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2/05 p:/ClRC/DateDue.indd-p.1

THE ROLE OF INDIRECT TRANSMISSION IN THE EPIDEMIOLOGY 0F
BOVINE TUBERCULOSIS IN CATTLE AND WHITE-TAILED DEER IN
MICHIGAN
By

Amanda Elizabeth Fine

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Large Animal Clinical Sciences

(Epidemiology)

2006

ABSTRACT

THE ROLE OF INDIRECT TRANSMISSION IN THE EPIDEMIOLOGY OF BOVINE
TUBERCULOSIS IN CATTLE AND WHITE-TAILED DEER IN MICHIGAN

By

Amanda Elizabeth Fine

Understanding the epidemiology of Mycobacterium bovis transmission in
Michigan is an essential component of nationwide efforts to control and eradicate bovine
tuberculosis (TB). Determining the role of indirect transmission in bovine TB dynamics
is a key to the application of epidemiologically effective methods of disease control in
both livestock and wildlife populations. The objective of this dissertation was to
characterize the persistence of M. bovis in the environment and its potential role in the
indirect transmission of disease among and between cattle (Bos taurus) and white-tailed
deer (Odocoileus virginianus) in Michigan.

Optimized techniques for isolating M. bovis ﬁ'om environmental substrates were
developed. These were applied to the testing of samples collected from cattle farms with
a known history of M. bovis infection. Samples collected opportunistically from
locations within areas with a high prevalence of M. bovis infection in white-tailed deer
were also tested. Though mycobacterial isolation was successful, none of the isolates
were identiﬁed as M. bovis. The inability to detect M. bovis from sites of known bovine
TB transmission suggests that the pathogen is not distributed broadly across the
landscape, making the identiﬁcation of a speciﬁc site of contamination difﬁcult.

To address the question of M. bovis persistence in the environment, given the

limitations of detecting the bacilli under natural conditions, a 12-month long experiment

was designed. Environmental substrates were experimentally inoculated with M. bovis
and exposed to natural weather conditions. The effects of environmental conditions on
the survival of M. bovis over time were assessed.

Persistence of M. bovis in the Michigan environment under natural weather
conditions was recorded for an average of 30 days in the cooler months of the year
(November -— May), and an average of 7 days in the warmer months (May - August).
This recorded persistence strongly suggests an important potential role for indirect
transmission in the epidemiology of bovine TB in the region. These data supplement
those produced through experimental M. bovis disease transmission studies that have
proven the feasibility of indirect transmission of M. bovis among and between cattle and
white-tailed deer. They also support the analyses of observational data on M. bovis
infection in cattle and white-tailed deer in Michigan that indicate the importance of
indirect transmission in the interspecies transmission of M. bovis.

Local, State and Federal bovine TB control and eradication policy needs to
consider indirect transmission of M. bovis through contaminated environmental substrates
in the creation and implementation of epidemiologically appropriate disease management
plans. In the bovine TB endemic region of Michigan, interspecies transmission of bovine
TB should be considered by both wildlife and livestock health agencies. If this
component of the epidemiology of M. bovis transmission in the region is ignored, efforts

to control and eventually eradicate the disease will fail.

To the many individuals and institutions dedicated to the global control and eradication

of bovine tuberculosis.

iv

ACKNOWLEDGEMENTS

I have the deepest appreciation for all of the individuals who have helped to make
this project possible. Like the approach to the control of bovine tuberculosis (TB) in
Michigan, I have beneﬁted greatly from the assistance and guidance of a
multidisciplinary and team. I would like to thank my two major advisors, Dr. John Baker
who created the opportunity for me to study TB in Michigan and pursue this PhD, and
Dr. John Kaneene who supervised the development of this research project and provided
expertise at every step of its progress toward completion. I would also like to thank the
members of my graduate committee, Dr. Carole Bolin, Dr. Joseph Gardiner, Dr. Dan
O’Brien, Dr. Scott Winterstein, and Dr. Gary Witmer for their guidance, support and the
valuable contributions to this research.

I would like to acknowledge the assistance of Dr. Charles Thornton (Integrated
Research Technology), Mr. Dale Berry (Michigan Department of Community Health)
and the staff at the National Veterinary Laboratories in Ames, Iowa, who were willing to
pick up the phone and give me advice on the development of the laboratory techniques
used in this research. For assistance in the laboratory at Michigan State University
(MSU) Diagnostic Center for Population and Animal Health, I am indebted to Joseph
Hattey, bovine TB laboratory technician, and to students Kathleen Gray and Christy
Sampson. Desiree Oliver contributed in the laboratory and the ﬁeld and Kile Kucher

assisted with data entry in the ﬁnal stages of this work.

A large component of this research would not have been possible without the
participation of cattle farmers and landowners in the bovine TB endemic region of
Michigan. I am grateful for their willingness to take time out of their busy schedules to
participate in the project. I would also like to acknowledge the staff at the Michigan
Department of Agriculture (MDA) who assisted with the identiﬁcation of bovine TB
positive farms in the region, the members of the Gaylord, MI, United States Department
of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Wildlife
Services (W S) team and the Michigan Department of Natural Resources (MDNR) ﬁeld
staff in northeast Michigan who assisted with environmental sampling.

The Michigan Agricultural Experiment Station, and the Departments of Fisheries
and Wildlife (FW) and Large Animal Clinical Sciences at MSU supported this research
and program. The MDA, USDA/APHIS/W S, and the USDA Bovine TB Special Grant
provided additional research funding.

I would like to thank ﬁ'iends and colleagues from the FW and LCS departments at
MSU and the multi-agency Michigan bovine TB team, which includes staff from the
MDNR wildlife disease laboratory, Veterinary Services and WS of USDA/APHIS, the
MDCH and faculty and researchers at MSU. I feel privileged to have had the opportunity
to engage and interact with such a supportive, talented and dynamic group of individuals.
Finally, I would like to thank my parents for instilling in me what will undoubtedly be a

life-long love of learning.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES ........................................................................................................ xii
INTRODUCTION .............................................................................................................. 1
CHAPTER 1

LITERATURE REVIEW OF THE PERSISTENCE OF M YCOBA C TERIUM
BOVIS IN THE ENVIRONMENT AND THE POTENTIAL ROLE OF INDIRECT
TRANSMISSION IN THE EPIDEMIOLOGY OF BOVINE TUBERCULOSIS IN

MICHIGAN ........................................................................................................................ 5
INTRODUCTION ...................................................................................................... 5
EVIDENCE OF THE PERSISTENCE OF MY COBACT ER] UM BO VIS IN THE
ENVIRONMENT ....................................................................................................... 8

THE ROLE OF INDIRECT TRANSMISSION IN THE EPIDEMIOLOGY OF
BOVINE TUBERCULOSIS IN CATTLE AND WHITE-TAILED DEER IN

NORTHERN LOWER MICHIGAN ........................................................................ 15
CONCLUSIONS ....................................................................................................... 30

