
infection that do not progress beyond the initial infection until much later in the
animal’s life. Typically such recrudescence is associated with concurrent illness or
immunosuppressive states, and is more commonly noted in mammals than in birds.
Fever and weight loss are typical in early active disease in both mammals and birds. If
infection is not “walled off” as a primary focus in cattle, dissemination into lymph
nodes draining areas of lesion formation is common. Progression therefore leads to
cachexia, respiratory distress, and enlarged or draining lymph node lesions.

Comparative Pathology

The pathology and pathogenesis of tuberculosis in each species is inherently linked
to the typical response to intracellular organism infections in each. Yet in order to
understand this response, some discussion of comparison of the basic cellular

inflammatory response is necessary. Although granulocytes (heterophils in birds, and

18

neutrophils in mammals) are called upon in the acute inflammatory response in both
species, in birds this phase progresses much more rapidly and is quite dissimilar to that
of mammals. A rapid influx of phagocytes in response to pathogens within the avian
respiratory and gastrointestinal tracts is common, largely due to the low numbers of
resident tissue macrophages available for defense (Qureshi et a1 2000). Within hours,
the avian heterophilic response is followed and enhanced by a macrophage response
that may not be seen for days to weeks, if at all, in mammals (Barnes 1996). The
formation of the heterophilic granuloma in birds is partially due to differences in
granule composition of the heterophil. Avian heterophil granules lacks
myeloperoxidase and alkaline phosphatase that are largely responsible for phagocytic
pathogen elimination in mammals (Harmon 1998; Montali 1988). This is not to
suggest that avian heterophils lack microbicidal activity, but this activity is limited to
lysosomal cationic killing. Because heterophils lack these proteinases, the
heterophilic granuloma tends to caseate centrally, whereas proteinases in neutrophils
create lesions which are centrally liquefactive (Montali 1988). Mononuclear cells are
recruited to the site of the developing lesion as events progress in both species,
although, again, this response is seen typically quite early on in birds (Barnes 1996).
CD4+ and CD8+ lymphocytes and natural killer cell involvement in delayed typed
hypersensitivity are present in both species (Gobel et al 1996; Arstila et a1 1994). As
cellular immunity progresses, activated macrophages become part of the response. In
birds, macrophages participating in heterophilic granulomas are ofien Langhans giant

cell types, due to an elicited foreign body type response during tissue caseation. In

19

mammals, resolution of suppurative inflammation may only involve lymphocytes and
plasma cells, with few macrophages participating. In birds, macrophage participation
is imminent as a primary defense against most immunostimulants (Qureshi et al 2000).
Aggregates of lymphocytes and plasma cells may or may not figure prominently in
avian granulomas, but these cells will usually be present in at least small numbers
(Barnes 1996; Montali 1988). Should the infection in a mammal be chronic enough to
elicit a granulomatous response, multinucleate giant cell formation is not necessarily
typical as it is in birds. Moreover, central caseation in mammals is more likely to
mineralize, an event not at all common in birds. When present in birds, mineralization
of caseogranulomas is most likely due to previous parasitic infection. In the latest of
chronic states in both species, whether granuloma formation had resulted in
mammalian tissue or not, complete resolution will usually involve eventual
granulation and/ or fibroplasia at the site of the initial lesion in both species, as
phagocytes continue to clear remnants of destroyed tissue parenchyma. Viable
tubercle bacilli may or may not remain within the caseous centers of such regressing
lesions (Barnes 1996; Thoen and Bloom 1995; Slauson and Cooper 1990).

Both similarities and differences between these species’ responses to intracellular
organisms exist as well. Initially, in each, mycobacteria are engulfed by resident
tissue phagocytes after inhalation of infective droplets or ingestion of contaminated
sources. As infection ensues, macrophages in each species produce cytokines which
signal development of memory T cells, which in turn activate macrophages further

through the production of further cytokines (see immune response discussion above).

20

These cytokines signal further phagocyte localization and blood monocyte adhesion
and colonization. By this point in both species, the lesion forming is the granuloma.
The histiocytic granuloma in birds consists of large, foamy to multinucleate giant
cells, sometimes with small numbers of heterophils. As these macrophages coalesce,
central caseation develops much as in heterophilic granulomas. Indeed, with
chronicity, it may be difficult to tell the difference between the two lesion types, the
primary difference being necrotic core cell composition, which can be amorphous and
almost amyloid-like, but rarely mineralized as is common in bovine tuberculosis
(Montali 1988). As mammalian granulomas develop, neutrophils are often featured
secondarily, but are featured primarily only in acute stages of infection. In bovine
tuberculosis, acute exudative lesions may be present, and these consist nearly entirely
of neutrophils with lesser numbers of necrotic macrophages. Such exudative lesions
are relatively typical in cervid M. bovis infections (Rhyan and Saari 1995). In both
birds and mammals, epithelioid macrophages and aggregates of lymphocytes and
plasma cells are featured in granulomas, particularly along the most peripheral edges
of lesions. Epithelioid macrophages and fibroblasts congregate around
caseogranulomas, forming a capsule that helps to prevent further spread of pathogens
into surrounding tissues (Slauson and Cooper 1990). Large numbers of acid-fast
bacilli are typically visible histologically in M. avium tuberculous granulomas. The
number of organisms associated with lesions due to the bovine variant is variable, but
often is minimal (Dungworth 1993; Mirsky et a1 1992). Lesion resolution with

replacement by fibrous tissue after infection is cleared, or pathogen dormancy in the

21

microaerophilic to anaerobic environments of encapsulated granulomas will then take
place unless these lesions expand to include vascular supplies allowing for
hematogenous spread (Thoen and Himes 1986). Such hematogenous spread without
the benefit of lymph node filtration leads to the commonly disseminated distribution
of lesions found in birds (Gerlach 1994). Since reticuloendothelial cell contributions
to clearing of infections are common, organs rich in both blood supply and in these
cell populations are often colonized by infected cells. These organs include, but are
not limited to, the spleen, liver, bone marrow, lungs, and kidney. Since mammalian
hosts do have the benefit of lymph nodes as defense, nodes draining the organ
originally infected will often develop lesions. In mammals, containment of infection
may take place in lymph nodes, or may progress further along lymphatics and into
other viscera, particularly in immunocompromised hosts (Greene and Gunn-Moore
1998; Thoen and Himes 1986).
Tuberculosis in Cattle and Deer

The primary agent causing tuberculous lesions in both cattle and deer is
Mycobacterium bovis. And although the basic modes of transmission and the
molecular events which follow infection in both cervid and bovine tuberculosis are
relatively similar, the cervid cellular response can be considered more dramatic and
acute in some respects. Both species are known to develop primarily pharyngeal area
and thoracic cavity lesions, particularly when aerogenously exposed (Griffin and
Buchan 1994). Yet the suppurative component to lesions in deer is usually more

pronounced, particularly when compared to the granulomatous to caseous lesions

22

present in most cases of tuberculosis in the bovine (Palmer et al 2000a; Clifton-Hadley
and Wilesrnith 1991). Cattle are typically found to have at least partially encapsulated
granulomas with thick, caseous exudate, which is often gritty due to partial
mineralization, in either the lung or lymph nodes draining the site of infection.
Grossly, lesions in deer may often be large abscesses containing thick purulent
exudate (see Figures 1.1-1 .4). It should be pointed out that within the various cervid
species known to be affected by bovine tubercle bacillus thus far, a wide range of
lesion types have been reported, both between and within any given cervid species
(Rhyan and Saari 1995; Griffin and Buchan, 1994). Lesions ranging from nearly
entirely suppurative with fistulous tracts draining externally from affected lymph
nodes, to more classically granulomatous and mineralized, can be identified in
tuberculosis cases in cervidae (Palmer et al 1999; Rhyan and Saari 1995). Yet
generally speaking, and even more importantly perhaps, speaking from personal
observation of white-tailed deer tuberculosis cases identified in Michigan’s ongoing
surveillance program, deer often develop severely necrotizing, pyogranulomatous,
liquefied to partially caseogranulomatous, occasionally cavitating or partially
mineralized abscesses in response to infection with M. bovis. Such lesion
development suggests poorer CMI in cervidae, as more liquefactive lesions are the
result of the more tissue-damaging, cytotoxic DTH being more involved in the defense
against mycobacteria than that of CMI (Dannenberg 1999). Also indicating poor CMI
is the larger number of bacilli typically found present in cervine tuberculosis lesions

compared to the often rare bacilli noted in bovine cases (Mirsky et al 1992). When

23

CMI is the primary mechanism by which mycobacteria are eliminated from the
developing lesion, the result is usually a more “pure” granuloma. Such lesions are
composed of large numbers of activated macrophages signaled to the area, often
forming Langhans giant cells. These lesions will often have central caseation and
mineralization also resulting from the cell death caused by DTH, but with containment
of bacillary growth and fibrous capsule formation, as is seen in most cases of
tuberculosis in cattle (Dannenberg 1999; Rhyan and Saari 1995; Dungworth 1993). It
would logically follow to assume that deer are then somewhat more “susceptible” to
the bovine tuberculosis agent when exposed than are the more “resistant” cattle when
the general presentation of lesions are compared. Interestingly, studies have shown
that deer, particularly those with disseminated disease and truly liquefactive lesions,
tend to mount a larger humoral response when antibody production is measured
(Palmer et al 2000b). Cattle rarely mount significant antibody responses until late in
the course of the disease (F ifis et a1 1994). Evolutionarily, it makes sense that the
species most historically affected by the agent (the bovine) would have generated less
dramatic and potentially less fatal inflammatory responses to the infectious agent, by
having better genetically-established CMI towards M. bovis antigens. Perhaps most
importantly however, is the noted potential for differences in lesion presentation both
between and within any species infected with M. bovis. The differences suggest that
there may not be any true “classic” presentation for this disease, and it should be
considered a differential in all susceptible species wherein the described spectrum of

lesions are found present at necropsy.

24

Surveillance Program Background-M. bovis in Michigan
Mycobacterium bovis

Tuberculosis in humans was suspected as being potentially linked to that in cattle
several hundred years before the actual link was discovered by Robert Koch in 1882.
(Grange 1995). Respiratory disease (“consumption”) was linked to bovine wasting
disease, cervical lymphadenitis (“scrofula”) was noted as being present more
commonly in those persons that were known to drink cow’s milk. Even cutaneous
mycobacterial lesions were suspected as being caused by animal carcass contact in
those people who were in professions that would require potential contact with such
sources, but no clear scientific proof existed for such connections prior to Koch’s
work in 1882 (Grange and Yates 1994). Initially, he isolated bacilli from both human
and animal sources, but failed to distinguish any difference between the two. It wasn’t
until 1898 that Smith defined differences between the bacilli (Grange 1995). After
Smith’s reports, the differences in the strains were thought to be significant enough
that each would be host-specific in its ability to cause disease. The primary
researchers in mycobacterial work were all under the assumption that bovine strains
were not pathogenic in humans. In fact, some of these scientists believed that animal
strains may have been capable of providing protection from disease when used as a
live vaccine in humans, and vice versa. It was actually Koch that first spearheaded
such beliefs in the scientific community (Collins 2000). Fortunately, researchers

(many of them veterinarians) that had suspected transmissibility of the organisms

25

between these hosts did not rely on Koch’s ideas at that time and went on to
demonstrate human cases of M. bovis. The work of these same researchers led to
subsequent reports that the organism, in fact, was a potentially serious threat to
humans. It was based on these findings that measures to eradicate the disease in cattle
were eventually established by the United States Department of Agriculture, albeit
several decades later in 1917 (Collins 2000; Grange 1995; Grange and Yates 1994).
Tuberculosis in Michigan Deer

Michigan was certified free of Mycobacterium bovis in its cattle herds in 1979, the
conclusion of intense eradication efforts established in the early 1970’s. Prior to the
eradication of tuberculosis in cattle, M. bovis was only reported in a single deer park in
Michigan in 1962 (Essey and Vantiem, 1995). During the time span of the cattle
tuberculosis eradication program, there was a single reported case of M. bovis
tuberculosis in a white-tailed deer (Odocoileus virginianus), identified in a hunter-
killed deer in the northeastern part of the lower peninsula of the state in 1975. This
case was not pursued further by the Michigan Department of Natural Resources
(MDNR) at that time, as it was a rare disease in deer, and when found was typically
associated with exposure to tuberculous cattle. The thought pattem at that time was
that eradication from cattle would eliminate any further risk to deer in the state and as
such, wildlife reservoirs of this disease were not a concern (Sikarskie et al 1999;
Schmitt et al 1997).

Concern was raised when the second hunter-killed deer with lesions suggestive of

tuberculosis was submitted to the Animal Health Diagnostic Laboratory, Michigan

26

State University (AHDL, MSU) in 1994. This deer was from the same area in the
state as was the deer identified in 1975, and was also confirmed as positive for M.
bovis (Schmitt et a1 1997).

Surveillance for additional infected deer began in 1995 as an attempt to determine
whether M. bovis infection was in fact a rarity in the Michigan free-ranging white-
tailed deer population, or whether this species could actually have been maintaining
the disease in the wild since the eradication program for cattle had been discontinued
due to its postulated success decades earlier. The diagnostic procedures involved in
surveillance primarily involve gross inspection of cranial lymph nodes followed by
culture and microscopic evaluations of tissue determined as suspect of tuberculosis
infection based on gross inspection at the AHDL. Parotid, submandibular, and medial
retropharyngeal lymph nodes are those inspected in all cases. Each year, a variable
number of carcasses and/ or additional organs (largely liver and lungs) are submitted
along with the heads of the deer to the AHDL from hunters or MDNR staff.
Typically, carcasses or tissues other than heads are submitted only if questionable
lesions or discoloration are found present during field inspection.

The presentation of tuberculosis has varied in Michigan surveillance deer cases, as
is typical of most cervid species (Rhyan and Saari 1995). Yet by far, the medial
retropharyngeal lymph node has been the most common site in which lesions are
found (O’Brien eta12001; Fitzgerald et al 2000). Typically, the gross lesion in these
deer is that of a unilaterally or bilaterally enlarged and abscessed retropharyngeal

lymph node, featuring purulent exudate which varies in color and consistency from

27

thick and yellow-green to more fluctuant and tan. True caseation and mineralization
are present less commonly (Figures 1.1-1.4). Histologically, these lesions often
feature multinucleate giant cells and epithelioid macrophages surrounding variably-
sized zones of caseous necrosis, with or without mineralization. A suppurative
component to the lesions is common, with degenerate neutrophils accenting the more
typical tuberculosis granuloma features mentioned. Acid—fast bacilli are present on
histologic examination of tissues in nearly 90 percent of the positive cases (Fitzgerald
et al 2000), although usually these are only found in low numbers.

The original year of surveillance indicated that deer were very much a potential
reservoir for bovine tuberculosis, at least in the limited area of the state in which it had
been performed (see Figure 1.5). Geographic expansion of surveillance has continued
each year since 1995 from the original “DMU 452” zone in the lower peninsula to
include surrounding counties, and eventually began to include representative samples
of deer from every county in the state annually (Figures 1.6-1.12).

Prevalence of the disease in deer in Michigan has apparently decreased from its
peak in 1997 over the past several years, and appears to be somewhat stable at the
present time (Table 1.1). The dramatic decrease in prevalence indicated by the 1998
surveillance results is likely the goal result of management procedures instituted and
enforced largely by the MDNR, including an increase in antlerless deer permit
distribution and the placing of restrictions on supplemental feeding and baiting of
deer. Both management tools appear to have contributed to decreased disease

prevalence by helping to decrease overall deer numbers in the wild, with subsequent

28

decreases in contacts between infected and noninfected animals. The elimination of
feeding and baiting in areas of highest prevalence has served to decrease incidences of
deer concentrated deer around feed piles, an activity which allows for an increase in
potential transmission of disease and which may lead to disease spread. Feeding
restrictions also serve to decrease the overall population of deer in the state, as cases
of starvation increase as supplemental feeding decreases, particularly during severe
winters (Sikarskie et a1 1999).

A trend towards decreased prevalence of tuberculosis in deer in Michigan should
continue as population management programs continue to be enforced. Allowing deer
numbers to match rather than exceed, or more ideally to be lower than, the biologic
carrying capacity of this species in the state is the primary goal towards which these
efforts are directed. Complete eradication may never be achieved realistically, but if
the disease can be reduced to a prevalence in deer such that domestic species
(particularly cattle) are no longer at significant risk, successful management may
eventually be realized. Continued surveillance to document the disease prevalence in
deer will be necessary at least until the time such a goal is met.

Tuberculosis in Other Michigan Wildlife Species

Non-cervid reservoir hosts for M. bovis tuberculosis are very important in
maintaining this disease in the wild in other countries. Because species such as the
badger, ferret, and brushtail possum are implicated as reservoir hosts in other countries
(0’ Reilly and Dabom 1995), examining the potential for similar species that are

native to Michigan which may serve as reservoirs became an important objective of

29

surveillance very soon after the disease was proven to be well-established in the white-
tailed deer population.

The search for additional potential reservoir hosts in Michigan began in 1996, when
various species of wild omnivores and carnivores were added to the surveillance
program, albeit on a much smaller scale than that of deer. Because these hosts may
often be infected with mycobacteria without having any gross evidence of disease,
surveillance in these species is relatively more involved than is that of deer. All
animals submitted as part of the carnivore and omnivore project have a complete
necropsy performed. Not only are those lymph nodes from cranial sites collected for
histologic and culture evaluations, but also those nodes from the abdominal and
thoracic cavities, as well as portions of any other organ with suspect lesions noted on
gross examination of the carcass (Buddle et al 1999).

Each year, new species are added to the small mammal surveillance program. To
date, fifteen potential hosts have been investigated (Table 1.2). Since submissions are
primarily those found dead or trapped, the total surveyed vary significantly between
species. All hosts investigated thus far appear more likely to be “spillover hosts”
rather than true reservoirs. Lesions, when present, are primarily found in mesenteric
lymph nodes in these animals. Such lesion distribution suggests ingestion of materials
infected with M. bovis as the primary mode of transmission, and does not lead to
further spread of disease by animals infected as such, as mesenteric node lesions
would tend to contain bacilli within granulomas unless hematogenous spread to other

viscera occurred. Very few cases of disseminated disease have been noted in any of

30

the species examined in this surveillance program. In contrast, disseminated disease is
common in non-cervid reservoir hosts in other areas of the world (O’Reilly and
Dabom 1995).