CHAPTER 2

OPTIMIZING THE ISOLATION OF MYCOBA C TERI UM BO VIS FROM

ENVIRONMENTAL SAMPLES ................................................................................... 32
ABSTRACT .............................................................................................................. 32
INTRODUCTION .................................................................................................... 33
MATERIALS AND METHODS .............................................................................. 38
RESULTS ................................................................................................................. 48
DISCUSSION ........................................................................................................... 57

CHAPTER 3

AN INVESTIGATION OF BOVINE TUBERCULOSIS TRANSMISSION SITES

(CATTLE FARMS AND WILDLIFE AREAS) IN MICHIGAN, USA ..................... 62
ABSTRACT .............................................................................................................. 62
INTRODUCTION .................................................................................................... 63
MATERIALS AND METHODS .............................................................................. 65
RESULTS ................................................................................................................. 73
DISCUSSION ........................................................................................................... 89

vii

CHAPTER 4
A STUDY OF THE PERSISTENCE OF M YCOBA C TERI UM BOVIS IN THE

ENVIRONMENT ............................................................................................................. 96
ABSTRACT .............................................................................................................. 96
INTRODUCTION .................................................................................................... 97
MATERIALS AND METHODS ............................................................................ 100
RESULTS ............................................................................................................... 1 12
DISCUSSION ......................................................................................................... 127

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS ............................... 134

APPENDIX A ................................................................................................................. 138

APPENDIX B ................................................................................................................. 139

APPENDIX C ................................................................................................................. 149

LITERATURE CITED ................................................................................................. 159

viii

LIST OF TABLES

Table 2.1 Inoculation Experiment 1: Record of contamination and culture
results of sterilized and non-sterilized ha , water and soil
samples inoculated with 1 X 106 to l X 10 CFU of M. bovis
and processed with the CB-18 or NaOH-based method ............................ 51

Table 2.2 Inoculation Experiment 2: Record of contamination and culture
results of sterilized and non-sterilized hay, water and soil
samples inoculated with either Dilution 1 (>10,000 CFU);
Dilution 2 (> 1,000 CFU); Dilution 3 (>100 CFU) or Dilution 4
(<10 CFU) of M. bovis and processed with the CB-18 or
NaOH-based method .................................................................................. 52

Table 2.3 Inoculation Experiment 3: Record of contamination and culture
results of sterilized and non-sterilized hay, water and soil
samples inoculated with either Dilution 1’ (120 CFU); Dilution
2' (0 CFU); Dilution 3' (0 CFU) and Dilution 4' (0 CFU) of M.
bovis and processed with the CB-18 or NaOH-based method ................... 53
.100 fold less than the intended concentration of M. bovis

Table 2.4.1 Inoculation Experiment 4: Record of contamination and culture
results of sterilized hay, water and soil samples inoculated with
either Dilution 1 (6 X 103 CFU) or Dilution 2 (3 X 103 CFU) of
M. bovis and processed with CB-18 immediately (Time 0);
after storage for 72 hr. at Room Temperature; after storage for
72 hr. at 4° C and after storage for 19 days at —20 ° C. “L”
denotes samples processed with liquefaction and “W” indicates
the substitution of water for liquefaction in the mixing process ................ 54

Table 2.4.2 Inoculation Experiment 4 continued: Record of contamination
and culture results of non-sterilized hay, water and soil samples
inoculated with either Dilution 1 (6 x 103 CFU) or Dilution 2
(3 X 103 CFU) of M. bovis and processed with CB-18
immediately (Time 0); after storage for 72 hr. at Room
Temperature; aﬂer storage for 72 hr. at 4° C and after storage
for 19 days at —20 ° C. “L” denotes samples processed with
liquefaction and “W” indicates the substitution of water for
liquefaction in the mixing process ............................................................. 55

ix

Table 2.5

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Table 3.6

Table 3.7

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Inoculation Experiment 5: Record of contamination and culture
results of non-sterilized hay, water and soil samples inoculated
with either Dilution l (5 X 103 CFU) or Dilution 2 (8 X 102
CFU) of M. bovis and processed with CB-18 immediately
(Time 0); after storage for 24 hr. at Room Temperature; aﬁer
storage for 24 hr. at 4° C and after storage for 16 days at —20 °
C ................................................................................................................. 56

Time lag between farm investigation (environmental
sampling), ofﬁcial TB positive date, and investigation date of
cattle depopulation ..................................................................................... 82

General characteristics of bovine TB positive farms selected
for environmental sampling ....................................................................... 83

A summary of cattle management practices on bovine TB
positive farms selected for environmental sampling ................................. 84

A summary of seed storage and fencing practices on bovine TB
positive farms selected for environmental sampling ................................. 85

A summary of reported white-tailed deer incursion on bovine
TB positive farms selected for environmental sampling ........................... 86

Mycobacterial culture results of environmental substrates
collected ﬁom bovine tuberculosis positive farms .................................... 87

Mycobacterial culture results of environmental substrates
collected from potential wildlife bovine tuberculosis
transmission sites ....................................................................................... 88

The make up of one sample set used in the bovine TB
environmental persistence study .............................................................. 108

Weather conditions recorded over the three sampling periods of
the Mycobacterium bovis environmental persistence study ..................... 116

Duration of M. bovis persistence in the environment on corn,
hay, water and soil samples in season “A”, “B” and “C” ........................ 117

Mean duration of M. bovis persistence in the environment on
com, hay, water and soil samples in season “A”, “B” and “C”
in shaded and non-shaded conditions ...................................................... 123

Table 4.5

Table 4.6

Table 4.7

Cox’s proportional hazard regression model for persistence of
M. bovis over three different sampling periods (fall/winter “A”,
winter/spring “B” and spring/summer “C”) or seasons ........................... 125

Univariable analysis of the inﬂuence of each weather related
variable on the hazard (survival) of M. bovis in the
environmental using the score test in Cox regression .............................. 126

Multivariable Cox proportional hazards model of weather related

factors associated with the survival of M. bovis in the
environment ............................................................................................. 126

xi

LIST OF FIGURES

 

Figure 3.1 Map of Michigan indicating the bovine tuberculosis State
Status Designations: 1) Modiﬁed Accredited (Infected Zone),
and 2) Modiﬁed Accredited Advanced (Disease Free Zone).
Source: Michigan Department of Agriculture

www.n1ichigan.gov/bovinetb ................................................................... 79

Figure 3.2 Map of the bovine TB “core” area within the Counties of
Montmorency, Alpena, Oscoda and Alcona, Michigan ............................. 80

Figure 3.3 Three photographs of sampling locations selected during
investigations of bovine tuberculosis positive cattle farms: 1)
an unprotected hay bale with evidence of deer feeding activity;
2) a pond in a pasture that both deer and cattle have access to;
and 3) an example of feeding cattle hay on the ground in the
woods ......................................................................................................... 81

Figure 3.4 Three photographs of sites selected for sampling in the wildlife
bovine TB transmission areas: 1) an oak forest where deer
fecal pellets were collected; 2) a deer feeder and a plot of
forage planted for deer; and 3) a pond near the trap location of
a bovine TB positive small mammal ......................................................... 81

Figure 4.1 A diagram of the location of the enclosure used for the bovine
TB environmental persistence study on the campus of
Michigan State University ....................................................................... 109