Free-ranging elk have been included in surveillance since 1996. Elk surveillance is
essentially identical to that performed on white-tailed deer in that gross inspection of
cranial lymph nodes is performed, followed by additional testing of suspect tissues
only. The first positive elk case was identified in surveillance for the year 2000 (Table
1.3). Interestingly, this animal had a bilateral suppurative tonsilitis detected grossly.
Tonsilar lesions have not been widely featured in positive white-tailed deer cases
identified in Michigan to date. In elk, as in deer, the primary site for lesions of
tuberculosis are reportedly the retropharyngeal, mesenteric, and mediastinal lymph
nodes (Rohonsky et al 1996). This elk also did not have acid-fast organisms detected
histologically. Apparently, this is often the case in elk, as the positive predictive value
of necropsy or histologic results identifying a true culture positive case can range from
44 to 80 percent, depending which results are used, and depending on if these are used
in combination either serially or in parallel (Rohonsky et a1 1996). In deer, the
positive predictive value for gross lesion identification alone has not been reported, yet
that of histology performed on tissues found suspect on gross examination is much

higher than that reported for elk (Fitzgerald et al 2000).

31

Objectives for Research

The primary goal of the research topics presented in this dissertation is to, in
combination, provide new insight to some very basic questions that have to date
remained unanswered regarding M. bovis tuberculosis in wildlife species, particularly
those in Michigan.

Specifically, these objectives can be divided into three categories. The first of
these is to address whether or not certain animals may be potential vectors of disease,
serving as either capable transmission hosts or as reservoirs similar to white-tailed
deer. Two hypotheses were generated from this objective. The first was that
American crows and European starlings could become infected with M. bovis by oral
and intraperitoneal inoculation with the organism. The second was that North
American Opossums could become infected with M. bovis by oral and intramuscular
inoculation routes.

The second objective was to explore potential antibiotic resistance patterns in the
endemic strain of M. bovis in Michigan wildlife. The hypothesis to be tested based on
this objective was that wild animal strains of M. bovis from tuberculosis cases in
Michigan would differ in their resistance patterns from human cases of M. bovis
tuberculosis diagnosed in the state, and that these animal strains would not have
acquired resistance to any of the drugs commonly used to treat M. bovis tuberculosis.

The third objective was to assess the reliability and accuracy of a novel, cytology-

based diagnostic test for mycobacterial infection that may have applications in human

32

medicine. The hypothesis for this objective was that M. bovis organisms would be
detectable in cells gathered from several sites (oral, nasal, and tonsilar mucosae) of
infected white-tailed deer, when cytology preparations made using ThinPrep 2000
technology were examined using light microscopy

Although the presented objectives may appear to be separately quite broad in their
scope, together they most definitely aim to expand the existing knowledge base of that
which exists concerning the transmission and pathology of M. bovis tuberculosis in

wildlife.

33

Figure 1.1: Tuberculosis in a white-tailed deer (Odocoileus virginianus). Gross
photograph of lung, cut section. Numerous tan to yellow-green, purulent to
fluctuant abscesses, ranging in size from approximately 3 mm to 3 cm diameter,

distributed throughout the pulmonary parenchyma.

34

Figure 1.1. Tuberculosis in the lung of a Michigan white-tailed deer
(Odocoileus virginianus).

    

35

Figure 1.2: Tuberculous granuloma on the pleural surface of the lungs of a
Michigan white-tailed deer. The nodule is singular, smooth, raised, and tan to
white in color in this case. Similar nodules are often multiple and variably-sized,
and distributed over both the parietal and visceral pleurae in more advanced cases

of M. bovis tuberculosis involving the thoracic cavity in deer.

36

Figure 1.2. Tuberculous granuloma on the pleural surface of the lungs of a
Michigan white-tailed deer.

 

37

Figure 1.3: Purulent tuberculous abscesses in the lymph nodes of a Michigan
white-tailed deer. Presentation is typical for M. bovis tuberculosis in cranial
lymph nodes of white-tailed deer. Centrally, lesions contain vast amounts of thick,
green-tan to yellow, purulent to viscous fluid, which may be contained by a fibrous
capsule. Variable amounts of caseation or mineralization are often featured, either

within or subtending such nodal parenchyma] lesions.

Figure 1.4: Caseous tuberculous abscess in the lymph node of a Michigan white-
tailed deer, cut section. Although such true caseogranulomas featuring thick,
gritty, mineralized inflammatory debris encased within a well-defined fibrous
connective tissue capsule are less commonly encountered as cranial lymph node
lesions in white-tailed deer, they do still occur frequently, particularly in smaller

lesions or in lesions affecting other tissues.

38

Figure 1.3. Purulent tuberculous abscesses in the
lymph nodes of a Michigan white-tailed deer.

 

 

 

Figure 1.4. Caseous tuberculous abscess in a lymph
node of a Michigan white-tailed deer, cut section.

 

39

Figures 1.5-1.12: Suveillance maps for M. bovis in Michigan’s white-tailed deer
population. Figure 1.5 shows the location of the initial tuberculous white-tailed deer
(a 9 year old female) identified in 1975. Not until 1994 was another hunter-killed deer
with tuberculosis found (Figure 1.6). Surveillance began in Deer Management Unit
(DMU) 452 in 1995 (Figure 1.7). Each year, surveillance expanded. In 1996,
intensive surveillance was performed in the 4 counties comprising DMU 452 and
lesser numbers of deer were examined fiom other counties in the state (Figure 1.8). In
1997, surveillance was primarily in the 5-county area composed of the 4 counties
surveyed in 1996 and that county outside of the original 4 in which a positive case had
been found the year prior (Figure 1.9). Surveillance in 1998 included those 5 counties
surveyed intensively in 1997 and a buffer zone extending to all sides of this block of
counties in the Northeast portion of the Lower Peninsula (Figure 1.10). Statewide
surveillance began in 1999 and continues to date, with larger numbers of deer taken
each year from all counties, particularly in those from which positive cases are found

for the first time (Figures 1.11 and 1.12). Images are presented in color.

40

Figure 1.5. Geographic location of initial M. bovis case in
Michigan, 1975.

”VIN! TUIIIGJLOQO IWVEY IEIULTI

I.“ w . .‘i .,.. mu—

 

 

 

 

k mammal“ 1 v' .
, ‘7‘“ ' ‘ 5‘, ' i
r can. It. ‘ F“, ‘
up l L
‘ . " an:
:“ any: .I‘
m.- in: 30th .' 1
m...

Figure 1.6. Geographic location of current propositus of
endemic M. bovis in Michigan wildlife, identified in 1994.

”VINE TUIIICULOII OLIVEY REDULT.
a lea.-

I n
X75. - ‘ gees:-
\ - I» Dun-u-

sfi. Elm-

Aw.

 

 

 

 

 

a s
‘i‘ K ' 1 >
hmmnm 1 , ‘ .
.n‘ .. ' - J-
' 5
mm [In A '
u—gl F
I V "I
u PH‘W'. can 1.
“1’ . .— .5
iv .
mm in: ‘1
I

.‘n—m—

41

Figure 1.7. White-tailed deer M. bovis cases identified in first year of
surveillance, 1995.

”VIII TUIIICULOCI IUIVEY RIIULTI

xii" 't ~ 3&9"
I; _...‘ 3...-

”m m1.-
a

nun . . ':' r: , i
"T. ' v ' ‘ \
hmmnmn a , . I
. , .

 

 

 

 

Figure 1.8. White-tailed deerM. bovis cases identified in 1996.

”VINE TUIIICULOUI MVEY RESULT.

' ' , anal-n
. .5 n 0‘ Dummy.-
o Dunn-
/\ - ~
f ' amn—

 

F
a.
III! ' T). w L a i
h 1.0ma1'lnlu ‘7 V" V I '
f‘a‘.‘
\ l t’T" E
x 15
“I" m ,
'0. -. l
' no
.- v- I-w-fa nun —-

 

 

 

 

42

Figure 1.9. White-tailed deer M. bovis cases identified in 1997.
”VINE TUIEIGJLOI. CURVEY IEIULTI
’ Elect-I
1997 "3' - eat-
/" ‘- a . gm
3’ .0 ._..,._. ,' '- ‘
m '

y .

“summon: rs , g, 3.1- ,:

 

 

 

if can. fit- tf-‘L ’
, .
up. ‘ '
w
‘u on... m
» . on
I...“ In! I” :
.1-

 

Figure 1.10. White-tailed deerM. bovis cases identified in 1998.
”WI TUIIRCULOU. SURVEY mun:

.t’d’ 5.; _ 3"}.
-fl:‘. ”,1- ‘I I I Eg‘i

“savour-man ; .7 . ‘1“ ' g ' i

 

 

 

 

.e
u.- ‘ l ' ’
' l " I.
u .err W um
," : '9
TI .
my. II- ‘

 

43

Figure 1.11. White-tailed deer M. bovis cam identified in 1999

IOVHI MIRCULOCR OW" RIIULTI
I

lilti- ... EH“.

Inl-
:I’IILA-
m
IN MI-
1‘ summon! u r ' "
f ~ ‘ so i
.' um .- uayfl‘ ’
p... ““157?" "
‘ “ III
Inna. I “

 

 

 

 

Figure 1.12. White-tailed deer M. bovis cases identified in 2000.

IOVIN! TUMOULOOII SURVEY REIULTO

_. g 82““...

a . ‘1'
.\ 's '
' \ 55 s
‘ '. N
.. J, "“ . . .
- ‘ I.
/ w
i ..
mu ‘1'" "

 

 

 

 

44

Figures 1.13-1.l4: Surveillance data from first (Figure 1.13) and most recent
(Figure 1.14) years of carnivore/ omnivore project, 1996 and 2000 respectively.
The first M. bovis-positive animal identified in this project was a coyote found in
DMU 452. An interesting trend to note is the close geographic proximity of these
positive small mammal cases to the positive white-tailed deer cases. For the most
part, positive camivore/ omnivore cases have been found in areas in which the
highest concentrations of positive deer cases exist. The presence of positive
animals in “outlying” areas in which even small numbers of positive deer have
also been found is intriguing as well. These geographic data seem to support the
idea that transmission to these species is the result of contact with infected deer or
deer carcasses (spillover from the deer reservoir of disease) rather than these

species being true reservoir hosts themselves. Images are presented in color.

45

Figure 1.13. Geographic. location of index case of M. bovis identified in
carnivore surveillance, a coyote from DMU 452, 1996.

”VII museum." W“ REIULTI

fl" - a“:
\‘ ESE“;-
I, j... H

 

 

 

 

 

.1 .... :3"-
m ’ _ v LB
hmmeovm 1 ‘ 3... “v ' %

 

 

 

 

 

 

 

 

 

 

Figure 1.14. Camivorel omnivore surveillance data (positive M. bovis
cases) from the most current year of surveillance, 2000.

”VIN! TUIIRCULOCI IURVEY RIIULT'

' '3‘ Ducal—
um“...-
. ‘ ' [:lnlanu-
i‘ ~ . ‘ turn.
'i m1

 

 

 

 

. w mu-
0‘ mm A'l' ‘

mun ,_
baseman" 4 . , .1» - i
human-ear 1 -- and '~ _
human": 1 » -.
mammalian. 2 ‘ in.” 5,, E
slum-Lacuna r

 

 

 

 

 

 

 

 

46

Table 1.1: Yearly prevalence rates for M. bovis tuberculosis in white-tailed deer
in Michigan, 1995-2000.

A Number of deer cases included in surveillance for the year listed.

3 Total number of deer found positive for M. bovis for the year listed.

C Number of positive cases divided by total number surveyed, multiplied by 100.
0 Prevalence rate for M. bovis in originally surveyed DMU 452 area. Note this
was the only area surveyed in 1995.

E Prevalence rate for M. bovis in the 5-county intensive surveillance zone.

47

Table 1.1. Yearly prevalence rates for M. bovis tuberculosis in deer in

 

 

Michigan, 1995-2000.
YEAR TOTAL NO.“ TOTAL POS.F PREV. RATEr CORE S-CO
PREV.” PREV};
1995 814 27 3.32 3.32 NA
1996 3718 47 1.26 2.32 1.26
1997 3681 73 1.98 4.42 1.98
1998 9067 78 0.86 2.43 0.94
1999 19503 58 0.29 2.16 0.69
2000 25706 53 0.21 2.32 0.83

 

48

Table 1.2: Positive cases of M. bovis tuberculosis in Michigan camivore/
omnivore surveillance through the year 2000.

A Number of this species surveyed through the end of the year 2000.

B Total number of culture-positives for M. bovis in this group.

C Number of positive cases in this species divided by total number surveyed,

multiplied by 100.

49

Table 1.2. Positive cases of M. bovis tuberculosis in Michigan carnivore/
omnivore surveillance through the year 2000.

 

 

SPECIES SCIENTIFIC NAME TOTAL TOTAL PREVALENCE
NUMBER“ POSITIVE 3 RATE C
BADGER T axidea taxus 25 0 0.0
BEAR, Ursus americanus 163 4 2.5
BLACK
BOBCAT F elis rufus 40 2 5.0
COYOTE Canis latrans 249 11 4.4
FOX, GRAY Urocyon 4 0 0.0
cinereoargenteus
FOX, RED Vulpes vulpes l6 2 12.5
MINK Mustela vison 2 0 0.0
OPOSSUM Didelphis virginiana 228 2 0.9
PORCUPINE Erethizon dorsatum 1 0 0.0
RACCOON Procyon lotor l 94 2 l .0
SKUNK Mephistis mephistis 12 0 0.0
WEASEL Mustela sp. 1 0 0.0
TOTALS 935 23 2.5

 

50

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Mycobacterium. In: Hagen and Bruner’s microbiology and infectious diseases of
domestic animals, 8th edition. JF Timoney, JH Gillespie, FW Scott, JE Barlough,
eds. Cornell University Press, Ithaca. pp. 270-289

Van Embden JDA, Schouls LM, Van Soolingen D. Molecular techniques:
applications in epidemiologic studies. In: Mycobacterium bovis infection in
animals and humans. CO Thoen and JH Steele, eds. Iowa State University Press,
Ames. pp. 4-28.

Vanham G, Toossi Z, Hirsch CS, Wallis RS, Schwander SK, Rich EA, Ellner JJ.
1997. Tuber. and Lung Dis. 78: 145-158.

Weinrich OA, van Pinxteren LA, Meng Okkels L, Birk Rasmussen P, Andersen
P. 2001. Protection of mice with a tuberculosis subunit vaccine based on a fusion

protein of antigen 85B and esat-6. Infect. Immun. 69: 2773-2778.

Wilkins EGL, Griffiths RJ, Roberts C. 1986. Pulmonary tuberculosis due to
Mycobacterium bovis. Thorax 41: 685-687.

57

Chapter 2
Experimental Inoculation of European Starlings (Sturnus vulgaris)
and American Crows (Corvus brachyrhynchos) with Mycobacterium

bovis

58

Abstract

The purpose of this series of pilot studies was to determine whether the
passerine species studied are susceptible to infection with M. bovis. Separate
experiments were conducted on wild-caught starlings (Sturnus vulgaris) and American
crows (Corvus brachyrhynchos). In each experiment, four birds were challenged
intraperitoneally and four were challenged orally with microorganisms. Challenge
dose was 1 x 105 colony forming units of M. bovis cultured from a white-tailed deer
(Odocoileus virginianus) case in Michigan. Birds were sacrificed at one and two
months post-inoculation. Histologic lesions suggestive of mycobacteriosis, without
the presence of acid-fast bacilli, were noted in all experimental groups. Mycobacterial
cultures performed on pooled tissue samples were positive for M. bovis only in some

of the intraperitoneal inoculates of each species.

Introduction

Mycobacterium bovis is endemic in Michigan’s free-ranging white-tailed deer
(Odocoileus virginianus) population in the northeastern section of the Lower
Peninsula of the state (Schmitt et a1 1997). Surveillance to assess the spread of this
disease within the deer population and into additional species has been ongoing since
1995. Through this surveillance, M. bovis has been detected in several camivore and
omnivore spillover host species. Avian species have not been included in the

surveillance program and as such, have not been assessed as potential reservoirs of M.

59

bovis. The foundation for the described research involves the ideas that the passerine
species used in the project are known to gather in agricultural settings in Michigan and
also commonly feed on organic materials (feed, fecal matter, viscera) that may harbor
M. bovis. Exposure of starlings or crows in such a manner would allow for
considerable widespread transmission, if the birds can, in fact, become infected with or
mm M. bovis agent and shed microorganisms in distant sites. No published reports of
M. bovis infections in passerines exist to our knowledge, although some citations do
report occurrence(s) in psittacine species (Gerlach 1994; Ackerman et a1 1974). The
closely related organism, Mycobacterium tuberculosis, is well-documented as being
infectious in psittacines (Hoop et a1 1996; Gerlach 1994; Ackerman et a1 1974). Yet
very little is known about the infectivity of M. tuberculosis complex microorganisms
in other avian orders/ species. The objective of this study was to investigate the
potential for native Michigan passerine species to become infected with M. bovis when
experimentally exposed orally or intraperitoneally.
Materials and Methods

Birds

A total of eleven starlings were caught in mist nets placed strategically in

doorways of dairy bams on the campus of Michigan State University. Three of the
eleven birds were used as controls. These birds were caught and sacrificed prior to the
capture of the eight birds to be inoculated with M. bovis. Twelve crows were caught

in one capture with a rocket net after baiting birds to a specific site on the mink farm

60

of Michigan State University for several weeks prior (Federal Fish and Wildlife Permit
number MB000836-0; State Scientific Collector’s Permit for Michigan number
SCI 05 7). Control and inoculated birds were housed in separate wards. All inoculated
birds were housed in biosafety cabinets in hepa-filtered rooms (biolevel 3 conditions)
at the University Laboratory Animal Research Facility.
Inoculum

M. bovis culture isolates from positive white-tailed deer cases resulting from
surveillance activities in Michigan are maintained by the tuberculosis laboratory at the
Michigan Department of Community Health (MDCH). Inocula for this study were
prepared using a typical strain common to all cases. Seven day growth in
Middlebrook 7H9 broth was adjusted to a 0.5 McFarland turbidity standard, diluted
1:100 with sterile water and tested by plate counts to determine the CFU’s (colony
forming units) per unit of volume. Single dose, 1 ml, aliquots containing
approximately 1 X 105 CFU/ml were used. The dose CFU/ml was confirmed again at
the time that the birds were inoculated. Single dose oral inoculations were
administered to birds via a tomcat catheter gavage. Intraperitoneally (IP) inoculated
birds were injected using lcc tuberculin syringes.

Experimental Design

Starling experiment

Prior to inoculation of experimental birds, three control birds were caught and
euthanatized for necropsy. Euthanasia of all birds in these studies was performed via

intracardiac injection of sodium pentobarbital solution (Fatal Plus®,Vortech

61

Pharmaceuticals, Dearbom, MI). Data recorded at necropsy included body weight of
each bird, individual organ weights (spleen, liver and lung), and gross lesions where
present. Sections of lung, liver, kidney, and spleen were collected and pooled for
mycobacterial isolation and identification at necropsy. Sections of brain, lung, heart,
kidney, liver, spleen, pancreas, small and large intestines, and ceca were collected at
necropsy and fixed in 10% formalin solution for histologic examination. Tissues were
stained with both routine hematoxylin and eosin (H & E) and either Ziehl-Neelsen or
peanut oil acid-fast stains prior to histologic examination. Eight birds were randomly
divided into equal groups of four orally inoculated and four 1P inoculated animals.
Fecal samples were collected from birds designated as those to be inoculated on the
day prior to inoculation, these samples were submitted for mycobacterial isolation and
identification. Inoculated birds were weighed on the day of inoculation and every two
weeks thereafter throughout the length of the experiment. Fecal samples of all birds
were collected twenty-four hours post inoculation (PI) and submitted for
mycobacterial isolation and identification. Birds were fed a diet consisting of ad
libitum access to pelleted zoo fowl maintenance diet 9037 (Master Mix Feeds, Fort
Wayne, IN) which was supplemented with canned lamb and rice-based dog food (Iarns
Co., Dayton, OH) once weekly. Birds were monitored for inappetence and for
inadequate fluid intake throughout the study. The clinical condition of each bird was
recorded daily as well. Half of each inoculation group (two birds from each group)

were euthanatized and necropsied one month after inoculation, with tissue processing

62

performed as described for control birds. The remaining birds were euthanatized and
necropsied at two months PI.

Crow experiment

The study design for the crow experiment was similar to that described for the
starlings, save for the inclusion of four control birds which were maintained as an
experimental group throughout the length of the experiment. No control birds were
sacrificed prior to inoculation of experimental birds. The four control crows were
housed separately from the eight inoculated crows, which were kept in biosafety level
3 housing identical to that of the starlings. All birds received ad libitum access to the
same pelleted diet as the starlings. Crows were supplemented twice weekly with
canned rabbit and rice canine diet (Nature’s Recipe Foods, Corona, CA). Fecal
collections and recordings of body weights were canied out as described for the
starlings. Necropsy dates were also at one and two months P1, with half of each group
(two birds from each: control, oral, and IP) randomly chosen and euthanatized.
Necropsy tissue collections for mycobacterial culture were identical to those of the
starlings. Histopathologic analyses of those tissues collected from starlings as well as
of proventriculus and ventriculus were performed on crow tissues. Both H & E and
Ziehl-Neelsen acid-fast stained sections of each tissue were examined.

Mycobacterial Isolation and Identification

Mycobacterial cultures were performed at the MDCH, Tuberculosis Laboratory.

Tissue specimens were ground, digested, and concentrated in a manner previously

described (Kent and Kubica 1985). Fecal samples were cultured directly after

63

digestion and concentration. One each of a Lowenstein-Jensen medium slant (Becton-
Dickinson, Cockeysville, MD), a Middlebrook 7H11S medium slant (Becton-
Dickinson, Cockeysville, MD), and a Bactec12B broth vial (Becton-Dickinson,
Sparks, MD), were inoculated with the resultant material. Media were examined for
growth on a weekly basis for eight weeks. Bacterial growth, determined to be acid-
fast by slide examination (Kent and Kubica 1985), were tested by genetic probe
(Accuprobes, Gen-Probe, San Diego, CA) to determine whether the bacteria were
members of the M. tuberculosis complex (Reisner et al 1994). Complete species
identification was performed using biochemical testing and high performance liquid
chromatography to differentiate M. bovis from the other members of the M.
tuberculosis complex and to speciate other mycobacteria (Nolte and Metchock 1995;
Butler et al 1991; Kent and Kubica 1985).

Statistical Analysis

SAS Version 8 statistical software was used for all calculations (Statistical

Analysis Systems, SAS Institute, Inc., Cary, NC). The Wilcoxon Rank-Sum test was
used to assess whether there were statistically significant (p < 0.05) changes in overall
body weight and in individual organ weights within each treatment group and between
groups. Associations between routes of exposure and the outcomes of interest (gross
and histologic lesions and mycobacterial culture results) were assessed using the two-
tailed Fisher’s Exact test, and associations were expressed as odds ratios with 95%

confidence intervals (CI).

 

Results
Clinical Condition of Birds
All birds remained clinically healthy throughout the trials. No depressions in
food or water consumption were noted. No mortality prior to assigned sacrifice dates
occurred.
Weight Change Data
No treatment group had significant changes in weight, though weight changes in
orally inoculated starlings approached significance (X2 = 9.33, p = .0533).

In crows, there were statistically significant differences in kidney weight, when
comparing intraperitoneal exposure (mean = 3.2 g) to controls (mean = 4.05 g;
Wilcoxon Rank-Sum X2 = 4.08, p = 0.0433), and in spleen weights when comparing
oral exposure (mean = 0.75 g) to controls (mean = 0.43 g; Wilcoxon Rank-Sum X2 =
5.4, p = 0.0202). There was also a statistically significant difference in lung weights
when comparing intraperitoneal exposure (mean = 7.85 g) with oral exposure (mean =
5.63 g; Wilcoxon Rank-Sum X2 = 4.08, p = 0.0433). There were no statistically
significant differences in organ weights for starlings.

The small numbers of subjects included in each experimental group may have
influenced the power of statistical analyses, so general trends in body weight were also
recorded. Changes in overall body weights are illustrated in Figure 2.1 (starlings) and
Figure 2.2 (crows). In both experiments, both IP inoculated groups (one and two

month sacrifice dates) showed reductions in mean group body weights over time. The

65

trend toward weight loss in IP inoculated birds was more steady in crows (Figure 2.2)
than it was in starlings (Figure 2.1). Weight gain of all oral inoculated birds and
control birds was the trend noted in the starlings (Figure 2.1). In crows, overall weight
gain was also noted in all groups save for those orally inoculated birds sacrificed at
one month (Figure 2.2). In these crows, mean body weight of the group increased
during the first two weeks of the experiment, but dropped slightly from two to four
weeks. Final mean weight of the group was still higher at the sacrifice date than at the
inoculation date, however. A similar trend of gain until two weeks followed by slight
loss until six weeks was noted in the two month sacrifice date, orally inoculated crows.
These birds gained weight during the last two weeks of the experiment, however, and
were greater in weight at the termination of the study than they were at inoculation.
Gross Necropsy Findings

Gross lesions suggestive of mycobacteriosis were not present in any starlings.
One IP inoculated starling sacrificed at one month PI had a single, 0.5 cm diameter,
green to brown, friable nodule present in the mesentery subtending the proventriculus.
Dissemination of granulomas was not noted. The other IP inoculated starling
sacrificed at one month had grossly detectable splenomegaly. Hepatomegaly and
splenomegaly were found at necropsy in one IP inoculated bird sacrificed at two
months. The other IP inoculated starling sacrificed at this time had only
hepatomegaly.

No gross lesions suggesting disseminated mycobacteriosis were present in any

crows. One IP inoculated crow sacrificed at one month PI had a prominent keel bone

66

with breast muscle atrophy and an enlarged spleen. The other IP inoculated crow
sacrificed at this time had fibrinous adhesions attaching the gall bladder to the small
intestine. Splenomegaly was noted in one orally inoculated bird sacrificed at two
months. One IP inoculated crow sacrificed on this date had a prominent keel bone and
breast muscle atrophy.

Microscopic Lesions

All tissues examined histologically from all birds were scored as either having

lesions suggestive of mycobacteriosis or not having lesions consistent with a diagnosis
of mycobacteriosis. Presence of bacilli on acid-fast stained tissue sections was also
recorded as positive or negative. These results are compiled for the starlings in Table
2.1 and for the crows in Table 2.2. Lesions considered histologically consistent with
mycobacterial infection included histocytic granulomas, with or without giant cells,
featuring central caseation but not associated with central mineralization and either
containing small numbers of heterophils or no heterophils. Often such granulomas
featured mild fibroplasia, which is also typical of tuberculous lesions in birds.
Primarily heterophilic granulomas were regarded as negative lesions, as were
granulomas with or without giant cells that featured central mineralization (Figures 2.3
and 2.4). In starlings (Table 2.1), primarily heterophilic lesions were present in the
liver or pancreas of three IP inoculated birds and in one control bird. No acid-fast
bacilli were histologically noted in any sections of tissues from these birds, however.
Additional non-specific lesions noted in starlings included reactive, hyperplastic

bronchial- associated lymphoid tissues (BALT); hepatic granulomas with moderate to

67

large numbers of associated heterophils (in three orally inoculated birds; Figure 2.3);
foci of primarily heterophilic hepatic necrosis with small numbers of associated
mononuclear inflammatory cells; and multifocal granulomatous hepatitis with giant
cells and central mineralization of necrotic hepatocytes, associated with histologically
detectable portions of a necrotic cestode parasite larva (Figure 2.4). The liver of this
same bird was marked by a mild, diffuse, vacuolar hepatopathy consistent with fatty
degeneration. One IP inoculated starling also had a moderate, multifocal,
nonsuppurative interstitial nephritis noted histologically.

In crows, three inoculated birds (one oral and two IP), and one control had
lesions that could be considered histologically consistent with mycobacterial infection
(Table 2.2). Small numbers (one to two bacteria) of acid-fast bacilli were noted within
cytoplasms of cells forming one granuloma (Figure 2.5), in one specially-stained
section of liver harvested from an IP inoculated crow. Lesions additionally noted that
were not specific to the diagnosis included hyperplasia of gut associated lymphoid
tissues (in orally and IP inoculated crows); splenic follicular reactivity with
reticuloendothelial cell hyperplasia (in birds from all groups); multifocal heterophilic
hepatic necrosis; and multifocal, primarily nonsuppurative hepatitis (also found in all
groups). Granulomas consisting of giant cells with or without central mineralization,
associated with fragments of degenerate cestode parasite larvae were present in
sections of liver, kidney, and pancreas from several birds. One IP inoculated crow had
adult cestodes noted within the lumen of sections of small intestine noted

histologically. One 1P inoculated crow had a mild, multifocal, heterophilic to

68

nonsuppurative interstitial nephritis. A mild, focal nonsuppurative myocarditis was
present in one orally inoculated crow and a moderate, multifocal, necrotizing
myocarditis and myocardial vasculitis was present in one IP inoculated crow.
Mycobacterial Isolation and Identification

All birds were scored as positive or negative for the presence of M. bovis in fecal
and tissue cultures (Tables 2.1 and 2.2). Mycobacterium bovis was not recovered from
the fecal samples of any birds, from either pre- or post inoculation sampling.
Mycobacterium gordonae was cultured from the pooled tissue culture of a single
control starling. Three IP inoculated starlings cultured positive for M. bovis, two after
one month and one after two months. No further starlings cultured positive for any
mycobacteria (Table 2.1). One [P inoculated crow cultured positive for M. bovis after
one month, and one after two months. No further crows cultured positive for any
mycobacteria.

Statistical Analysis

Compared to control birds, birds with intraperitoneal exposure were significantly
more likely to have positive mycobacterial culture (Fisher’s Exact p =0 .0256; odds
ratio = 3.46, 95% CI = 1.25 - 9.55) and gross or histologic lesions (Fisher’s Exact p =
0.0256, odds ratio = 3.49, 95% CI = 1.29 - 9.45). No significant effects were seen
comparing controls to orally exposed birds. When comparing oral exposure to
intraperitoneal exposure, birds with intraperitoneal exposure were significantly more
likely to have a positive mycobacterial culture (Fisher’s Exact p = 0.0256; odds ratio =

13.61, 95% CI = 1.19 - 155.9).

69

Discussion

In both bird species studied, M. bovis infection was apparently capable of being
established via [P inoculation. In three of four starlings and in two of four crows
inoculated by this route, infection was supported by microbiologic isolation of the
organism. All birds that cultured positive for M. bovis had histologic lesions
suggestive of mycobacterial infection. The experimental group that had the largest
number of birds with histologic lesions and positive cultures in each species, the IP
group, was also that which exhibited the most regular trend in weight loss. Yet in only
one crow were all criteria typically considered as suggestive of positive infection
(positive culture, gross lesions, histologic lesions, and acid-fast bacilli detection
histologically) noted. No further inoculates had gross lesions suggesting
mycobacterial infection. This bird had a mesenteric granuloma, which may or may not
have been related to intraperitoneal infection. No acid-fast bacilli could be detected
within this lesion histologically, yet histologic features were consistent with a
mycobacterial, centrally caseous, histiocytic granuloma. If this lesion was exclusively
a foreign-body type injection reaction, a more heterophilic granuloma would be
expected (Barnes 1996; Montali 1988). Late in their development, both types of these
granulomas may appear quite similar histologically, so the true nature of its
development cannot be determined. Some birds did have nonspecific changes such as
liver and spleen enlargement noted grossly, yet neither of these changes are restricted

to being typical of mycobacterial infections alone. No birds other than this single

70

inoculated crow had histologically detectable acid-fast organisms in any sections of
specially-stained tissues.

Organ weight analyses did have significant results in some groups. Yet
differences in individual animal weights may have contributed to these data. No clear
explanation exists as why 1P inoculated crows had smaller kidney weights. No gross
or histologic basis for this finding was present. Lymphoid infiltrates in the spleens of
inoculated animals were present histologically in some cases, possibly explaining the
relative heaviness of these organs in some groups in comparison to controls. Yet no
clear reason exists that explains why orally inoculated birds had significant differences
in weights of these organs compared to controls while 1P inoculates did not.
Lymphoid GBALT) hyperplasia noted more frequently in the lungs of IP inoculated
crows could reasonably explain heavier lungs in these animals in comparison to orally
inoculated crows.

Some birds that did not culture positive for M. bovis had histologic lesions
suggestive of mycobacterial disease. In one control starling, this result appears to be
the result of infection by M. gordonae, which was cultured from pooled tissues. In
one control crow, these lesions are most likely in response to the same cestode parasite
larvae which were identified in tissues of other crows, although actual parasite
remnants no longer remained in the sections of tissues harvested for histology. Clear
speciation of these parasites was not achieved, but literature reviews suggest that
Plagiorhynchus cylindraceus is a likely agent in these birds, particularly in starlings

(Moore and Bell 1969). The similarity between mycobacterial-type granulomas and

71

parasite-induced granulomas in birds can be striking. Central mineralization is much
more suggestive of parasite involvement (in lieu of, or in addition to, finding actual
parasites in section) than are histiocytic granulomas alone (Barnes 1996; Montali
198 8). Another reasonable explanation for such lesions could be that other bacteria
not microbiologically detected (only cultures for mycobacteria were requested, due to
limited project funding) were involved in lesion formation. Additional bacteria that
may produce granulomas similar to those noted in mycobacterial infections include
salrnonellae, enterococci, and E. coli, amongst others (Goodwin 1996). Unidentified
bacterial infections were likely responsible for most of the primarily heterophilic
lesions detected and reported in several birds of both groups as well. Histologic
changes such as lymphoid tissue hyperplasia (in the spleen, gastrointestinal tract, and
gut) were noted in many birds. Such changes suggest nonspecific immune
stimulation, which could be attributed to any number of infectious agents, systemic or
otherwise, but are certainly not specific to mycobacteriosis. Failure of the systems
used to detect mycobacteria may have been involved in some instances in which
inoculated birds with lesions could not be definitively diagnosed with M. bovis
infection. Even the “gold standar ” of mycobacterial isolation and identification will
not detect all positive infections (Fitzgerald et a1 2000).

One 1P inoculated starling cultured positive, but did not have any histologic
lesions suggestive of mycobacteriosis. Since this bird was sacrificed at one month PI,
lesion formation may not have progressed to those histologically detectable in

randomly harvested sections of tissue. Immune status plays an important role in the

72

progression of intracellular bacterial infections in birds, with lymphoid depletion in
important organs such as the spleen being part of the pathophysiology (Gerlach 1996).
This animal may have had a stronger overall immune status than others that had
histologically detectable lesions in combination with being culture positive at the same
sacrifice date. Also possible is that a small number of non-grossly detectable lesions
had developed in one or more tissue sections not harvested for histologic examination,
but instead submitted for mycobacterial isolation, in this bird’s case.

In both crows and starlings, oral gavage with mycobacteria did not result in
mycobacterial infection that could be discerned grossly, histologically or by pooled
tissue cultures run on specimens collected from these animals. Body weight trends
throughout the experiment in both orally inoculated crows and starlings suggest that
overall weight loss was not featured as a result of such inoculation, unlike those results
seen in IP inoculated birds. However, this observation could not be confirmed
statistically. Fecal cultures run immediately PI suggest that even large doses of
mycobacterial organisms are inactivated in the gastrointestinal tracts of the species
studied, as no shedding could be detected by these cultures. Serial fecal culture results
obtained throughout the experiment might have allowed this to be a more definitive
conclusion, but the combination of negative fecal and tissue cultures obtained in all
orally inoculated birds is supportive of resistance to M. bovis infection when
organisms are introduced by this route in both of these species.

Orally inoculated birds appeared resistant to M. bovis infection, yet IP

inoculated birds did develop lesions suggestive of mycobacterial infection in

73

statistically significant numbers. Perhaps even more importantly, many of these
animals actually cultured positive for the organism to explain the true nature of these
lesions, giving proof that this organism can indeed survive within the realms of avian
physiology when inoculation is used to bypass natural protective mechanisms. Oral
exposure in the wild is undoubtedly a more likely route by which these birds could be
infected than is the IP route. But survival of M. bovis intraperitoneally in these species
may warrant additional investigation into other potential exposure routes that might
commonly occur in the wild, namely inhalation of infectious mycobacterial droplet
nuclei.

Overall, these experiments demonstrate the remarkable resistance that several
avian species have to infection with M. bovis. Both studied species are quite
susceptible to avian tuberculosis and are implicated as potential sources of this disease
in both wild and domestic avian and mammalian populations (Bickford et a1 1966,
Mitchell and Cuthrie 1950). Yet these passerines may be less susceptible to all of the
genetically related M. tuberculosis complex organisms (M. tuberculosis, M. bovis, M.
microti, and M. africanum) than are psittacines, as several case reports of M.
tuberculosis in psittacines exist, yet none have been noted in passerines (Washko et al
1998; Hoop et a1 1996; Gerlach 1994; Ackerman 1974). Alternatively, all bird species
may be relatively resistant to infection by M. bovis, as is the commonly held belief. It
is generally accepted that the higher core body temperatures of birds may prevent
active infection with M. tuberculosis complex organisms in these animals. No

convincing reports of infection with M. bovis in any bird species exist. Fecal shedding

74

appears to be quite uncommon in these birds after oral gavage with a large number of
microorganisms, a dose of which they would be very unlikely to encounter in nature.
It is therefore a reasonable conclusion that these animals do not play a large role, if
they play any at all, in the dissemination of bovine tuberculosis within and between
Michigan wildlife and domestic livestock populations. Fecal shedding did not occur
in IP inoculated birds either, suggesting that even if a bird could somehow become
intraperitoneally “inoculated” with infected materials in the wild, that spreading
disease in such a manner is not of major concern. Further exposure routes such as
inhalation that may lead to droplet spread after infection by these birds would be
reasonable exceptions to this statement, as they have not yet been studied.