Figure 4.2 A diagram of the placement of samples and weather station
within the bovine TB environmental persistence study
enclosure .................................................................................................. 110

Figure 4.3 Diagram of timing of sampling periods: Monthly over 1 year
and weekly over 3 seasons ....................................................................... 1 1 1

Figure 4.4 The mean and maximum survival of M. bovis in corn, hay, soil
and water exposed to environmental conditions in F all/Winter
“A”, Winter/Spring “B”, and Spring/Summer “C”. Error bars
represent standard deviation .................................................................... 118

Figure 4.5 The overall mean and maximtun survival time (persistence) of
M. bovis in all substrates exposed to natural environmental
conditions in Fall/Winter “A”, Winter/Spring “B”, and
Spring/Summer “C”. Error bars represent standard deviation ................. 119

xii

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

 

The percent (n=4 of each substrate) of M. bovis positive
replicates of corn, hay, soil and water at each sampling point in
Fall/W inter “A”, Winter/Spring “B”, and Spring/Summer “C” .............. 120

The average number of M. bovis colony forming units isolated
from processed replicates of corn, hay, soil and water samples
over time in Fall/Winter “A”, Winter/Spring “B”, and
Spring/Summer “C” ................................................................................. 121

Survival probabilities of M. bovis in water, corn, hay and soil
across all seasons tested ........................................................................... 122

Survival probabilities of M. bovis exposed to natural

environmental conditions in “A”, “B”, and “C” sampling
periods ...................................................................................................... 124

xiii

INTRODUCTION

PROBLEM STATEMENT

Bovine tuberculosis (TB) caused by Mycobacterium bovis has been recognized as
a threat to the livestock industry and public health in North America since the late 1800’s.
The bovine TB eradication program, which began in the United States in 1917, has
resulted in a dramatic reduction in the prevalence of bovine TB in cattle (Bos taurus)
herds across the country, but nationwide TB-free status has not been accomplished (Frye,
1995). A tremendous threat to the eradication effort in the United States today is the
emergence of bovine TB in a population of free ranging white-tailed deer (Odocoileus
virginianus) in northeast Lower Michigan and the subsequent detection of bovine TB in
cattle herds in the same region (Schmitt et al., 2002). It is believed that this is the ﬁrst
time a reservoir for bovine TB has been established and maintained in a wildlife
population in the mainland United States (Schmitt et al., 1997).

The emergence of a wildlife reservoir for bovine TB in North America, and
evidence of disease transmission between infected free-ranging white-tailed deer
populations and domestic cattle, has forced a reevaluation of our understanding of the
epidemiology of bovine TB. The long-standing notion, that bovine TB is exclusively a
directly transmitted disease requiring very close contact between an infected and
susceptible host for disease transmission to occur, is under scrutiny. This reevaluation of
the epidemiology of bovine TB will have necessary and important impacts on disease

management and prevention strategies in both domestic and wildlife populations.

STUDY RATIONALE

The role and relative importance of the indirect transmission of M. bovis in the
epidemiology of bovine TB in Michigan cattle and wildlife populations is unknown. A
clearer understanding of this component of the epidemiology of bovine TB is essential
for the improvement of protocols for cattle farm bio-security, the maintenance of
appropriate restrictions on feeding and baiting free-ranging white-tailed deer and other
wildlife, and for informing human health and safety regulations regarding the potential
public health hazards of bovine TB.

An understanding of the presence and persistence of M. bovis in the environment
is required to assess the role of indirect transmission in the epidemiology of M. bovis.
Until this PhD project was implemented, little work had been done to test environmental
substrates for the presence of M bovis or to characterize the persistence of the Michigan
strain of M. bovis in the environment under conditions typical of the bovine TB endemic
region. Earlier attempts to detect and characterize M. bovis persistence in the
environment were constrained by the limits of standard methodologies, time and ﬁnancial
resources.

Information regarding the persistence of M. bovis in the environment in Michigan
is by stakeholders (State and Federal regulatory agencies, policy makers, cattle producers,
recreational hunters, and the academic community) who are working toward the control

and eventual eradication of bovine TB in Michigan.

OBJECTIVES

This PhD project was designed to further the understanding of the role of indirect
transmission in the epidemiology of bovine TB in northeast Michigan. To accomplish
this goal the speciﬁc objectives included: 1) Optimizing a technique for processing
environmental samples for M. bovis detection by mycobacterial culture; 2) Investigating
and testing environmental samples from sites of natural bovine TB transmission; and 3)
Characterizing the persistence of M. bovis in the environment under conditions typical of

the bovine TB endemic region of Michigan.

OVERVIEW

Chapter 1 is a review of the literature addressing the role of indirect transmission
of bovine TB in domestic and wild populations of animals and a presentation of studies
that have examined the persistence of M. bovis in the environment in Michigan and other
regions of the world. Chapter 2 describes the difﬁculties associated with the detection
of M. bovis in the environment and the methods and techniques developed for processing
environmental samples and detecting M. bovis during the course of this dissertation work.
Chapter 3 presents the results of a series of ﬁeld investigations designed to detect M.
bovis in environmental samples ﬁ'om known TB transmission sites including TB positive
cattle farms and areas of the Michigan with the highest apparent prevalence of M. bovis
in free-ranging white-tailed deer. Chapter 4 describes an experimental study designed to
determine the length of survival of M. bovis on a range of substrates (feed, soil, and
water) exposed to environmental conditions in Michigan throughout a calendar year and

the factors that inﬂuence the likelihood of survival.

The hypothesis being tested is that M. bovis can survive in environmental substrates
for sufﬁcient lengths of time to serve as a source of infection for cattle and/or free-ranging
white-tailed deer and that speciﬁc conditions inﬂuence the persistence of M. bovis in the
environment. The implications of the ﬁndings presented in Chapters 1 through 4 are
discussed in the “overall conclusions” section at the end of this dissertation. The
prospects for the control and eradication of bovine tuberculosis in Michigan, the
effectiveness of the century-old bovine TB eradication program in the United States and
our understanding of wildlife/livestock disease transmission dynamics are all inﬂuenced
by this closer examination of the persistence of M. bovis in the environment and the role

of indirect transmission in the epidemiology of bovine tuberculosis.

Chapter 1

LITERATURE REVIEW OF THE PERSISTENCE OF M Y CUBA CT ERI UM
BO VIS IN THE ENVIRONMENT AND THE POTENTIAL ROLE OF INDIRECT
TRANSMISSION IN THE EPIDEMIOLOGY OF BOVINE TUBERCULOSIS IN

MICHIGAN

1.0 INTRODUCTION

The role of indirect transmission of Mycobacterium bovis in the epidemiology of
bovine tuberculosis (TB) in Michigan has been a point of discussion since the current TB
epidemic in Michigan was ﬁrst described in 1997 (Schmitt et al., 1997). Spillover of
bovine TB infection from domestic cattle to white-tailed deer in Michigan is thought to
have occurred during the late 1950’s when large numbers of cattle infected with M. bovis
were present in the State (Frye, 1995). The establishment and persistence of M. bovis in
free-ranging white-tailed deer in northeast Michigan today is thought to have been
inﬂuenced by the long-term practice of winter feeding of deer in the region (Schmitt et
al., 1997). The feed, set out to attract deer and improve their productivity and winter
survival, is thought to contribute to the transmission of TB among white-tailed deer by 1)
increasing local density and direct contact between animals, 2) increasing overall density
of deer above biological carrying capacity making deer more susceptible to M. bovis
infection, and 3) providing a site for the indirect transmission of TB through

contamination of the feed by infected deer shedding M. bovis in their saliva or nasal

discharges and the subsequent infection of a naive deer by consumption of contaminated
feed (Hickling, 2002; Schmitt et al., 1997).