Also of consideration in this particular set of experiments was the timing
between inoculation and sacrifice. Because the development of tuberculosis in most of
the species it affects is often realized in its most chronic stages, longer time periods
between inoculation and sacrifice of birds may still prove to be worthy of
investigation. Funding was unfortunately available neither for a more chronic study
nor for serial culture work in this experimental series. Multiple fecal cultures of living
birds throughout the experiment may have given a better assessment of potential
intermittent fecal shedding of organisms that may have gone unnoticed in the more

restricted culture work included in the described protocol.

75

Table 2.]: Laboratory findings in starlings. A Number of birds tested/ number of birds
positive for feature. Mycobacterial culture positives indicate M. bovis cultured from

pooled tissue samples.

Table 2.2: Laboratory findings in crows. A Number of birds tested/ number of birds

positive for feature. Mycobacterial culture positives indicate M. bovis cultured from

pooled tissue samples.

76

Table 2.1. Laboratory data for starlings

 

 

 

 

 

 

 

 

 

 

 

 

 

Mycobacterial Histologic AFB Noted
Experimental Gflrp M Lesions A On Histology A
Gm Siz_e Positive Result A
Control 3 0/ 3 1/ 3 0/ 3
Oral 4 0/ 4 0/ 4 0/ 4
IP 4 3/ 4 2/ 4 0/ 4
Table 2.2. Laboratory data for crows
Mycobacterial Histologic AFB Noted On
Experimental @922 9.1325? Lem A Histology A
§r_oup _S_igc_: Positive Result A
Control 4 0/ 4 1/ 4 0/ 4
Oral 4 0/ 4 1/ 4 0/ 4
IP 4 2/ 4 3/ 4 1/ 4

 

77

Figure 2.1: Weight data for starlings. X axis: weeks post inoculation that weight
was recorded. Y axis: MEAN body weight for entire group in grams (indicated in

legend) at given date.

Figure 2.2: Weight data for crows. X axis: weeks post inoculation that weight
was recorded. Y axis: MEAN body weight for entire group in grams (indicated in

legend) at given date.

78

Figure 2.1 Weight data for starlings

 

 

 

 

A 100 ~
in _
E
g 90 -. --<>--Oral
a 80 - ,
'8 i—D—IP
3
.5: 7O - .

j _ _ S
8 A A i -A Controli
: 60 -
ll
0 .
E 50 _ z._______.__ _.__--_-_ .zi____.

O 2 4 6 8
Weeks Post Inoculation
Figure 2.2. Weight data for crows

A 550
U)
E t ___ _ __ _ -
S2.
3 0 -Oral
.C
,9 2
a .
3 3—D—IP
>4 .
1: .
o f
I: 3 - -A- Control
a .‘
G
E

 

 

0 2 4 6 8

Weeks Post Inoculation

79

Figure 2.3: Photomicrograph of liver from a starling orally inoculated with M. bovis.
Heterophilic hepatic granuloma not consistent with mycobacterial infection.

Hematoxtlin & Eosin (H & E); 40X.

Figure 2.4: Photomicrograph of liver from a starling orally inoculated with M. bovis.
Histiocytic hepatic granulomas with central mineralization (white arrows) and central
necrosis associated with cestode larvae (black arrows); diffuse microvesicular

hepatopathy consistent with hepatic lipidosis. H & E; 20X.

Figure 2.5: Photomicrograph of liver from a crow intraperitoneally inoculated with M.

bovis. Histiocytic hepatic granuloma with giant cells associated with mild

nonsuppurative inflammation and fibroplasia. H & E; 40X.

80

3

2

Figure

in“... .m
L C— r.. .
If.

we...

          

Figure 2.4

 

Figure 2.5

 

81

References

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infection in a parrot (Amazona farinosa). Am. Rev. Resp. Dis. 109: 388-390.

Barnes HJ. 1996. Hemic system. In: Avian histopathology, 2nd ed. C. Riddell,
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Bickford AA, Ellis GH, Moses HE. 1966. Epizootiology of tuberculosis in
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Butler WR, Jost KC Jr., Kilbum JO. 1991 Identification of mycobacteria by high
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Fitzgerald SD, Kaneene JB, Butler KL, Clarke KR, Fierke J S, Schmitt SM,
Bruning-Fann CS, Mitchell RR, Berry DE, Payeur JB. 2000. Comparison of
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deer (Odocoileus virginianus). J. Vet. Diagn. 12: 322-327.

Gerlach H. 1994. Bacteria. In: Avian medicine: principles and application. B.
W. Ritchie, G. J. Harrison, and L. R. Harrison, eds. Wingers Publishing, Lake
Worth. pp. 949-983.

Goodwin MA. 1996. Alimentary system. In: Avian histopathology, 2nd ed. C.
Riddell, ed. American Association of Avian Pathologists, Saskatoon. pp. 111-
142.

Hoop RK, Bottger EC, Pfyffer GE. 1996. Etiological agents of mycobacterioses
in pet birds between 1986 and 1995. J. Clin. Microbiol. 34: 991-992.

Kent PT and Kubica GP. 1985. Public health mycobacteriology. A guide for the
level III laboratory. US. Department of Health and Human Services. Atlanta,
Georgia.

Merchant IA and Packer RA. 1961. Mycobacterium. In: Veterinary bacteriology
and virology, 7th ed.. Iowa State University Press, Ames. pp. 448-449. 1961.

Mitchell CA and Duthie RC. 1950. Tuberculosis of the common crow. Can .J.
Comp. Med. 14: 109-117.

Moore J and Bell DH. 1969. Pathology of Plagiorhynchus cylindraceus in the
starling, Sturnus vulgaris. J. Parasitol. 69:387-390.

82

Montali RJ. 1988. Comparative pathology of inflammation in the higher
vertebrates (reptiles, birds, and mammals). J. Comp. Path. 99: 1-26.

Nolte FS and Metchock B. 1995. Mycobacterium. In: Manual of Clinical
Microbiology. P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H.
Yolken, eds. American Society for Microbiology Press, Washington, DC, pp.
400-437.

Reisner BS, Gatson AM, Woods GL. 1994. Use of gen-probe accuprobes to
identify Mycobacterium avium complex, Mycobacterium tuberculosis complex,
Mycobacterium kansasii and Mycobacterium gordonae directly from Bactec TB
broth cultures. J. Clin. Microbiol, 32: 2995-2998.

Schmitt SM, Fitzgerald SD, Cooley TM, Bruning-Fann CS, Sullivan L, Ben-y DE,
Carlson T, Minnis RB, Payeur JB, Sikarskie J. 1997. Bovine tuberculosis in free-
ranging white-tailed deer from Michigan. J. Wildl. Dis. 33: 749-758. 1997.

Washko RM, Hoefer H, Kiehn TE, Armstrong D, Dorsinville G, Frieden TR.
1998. Mycobacterium tuberculosis infection in a green-winged macaw (Ara
chloroptera): report with public health implications. J. Clin. Microbiol. 36: 1101-
1102.

83

Chapter 3
Experimental Inoculation of North American Opossums (Didelphys

virginiana) With Mycobacterium bovis

84

Abstract

In order to investigate their potential as reservoir hosts for bovine tuberculosis in
Michigan, eight North American opossums (Didelphis virginiana) were inoculated
with 1 X 105 CFU (colony forming units) of Mycobacterium bovis. Half of the
animals (four) received this dosage orally, and half were inoculated intramuscularly
(IM). In each group, two animals were euthanatized at one month post inoculation
(PI) and two at two months P1. Four control animals were housed separately and
sacrificed in the same manner as those inoculated. One of the four orally inoculated
opossums, and three of the four intramuscularly inoculated opossums were positive for
M. bovis on tissue specimen cultures obtained at necropsy. The oral recipient had
positive tissue cultures on both intestine and pooled lymphoid samples. All IM-
inoculated animals that had positive tissue cultures had these positive results from
pooled lymphoid samples. Two of the three tissue culture-positive 1M recipients also
had positive liver and lung samples. One animal with gross and histologic lesions

compatible with tuberculosis did not have any positive tissue cultures.

Introduction

In Michigan, surveillance for Mycobacterium bovis tuberculosis in wild and
captive cervids has been ongoing since 1995 (Schmitt et a1 1997). As an attempt to
identify spillover hosts in the wild, and also to search for potential reservoir hosts for
this disease other than white-tailed deer (Odocoileus virginianus), additional carnivore

and omnivore wildlife species have been added to the surveillance program over the

85

past six years (Bruning-Fann et al 2001). No additional reservoir hosts have been
conclusively defined, yet positive cultures representing spillover infections have been
identified in black bears (Ursus americanus), bobcats (F elis rufus), coyotes (Canis
latrans), raccoons (Procyon lotor), red foxes ( Vulpes vulpes), and North American
opossums (Didelphis virginiana) via surveillance through the end of the year 2000.
For the first several seasons of carnivore/ omnivore surveillance, no opossums were
identified as positive, although they, along with coyotes, represented the largest in
numbers of cases submitted (over one hundred each at that point). Although North
American opossums are only distantly related to the New Zealand brushtail possum
(T richosurus vulpecula), the possibility of their resistance to M. bovis tuberculosis in
comparison to such a susceptible reservoir host species was speculated upon. In order
to determine whether North American opossums were uniquely resistant to M. bovis, a
pilot study was designed to challenge these animals with the bovine tuberculosis agent
by either the oral or intramuscular routes.
Materials and Methods
Opossums

The total number of animals to be used for the study was, based on available
funding and biolevel-3 laboratory space restrictions, limited to twelve. Initially, five
adult North American opossums were humanely live-trapped in Ingharn County,
Michigan. (State Scientific Collector’s Permit for Michigan number SC1057). Upon

collection, one of these adults was noted to be a late-term pregnant female. This

86

female was observed for several days when she was noted to have given birth to seven
offspring. These young were captive-raised, weaned as juveniles, and included as
subjects in this study, necessitating fewer live wild captures. Juveniles and adults
(seven females and five males) were acclimated to the diet to be used throughout the
experiment. During this diet acclimation period, each animal received 100 mg/kg
fenbendazole (PanacurQ, Hoechst-Roussel Agri-Vet Co., Somerville, NJ) orally, once
daily, for three consecutive days. Control and inoculated opossums were moved to
separate wards one week prior to inoculation to allow acclimation to the new
laboratory. One adult male became suddenly and severely clinically depressed and
anorectic one week after intramuscular inoculation and was found dead the next
morning. This animal was replaced with a new, wild-caught juvenile male.
Inoculum

M. bovis culture isolates from positive white-tailed deer cases resulting from
surveillance activities in Michigan are maintained by the Tuberculosis Laboratory at
the Michigan Department of Community Health (MDCH, Lansing, MI). Inocula for
this study were prepared using a typical strain (evaluated by the restriction fragment
length polymorphism analyses, RFLP) common to all cases. Seven-day growth in
Middlebrook 7H9 broth was adjusted to a 0.5 McFarland turbidity standard, diluted
1:100 with sterile water and tested by plate counts to determine the CFU’s (colony
forming units) per unit of volume. Single dose, 1 ml, aliquots containing
approximately 1 X 105 CFU/ml were used for intramuscular (IM) recipients and 0.2 ml

containing the same CFU were used for oral recipients. A more concentrated solution

87

was used for these oral recipients to assure complete delivery of the entire inoculum
dosage. The dosage CF U/ml was confirmed again at the time that the opossums were
inoculated. Single dose oral inoculations were administered to opossums mixed in a
teaspoon of strawberry preserves, served on a one inch square of bread. Animals were
monitored to assure the complete inoculation dosage was consumed. Intramuscular
(IM) recipients were injected in the musculature of the left hind limb using lml
tuberculin syringes.

Experimental Design

Twelve opossums were randomly divided into groups of four receiving oral

inoculation, four receiving IM inoculation, and four controls. All inoculated opossums
were housed in separate Horsfal units in hepa-filtered rooms (biolevel 3 conditions) at
the Michigan State University Containment Facility until the date of sacrifice. Control
opossums were housed in standard rabbit cages in a separate ward in the Facility.
Animals received a diet of approximately 4 ounces of Hill’s Science Diet Canine
Maintenance Dry daily and approximately 2 ounces of Hill’s Canine p/d Canned every
other day (Hill’s Pet Nutrition, Inc., Topeka, KS). This primary diet was
supplemented with fresh fi'uit and strawberry preserves weekly prior to inoculation,
and weekly with fruit alone post inoculation (PI). The clinical condition, food
consumption, and water consumption of each opossrun were recorded daily. Save for
the single aforementioned adult male, no opossums showed signs of debilitation prior

to sacrifice.

88

Animals were weighed on the day prior to inoculation and every two weeks
thereafter throughout the experiment. Fecal samples for mycobacterial culture were
collected fiom all animals on the day prior to inoculation and again at 24 hours post
inoculation in order to assess shedding of microorganisms. Fecal samples were
collected for mycobacterial culture from all animals again at 31 days PI. Half of the
animals in each experimental group were euthanatized at thirty-one days PI and the
remaining animals were sacrificed at 61 days PI. Euthanasia was performed via
intracardiac injection of sodium pentobarbital solution at a dosage of 120 mg/kg (Fatal
Plus®,Vortech Pharmaceuticals, Dearbom, MI) after initial sedation was achieved
using 5 mg/kg xylazine (Rompun®, Miles, Inc., Shawnee, KS) injected
intramuscularly. Fecal samples for mycobacterial culture were submitted at the same
time as tissue samples collected from those animals sacrificed on that day. Fecal
cultures were performed for all remaining opossums sacrificed at 61 days PI, also with
pooled tissue cultures.

Data recorded at necropsy included total body weight of each animal,
individual organ weights (spleen, liver, kidneys and lung), and gross lesions where
present. Tissues harvested and submitted for mycobacterial isolation and
identification included pooled liver and kidney, pooled small and large intestine,
pooled spleen and lymph nodes, and lung. Sections of brain, heart, lung,
tracheobronchial lymph node, stomach, small intestine, pancreas, large intestine,
mesenteric lymph node, liver, kidney, adrenal gland, spleen, urinary bladder, teste or

ovary, skin, hindlimb musculature, and an entire eyeball were collected from each

89

animal at necropsy and fixed in 10% neutral buffered formalin solution for histologic
processing and examination. Tissues were stained with both routine hematoxylin and
eosin and Ziehl-Neelsen acid-fast stains prior to histologic examination (Prophet et a1
1992)
Mycobacterial Isolation and Identification

Mycobacterial cultures were performed at the MDCH Tuberculosis Laboratory.
Tissue specimens were ground, digested, and concentrated in a manner previously
described (Kent and Kubica 1985). One each of a Lowenstein-Jensen medium slant
(Becton-Dickinson, Cockeysville, MD), a Middlebrook 7H11S medium slant (Becton-
Dickinson, Cockeysville, MD), and a Bactec12B broth vial (Becton-Dickinson,
Sparks, MD), were inoculated with the remaining material. Media were examined for
growth on a weekly basis for eight weeks. Bacterial growth, determined to be acid-
fast by slide examination (Kent and Kubica 1985), were tested by genetic probe
(Accuprobes, Gen-Probe, San Diego, CA) to determine whether the bacteria were
members of the M. tuberculosis complex (Reisner et a1 1994). Complete species
identification was performed using biochemical testing and high performance liquid
chromatography to differentiate M. bovis from the other members of the M.
tuberculosis complex (M. bovis, M. tuberculosis, M. microti, and M. africanum), and
from other mycobacteria (Butler et a1 1991; Kent and Kubica 1985; Nolte and

Metchock 1995).

90

 

Surveillance Data

Carnivore and omnivore surveillance has continued in a method previously
described (Bruning-Fann et a1 2001), through the end of the year 2000. Cranial,
thoracic, and abdominal lymph nodes from animals collected by the Michigan
Department of Natural Resources (MDNR) were examined grossly and histologically
as previously described. In cases where animals had lung lesions noted grossly, lung
tissue was also harvested, fixed in 10 % neutral buffered formalin, paraffin embedded
and sectioned for routing hematoxylin and eosin and Ziehl-Neelsen staining prior to
histologic examination. Tissue mycobacterial cultures (pooled lymph nodes fi'om all
animals, and in those cases with lung lesions, pooled lymph nodes and lung) were
performed as described for experimental subjects in this manuscript, at the MDCH
Tuberculosis Laboratory.
Statistical Analysis

SAS Version 8 statistical software was used for all calculations (Statistical
Analysis Systems, SAS Institute, Inc., Cary, NC). The Wilcoxon Rank-Sum test was
used to assess whether there were statistically significant (p < 0.05) changes in weight
within each treatment group over time. Associations between routes of exposure and
the outcomes of interest (gross lesions, histologic lesions, mycobacterial cultures from
fecal material, and mycobacterial cultures from organ samples) were assessed using
the two-tailed Fisher’s Exact test, and associations were expressed as odds ratios (OR)

with 95% confidence intervals (CI).

91

 

Results

All animals gained weight during the experiment. The amount gained in
controls was similar to that gained in inoculation groups. No statistically significant
differences in organ weights were noted at necropsy. Gross lesions included
hepatomegaly in one oral and one intramuscular recipient, splenomegaly in two of
each inoculation group, and numerous gastric Physaloptera turgida_in one 1M
recipient, one oral recipient, and one control animal. The same IM recipient had large
numbers of cestodes throughout the entire gastrointestinal tract. At necropsy, the adult
male replaced early in the experiment due to decline in clinical condition was found to
have gastric ulceration and rupture with a resultant hemoperitoneum due to a severe
Physaloptera turgida infection. One oral recipient had numerous, randomly scattered,
firm, one to four millimeter diameter, pale tan to white nodules scattered throughout
both the pulmonary and renal parenchyma. Two IM recipients had lesions in both the
lungs and at the injection site. The lesion in the first of these animals was a 4.0 mm
diameter, irregular, yellow to tan, firm nodule within the parenchyma of the caudal-
dorsal right lung lobe. The lung lesion noted grossly in the second animal was a 0.5
mm diameter, irregular, gray to tan, firm nodule, also within the caudal right lung
lobe. Both of these animals had large (3 cm X 2 cm and 3 cm X 3 cm, respectively)
abscesses within the musculature of the left hind limb. The abscesses contained

approximately three to five mls of exudate which ranged fi'om thick and yellow-green

92

to watery and straw-colored. The abscesses extended into a deep cellulitis and fasciitis
in one of the animals.