Michigan received its United States Department of Agriculture (USDA)
“accredited-ﬂee of TB” status in 1979. Concern surrounding the role of indirect
transmission in the epidemiology of bovine TB in Michigan increased when the ﬁrst case
of bovine TB in cattle was conﬁrmed in northeast Michigan in 1998 (State of Michigan,
2005). Since 1998, thirty-seven cattle herds in Michigan have been conﬁrmed as TB-
positive and two of these herds have been found to be positive on two separate occasions
(Judge, 2006). The spatial and temporal relationship between bovine TB in deer and
cattle in Michigan, coupled with data from DNA and restriction fragment length
polymorphism (RF LP) analyses of M. bovis isolates from both populations, indicate that
deer and cattle are infected with the same strain of TB (Whipple et al., 1999a). Close
“nose-to-nose” contact and interaction, necessary for aerosol transmission of M. bovis,
are very rarely observed between wild white-tailed deer and cattle (DeLiberto et al.,
2004). Indirect transmission of M. bovis has, therefore, been suggested as a possible
mechanism for interspecies (deer to cattle or cattle to deer) TB transmission in Michigan.

Control of bovine TB in Michigan is of interest to individuals and agencies in the
natural resources, veterinary and public health sectors. The presence of TB in free-
ranging wildlife and domestic cattle is associated with economic costs at the local and
national level (Comer, 2006; Holecek and Bristor, 2003; Thornton et al., 1998c). The
economic costs in Michigan have included losses associated with the restriction of cattle
trade, the cost of disease surveillance and loss of genetic stock. In addition economic

losses have been noted in the tourism sector due to concerns regarding bovine TB in the

white-tailed deer population. The economic impact of bovine TB in Michigan has also
extended to the public health and companion animal ﬁelds in which the risk of zoonotic
disease transmission is being monitored (Kaneene et al., 2002a; Wilkins et al., 2003).

To address this multifaceted disease issue, scientists and disease control ofﬁcials
from Michigan State University (MSU) and MSU’s Diagnostic Center for Population and
Animal Health (DCPAH), the Michigan Departments of Natural Resources (MDNR),
Agriculture (MDA) and Community Health (MDCH), and the USDA, Animal Plant
Health Inspection Service (APHIS), Veterinary Services (VS) and Wildlife Services
(W S), have initiated an extensive cooperative TB research and control program
addressing the disease in both domestic and wildlife populations. Understanding the role
of indirect transmission in the epidemiology of bovine TB in Michigan will assist policy
makers in their efforts to control and eradicate bovine TB. Improved understanding of
bovine TB transmission should increase public conﬁdence in the policies developed and
ensure their continued cooperation in efforts to eradicate bovine TB in Michigan.

The potential role of indirect transmission in the epidemiology of bovine TB in
Michigan is explored below through a review of: 1) experimental and observational
studies describing evidence of the persistence of Mycobacterium bovis in the
environment and the role this may or may not have in inter- and/or intra-species
transmission of bovine TB and; 2) experimental and observational studies examining the
potential role of indirect transmission in the epidemiology of bovine TB in cattle and deer

in Michigan.

1.1 EVIDENCE OF THE PERSISTENCE OF MYCOBACTERIUM BOVIS IN

THE ENVIRONMENT

Attempting to detect and characterize the persistence of M. bovis in the
environment has historically been the approach to elucidating the role of indirect
transmission in the epidemiology of bovine tuberculosis in systems around the world.
The role of indirect transmission in the epidemiology of M. bovis is of particular concern
in systems in which a wildlife reservoir of M. bovis has been identiﬁed with the threat of
the spillover (or spill back) of infection into domestic cattle populations (Corner, 2006).
The role of indirect transmission in bovine TB epidemiology has been studied by
attempting to detect M. bovis in the environmental substrates in a location where a M.
bovis transmission event has occurred, and in experimental studies in which either the
substrates or the species under investigation are infected with M. bovis and the
persistence of M. bovis is assessed under varying conditions designed in a simulated

setting.

1.1.1 Detection of Mycobacterium bovis under natural conditions

F ew studies have been published that address the detection of M. bovis under
natural conditions or discuss attempts to investigate environmental sources of M. bovis.
In one study, Pillai and coworkers (2000) described an investigation of 14 dairy herds in
El Paso County, Texas, with a prior history of bacteriologically conﬁrmed M. bovis
infection in cattle. Extensive soil, water and air sampling was carried out on each farm

and the samples were processed for mycobacterial culture, but M. bovis was not isolated.

Young and coworkers (2005) detected M. bovis in the farm environment using
molecular techniques. Speciﬁc polymerase chain reaction (PCR) primers targeting the
M. bovis 16S rRNA gene were used to detect M. bovis in environmental soil samples
collected from a cattle farm in Ireland with a history of bovine tuberculosis. The
pathogen was detected in soil from the farm at 4 and 12 months after possible
contamination. The source of contamination in this farm was presumably M. bovis shed
by infected badgers (Meles meles) present on the property before the removal of all cattle
and badgers according to disease control protocols in the region.

Other attempts to identify and isolate mycobacterial species from environmental
substrates, i.e. soil and stream water, have failed to detect M. bovis (Cooney et al., 1997;
Livanainen, 1995; Livanainen et al., 1999). Failure to detect, and therefore report, the
presence of M. bovis in the environment is related to difﬁculties associated with isolating
M. bovis from environmental substrates. An efﬁcient and effective method for
processing environmental samples for mycobacterial isolation is needed to support efforts
to understand M. bovis transmission.

The challenges associated with the detection of M. bovis in the environment fall
into three major categories. First, environmental samples (soil, feed, fecal material,
pond/stream water, etc.) contain large numbers of saprophytic bacteria, molds and other
infectious organisms. Mycobacterium are slow- growing in culture and other organisms
interfere with the isolation of mycobacteria by overgrong and out-competing the
mycobacteria during the bacterial culture process. The essential decontamination

process, designed to eliminate saprophytic organisms, also reduces the viability of

mycobacteria in the specimen and therefore interferes with the sensitivity of detection of
M. bovis by bacterial culture methods (Kent and Kubica, 1985; Yajko et al., 1995).

Secondly, evidence suggests that the shedding of M. bovis from both infected deer
(Palmer et al., 2001) and infected cattle (Goodchild and Clifton-Hadley, 2001; Neill et
al., 1988) is intermittent. Intermittent or low level shedding of M. bovis coupled with the
relatively small size—when compared with the scale of the potentially contaminated
environment—of sample that can be collected and processed for mycobacterial culture
results in a greatly reduced chance of collecting a contaminated substrate in the
environment.

Thirdly, the properties of M. bovis make it particularly difﬁcult to process and
culture. Like other mycobacteria, M. bovis has a tendency to clump and form cords so it
is often not evenly distributed in a processed sample. Its thick, waxy cell wall makes it
buoyant and reduces the success of centrifugation methods aimed at concentrating the
organism in the sample. In addition, M. bovis requires 6-8 weeks for growth on solid
media, prolonging the time the specimen must be maintained at optimal conditions (37°
C, moist and free of other microbial contamination).

Improved protocols for processing samples collected from the environment for
mycobacterial culture are necessary to further the investigation of M. bovis persistence in
the environment and our understanding of the role of indirect transmission in the

epidemiology of bovine TB in Michigan and other regions of the world.

10

1.1.2 Experimental Mycobacterium bovis persistence studies

A number of reports have been published that examine the persistence of
Mycobacterium bovis in the environment by utilizing various experimental study designs.
Early studies were concerned primarily with food hygiene and examined the persistence
of M. bovis in meat and dairy products experimentally contaminated, or known to be
contaminated, with M. bovis ﬁom infected cattle (Mitscherlich and Marth, 1984).
Interestingly, one of the studies cited in the Mitscherlick and Marth textbook chapter on
mycobacterial survival in the environment was an experiment performed by Soparkar in
1917 with “a purulent mass obtained from the lung of a deer which had died of bovine
tuberculosis”. Soparkar reported that M. bovis in the purulent mass survived for 10 but
not 12 hours in direct sunlight and 30 days in diffuse sunlight.

Early studies of more relevance to concerns surrounding the persistence of M.
bovis in the environment and the potential role of indirect transmission in the
epidemiology of bovine TB in Michigan, are a series of studies published in the Journal
of Hygiene in the early 1930’s (Maddock, 1933, 1934, 1936; Williams and Hoy, 1930).
William and Hoy were concerned with the persistence of M. bovis in feces shed by
tuberculous cows in the south of England. Their attempts to isolate M. bovis from feces
on pasture grazed by a naturally infected cow failed, despite the fact that the cow had
culture-conﬁrmed M. bovis in its feces at the start of the experiment. M. bovis was
reported to survive for four months in naturally infected feces that was subsequently
spread on pasture and monitored. The Williams and Hoy experiments with artiﬁcially

infected cattle feces spread on pasture and monitored indicate that M. bovis can

11

predictably be recovered from feces on pasture for two to four months. Survival was
reported to be longer during the winter (5 months) and shorter during the summer (< 2
months) but these conclusions must be accepted with caution given, 1) the fact that the
source of the inoculums was emulsiﬁed tuberculous lungs; and 2) the fact that the dose of
M. bovis inoculums used for the Williams and Hoy autumn, winter and spring
experiments were 20 to 100 times larger than those used for summer experiments.
Williams and Hoy also reported the recovery of M. bovis from naturally infected feces
stored in cool dark conditions after 12 months and recovery of M. bovis from
experimentally infected feces stored in a similar manner for 2 years (Williams and Hoy,
1930).

Maddock expanded on the work of Williams and Hoy by examining the survival
of M. bovis in soil and on pasture grass as well as feces, and by using guinea pig
inoculation to assess the viability of the M. bovis recovered from samples (Maddock,
1933). In the 1933 study, Maddock reported a 6-month survival time for M. bovis in soil,
soil and feces mixtures and in feces exposed to the environment on pastures in south
England. In 1934, Maddock reported the failure to infect guinea pigs or calves grazed on
pasture known to be contaminated by the fecal shedding of M. bovis in the feces of cows
with generalized TB (Maddock, 1934). The 1934 study was followed by one of similar
design but in which pastures were artiﬁcially contaminated with an emulsion of
tubercular bovine lungs. Both guinea pigs (Cavia porcellus) and calves that grazed on
these M. bovis contaminated pastures and that were fed M. bovis contaminated grass

indoors, contracted tuberculosis (Maddock, 1936).

12

The development of intensive livestock farming, and the disposal of animal waste
from these operations, raised concerns about the survival and spread of pathogenic
bacteria, including M. bovis, in the environment (Wray, 1975). The transmission of
infectious mycobacteria in sewage efﬂuent or sludge used in crop irrigation led to a study
in the 1970’s designed to determine the survival of M. bovis BCG in soil and on
vegetables irrigated with efﬂuent or sludge containing BGC. Reported ﬁndings indicated
that BCG persisted for 11 days in efﬂuent sprayed soil, 8 days in sludge treated soil, 4- 6
days on the surface of radishes and only intermittently on lettuce (Van Donsel and
Iaﬂdn,1977)

Although the studies outlined above do indicate that M. bovis survives for
signiﬁcant periods of time on soil or pasture, removal of tuberculous and exposed cattle
from the premises, coupled with efforts to remove or expose any remaining organic
matter to sunlight, should ensure that the farm is free of M. bovis in the environment
within a 12-month period. This is, however, only true when cattle are the only source of
M. bovis infection. In disease systems in which a wild animal population has been
identiﬁed as a reservoir of infection for domestic cattle, traditional “cow-centered”
disease management protocols alone will not protect a farm from TB re-infection. Recent
studies designed to examine the persistence of M. bovis in the environment and the role
of indirect transmission in the epidemiology of bovine TB have been focused in New
Zealand, Australia, Kruger National Park in South Aﬁica, Ireland and Great Britain,
where a wildlife species has been identiﬁed as a reservoir of M. bovis infection (Corner,

2006).

13

Minimal environmental persistence of M. bovis was reported in an experimental
study of M. bovis infection in badgers (Mesles meles) and cattle conducted over a four
year period in south England (Little et al., 1982). TB transmission among badgers and
between badgers and cattle was conﬁrmed in these experiments but repeated attempts to
isolate M. bovis ﬁom environmental samples were negative. Attempts were made to
isolate M. bovis from the soil, hay, badger feces, scrapings from feed bowls and water in
the experimental enclosures. M. bovis was isolated from badger feces and water on one
occasion, and the authors concluded that M. bovis did not exist for long periods in the
environment.

An experimental study of the survival of M. bovis in soil and bovine feces
exposed to environmental conditions in north Queensland, Australia, indicated that M.
bovis survived 4 weeks but less than 8 weeks (Dufﬁeld and Young, 1985). Rapid death of
M. bovis was noted as a result of exposure to sunlight and the higher temperatures
recorded in north Queensland were thought by the authors to be the reason behind the
shorter M. bovis survival times in their study as compared to similar studies performed by
Williams and Hoy (1930) and Maddock (1933, 1934, 1936) in south England.