Histologic lesions attributable to M. bovis were not present in any of the
routinely stained or acid-fast stained tissue sections examined in either control or oral
inoculation groups (Table 3.1). In the IM inoculated group, two animals had acid-fast
bacilli noted histologically in skeletal muscle tissue sections. Each case featured a
severe, multifocal and coalescing to dissecting, chronic-active, pyogranulomatous to
caseogranulomatous myositis, cellulitis and fasciitis, with foci of mineralization and
featuring multinucleate giant cells (Figure 3.1). Acid-fast bacilli were present both
within the cytoplasm of inflammatory cells in these lesions, and free within centers of
caseous exudate. An additional 1M recipient had similar skeletal muscle lesions
present without histologically confirmed presence of bacilli. This same animal did,
however, have acid-fast organisms within the cytoplasms of macrophages in sections
of lung. Lesions in the lung of this animal were histologically characterized as a
moderate to severe, multifocal, chronic, granulomatous interstitial pneumonia with
foci of caseation and with multinucleate giant cells. Acid-fast bacilli within
inflammatory cells in the lung, associated with a similar caseogranulomatous
pneumonia were present in one additional 1M recipient (Figures 3.2 and 3.3). This
second case was one of the two recipients with a granulomatous myositis that featured
acid-fast organisms histologically. No further significant histologic lesions were noted

in any animals.

93

 

 

Compared to controls, opossums that were intramuscularly exposed to M. bovis
were at increased risk for being histopathology positive (OR = 3.00, 95% CI = .97 -
9.30), having visible lesions (OR = 2.33, 95% CI = 0.99 - 5.49), and having positive
culture results for organ tissues (OR = 9.0, 95% CI = 0.64 - 126.84), but none of these
results were statistically significant (p > 0.05). Similarly, opossums that were orally
exposed to M. bovis were at increased risk for having positive culture results from
feces (OR = 3.0, 95% CI = 0.97 - 9.30) and organ tissues (OR = 2.33, 95% CI = 0.99 -
5.49) compared to controls, but none of these results were statistically significant (p >
0.05).

When comparing the two different routes of exposure, there were no statistically
significant differences (p > 0.05) in body weight, organ weights, or any of the
outcomes of interest. Compared to oral exposures, opossums exposed intramuscularly
were at increased risk for having gross lesions (OR = 2.33, 95% CI = 0.99 - 5.49),
positive fecal culture (OR = 3.0, 95% CI = 0.97 - 9.30) and positive tissue culture (OR
= 6.0, 95% CI = 0.43 - 83.54), but were at decreased risk for positive histopathology
(OR = .26, 95% CI = .02 - 3.46). None of these relationships were statistically
significant (p > 0.05).

Several histologic lesions were noted in all groups, including control animals.
Most common was a mild to moderate, diffuse, microvacuolar to granular hepatopathy
which was occasionally associated with a mild, multifocal, nonsuppurative, portal
hepatitis. Also common was a mild to moderate, diffuse, eosinophilic to

nonsuppurative enterocolitis, occasionally associated with intraluminal nematode or

94

cestode parasites in cross-section. In one control animal, two oral recipients, and one
IM recipient, a moderate to severe, multifocal and coalescing, chronic alveolar
histiocytosis with a mild to moderate, multifocal, chronic nonsuppurative interstitial
pneumonia was present within the pulmonary parenchyma when examined
histologically. The macrophages comprising the lesion in these cases were large and
lipid-laden and most consistent with a diagnosis of endogenous lipid pneumonia.
Further nonspecific lesions common to all groups included splenic, pulmonary, and
hepatic congestion, and nonspecific lymphoid reactivity in the spleen, lymph nodes,
bronchial-associated lymphoid tissue (BALT), and gut associated lymphoid tissues
(GALT). One control opossum had Besnoitia darlingi protozoa] cysts distributed
throughout the renal parenchyma. Similar cysts were also noted histologically within
the skeletal muscle of one oral recipient, within the lung of one IM recipient, and
within the adrenal gland, skeletal muscle, and the lung of another oral recipient.
Tissue reaction to these cysts ranged from minimal, with little associated
inflammation, to severe, with fibrous encapsulation of necrotic to mineralized cysts or
with large numbers of eosinophils and mononuclear inflammatory cells associated
with variable degrees of granulation in some instances. Sarcocyst spp. cysts were
present in the skeletal muscle of one oral recipient with no noted tissue reaction.
Fecal cultures were negative for the presence of mycobacteria in all animals
prior to inoculation. One oral recipient had M. bovis cultured from feces collected 24

hours after inoculation. A different oral recipient had positive M. bovis cultures in

95

 

feces collected at 31 days PI. No additional positive fecal cultures were obtained from
any animal (Table 3.2).

Tissue cultures were positive in one oral recipient and in three IM recipients
(Table 3.2). All animals with positive cultures were positive in the pooled spleen and
lymph node submission. The oral recipient was also positive for M. bovis in the
pooled small and large intestine submission. Two 1M recipients were positive for M.
bovis in the lung and in pooled liver and kidney in addition to being positive on the
spleen and lymph node submission. Interestingly, the only animal with a combination
of gross and histologic lesions compatible with tuberculosis did not have any tissue
cultures from which mycobacteria could be isolated.

Updated carnivore/ omnivore surveillance activity results since the project’s
inception, and including the two opossums that have cultured positive for M. bovis are
included in Table 3.3. Each of these animals had gross lesions noted in the lungs. A
moderate to severe, multifocal to coalescing, caseogranulomatous pneumonia with
intralesional acid fast bacilli noted on specially-stained tissue sections was present in

each of these two cases.

Discussion

Most of the gross lesions found at necropsy in experimental subjects could not
be attributed to inoculation. Gastrointestinal parasitism, hepatomegaly, and
splenomegaly were present in all groups and therefore did not seem to be related to

mycobacterial disease. One opossum with lung lesions that were suggestive of

96

granulomas grossly was determined to have pulmonary besnoitiosis based on
histologic examination of the tissue. Also noted histologically in some animals was an
endogenous lipid pneumonia. When accumulations of lipid-laden macrophages are
subpleural, they may give a gross appearance of small granulomas of undeterrninable
pathogenesis based on gross inspection alone. This contributed to the array of lesion
descriptions noted in the three animals with grossly visible lung lesions. Only one of
these three animals had histologically confirmed evidence of gross lung lesions being
attributable to tuberculosis. In both cases with myositis noted grossly nearly the
injection site, the infection was directly caused by the injected organisms based on
histologic examination of the tissues, rather than by a foreign-body-type injection
reaction.

Nearly all animals in this study had some degree of hepatic glycogen or lipid
storage detected histologically. Although animals were not fed ad libitum, access to
food was relatively easier and more nutritious than that which they may encounter in
the wild, and physical exercise was limited due to cage restrictions. Although no
animal showed clinical evidence of hepatic disease, and no case was histologically
severe, dietary restrictions for opossums in captivity may need to be considered based
on these findings.

Interesting was the common finding of endogenous lipid pneumonia in these
animals. This process is most typically reported in lab rodents and may be
multifactorial in origin (Dungworth, 1993). There is no apparent explanation for its

presence in these opossums, although a report describing it as being a rather common

97

 

finding in wild opossums exists (Brown, 1988). This report suggests lipid pneumonias
in opossums may be related to pulmonary nematodiasis, but there were no instances in
which pulmonary nematodes were found present on necropsy in this study.

Besnoitia darlingi protozoa were noted histologically in all groups. Typically,
Besnoitia darlingi is not implicated as causing disease in opossums, yet the large
numbers of cysts noted, particularly in some of the treatment group animals, is
suggestive of some degree of pathogenicity in some animals in the study (Davidson
and Nettles 1997). In fact, these protozoa, cestode, and nematode enteric parasites
were present more frequently in inoculated animals than in control animals, suggesting
possible immunosuppressive effects of M. bovis. Stress of capture and captivity alone
may have been partially responsible for contributing to immunosuppression and thus,
potentially, to parasitism in both control and treatment group animals. The Sarcocyst
spp. present in one inoculated opossum was determined to be an incidental finding
based on the limited tissue reaction and on the typical lack of pathogenicity of this
organism in this species (Davidson and Nettles 1997).

At the time of completion of this study, no positive cases of M. bovis
tuberculosis in North American opossums had yet been identified through wildlife
surveillance activities in Michigan. Surveillance data through the end of the year 2000
now includes two positive cases of M. bovis tuberculosis out of 228 animals tested.
This calculates to the one of the lowest prevalence rates (0.9 %) in any of the wild
carnivore/ omnivore species with identified positive cases in surveillance activities to

date (Table 3.3).

98

It was the original intent of the investigators to determine whether the North
American opossum was at all susceptible to infection by M. bovis, as there had been
no reports of the disease in this species. Also of interest was whether this species
shared any similarity in infectivity with its distant marsupial relative, the brushtail
possum (T richosurus vulpecula), after M. bovis challenge. Along with the ferret
(Mustelafuro), the brushtail possum is known to be a potent, non-cervid wildlife
reservoir host for M. bovis in New Zealand (0’ Reilly and Dabom 1995). Both the
laboratory and surveillance data presented here support the hypothesis that the North
American opossum is indeed susceptible to infection, yet apparently to a less dramatic
degree than either of these species.

In our experiment, opossums that were inoculated intramuscularly had more
histologic evidence of lesions consistent with tuberculosis and had larger numbers of
positive tissue cultures as a group than did those receiving oral inoculation. However,
only those orally inoculated had positive fecal cultures at any time throughout the
experiment, suggesting this transmission route as a plausible one in the wild.
Interesting was the fact that two of the intramuscularly inoculated opossums
developed pulmonary lesions consistent with tuberculosis. At least a partial affinity of
the organism for the lung in this species is suggested by this finding. Additionally
intriguing is the fact that the two opossums identified as positive for M. bovis
tuberculosis in Michigan wildlife surveillance also had identifiable lesions limited to
the thoracic cavity. This finding is especially interesting considering most of the

additional camivore and omnivore surveillance cases identified to date have had

99

abdominal cavity lesions (mesenteric lymph nodes in particular), if lesions were
present at all (Bruning-Fann et a1 2001).

Only one animal had met all three diagnostic criteria (gross lesions, histologic
lesions, and positive mycobacterial isolation/ identification) for M. bovis tuberculosis.
This stresses the importance of combining laboratory findings before making a final
determination as to the presence or absence of this disease. Most obvious is the case
of the “missed” histologic evidence of disease when only small sections of tissue may
be available for examination. Yet equally important is the potential for problematic
culture results, particularly when stringent tissue preparations/ digestions are necessary
as is the case in some mycobacterial work performed on animal tissues, where
microorganism recovery can prove quite difficult.

While this species is susceptible to M. bovis, the likelihood of it serving as a
reservoir host under natural conditions may not be as great as is that of the brushtail
possum. The dosage of microorganisms used in this pilot study was much higher than
that used in studies with the brushtail possum, and at similar times post-inoculation,
necropsy of North American opossums revealed far less extensive lesions than those
that have been published for the brushtail possum (Corner and Presidente 1981). In
fact, both experimentally and in the wild, brushtail possums are known to develop
severe, generalized lesions, oftentimes including fistulous tracts that drain externally
from infected lymph nodes, in response to exposure to M. bovis (Comer and
Presidente 1981; Buddle et a1 1994; Pfeffer et a1 1995; Cooke et a1 1995). When the

lesions in either these laboratory inoculated, or in wild opossums that have cultured

100

 

 

 

positive through routine, ongoing carnivore/ omnivore surveillance are compared to
those found naturally in brushtail possums (Cooke et a1 1995), the difference in
resistance to generalized infection after exposure to this organism becomes striking
between species. In our experiment, even those animals inoculated with very high
doses of M. bovis that could arguably be difficult for any animal to encounter in the
wild had limited tissue distributions of lesions that could be definitively diagnosed as

resulting from mycobacterial infection. In the presently identified two surveillance

n or an” «1:: In...

_-‘.._

cases of bovine tuberculosis found in North American opossums in Michigan, similar ‘3;
limitations in both lesion severity and in distribution to the organs of the thoracic
cavity have been noted.

In contrast to the brushtail possum is the badger (Meles meles), which is a
known reservoir host for M. bovis tuberculosis in the United Kingdom and in Ireland
(Little et a1 1982). These animals, although very efficient reservoir hosts, apparently
often shed M. bovis organisms in the wild without any evidence of clinical disease or
debilitation (Gallagher et al 1998). Even in laboratory inoculation studies, the badger
may live several years without evidence of clinical disease that would be typical of
that in the brushtail possum given similar inoculation routes and organism dosages
(Comer and Presidente 1980; Pritchard et a1 1987; Buddle et a1 1994). Soto speculate
that a spectrum of potential reservoir hosts for bovine tuberculosis may exist, some of
which may become severely affected by the infection, and some of which may be able
to carry and shed organisms without apparent clinical disease, seems reasonable.

Whether or not the North American opossum has a place in this spectrum remains to

101

be seen. More likely, this species represents a “spillover host” with relatively high
resistance to infection when compared to some spillover species, and indeed to any
true reservoir host.

Additional studies that further explore the transmission and excretion routes of
M. bovis in this species are, however, warranted. Based on lesion distribution in this
study and in surveillance cases, the North American opossum seems more apt to

potentially spread tuberculosis by the more efficient respiratory route when compared 1"

if: '. a“ a.“

to other spillover species in Michigan. Opossums, both in surveillance and in these
lab-based analyses, have shown a tendency towards respiratory infections, whereas all
other spillover host species identified at this time in Michigan have had enteric
disease. As such, intratracheal inoculation study results may prove useful in providing

a better understanding of the disease process in this species.

 

102

 

Table 3.1: Laboratory results. Histology and culture results. Histologic examination
based on the presence or absence of acid-fast bacilli on Ziehl-Neelsen acid-fast stained
tissue sections. Culture results are those animals with positive tissue cultures for M.

bovis .

Table 3.2: Summary of cases with evidence of M. bovis tuberculosis. Specific
descriptions of those animals positive for M. bovis on necropsy.

A J = juvenille; A = adult; 8 F = female; M = male;

C Mu = muscle, Lu = lung, I = pooled small and large intestine, Ln = pooled spleen and

lymph nodes, Liv = pooled liver and kidney.

103

Table 3.1. Laboratory results.

 

 

Inoculation Number of Acid-Fast Mycobacterial

Group Animals In Organisms Noted Culture Positives
Group Histologically

Control 4 0/ 4 0/ 4

Oral 4 0/ 4 1/ 4

Intramuscular 4 3/ 4 3/ 4

 

Table 3.2. Summary of cases with evidence of M. bovis tuberculosis.

 

AgeA Sex“ Inoculation Gross Positive Positive on Positive on

 

Route/ Lesions/ on Fecal Culture/
Days PI OrgansC Histology/ Culture/ OrgansC
Sacrificed OrgansC Days PI
Cultured
Positive
J M Oral/ 31 No No Yes/ 31 days Yes/ 1, Ln
days
J F M 31 days Yes/ Yes/ Mu No Yes/ Lu, Liv, Ln
Mu, Lu
J M IM/ 31 days No Yes/ Mu, No Yes/ Lu, Liv, Ln
Lu
J F IM/ 61 days Yes/ Yes/ Mu, No No
Mu, L Lu
A F M 61 days No - No No Yes/ Ln
J M ' Oral/ 61 No No Yes/ 1 day No
days

 

104

%m

 

Table 3.3: Carnivore and omnivore surveillance results for M. bovis in Michigan
(1996-2000).

2’Number of this species surveyed through the end of the year 2000;.

b Total number of culture-positives for M. bovis in this group;

C Number of positives in this species divided by total number surveyed multiplied by

100.

105

Table 3.3. Carnivore and omnivore surveillance results for M. bovis in Michigan

 

 

 

(1996-2000).

M Scientific Name 1933]: Numberb Prevalencec
Badger T axidea taxus 25 0 0.0
Bear, Ursus americanus 163 4 2.5
Black

Bobcat F elis rufus 40 2 5.0
Coyote Canis latrans 249 11 4.4
Fox, Gray Urocyon 4 0 0.0
Fox, Red Vulpes vulpes l6 2 12.5
Mink Mustela vison 2 0 0.0
Opossum Didelphis virginiana 228 2 0.9
Porcupine Erethizon dorsatum l 0 0.0
Raccoon Procyon lotor 194 2 1 .0
Skunk Mephistis mephistis 12 0 0.0
Weasel Mustela sp. 1 0 0.0
TOTALS 935 23 2.5

 

106

 

Figure 3.1: Section of skeletal muscle from an opossum inoculated intramuscularly
with M. bovis. Severe caseogranulomatous myositis with central mineralization is

featured. Hematoxylin & Eosin (H & E); 4X.

Figure 3.2: Section of lung from an opossum intramuscularly inoculated M. bovis.
Note large numbers of macrophages filling alveoli, coalescing as early granulomas. H

& E; 40X.

Figure 3.3: Section of lung from an opossum intramuscularly inoculated with M.
bovis. Acid-fast bacillus within the cytoplasm of one of several large macrophages in

an alveolus (arrow). Ziehl-Neelsen acid-fast; 100X.

107

gesin

Figure 3.1

 

Figure 3.2

     
 
  

"”\ ‘ -
@' 1‘44 .3

t.
' :61» ‘-
z‘tf'tr'i}? '1' ‘

108

 

References

Brown CC. 1988. Endogenous lipid pneumonia in opossums from Louisiana. J.
Wild. Dis. 24: 214-219.

Bruning-Fann CS, Scmitt SM, Fitzgerald SD, Fierke J S, Friedrich PD, Kaneene JB,

Clarke KA, Butler KL, Payeur JB, Whipple DL, Cooley TM, Miller JM, Muzzo DP.
2001. Bovine tuberculosis in free-ranging camivores from Michigan. J. Wild. Dis.

37: 58-64.

Buddle BM, Aldwell FE, Pfeffer A, DeLisle GW. 1994. Experimental
Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula):
pathology, haemotology and lymphocyte stimulation responses. Vet. Micro. 38: 241-
254.

Buddle BM, Skinner MA, Chambers MA. 2000. Immunological approaches to the
control of tuberculosis in wildlife reservoirs. Vet. Immun. Immunopath. 74: 1-16.

Butler WR, Jost KC Jr., Kilburn JO. 1991. Identification of mycobacteria by high
performance liquid chromatography. J. Clin. Micro. 29: 2468-2472.

Cooke MM, Jackson R, Coleman JD, Alley MR. 1995. Naturally occurring
tuberculosis caused by Mycobacterium bovis in brushtail possums (Trichosurus
vulpecula): II. Pathology. N. Z. Vet. J. 43: 315-321.

Comer LA and Presidente PJ A. 1980. Mycobacterium bovis infection in the brush-
tailed possmn (Trichosurus vulpecula): 1. Preliminary observations on experimental
infection. Vet. Micro. 5: 309-321.

Davidson WR and Nettles VF. 1997. Opossums. In: Field manual of wildlife
diseases in the southeastern United States. Second Edition. Southeastern Cooperative
Wildlife Disease Study. Athens, GA, pp. 211-219.

Dungworth DL. 1993. The respiratory system: special forms of
pneumonia. In: Pathology of domestic animals, volume 2, 4th edition. KFV Jubb, PC
Kennedy, and N Palmer, eds. North Academic Press, Inc., San Diego. pp. 610-613.