A study performed in New Zealand, where the brushtail possum (T richosurus
vulpecula) is the wildlife reservoir of M. bovis, concluded that environmental
contamination of pasture, the forest ﬂoor and possum dens was unimportant in the
epidemiology of tuberculosis in cattle, deer (Cervus elaphus) and possums in New
Zealand (Jackson et al., 1995). Cotton ribbons impregnated with M. bovis and set out at
various sites in a typical farm environment were used to assess the environmental

persistence of M. bovis. Weather data were collected to determine the effects on M. bovis

l4

survival. M. bovis survived 2 days on pasture and 14 days on the forest ﬂoor and in
possum dens. Survival time increased as daily temperatures decreased across all sites.
Researchers at Kruger National Park in South Aﬁica, concerned about disease
transmission among multiple species of wildlife, designed a study to determine the
maximum survival time of M. bovis in tissues and feces (Tanner and Michel, 1999).
Portions of tuberculous lung and lymph nodes from a naturally TB-infected African
buffalo (Syncerus caﬂer), and cattle feces inoculated with M. bovis, were exposed to a
range of environmental conditions throughout a 12-month period. Isolation of M. bovis
was possible from infected tissues for 6 weeks, and ﬁ'om experimentally contaminated
feces for 4 weeks, during winter months. The survival of M. bovis was limited to less
than 2 weeks during the warmer seasons of the year and in the absence of moisture.
Although indirect disease transmission seems feasible over the survival times reported,
the authors conclude that the limiting factors present under natural conditions
(withdrawal of water, predation, and the presence of competitive bacteria) would reduce
the survival time below that recorded in their experimental studies to a point that M. bovis
in the environment would not likely serve as a source of infection for cattle and wildlife.

in Kruger National Park.

1.2 THE ROLE OF INDIRECT TRANSMISSION IN THE EPIDEMIOLOGY
OF BOVINE TUBERCULOSIS IN CATTLE AND WHITE-TAILED DEER
IN NORTHERN LOWER MICHIGAN
Published reports on attempts to detect Mycobacterium bovis in the environment

in Michigan are limited to the efforts described by Pahner et a1. (2000), during a herd

15

depopulation process on a privately owned white-tailed deer farm in northeast Michigan
(Palmer et al., 2000). The attempt involved collecting feed, soil, fecal and water samples
for mycobacterial culture. Despite these efforts, M. bovis was not isolated from any of
the samples. The remainder of reports in the published literature have addressed the
issue of the environmental persistence and indirect transmission of M. bovis through
experimental disease transmission studies and a series of observational epidemiologic

studies. The relevant ﬁndings of these studies are discussed in the sub-sections below.

1.2.1 Experimental studies

Scientists and personnel ﬁom the Agricultural Research Service (ARS) of the
USDA have performed a series of experiments in the bio-security level III facilities at the
National Animal Disease Research Center (NADC) in Ames, Iowa, designed to
characterize the transmission of M. bovis among and between white-tailed deer and cattle.
Although the limitations of experimental transmission studies (stress and atypical
behavior related to conﬁnement of wild animals) apply, efforts were made to use white-
tailed deer ﬁom northeast Michigan and a strain of M. bovis isolated from a wild white-
tailed deer in northeast Michigan for these studies (Palmer et al., 1999).

An initial study was performed to develop an experimental model of natural M.
bovis infection in white-tailed deer (Palmer et al., 1999). The deer were infected by
instilling 1 X 103 colony forming units (CFU) of M. bovis (low dose) or 2 X 105 CFUs
(high dose) in each tonsilar crypt. Of importance in determining the potential role of
indirect transmission of M. bovis in the epidemiology of bovine TB, were the data that

conﬁrmed the presence of M. bovis in the saliva, nasal secretions and tonsilar swabs of

16

TB-infected deer. M. bovis shed in the saliva or nasal mucous of a T B-infected deer
could serve as a source of contamination of shared feeding or watering sites. If M. bovis
persists in the environment long enough, it could serve as a source of infection for
previously unexposed deer visiting the feeding or watering sites. Also of note, are the
data from this study that suggest that disseminated tuberculosis (gross lesions in multiple
sites) is not a prerequisite for the presence of M. bovis in saliva or nasal secretions of
infected deer.

The persistence of viable M. bovis on a variety of feeds oﬁen used for baiting
white-tailed deer in Michigan (alfalfa hay, shelled corn, sugar beets, apples, carrots and
potatoes) has been established in a laboratory setting (Whipple and Palmer, 2000). The
feeds were inoculated with an unknown amount of M. bovis and held for 16 weeks at
75°F, 46°F and 0°F and sampled at daily and weekly intervals. The study demonstrated
survival on all feed types stored at all temperatures at 1 week and for all feed types held
at 0°F for at least 12 weeks. These data indicate “short-term” survival of M. bovis at
temperatures consistent with the summer season in Michigan and prolonged persistence
of M. bovis on feed types at temperatures that would be found in Michigan during the
winter season.

Deer-to-deer transmission of M. bovis was studied by determining whether or not
M. bovis can be transmitted ﬁ‘om experimentally infected deer to uninfected in-contact
deer (Palmer et al., 2001). Transmission of M. bovis was 100% in this sample of 8 in-
contact deer indicating efﬁcient disease transmission and signiﬁcant susceptibility to M.
bovis in this species. Of importance in elucidating the potential role of indirect

transmission of M. bovis in this and other more natural situations are the following study

17

results: 1) Shedding of M. bovis was detected in the saliva and nasal secretions of
experimentally infected deer and less commonly in in-contact deer; 2) M. bovis was
detected in pelleted feed and hay, and the timing of the detection indicated that the feed
was most likely contaminated by the “naturally” infected in-contact deer; and 3) Bacterial
culture of feces from one pen and 1 of 8 fecal samples collected at necropsy and 1 of 4
urine samples collected at necropsy yielded M. bovis.

Although the TB transmission study described above produced data that indicate
that indirect transmission of M. bovis between deer through feed or a contaminated
environment might be possible, it was not designed to differentiate between routes of
exposure. A follow-up study was designed to explicitly examine shared feed as a means
of deer-to-deer transmission of M. bovis (Palmer et al., 2004b). Experimentally infected
and uninfected deer were housed separately with no direct contact between the groups.
Airﬂow was controlled to prevent room-to-room transfer of air and precautions were
taken to eliminate any transfer of M. bovis between rooms by personnel. Only uneaten
feed (complete pellet) was transferred ﬁ'om the pen holding the infected deer (4
individuals) to the pen holding the uninfected deer (4 individuals) for 136 days of feed
sharing. All of the deer exposed to the feed previously offered to experimentally-infected
deer exhibited gross and microscopic lesions consistent with TB at necropsy and isolation
of M. bovis was conﬁrmed from at least one cultured tissue in each animal. This study
clearly demonstrates indirect transmission of M. bovis through contaminated feed from
infected white-tailed deer to naive “non-contact” deer.

The role of indirect transmission of M. bovis in interspecies disease dynamics in

Michigan is of particular interest when attempting to explain deer to cattle and/or cattle to

18

deer transmission events. The presence of M. bovis infection in cattle from over V2 of the
cattle herds conﬁrmed as TB-positive in Michigan have had no relationships (fence-line
contact, or cattle contact through movement) with other domestic cattle herds affected by
bovine TB (O'Brien et al., 2006). It is presumed that the remaining herds have become
infected by interspecies transmission of M. bovis from deer (O'Brien et al., 2002).
Interspecies transmission of M. bovis from deer to cattle through indirect contact was
examined by exposing cattle (9 six-month-old calves) to pens previously occupied by
experimentally infected deer (Pahner and Whipple, 2000). The pen switching was
carried out for 80 days. At day 77, all of the calves reacted positively to the comparative
cervical skin test for bovine TB. At day 177, the calves were necropsied and all
demonstrated gross and microscopic lesions consistent with TB and M. bovis was isolated
from all calves. Neither the speciﬁc substrate (water, ﬂoor or feed) contaminated with
M. bovis nor the speciﬁc discharge (nasal secretions, saliva, urine or feces) containing M.
bovis can be determined by this study, but it conﬁrms that M. bovis can be transmitted
from deer to cattle through a contaminated environment and indirect contact.

An expansion of the study described above, was designed to determine whether or
not cattle become infected with M. bovis after oral exposure and whether or not cattle
become infected with M. bovis when exposed to feed previously offered to M. bovis
infected white-tailed deer (Palmer et al., 2004a). In the oral exposure study, cattle were
exposed to M. bovis by feeding shelled corn mixed with 8.6 X 105 CFU of M. bovis (high
dose) or 9.9 X 102 CFU (low dose) over 5 consecutive days. At 133 days after
inoculation, the calves were euthanatized and examined for signs of established M. bovis

infection and lesions consistent with TB. Ingestion of M. bovis resulted in TB lesions or

19

M. bovis was isolated in 3 of 4 calves in the “low-dose” group and 1 of 4 calves in the
“high dose” group, indicating that cattle are susceptible to infection via the oral route of
exposure and at doses of M. bovis lower than those previously considered necessary for
infection via ingestion (O'Reilly and Dabom, 1995; Palmer et al., 2004a). The ﬁnal phase
of the study, in which calves were exposed exclusively to feed previously offered to M.
bovis infected deer, also conﬁrmed the feasibility of contaminated feed acting as a
vehicle in the indirect and interspecies transmission of M. bovis between white-tailed
deer and cattle. Four of the nine calves exposed to the feed previously offered to M. bovis
infected deer had lesions consistent with TB or had tissues from which M. bovis was
isolated at the end of the study.

The studies presented in this section clearly demonstrate the feasibility of indirect
transmission of M. bovis between white-tailed deer and cattle through M. bovis
contaminated feed and/or environment. The application of the ﬁndings of these
transmission studies, performed in a laboratory environment, to disease dynamics in the
natural setting have been questioned given the fact that animals, both domestic and wild,
housed in conﬁnement are often under a level of stress that might compromise their
immune systems. An additional drawback of transmission studies carried out in conﬁned
housing (unavoidable given the bio-security concerns when working with M. bovis) is
that animals in conﬁned housing will exhibit behaviors different from those observed in a
natural setting and these behaviors in conﬁnement (primarily close contact and feeding
ﬂour a single source) will generally increase both direct and indirect transmission of M.
bovis. Regardless of the limitations of these transmission studies, they do conﬁrm the

shedding of M. bovis in the secretions of infected deer and the subsequent contamination

20

of feed and the environment. The studies conﬁrm the susceptibility of cattle to relatively
low doses of M. bovis via the oral route and they demonstrate indirect transmission of M.

bovis among white-tailed deer and between white-tailed deer and cattle.

1.2.2 Lesion distribution, character and route of disease transmission
1.2.2.1 Lesion Distribution in White-Tailed Deer

The distribution of tuberculosis lesions in an infected animal has often been used
as an indication of the route by which the animal became infected with M. bovis. The
majority of tuberculosis lesions in cattle occur in the respiratory lymph nodes and lungs
and the most common and efﬁcient route of disease transmission is thought to be the
inhalation of aerosolized M. bovis on respiratory droplets produced by an infected animal
(Drobniewski et al., 2003). The less common discovery of a tuberculosis lesion in the
mesenteric lymph nodes of cattle has often been attributed to infection via the oral route
(Neill et al., 1994). Attempts have, therefore, been made to examine the distribution of
gross and microscopic tuberculosis lesions in Michigan white-tailed deer to assess,
among other objectives, what can be determined about the route of pathogen
transmission. Studies assessing the distribution of lesions in domestic cattle from TB—
positive farms in Michigan have been more limited and instead research has been more
focused on perfecting and assessing TB infection detection methods in cattle (Dunn et al.,
2005; Norby et al., 2004; Norby et al., 2005).

The distribution of lesions and extent of tissues infected with M. bovis was
examined in a population of captive white-tailed deer in Michigan that had been naturally

infected with M. bovis (Palmer et al., 2000). Fourteen of the 116 captive deer examined

21

were determined to be TB-positive based on the isolation of M. bovis from one or more
tissues. Nine of the 14 deer had lesions consistent with TB. Lesions were most
commonly seen in the medial retropharyngeal lymph node followed by the lung. Lesions
were also noted in the mediastinal lymph node, hepatic lymph node, mesenteric lymph
node, small intestine and palatine tonsil. This distribution of lesions is similar to that
described in the population of free-ranging white-tailed deer examined during the initial
surveys for TB in northeast Michigan in 1995-1996 when the outbreak in the region was
ﬁrst described (Schmitt et al., 1997). The 1995-1996 survey relied on the submission of
hunter-harvested deer so full carcasses were rarely (3 of 354) available for examination.
However, the medial retropharyngeal lymph node was again identiﬁed as the tissue most
likely to contain gross lesions consistent with TB in M. bovis infected deer. The medial
retropharyngeal lymph node was also found to be the most commonly affected tissue
among white-tailed deer that tested positive (5 8) in a total of 19,500 deer submitted to the
Rose Lake Wildlife Disease Laboratory for M bovis testing in 1999, as part of the on-
going surveillance efforts of the Michigan Department of Natural Resources. The vast
majority of these submissions also consisted only of the head, but when extracranial
lesions were noted the thorax, speciﬁcally the costal pleura, was the most common lesion
site (O'Brien et al., 2001).