Gallagher J, Moines R, Gavier-Widen M, Rule B. 1998. Role of infected, non-

diseased badgers in the pathogenesis of tuberculosis in the badger. Vet. Rec. 142:
710-714.

109

Kent PT, Kubica GP. 1985. Public health mycobacteriology. A guide for the level III
laboratory. US. Department of Health and Human Services. Atlanta,Georgia.

Little TWA, Maylor PF, Wilesmith JW. 1982. Laboratory study of Mycobacterium
bovis infection in badgers and calves. Vet. Rec. 111: 0-557.

Mahmood KH, Rook GA, Stanford JL, Stuart FA, Pritchard DG. 1987. The
immunological consequences of challenge with bovine tubercle bacilli in badgers
(Meles meles). Epi. Infec. 98: 155-163.

Nolte F S, and Metchock B. 1995. Mycobacterium. In: Manual of Clinical
Microbiology. PR Murray, EJ Baron, MA Pfaller, F C Tenover, RH Yolken, eds.
American Society for Microbiology Press, Washington, DC, pp. 400-437.

O’Reilly LM, and Dabom CJ. 1995. The epidemiology of Mycobacterium bovis
infections in animals and man: a review. Tuber. Lung Dis. 76 Suppl. 1: 1-46.

Pritchard DG, Stuart FA, Brewer JL, Mahmood KH. 1987. Experimental infection of
badgers (Meles meles) with Mycobacteritnn bovis. Epi. Infec. 98: 145-154.

Prophet EB. 1992. Laboratory methods in histotechnology. B Mills, JB Anington,
LH Sobin, eds. Armed Forces Institute of Pathology, American Registry of
Pathology, Washington, DC, 279 pp.

Reisner BS, Gatson AM, Woods GL. 1994. Use of gen-probe accuprobes to identify
Mycobacterium avium complex, Mycobacterium tuberculosis complex,
Mycobacterium kansasii and Mycobacterium gordonae directly from Bactec TB broth
cultures. J. Clin. Micro. 32: 2995-2998.

Schmitt SM, Fitzgerald SD, Cooley TM, Bruning-Fann CS, Sullivan L, Berry DE,

Carlson T, Minnis RB, Payeur JB, Sikarskie J. 1997. Bovine tuberculosis in free-
ranging white-tailed deer from Michigan. J. Wildl. Dis. 33: 749-758.

110

 

 

Chapter 4

Antimicrobial susceptibility patterns of human and wildlife

Mycobacterium bovis isolates from Michigan

111

Abstract

In Michigan, a continuing endemia of Mycobacterium bovis in wildlife has
raised much concern for its potential impact on human health, particularly since the
disease has been found present in domestic beef and dairy cattle and captive cervid
herds in the state. No cases of human tuberculosis caused by the wildlife M. bovis
strain have been identified to date, but preparedness for such an event has been
warranted. As part of this preparedness, a better understanding of the strain in a

it
question, particularly that of its potential resistance to common antituberculous drugs g}
is desirable. Cultures of this strain, obtained from positive cases found through both
wild and domestic animal surveillance in Michigan, were tested by both agar plate and
Bactec broth methods for resistance to the most commonly used antimycobacterial
drugs. These results were compared to those from five human M. bovis tuberculosis
cases identified in Michigan to date. No similarities between human and animal
restriction fragment length polymorphism (RF LP) patterns were noted, and no cases
were multidrug resistant. Some colony growth was present in the presence of
isoniazid in several animal cases, and in the presence of cycloserine in one case.
Although these cases are not considered as being resistant to these drugs, the grth
patterns may be important in gaining a better understanding of this particular M. bovis

strain in Michigan.

 

112

Introduction

Tuberculosis (both Mycobacterium tuberculosis and Mycobacterium bovis)
infections in the world’s population have increased dramatically in the last decade,
necessitating declaration of the disease as a global emergency by the World Health
Organization (Kamholz 1996; Lee et a1 1995; Yew and Chau 1995). Risk factors
including immigration, increased spread of the human immunodeficiency virus and
other immunosuppressive diseases and treatments, poverty, and technological
developments allowing increased international mobilization and travel have
contributed to the spread of this disease worldwide, making tuberculosis the infectious
process most commonly associated with death in the world (Bloom and Small 1998;
Bradford and Daley 1998; Cohen 1997; Lee et a1 1995; Yew and Chau 1995).
Additionally problematic is the worldwide phenomenon of emergence of drug
resistant strains of microorganisms, including mycobacteria responsible for
tuberculosis infections (Castiglia and Smego 1997; Cohen 1997). The threat of
continuing and, even increasing , tuberculosis case fatalities has become a great
concern in the medical community largely because of the increasing number of multi-
drug-resistant (MDR) strains (Cohen 1997; Yew and Chau 1995). MDR tuberculosis
is defined as that which is resistant to both isoniazid (INH) and rifampin, with or
without resistance to additional antimicrobials (Lee et al 1995). Although MDR M.
tuberculosis is of primary concern as a human pathogen, human and wildlife M. bovis
cases have been of increasing concern as well, both MDR strains and susceptible

strains (Brett 1991).

113

 

Although relatively rare in humans, MDR M. bovis is of increasing concern due
to this overall rise in serious mycobacterial illness. In Michigan specifically,
awareness of M. bovis infections has been heightened dramatically in recent years due
to recognition of an enzootic strain of the organism in Michigan wildlife (Bruning-
Fann et al 1998; Schmitt et a1 1997). Transmission of M. bovis to humans fi'om
wildlife is well documented in locales with endemically-infected feral animal
populations (Brett 1991). In order to best eradicate M. bovis from endemically-
infected Michigan wildlife, gaining understanding of potential drug resistances in the
endemic strain of this microorganism is an important step in assuring safety for both
humans and animal species in the future.

Specifically, the objective of this project was to define drug susceptibility
patterns for isolates of Mycobacterium bovis obtained from Michigan human and
wildlife cultures. The antituberculous antimicrobials isoniazid (INH), streptomycin
(SM), rifampin (RA), ethambutol (EMB), ethionamide (EA), kanamycin (K),
ciprofloxacin (CIP), cycloserine (CS), capreomycin (CM), and pyrazinamide (PZA)
were tested by either BactecR methodology, proportion agar plate methodology, or
both.

Antimicrobial susceptibility profiles were compiled using mycobacterial
isolation and drug sensitivity testing procedures previously described (Kamholz 1996;
Siddiqi 1996) and currently employed by the Tuberculosis Laboratory at the Michigan

Department Of Community Health (MDCH).

114

Cultures from five human M. bovis cases, six carnivores (three coyotes, two
raccoons, and one black bear), thirty cervid (white-tailed deer), and three bovine cases,
all isolated in Michigan, were used for the study. Isolates were purified and were used
as inocula for both Bactec broth media and Middlebrook 7H10 Agar plates
impregnated with multiple antituberculous antibiotic discs for proportion plate
susceptibility testing (Kent and Kubica 1985).

Materials and Methods

Growth from all isolates was adjusted to a 0.5 MacFarland suspension to prepare
each for Bactec susceptibility testing (Nolte and Metchock 1995). The susceptible
control for both Bactec (non-PZA) and proportion plate tests was TMC 102
Mycobacterium tuberculosis. The resistant control used for the Bactec PZA test was
TMC 1011 M. bovis. The following antimicrobials: streptomycin, isoniazid, rifampin,
and ethambutol were added to Bactec lZB vials (Middlebrook 7H12 meditun, Bactec
460 TB system, Becton Dickinson and Co., Sparks, MD) resulting in final
concentrations of 2.0, 0.1, 2.0, 2.5 ug/ml respectively. The 0.5 MacFarland
suspensions were then inoculated into each of the drug containing vials (Siddiqi 1996).
A 1:100 dilution of each 0.5 MacFarland suspension was also inoculated into a
separate Bactec 128 vial to serve as a control vial for each test drug test panel. Bactec
12B vials were read daily for a growth index (G1) on the Bactec 460 TB system for a
maximum of 14 days or until the control vial reached a GI of greater than or equal to

30. Susceptible isolates are those with a AGI (the difference between the most recent

115

 

GI value and the GI from the previous day) less than the AGI of the control vial.
Resistant isolates are those with a AGI greater than the AGI of the control.

The Bactec method for PZA susceptibility testing was also implemented for
each isolate. This required a Bactec 12B vial inoculated from the original 0.5
MacFarland with a GI of 300-499 to be inoculated into two separate, modified
Middlebrook 7H12 medium (PZA test medium) vials with the pH adjusted to 6.0.
PZA drug was added to one of these two vials to result in a final concentration of
100ug/ml. The other vial was maintained without PZA to serve as the test control vial.
Results were interpreted when the GI of the control vial reached 200 or more.

Interpretations of Bactec PZA results were calculated as a % susceptibility using
the following formula:

GI DrugVial x 100 = % Susceptibility
GI Control Vial

 

Susceptible isolates were those resulting in percentages less than 9%, resistant isolates
were those resulting in percentages greater than 11%, "borderline" isolates were those
resulting in percentages between 9-1 1%.

Proportion plate susceptibility testing (1% method) was performed on each
culture isolate as well. Middlebrook 7H10 agar plates (Becton Dickinson and Co.,
Sparks, MD) were prepared using drug-impregnated discs (Becton Dickinson and Co.,
Sparks, MD) resulting in the following final drug concentrations: INH (0.2 and 1.0 pg/
ml); SM (2.0 and 10.0 ug/ ml); RA (1.0 ug/ ml); EMB (5.0 ug/ ml); EA (5.0 ug/ ml);
K (6 ug/ ml); and CIP (2.0 ug/ ml). Powdered CS, CM, and thiophen-2-carboxylic

acid hydrazide (TCH) suspensions were used to prepare media plates at concentrations

116

 

 

of 30.0, 10.0, and 5.0 ug/ml, respectively. TCH testing was included as a part of the
routine mycobacterial species determining protocol. Isolates were subcultured into
Middlebrook 7H9 broth (Becton Dickinson and Co., Sparks, MD), and incubated for 2
to 5 days each. These broths were inoculated onto 7H10 Agar plates in two dilutions
determined on a case-by-case basis for each culture, such that a colony count of 100 to
300 results from one of these dilutions. Dilutions were determined based on
comparisons to MacFarland standards and were made with phosphate buffered saline
(Nolte And Metchock 1995). Plates were examined weekly for three weeks, using a
lighted magnification lens. Growth was recorded as 4+ (>500 colonies), 3+ (200-500
colonies), 2+ (100-200 colonies), or 1+ (50-100 colonies). Quadrants with less than
50 colonies present had the actual colony number counted and recorded. Inocula with
no dilution showing 100-300 colonies in one of the quadrants were retested. The
dilution that showed colony (desired) growth nearest to 200 was then used to
determine resistance patterns. A percent resistance was calculated as follows:

Number of colonies on drug quadrant of plate X 100 = % Resistance
Number of colonies on control quadrant of plate

 

A culture population with a percent of resistance greater than or equal to 1% was
considered resistant to the tested drug. Cultures with less than 1% resistance were
considered susceptible.

Results

All isolates were found to be PZA resistant, as is expected of M. bovis. Unlike
other members of the M. tuberculosis complex (M. tuberculosis, M. microti, and M.

africanum), M. bovis lacks the enzyme pyrazinarnidase which is necessary to activate

117

this drug, rendering all strains of the organism resistant to its activity. No isolates
were found to be resistant to any of the other antimicrobials tested by Bactec
methodology. The results of isolates showing growth on the susceptibility test agar
plates in this study are provided in Tables 4.1 (animal isolates) and 4.2 (human
isolates). No animal isolates were, by definition, resistant to any antimicrobials on
agar plate protocols either. Several isolates did have slight growth noted, particularly
in the presence of INH, but only when reading the inoculation dilution that generated
4+ control growth (Table 4.1). No growth was noted from these same isolates at the
inoculation dilution that generated only 2+ control growth. Human isolates varied
widely in growth patterns on agar plate testing (Table 4.2). Two human isolates were
found to be single-drug resistant, one to EA, and one to CM. No human isolate tested
was multidrug resistant.

Restriction fragment length polymorphism (RF LP) analysis was canied out on
all isolates in this study using methods previously described (Chaves et al 1996; van
Embden et al 1993). These “genetic fingerprints” were identical for all animal
isolates, but varied widely both between those human isolates tested and between
human and animal isolates.

Discussion

This is the first published report describing antimicrobial resistance patterns in
Michigan’s endemic strain of M. bovis. Except for PZA, no resistance was identified
through conventional testing methods. The lack of resistance is possibly best

attributed to the lack of “treatment pressure” that is often associated with

118

development of resistance in mycobacteria. Since positive animal cases are
slaughtered rather than treated, the potential for selection of resistant mutant strains of
the organism is eliminated. Although drug resistance does not appear to be a problem
currently, some speculation can be given as to whether development of resistance in
the future may be of concern based on the limited observed growth.

At-risk human populations in this endemic zoonosis include hunters, farmers,
butchers, taxidennists, laboratory personnel, veterinarians, and veterinary professional
students and their families. To date, no human cases of M. bovis have been caused by
the endemic strain, as evidenced by both susceptibility and genetic typing (RF LP)
assays. Only one additional case of M. bovis has been identified in Michigan since the
time of conclusion of this study, and this case was found to be unrelated to all other
human and animal cases as well. The human cases identified in the state thus far have
been linked to exposure on farms in foreign countries, recrudescence of childhood
infections established prior to eradication in Michigan, or from cancer therapy-induced
infection by the bacillus Calrnette-Guerin (BCG) strain of M. bovis.

Widespread education of hunters and farmers through outreach programs in
Michigan has seemingly been of great benefit in controlling the spread of bovine
tuberculosis in the state. These programs have included literature distribution, well-
maintained web pages, lecture series, public radio broadcast updates, and well-
advertised meetings, both local and statewide, with open invitations to concerned
individuals. Yet certainly, careful monitoring of any new cases for potential

relatedness to the endemic strain is of utmost necessity.

119

Colony grth in the presence of INH in the wildlife strain was minimal where
observed. The clinical relevance of the growth, which was also typically noted at the
more concentrated inoculum dilutions, is therefore insignificant from a therapeutic
standpoint. Some below-standard growth is typical in tuberculosis susceptibility tests,
so it is not the intent of this report to over-interpret the noted grth as definitive
resistance. In fact, resistance in the endemic strain should not be problematic in
Michigan at any time in the near future. Yet periodic susceptibility testing of the
wildlife strain of M. bovis would nonetheless appear to be warranted. Such testing
would be useful not only to assess potential development of resistance over time
which exists in any strain of microorganism, but also that which may be already
suggested by the findings in this report. And should any human case result from the
described contact with infected animals or products, having a pre-existing
understanding of which antimicrobials may be most efficacious for immediate
administration of treatment rather than having to speculate and wait for final

laboratory results would most certainly be beneficial.

120

 

 

Table 4.1: Wildlife-source M. bovis cases/ isolates from Michigan showing growth on
proportion plate drug susceptibility testing.
' Case numbers assigned by Michigan Department of Community Health.
b INH = isoniazid; CS = cycloserine.
° Growth was recorded as 4+ (>500 colonies), 3+ (200-500 colonies),
2+ (100-200 colonies), or 1+ (50-100 colonies).

d All readings at —1 inoculum dilution log.

Table 4.2: Human—source M. bovis cases/ isolates from Michigan showing growth on
proportion plate drug susceptibility testing. Bold italicized samples (cases 1201 and
252) are those determined resistant.

' Case numbers assigned by Michigan Department of Community Health.

b CM = capreomycin; EA = Ethionarnide; INH = isoniazid; EMB = ethambutol.

c Growth was recorded as 4+ (>500 colonies), 3+ (200-500 colonies),

2+ (100-200 colonies), or 1+ (50-100 colonies).

121

Table 4.1: Wildlife-source M. bovis cases/ isolates from Michigan showing growth
on proportion plate drug susceptibility testing.

 

Sample Species and Drug With Colony Resistance Score " " Growth of Control

 

Case Number ' Growth Identified Or Number of At Same Dilution
on 7H10 Agar b Colonies Identified
Deer 5352 INH 0.2 ug/ ml < 1+ (4 colonies) 4+
Deer 2673 INH 0.2 pg/ ml < 1+ (3 colonies) 4+
Deer 7278 INH 0.2 ug/ ml < 1+ (3 colonies) 4+
INH 1.0 ug/ ml < 1+ (4 colonies) 4+
Deer 5640 INH 0.2 pg/ ml < 1+ (6 colonies) 4+
Coyote 1251 CS 1+ 4+
Coyote 1865 INH 0.2 ug/ ml < 1+ (5 00101435) 4+
INH 1.0 ug/ ml < 1+ (6 colonies) 4+
Raccoon 1966 INH 0.2 ug/ ml < 1+ (3 colonies) 4+
Raccoon 1779 INH 0.2 ug/ ml < 1+ (38 001011165) 4+

INH 1.0 ug/ ml < 1+ (31 colonies) 4+

 

Table 4.2: Human-source M. bovis cases/ isolates from Michigan showing growth
on proportion plate drug susceptibility testing.

 

 

Sample Drug With Resistance Dilution Log of Growth of
Number ' Colony Growth Score ° Or Inoculum Control At
Identified on Number of Same Dilution
7H10 Agar” Colonies
Identified
1201 CM 2+ -1 3+
252 EA 3+ -2 4+
EA 1+ -4 2+
INH 0.2 ug/ ml < 1+ (25 -2 4+
colonies)
671 EMB 1+ -1 4+
INH 0.2 ug/ ml < 1+ (9 -1 4+
colonies)
INH 1.0ug/m1 <1+(4 -1 4+
colonies)
1378 INH 0.2 ug/ ml < 1+ (7 -1 4+
colonies)
INH 1.0 rig/m1 <1+(3 -1 4+
colonies)

 

122

References

Bloom BR and Small PM. 1998. Editorials: The evolving relation between humans
and Mycobacterium tuberculosis. N. Eng. J. Med. 338: 677-678.

Bradford W2 and Daley CL. 1998. Multiple drug-resistant tuberculosis. Infect. Dis.
Clin. N. Am. 12: 157-172.

Brett JL. 1991. Incidence of human tuberculosis Caused by Mycobacterium bovis. N.
Z. Med. J. 104: 13-14.

Bruning-Fann CS, Schmitt SM, Fitzgerald SD, Payeur JB, Whipple DL, Cooley TM,
Friedrich P. 1998. Mycobacterium bovis in coyotes from Michigan. J .Wild. Dis. 34:
632-636.

Castiglia M and Smego RA Jr. 1997. The global problem of antimicrobial resistance.
J. Amer. Phann. Assoc. (Wash) NS37: 383-387.

Chaves F, Yang Z, El Hajj H, Alonso M, Burman WJ, Eisenach KD, Dronda F, Bates
JH, Cave MD. 1996. Usefirllness of the secondary probe pTBN12 in DNA
fingerprinting of Mycobacterium tuberculosis. J. Clin. Microbiol. 34: 1118-1123.

Cohen ML. 1997. Epidemiological factors influencing the emergence of
antimicrobial resistance. Ciba Found. Symp. 207: 223-231.

Kamholz SL. 1996. Resurgence of tuberculosis. J. Assoc. Acad. Minor Phys. 7: 83-
86.

Kent PT and Kubica GP. 1985. Public Health Mycobacteriology/ A Guide for the
Level III Laboratory. US. Department of Health and Human Services. Public Health
Service (CDC), Atlanta.

Lee SK, Tan KK, Chew SK, Snodgrass I. 1995. Multidrug-resistant tuberculosis.
Ann. Acad. Med. Singapore 24: 442-446.

Rao GG. 1998. Risk factors for the spread of antibiotic-resistant bacteria. Drugs 55:
323-330.

Schmitt SM, Fitzgerald SD, Cooley TM, Bruning-Fann CS, Sullivan L, Berry DE,
Carlson T, Minnis RB, Payeur JB, Sikarskie J. 1997. Bovine tuberculosis in free-
ranging white-tailed deer from Michigan. J. Wildl. Dis. 33: 749-758.

Siddiqi SH. 1996. Bactec 460 TB System Product and Procedure Manual
(Publication No. MA-0029). Becton Dickinson and Company, Sparks, Maryland.

123

van Embden JDA, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B,
Hennans P, Martin C, McAdam R, Shinnick TM, et al. 1993. Strain identification of
Mycobacterium tuberculosis by DNA fingerprinting. J. Clin. Microbiol. 31: 406-409.

Yew WW and Chau CH. 1995. Drug-resistant tuberculosis in the 19905. Eur. Resp.
J. 8: 1184-1192.

 

124

 

Chapter 5

A Novel Cytologic Evaluation Technique for the

Detection of Mycobacteria

125

Abstract

Mycobacterial culture and identification is currently the “gold standar ”
technique for the diagnosis of mycobacteriosis in animals. Unfortunately, confirming
infection using this technique can be time-consuming and cost-prohibitive in the
veterinary setting, particularly when large numbers of samples require testing, as in a
herd situation. The objective of this study was to compare results from a novel,
cytology-based procedure for the diagnosis of tuberculosis in deer to culture results
from these same animals. In order to test the new technique, cell-rich samples were
collected from nineteen white-tailed deer (Odocoileus virginianus) inoculated by
intratonsilar instillation of 2 x 108 colony forming units (CFU) Mycobacterium bovis.
These cell samples were processed using both this new cytology method and by
routine mycobacterial culture. The new test was found to be both less sensitive and
less specific than is the culturing of samples for M. bovis tuberculosis, but was also
found to be more time and cost-efficient. Moreover, this test may have substantial
value in those species in which skin testing is an unreliable method for the antemortem
diagnosis of tuberculosis.

Introduction

The diagnosis of mycobacterial disease has received much attention clinically in

recent years, particularly due to the resurgence of tuberculosis. Tuberculosis is the

only disease to be declared a global health emergency by the World Health

Organization. Failures in both detection of the disease and in treatment of diagnosed

126

cases have been cited as contributing causes to this worldwide epidemic (Maher and
Raviglione 1999).

Concerns over increased numbers of both Mycobacterium tuberculosis and
Mycobacterium bovis tuberculosis cases in humans have been raised recently,
although in industrialized countries, the prevalence of M. bovis tuberculosis is
typically less than one percent of all diagnosed tuberculosis cases (Wilkins et al 1986).
Many human M. bovis cases diagnosed in these countries in recent years have been
found to be reactivated infections established prior to eradication programs in cattle
and universally accepted milk pasteurization practices. Nonetheless, the disease
remains a significant threat in third-world nations, along with that caused by M.
tuberculosis. This increased prevalence in poorer countries is largely due to less
adequate health care in both humans and animals, allowing the spread of this zoonotic
disease to occur and oftentimes to go undetected.

M. bovis tuberculosis has recently regained much attention in the state of
Michigan (Schmitt et al 1997). In Michigan, an endemic strain of M. bovis
tuberculosis established in free-ranging white-tailed deer has become a major threat to
livestock producers and has resulted in the loss of the tuberculosis-free status of the
state. The need for efficient and reliable diagnostic testing for tuberculosis has been
highlighted in the face of this endemic situation.

Currently, the gold standard diagnostic test for tuberculosis remains
mycobacterial culture (isolation and identification), which, although very sensitive and

specific, often takes weeks to complete. Histopathologic examination of acid-fast

127

stained tissue sections can be a very specific diagnostic tool, but depends on gross
suspicion or actual detection of lesions and the number of bacilli associated with the
section of tissue examined. Without histologic identification of actual bacilli, a
diagnosis of tuberculosis cannot be confirmed on light microscopy alone and culture
results are still necessary. Perhaps most importantly, in the veterinary world, both of
these diagnostic procedures rely primarily on the collection of tissues or fluids during
the post-mortem examination. The gathering of tissues by biopsy in order to diagnose
or to treat the tuberculosis patient is a technique that is applied much less frequently in
the veterinary clinical setting than in that of human medicine. As such, very few
attempts at novel ante-mortem diagnostic techniques for tuberculosis in animals have
been made in recent years, and those that do exist are predominantly serologic assays.
The widely accepted tuberculin (purified protein derivative, PPD) skin test
varies in reported sensitivities and specificities, but still remains one of the best
antemortem detection methods for the disease. Yet it is an accepted fact that the
slaughter of healthy animals may be the result of false positives readings. And equally
as important are the false negative readings that may overestimate sensitivity estimates
of this test, and allow unhealthy cattle to remain sources of infection for other animals.
Reported specificity and sensitivity values for tuberculin testing vary widely according
to a review of the literature, from approximately 72% to over 99% in cattle, with an
even wider range reported for deer (Pahner et al 2000; O’Reilly 1995). Results are

often based on the type of skin test applied (single cervical, comparative cervical, or

128

caudal fold), whether this result is used in series with other diagnostic techniques, and
on the geographic location of the cattle herd (O’Reilly 1995).

Lymphocyte transformation assays performed on blood collected from suspect
cases, which measure the immune response to mycobacterial proteins have been of
some use, but can be difficult to standardize, particularly when application across
species lines is attempted. The blood tuberculosis test (BTB) test, a combination of
the lymphocyte transfonnation test and an ELISA test run on blood of suspect cases,
seems to offer higher sensitivities and specificities. However, this assay may produce
variable results based on the species being tested, the antigens used in the assay, and
the stage of disease or immune status of the patient (Pahner et a1, 2000). Also being
developed rapidly are gamma interferon (IFN-y) measurement assays, which show
promise in application to both cattle and cervid tuberculosis diagnosis. But at the
present time, any immune response measurement test is best used in conjunction with
skin tuberculin testing, based on the reasons listed above (Thoen et a1 1995).

Gaining interest in the diagnosis of tuberculosis are those molecular techniques
which incorporate genome amplification of the organism coupled with either
chemiluminescent or colorimetric probes specific for the mycobacterial species being
investigated. These tests have shown great promise in their application in human
medicine, and are quite rapid, highly specific and reasonably sensitive when used as
intended (Zheng and Roberts 1999). However, due to the fact that they are best
applied on smear-positive human respiratory specimens (tracheal washes, etc.), their

application in the veterinary field may be limited. Currently these tests are also much

129

more costly than is routine mycobacterial culture and identification, which would be
somewhat prohibitive in the screening of large numbers of samples in a herd situation
(Thoen et a1, 1995).

One simple and relatively inexpensive technique which is rarely used in
diagnosing tuberculosis in animals is cytology. Cytologic evaluations of proposed
shedding sites have not been investigated prior to this report. Since mycobacteria are
intracellular organisms, if present in adequate numbers in cells near shedding sites,
they should be detectable within cells harvested from these sites. It was the purpose of
this study to investigate whether such detection was possible. Moreover, since
knowledge on transmission routes of tuberculosis in white-tailed deer is limited, this
study sought to better define these potential shedding routes while investigating the
potential applications of a novel cytologic mycobacterial preparation method
previously used only in human medicine, the ThinPrep 2000 automated cytology
equipment (Cytyc Corp., Boxborough, MA). This technology is currently applied
primarily in the diagnosis of malignancy in gynecologic specimens (Pap-smears) in
the human clinical setting. Reports of its use in clinical veterinary medicine, or for
that matter for the diagnosis of infectious disease in either humans or animals, do not
exist. This report describes the application of the device in preparing cytology

samples used to detect M. bovis infection in white-tailed deer.

130

Materials and Methods
Deer subjects

Nineteen six month-old, white-tailed deer (Odocoileus virginianus) were
experimentally inoculated with Mycobacterium bovis as part of a high-dose, short term
inoculation study at the National Animal Disease Center (NADC) in Ames, Iowa.
These eight castrated males and 11 females were experimentally inoculated by
intratonsilar instillation of 2 x 108 colony forming units (CFU) of M bovis as
previously described (Palmer et al 1999). Deer were housed inside a biosecurity level
3 building with directional airflow such that air from the animal pens was pulled
towards a central corridor and passed through high efficiency particulate air (I-IEPA)
filters before exiting the building. Airflow velocity was controlled to provide 10.4 air
changes/minute in the animal pens. Deer in each pen had access to a circulating
watering device and were fed a pelleted feed (deer and elk complete feed 55P3, Purina
Mills, St. Louis, MO) and alfalfa hay. Pens were cleaned once daily, one at a time, by
transferring deer to a holding pen with thorough washing of the floor and lower walls
of the empty pen with a high—pressure hose. During cleaning, deer had contact with
penmates only and not with deer from other pens.

Between days 21 and 63 after inoculation, 3 deer were euthanized due to injuries
acquired during handling. Four deer were euthanized between 63 and 90 days after
inoculation due to poor condition from advanced tuberculosis, and another 3 deer were
euthanized due to injuries from handling. Between 90 and 113 days after inoculation,

1 deer was euthanized due to advanced tuberculosis and another due to trauma related

131

injuries. One hundred twenty days after inoculation all seven remaining
experimentally inoculated animals were euthanized. Consequently, as the study
progressed, the number of animals available for sampling decreased (Table 5.1).

For inoculation and sampling, deer were anesthetized with a combination of
xylazine (Mobay Corporation, Shawnee, KS) (2 mg/kg of body weight) and ketamine
(Fort Dodge Laboratories, Fort Dodge, IA) (6 mg/kg) injected intramuscularly. The
effects of xylazine were reversed with tolazoline (Lloyd Laboratories, Shanandoah,
IA) (4 mg/kg) injected IV.

Mycobacterial Strain

The strain of M.bovis used was strain 1315, originally isolated from a free-
ranging tuberculous white-tailed deer killed by a hunter in Alpena county, Michigan in
1994. The isolate was incubated at 370 C for 6 weeks on Middlebrook 7H9 liquid
media with 10% oleic albumin dextrose citrate enrichment (Bacto Middlebrook
OADC Enrichment, DIFCO Laboratories, Detroit, MI). After incubation, the bacteria
were harvested by centrifugation, and washed twice with 0.01M phosphate buffered
saline (PBS) solution, pH 7.4. After resuspension in PBS solution, serial 10-fold
dilutions were inoculated on Middlebrook 7H10 agar slants supplemented with OADC
to determine the number of CFU. Inoculum was then frozen at -80 C for future use.
Specimen Handling

Nasal and oral swabs were collected for cytologic evaluation and bacteriologic
culture on days 21, 63, 90 and 113 after inoculation. Swabs of the tonsilar crypts were

collected 21 days after inoculation and at the time of necropsy. Swabs for cytologic

132

evaluation and bacteriologic culture were collected using a sterile 18 cm cytology
brush (Puritan Medical Products, Guilford, ME). For bacteriologic culture, swabs
were rinsed thoroughly in 1.0 ml PBS. One half of the sample (0.5 ml) was added to
0.2% benzalkonium chloride solution (Zephiran chloride, concentrate 17%, Sterling
Drug Inc., New York, NY) and let sit at room temperature for 15 minutes. After
decontamination with benzalkonium chloride, samples were centrifuged for 20 min. at
2000 rpm and the supernatant decanted. To the sediment was added 0.5 ml of Bacto
egg yolk enrichment 50% (DIFCO Laboratories, Detroit, MI). Samples of the
sediment-egg yolk combination were inoculated onto separate agar slants containing
Stonebrink’s, Harrold’s egg yolk, Middlebrook 7H10, or Middlebrook 7Hll media.
Inoculated agar slants were incubated at 37° C for 8 weeks.

Cytology Preparation and Scoring

Tonsil swabs could not be obtained for either culture or ThinPrep 2000 sampling
from any animals at 63 days PI (Tables 5.2 and 5.3). Only two of nine available
animals could be sampled for cytologic evaluation at 90 days.

Samples were obtained from the NADC research deer described. Cytology
brushes were placed in 20 mL Preseeryt® solution (Cytyc Corp., Boxborough, MA)
contained in a 2 ounce plastic vial and were shipped to the Animal Health Diagnostic
Laboratory (AHDL), Michigan State University (MSU) for preparation. Specimens
were processed as recommended for mucoid samples according to the ThinPrep 2000
Operator’s Manual (Cytyc Corp., Boxborough, MA), in a method previously described

(Wang et a1 1996). Briefly, this procedure involves vortexing the collected brush

133

sample, running the sample on the ThinPrep 2000 machine, fixing the slides in 95%
ethanol for ten minutes or longer, and staining the slides with a modification of the
Ziehl-Neelsen technique for the identification of acid-fast bacteria in cytology
preparations (Prophet 1992). The modified staining procedure uses New Fuchsin blue
to replace carbol fuchsin, and acetic alcohol is used for differentiation. While in the
ThinPrep 2000, sample fluid in the plastic vial is first rotated to disperse cell materials
from debris. Cells are then collected from the fluid across a filtration membrane using
vacuum pressure. Filtered cells are then evenly pressed to a glass slide by the
machine, in a circular area of approximately 20 mm diameter. Prepared slides with
adherent cells are then placed in fixative for staining and evaluation.

In our study, slides were prepared and evaluated prior to any culture result being
reported and were read without knowledge of animal history, with light microscopy.
Subjective cellularity scores were assigned to each slide (poor, moderate or good
cellularity). Slides were graded for the presence of mycobacteria at 40X
magnification. Grades were assigned as follows: less than one acid-fast organism
noted per high power field (HPF) = 1+; two to ten organisms noted per high power
field = 2+; greater than ten organisms per HPF = 3+.

Statistical Analysis

Descriptive statistics (sensitivities, specificities, positive and negative predictive
values) were calculated using Microsoft Excel software (Microsoft Corp., Redmond,
WA). These values were generated by comparing the results of the ThinPrep

technique to those of swabs obtained fiom the same anatomic sites, cultured for

134

mycobacteria. A swab fi'om which M. bovis was cultured was considered a true
positive result. Values were calculated separately for each anatomic site and also for
the summed total of all results. Sensitivity defines the number of true positive samples
(percentage positive by cytology that were positive by culture). Specificity defines the
number of true negative samples (percent negative by cytology that were negative by
culture). Positive predictive value is the probability that positive cytologies were truly
positive when compared to the “gold standar ”, and negative predictive value is the

probability that negative cytology samples were truly negative on culture.

Results

The number of deer determined to be positive for M. bovis by both
mycobacterial culture (Table 5.2) and by the ThinPrep 2000 cytology technique (Table
5.3) are shown. Most samples that were positive had one or fewer organisms noted
per HPF, a score of 1+ (Table 5.4). Eight of the 21 (38.1 %) positive samples
examined from the tonsil had a score of 2+, and one of seven positive samples from
the nasal cavity had a score of 2+ (14.3%). No samples received a 3+ grade for
positivity. The new technique had an overall sensitivity of 73.17% and a specificity of
16.85 % when compared to the gold standard of mycobacterial culture (Table 5.5).
Cytology preps obtained fi'om the tonsil had the highest sensitivity and specificity of
any of the three sites calculated individually (84.21% and 55.56% respectively). The
overall positive predictive value for the test was 66.67%, and the overall negative
predictive value 87.06%. The site generating the highest individual positive predictive

value was the nasal cavity (85.71%), whereas the highest negative predictive value

135

resulted from those samples obtained from oral cavity swabs (91.18%). Subjective
cellularity grades for the oral cavity were poorest. All samples from other sites had
moderate to good cellularity reported for all slides, yet samples from the oral cavity
obtained at all dates post inoculation had poor scores to a variable degree (one at 21

days, four at 63 days, four at 90 days, and three at 113 days).

Discussion

The ThinPrep 2000 (Cytyc Corp., Boxborough, MA) is currently Food and Drug
Administration approved for use in diagnosis of multiple human samples, including
but not limited to superficial scrapings, fluids, needle aspirates, mucoid samples
(sputum, gastrointestinal, etc.), and gynecologic samples. To our knowledge however,
despite its wide range of potential clinical applications, use of the process for the
diagnosis of mycobacterial disease has never been evaluated. Use of this technology
for processing veterinary clinical/ diagnostic samples has not been reported to date
either, save for one reference mentioning its possible application in needle aspirate.
evaluation in research mice (Kobayashi et al 1997). In human literature, the technique
has been evaluated to be as good as or better than cytocentrifuge or direct smear
preparations (Wang et a1 1996; Papillo and Lapen 1994).

In our study, cellularity was quite good in all samples, save for some of the oral
cavity brush swabs. The likelihood that sampling technique was responsible for much
of the noted decreased cellularity in these samples remains plausible. Subjectively, all
slide preparations were easily read and did provide an adequate number of cells to

determine whether acid-fast organisms were present or not. The stain procedure

136

worked very well in highlighting organisms where present, and did not stain non-
mycobacterial organisms in any cases (Figures 5.1-5.3).

The more important shedding routes, both by culture and cytology, seem to be
the nasal and tonsilar routes, as oral samples were rarely positive by either technique.
Pathophysiologically, these results seem reasonable, as tuberculosis is primarily a
respiratory and/ or lymphoid system-based disease in many large animal species.
Since this is the first such study in deer, more data are necessary to confirm these
findings, particularly because initial inoculation was intratonsilar in the study.
Overall, of those sites assessed herein, the oral route appears to be the least
diagnostically rewarding in cases of tuberculosis based on the low frequency of
shedding and the relatively poor cellularity of oral cavity samples compared to tonsilar
and nasal samples. These results are not surprising, as previous inoculation studies in
white-tailed deer have suggested tonsilar and nasal shedding routes to be most
important based on the distribution of those sites most commonly developing lesions
after infection (Palmer et a1, 1999).

Our evaluation of the ThinPrep technique for diagnosing mycobacterial
infection resulted in an overall sensitivity comparable to that reported for detecting
malignancy in human specimens (Wang et a1 1996). Interestingly, the lack of
specificity of the technique was the primary concern in that study as it appears to be in
ours. The fact that the tonsil was the tissue with the highest sensitivity and specificity
throughout the study was most reasonably due to the fact that inoculation of deer was

intratonsilar, and shedding was most apparent in this tissue whenever samples were

137

taken. An intriguing finding is that tonsil swabs were positive by culture for all
animals only at 21 days P1, with no further deer having positive tonsil culture results.
Our technique detected several animals with intracellular acid fast organisms from
tonsil samples at 90 and 113 days PI. Using culture as the gold standard technique
would necessitate deeming these results as false positives. However, since cultures
were performed on swabs rather than on macerated tissues, potentially decreasing the
yield of organisms and overall specimen quality, it is proposed that perhaps all
negative cultures may not have been truly negative if actual tissues had been sampled.
One could similarly speculate that cultures performed on swabs from the oral cavity
were falsely negative at 21 days PI. Conversely, the high negative predictive value
does nevertheless suggest that shedding not detected by our test will not be able to be
detected by the gold standard technique, mycobacterial isolation and identification, in
most cases.

The number of animals used in this study was somewhat small to make
conclusions using definitive descriptive statistics, but the resultant values do show that
our technique may have some valid diagnostic application in the antemortem diagnosis
of tuberculosis in animals. The difficulty in restraining captive cervidae for testing
became apparent in the repeated collection of samples necessary for this study.
Limiting the number of times animals require restraint in order to obtain an accurate
diagnosis is of course warranted and is quite possible when samples or readings for
multiple tests can be taken at one time. It certainly then follows that this test could be

beneficial if used in conjunction with skin testing of deer, or even cattle. Results from

138

this technique, when used in conjunction with skin test results, would validate those
cases in which active shedding may be occuning regardless of the certainty of a
comparative cervical test result. And certainly in any animal species, this test could
help distinguish active shedders from those animals that may have established but
latent infections. Furthermore, in the case of many small, or even some exotic animals
suspect of having tuberculosis, this test may be able to help diagnose active disease as
well, and may even play a bigger role clinically in that no reliable skin testing protocol
exists for many of these species.

In any of these potential clinical applications, the described procedure certainly
surpasses culture for mycobacteria in cost-efficiency and timeliness of diagnosis. The
estimated tum-around time for this test approximates 48 hours, fi'om sample collection
to reporting. The cost in our laboratory was eighteen dollars per sample including
technician labor costs. When compared to the expected six to twelve weeks necessary
for mycobacterial culture and the cost of culturing a clinical sample, approximately
110 dollars based on the charges we incurred through contractual laboratory work,
clearly the ThinPrep method is the more rapid and cost-efficient technique. Of course,
there are sacrifices of specificity and sensitivity of results to some degree with the
technique, and no mycobacterial speciation can be performed without following
cytology examination with the more expensive and time-consuming culture
procedures. But when used as a screening process in cases with suspected active
infections, particularly when large numbers of samples may need to be processed (e.g.,

in a herd situation), the benefits of the test are readily apparent.

139

No test will match the sensitivity and specificity of the available post-mortem
methods of confirmation of mycobacterial infection. However, when used in
conjunction with other available antemortem tests, ThinPrep 2000 cytologic analysis
of veterinary samples may prove to be a quick and inexpensive tool for validation of

established mycobacterial infections in some instances.

140

Table 5.1: Number of deer available for sampling at various times after

inoculation.

Table 5.2: Deer which cultured positive for M. bovis. Ntunber of deer culturing
positive for listed tissue out of total number of deer sampled at that date, post

inoculation (P1), are given.

Table 5.3: Deer with positive cytology readings (acid-fast bacilli noted with light

microscopy). Number of deer with positive slide reading from listed tissue out of

total number of deer sampled at that date, post inoculation (PI), are given.

141

Table 5.1. Deer numbers at post-inoculation (PI) sampling dates.

 

 

 

Days PI Number of Deer
21 19

63 16

90

113 7

 

Table 5.2. Deer which cultured positive for M. bovis.

 

Culture Positive Tissues

 

 

 

Days PI m 9_ra_l Nasal
21 19/19 0/19 2/19
63 0/0 6/ 16 6/ 16
90 0/9 3/9 2/9
113 0/7 2/7 1/7

 

Table 5.3. Deer with positive cytology readings.

 

Cytology Positive Readings

 

 

 

Days PI Tonsil 9r_al Nasal
21 16/19 7/19 0/19
63 0/0 5/16 3/16
90 1/2 1/9 2/9
113 4/7 4/7 2/7

 

142

Table 5.4: Cytology scores for those samples with positive readings. Results are
given as number of samples with listed score over total number of positive samples for
listed tissue. A Scores assigned to average reading for entire slide as follows: 1+ = one

or fewer organisms per high power field (HPF; 40X); 2+ = 2 to 10 bacilli per HPF; 3+

= greater than 10 bacilli per HPF.

Table 5.5: Results of descriptive statistical analysis of Thinprep 2000 cytology
technique. Sensitivity defines the number of true positive samples (percentage
positive by cytology that were positive by culture). Specificity defines the number of
true negative samples (percent negative by cytology that were negative by culture).
Positive predictive value is the probability that positive cytologies were truly positive
when compared to the “gold standard”, and negative predictive value is the probability

that negative cytology samples were truly negative on culture.

. 143

Table 5.4. Cytology scores for those samples with positive readings.

 

Scores Assign_ed to Cytology:

 

 

 

ScoreA Tonsil (_)r_al Nasal
1+ 13/21 17/17 6/7
2+ 8/21 0/17 1/ 7
3+ 0/21 0/17 0/ 7

 

Table 5.5. Results of descriptive statistical analysis of ThinPrep cytoprep

 

 

 

 

 

technique.

leit Oral Nasal Overall (All sites)
Sensitivity 72.73 54.55 73.17

Specificity 22.50 2.50 16.85

Positive Predictive Value 76.19 47.06 85.71 66.67

Negative PredictiveValue 57.14 91.18 88.64 87.06

 

144

Figure 5.1. Positive (2+) cytology obtained using ThinPrep technique, tonsilar

sample. Intracellular acid-fast bacilli (arrows). New Fuchsin stain; 100X.

Figure 5.2. Positive cytology (2+) obtained using ThinPrep Technique, nasal
sample. Large group of intracellular acid-fast bacilli (arrow). New Fuchsin stain,

lOOX.

Figure 5.3. Cytology example with large numbers of intracellular cocci and no
acid-fast bacilli noted, ThinPrep technique. Photomicrograph displays clarity of
cellular preparation for bacteria other than mycobacteria. New Fuchsin stain,

lOOX.

145

 

v of

Figure 5.1. Positive (2+) cytology obtained using ThinPrep
technique, tonsilar sample.

 

Figure 5.2. Positive cytology (2+) obtained using ThinPrep
technique, nasal sample.

 

Figure 5.3. Cytology example with large intracellular
cocci and no acid-fast bacilli noted, ThinPrep techinique.

 

146

References

Kobayashi TK, Nakano Y, Sugimoto H, Saito K, Nishino H. 1997. Cellular
preparations of fine needle aspirates from transplanted tumour in nude mice using
ThinPrep technique. Cytopath. 8: 354-357.

Maher D and Ravi glione MC. 1999. The global epidemic of tuberculosis: a World
Health Organization perspective. In: Tuberculosis and nontuberculous mycobacterial
infections, 4th edition. D Schlossberg, ed. W.B. Saunders Co., Philadalphia. pp. 104-
129.

O’Reilly LM. 1995. Tuberculin skin tests: sensitivity and specificity. In:
Mycobacterium bovis infection in animals and humans. CO Thoen and JH Steele, eds.
Iowa State University Press, Ames. pp. 85-92.

Palmer MV, Whipple DL, Olsen SC. 1999. Development of a model of natural
infection with Mycobacterium bovis in white-tailed deer. J. Wild. Dis. 35: 450-457.

Pahner MV, Whipple DL, Olsen SC, Jacobson RH. 2000. Cell mediated and humoral
immune responses of white-tailed deer experimentally infected with Mycobacterium
bovis. Res. Vet. Sci. 68: 95-98.

Papillo JL and Lapen D. 1994. Cell yield. ThinPrep vs. cytocentrifuge. Acta Cytol.
38: 33-36.

Schmitt SM, Fitzgerald SD, Cooley TM, Bruning-Fann CS, Sullivan L, Berry DE,
Carlson T, Minnis RB, Payeur JB, Sikarskie J. 1997. Bovine tuberculosis in free-
ranging white-tailed deer from Michigan. J. Wildl. Dis. 33: 749-758.

Prophet EB. 1992. Laboratory methods in histotechnology. B Mills, JB Anington,
LH Sobin, eds. Armed Forces Institute of Pathology, American Registry of
Pathology, Washington, DC, 279 pp.

Thoen CO, Huchzenneyer H, Himes EM. 1995. Laboratory diagnosis of bovine
tuberculosis. In: Mycobacterium bovis infection in animals and humans. CO Thoen
and JH Steele, eds. Iowa State University Press, Ames. pp. 63-72.

Wang HH, Sovie S, Trawinski G, Garcia LW, Abu-Jawde GM, Upton M, Wemeke S.
1996. ThinPrep processing of endoscopic brushing specimens. Am. J. Clin. Pathol.
105: 163-167.

Wilkins EGL, Griffiths RJ, Roberts C. 1986. Pulmonary tuberculosis due to
Mycobacterium bovis. Thorax 41: 685-687.

147

Zheng X and Roberts GD. 1999. Diagnosis and susceptibility testing. In:
Tuberculosis and nontuberculous mycobacterial infections, 4th edition. D
Schlossberg, ed. W.B. Saunders Co., Philadalphia. pp. 57-64.

148

Chapter 6

Conclusions and Future Directions

149

 

The importance that the interaction of animals and man can have on the medical
community is well exemplified in the case of a zoonotic disease. Tuberculosis is one
of the most important zoonotic diseases both historically and currently. With the
reemergence of the human disease globally as a world health emergency, a need to
better understand the concepts of transmission and control of the disease in either an
animal or human population is essential.

Although advanced technologies are the current focus of much of the research
surrounding the diagnosis and treatment of tuberculosis, there are many essential,
more basic questions about the transmission and pathology of the disease, both in
animals and in man, that remain to be answered. Pathology is defined as the study of
the nature of disease. The knowledge base that composes the understood pathology of
any specific disease is best viewed as a continuum. This continuum constantly grows
as new technologies are developed and new ideas are formulated involving the
diagnosis and treatment of disease, the more clinical applications of medicine to which
pathology is always inherently linked. And likewise, as better technologies are
developed and implemented, the understanding of the nature of disease becomes more
complete.

Answers to basic questions about tuberculosis are particularly lacking in the
veterinary field, wherein understanding the true nature of the disease in a given animal
population can become easily obscured by test and slaughter control methods.

Although these methods are effective and probably even necessary to eradicate the

150

disease, they have to some extent eliminated much of the need for further study of the
pathology of the disease in animals, as treatment is not typically a viable option.

The research involving M. bovis tuberculosis in the veterinary setting can in
most instances be interpreted as primarily driven by need. When large outbreaks of
the disease occur, particularly in wild animal populations, threats to the health of
domestic livestock and humans become immediately important, and a need to answer
basic questions to better understand the outbreak becomes imminent. The preceding
chapters, in their simplest unified form, sought to answer some such basic questions
that became imminently important in the face of Michigan’s tuberculosis outbreak that
has now become endemic in white-tailed deer.

Although it has been established in other countries that small mammals
including badgers, ferrets, and brushtail possums can act very efficiently as reservoir
hosts for M. bovis in the wild in addition to or in combination with cervids, little was
known about transmission between deer and other wildlife hosts in Michigan. The
first experimental chapter in this body of work sought to determine whether two bird
species might reasonably be implicated in the transmission of M. bovis in Michigan.
The second experimental chapter examined the same question in a mammalian
species, the North American opossum.

The avian transmission study was perhaps most important due to the fact that no
reports, experimental or clinical, of M. bovis in any bird species existed prior to its
inception. European starlings and American crows were chosen because both occur

commonly in wildlife and agricultural settings, and could reasonably be exposed to

151

materials infected with M bovis. Oral inoculation of the animals sought to simulate
the natural ingestion of such materials (feed, carrion, feces, etc.), and intraperitoneal
inoculation was used as an altemative route and contrast the type or extent of infection
established, if any. It was found that these avian species are not likely to become
infected by ingestion of M. bovis, but that they could reasonably maintain organisms
intraperitoneally. The implication either of these species as viable wildlife reservoirs
is considered unlikely based on our study results.

The transmission experiment using opossums was in some ways an extension of
research carried out in its distantly related marsupial species, the brushtail possum, in
New Zealand. Since the brushtail possum is known as a primary wildlife reservoir for
M. bovis in New Zealand, it was reasonable to hypothesize that our own opossum
species might play some role in the maintenance of the disease in Michigan, or at least
be susceptible to experimental inoculation with the organism. Inoculation routes used
in this study were intramuscular and oral. Both routes resulted in established systemic
infections in some animals. Although it is unreasonable to assume that because they
are susceptible to large doses of mycobacteria experimentally, these animals are
reservoir hosts, there is a unique potential for infection in this species. Surveillance
data suggest their potential role as reservoirs. Although very few cases of M. bovis in
wild opossums have been identified to date through surveillance, it remains that they
may play some part in the maintenance of the disease on a very small scale, or may

become infected more readily than other small mammals when exposed.

152

 

The third project presented herein answered an essential question about
Michigan’s endemic strain of tuberculosis in wildlife: whether or not this strain
specifically had any inherent resistance to antimicrobials. Although it would not be
expected that the strain would have any secondary resistance due to treatment pressure
or therapeutic noncompliance, the possibility of a primary resistance certainly does
exist. A number of isolates from deer, cattle, and small mammal cases of M. bovis
tuberculosis gathered through surveillance over the years in Michigan were tested for
susceptibility to the standard panel of primary antimycobacterial drugs. As a
comparison, all human isolates of M. bovis diagnosed in Michigan until the time of the
realization of the study were tested for susceptibility to the same drugs. No animal
isolates were resistant to any antimycobacterial tested based on the standards used by
the Tuberculosis Laboratory at the Michigan Department of Community Health.
Human M. bovis isolates were variably resistant, but none were found to be multidrug-
resistant. All isolates, animal and human, were however resistant to pyrazinamide as
is expected of M. bovis. Genetic analysis of the isolates did confirm that no human
isolates were genetically identical, that these isolates differed substantially from
animal isolates, and that all tested animal isolates were genetically identical. None of
these resultant data were unexpected. Yet the study sets an important precedent for
understanding the strain of microorganism involved in an outbreak on all levels
including its resistance potential and genetic makeup, even one where treatment of
infected animals may not be important. Even if bovine tuberculosis is best controlled

by test and slaughter in animals, preparedness for the potential spread to humans,

153

because such an event would require treatment of human patients, remains an
important consideration.

The final research objective presented in this dissertation was also partially
inspired by an eventual application to human medicine. In this chapter, a new
diagnostic technique for the detection of mycobacteria in cell-rich clinical samples
was evaluated. Cellular swabs obtained from deer experimentally inoculated with M.
bovis were processed on the ThinPrep 2000 automated cytology device, one which
prepares very high quality cytologic slides, and were stained for acid-fast organisms.
Results were compared to culture results of swabs taken fi'om the same sites in the
inoculated deer. The technique was developed as a rapid, inexpensive, antemortem
test that would require relatively little technical training in its application. Although
this cytology examination-based test was found to be less sensitive and specific than
culturing clinical specimens, it has promise in its application in both human and
veterinary medicine. As an inexpensive adjunct test that can be prepared and run at
nearly any facility, without specialized knowledge of mycobacterial culture and
identification techniques, it holds promise for being a useful procedure in the
veterinary field.

While the work presented in this dissertation has contributed significantly to our
understanding of the nature of M. bovis in Michigan wildlife, questions that have
emerged from this work may prove equally as significant and will require additional,
specifically-focused studies. The promise of a new antemortem veterinary diagnostic

test for the detection of mycobacteria has been realized, but the extent to which it can

154

 

enhance existing and developing technologies remains to be seen. Larger numbers of
samples, and perhaps even those fiom different animals sources should be tested
before a final determination is made on the usefulness of ThinPrep processing of
cytology samples for the diagnosis of tuberculosis. Moreover, the application of this
technology for its use in the diagnosis of infectious diseases such as mycobacteriosis
in the human clinical setting should be examined.

More final in the application of its results was the study of the susceptibility
pattern of Michigan’s endemic strain of M. bovis. Although it was determined that at
present, no threat of drug resistance exists in the strain, tracking the isolate for genetic
drift, particularly for that of acquired resistance over time, is now possible. Because
there will always be a threat of transmission to humans as long as this disease remains
prevalent in wild and domestic animals species in Michigan, assurance that this strain
is not developing either primary or secondary, treatment pressure-based mutational
resistance over time is important. The results of our study serve as a very good initial
baseline in order to perform such monitoring.

The largest number of remaining questions was generated by our transmission
studies. A limited number of potential reservoirs for M. bovis in Michigan were
capable of being examined in the time period over which these studies were
completed, and in these animals, a limited number of inoculation routes were assessed.
Little remains known about the susceptibility of wild, non-passerine, avian species to
M. bovis. An interesting follow-up study would be to compare the infection potential

of certain herbivorous or non-passerine, native Michigan bird species. The reasoning

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behind why certain camivores and omnivores seem more easily infected with M. bovis
in the wild remains uncertain. Continued inoculation studies that not only explore
new inoculation routes in opossums, but also those in different species and the
variable immune responses of these species when challenged with bovine tuberculosis
will help to complete the understanding of the cycle of its transmission in and between
wild and domestic animal hosts. Specifically, inoculation studies in wild-caught
badgers, rodents, and other carnivores included in surveillance in Michigan are
warranted. Moreover, in those species appearing to have the potential of becoming
infected with M. bovis, inoculation studies determining specific doses of organisms
necessary to establish infection, and time required for these species to shed bacteria
from various sites after inoculation would certainly better our overall understanding of
the precise pathogenesis of tuberculosis in these animals.

In summary, this body of work demonstrates that several very important
objectives for researching various aspects of Michigan’s endemic strain of M. bovis
have been met. We have shown that European starlings, American crows, and North
American opossums do not appear to be active reservoir hosts for this disease in the
state at the current time. Yet it has been established that opossums have the potential
to become a reservoir for the disease in the wild. The endemic strain of M. bovis in
Michigan wildlife has been shown to be susceptible to most of the common
antituberculosis therapies. A new technique for quickly and inexpensively
documenting the shedding of mycobacteria from animals with active infection has

been developed. All of these findings illuminate areas of bovine tuberculosis research

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that had been ignored until the present time. The base of knowledge they provide for
the continuing study of M. bovis in wildlife is therefore invaluable, and in all hopes
will be built upon as future research continues to more completely define the

pathology and transmission of this disease.

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