The predominance of TB lesions in the medial retropharyngeal lymph nodes of
nattu'ally infected white-tailed-deer neither conﬁrms nor refutes the possibility that these
deer are infected with M. bovis indirectly via ingestion of M. bovis contaminated feed or
other substrate found in the environment. As has been pointed out by other authors,

involvement of medial retropharyngeal lymph node in deer is likely the result of drainage

22

ﬁom the palatine tonsil, and due to its anatomical location, the tonsil would process M.
bovis in the oropharynx whether it was initially inhaled or ingested (Palmer et al., 2002d).
A distinction, however, has been noted in TB lesions in white-tailed-deer infected with
M. bovis experimentally via the aerosol or “oral” route. The TB lesion distribution
recorded in deer known to be infected orally via intratonsilar instillation of M. bovis (low
and high doses) or those presumed to be infected orally via exposure to contaminated
feed, was similar to that seen in naturally infected deer in Michigan with lesions
predominantly found in the medial retropharyngeal lymph nodes with the tonsil culturing
positive for M. bovis throughout the course of infection (Pahner et al., 2002a, 2002b;
Palmer et al., 1999). The TB lesions in deer experimentally infected with a low dose of
aerosolized M. bovis were distributed primarily in the lungs, tracheobronchial and
mediastinal lymph nodes, with fewer lesions in the medial retropharyngeal lymph nodes
and fewer tonsils that cultured positive for M. bovis (Pahner et al., 2003).
1.2.2.2 Lesion Distribution in Cattle

The information available on the distribution of lesions in Michigan cattle
involved in the current outbreak of bovine TB is based on a review of the necropsy
records of the cattle culled ﬁom the ﬁrst eight farms identiﬁed as TB-positive since 1998.
Tuberculosis lesions were detected in 45 animals and the breakdown by location was
69% in the chest, 47% in the head and neck, and 24% in the abdomen. Animals with
lesions limited to only one site included 33% with lesions only in the chest, 20% with
lesions only in the head and neck and 2% with lesions only in the abdomen (Norby,
personal communication). This distribution of lesions is similar but not identical to

those reported in cattle ﬁom a TB-positive farm identiﬁed in the US. before the

23

Michigan outbreak (Whipple et al., 1996). The animals in the 1996 study were subjected
to a similar full postmortem exam with bacterial culture of tissues for M. bovis as that
performed for the ﬁrst 8 herds identiﬁed as TB-positive in the Michigan epizootic. The
breakdown of lesions by site in the 1996 non-Michigan herd was 60% in the chest, 26.7%
in the head and 13% in the abdomen. Reports of lesion distribution in naturally infected
TB-positive cattle from different regions of the world are based primarily on slaughter
surveillance data. A study of 2,886 cattle with conﬁrmed tuberculosis lesions identiﬁed
in the slaughter houses of Northern Ireland over 6 year period indicated that 57%
annually had lesions only in the chest, 23% annually had lesions only in the head and
3.2% annually had lesions only in the abdomen (mesenteric lymph nodes) (Neill et al.,
1994)

As with the data on lesion distribution in naturally infected white-tailed deer in
Michigan, the distribution of lesions in cattle identiﬁed as part of the current TB outbreak
in Michigan provides little deﬁnitive information on the route of disease transmission. It
is generally accepted that the presence of a primary tuberculosis lesion in the lungs and
thoracic lymph nodes is the result of aerosol exposure of cattle to M. bovis and that
lesions in the intestinal tract are the result of cattle ingesting food and/or water
contaminated with M. bovis (Kaneene et al., 2002a). This strict delineation between
lesions that are the result of ingestion of M. bovis in contaminated food vs. inhalation of
via exposure to aerosolized M. bovis has been challenged by a number of the
experimental transmission studies performed at the NADC in Ames, Iowa, and described
in Section 1.3.1 (Pahner et al., 2004a; Palmer and Whipple, 2000). Both cattle that were

inoculated orally with M. bovis in feed, and those presumed to be infected orally when

24

presented feed previously offered to M. bovis infected deer, developed lesions primarily
in the lungs and associated lymph nodes (tracheobronchial and mediastinal) with few
lesions reported in the medial retropharyngeal lymph nodes, no lesions reported in the
mesenteric lymph nodes and only one lesion, in a hepatic lymph node, reported in the
abdomen.
1.2.2.3 Lesion Character

Gross TB lesions in lymph nodes of experimentally infected white-tailed deer
(Palmer et al., 2002a) and some naturally infected deer (O'Brien et al., 2001; Schmitt et
al., 1997) in Michigan have been characterized as containing abundant purulent exudates
surrounded by a layer of inﬂammatory cells and a light ﬁbrous capsule. The frequent
presence of liqueﬁed lesions in the lymph nodes of the head of TB positive deer may be
important in the dissemination of disease and the indirect transmission of M. bovis by
increasing the likelihood that M. bovis is shed in the nasal exudates and oral secretions of
infected deer. However, liqueﬁed lesions are not consistently recorded, even in deer with
disseminated disease.
1.2.2.4 Route of Disease Transmission

The distribution of lesions described in naturally infected deer and cattle in the
Michigan TB epidemic support transmission of M. bovis through contaminated feed as a
potentially important means of TB transmission among and between wild white-tailed
deer and domestic cattle. The distribution of lesions seen in naturally infected white-
tailed deer in Michigan is similar to the distribution seen in orally infected white-tailed
deer, as compared to deer infected via inhalation of aerosolized M. bovis. This is an

indication that inhalation (necessitating close contact between animals) is not the only

25

important route of disease transmission among white-tailed deer. Lesions recorded in
cattle from TB farms in Michigan, and those experimentally infected through the oral
route, were located primarily in the lungs and associated lymph nodes. It is possible that
M. bovis can be aerosolized over feed piles or within the oral cavity during chewing and
eructation. But the distribution of lesions in the lungs and associated lymph nodes does
not necessarily indicate direct transmission of via nose-to-nose contact between animals.
In addition, the time point in the course of infection at which an animal is examined has
been shown to inﬂuence the pattern of TB lesion distribution in cattle (Goodchild and
Clifton-Hadley, 2001; Menzies and Neill, 2000). Conclusions about routes of disease
transmission based on the recorded distribution of TB lesions found within an animal
should be addressed with caution. This is of particular concern in the population of
cattle in northeast Michigan since they are ﬁ'equently tested for exposure to M. bovis and
will likely be identiﬁed as positive during the early stages of bovine TB disease. The
absence of extra-pulmonary lesions in Michigan cattle should not rule out their exposure

to M. bovis via the oral route.

1.2.3 Observational studies in Michigan

Epidemiological studies and disease modeling have been used to quantify the
relative importance of various sources of M. bovis transmission in cattle and other species
(Menzies and Neill, 2000). Observational case-control studies designed to identify risk
factors for disease transmission can, when coupled with the ﬁndings of experimental
studies, improve our understanding of disease dynamics. A number of studies have

focused on Michigan’s recent M. bovis outbreak and provide various clues to the

26

potential role of indirect transmission of bovine TB between and among cattle and
wildlife populations in the state.
1.2.3.1 Observation Studies of White-tailed Deer

A retrospective study of Mycobacterium bovis in white-tailed deer in Michigan,
based on data ﬁ'om 5 years of a ﬁeld surveillance program, provides information on
trends in apparent prevalence of disease and various factors associated with M. bovis
detection in this population (O'Brien et al., 2002). The risk factors associated with M.
bovis infection identiﬁed in this study were age, sex, age/sex interaction and geographic
location; older male deer (2 2 years), female deer (3 3 years) and location within the TB
“core”! area, signiﬁcantly increased the odds of a deer being TB positive. Explanations
for the disease risk factors identiﬁed do not necessarily point to indirect transmission of
M. bovis as an important means of disease transmission among this population, however,
the dispersal behavior of male deer would provide them the opportunity to contact a
larger number of potentially M. bovis contaminated feed sites in addition to more
potentially contaminated individuals during their lifetime. Data available on Michigan
elk (Cervus elaphus), also included in the TB ﬁeld surveillance program, provide
evidence of the occurrence of indirect disease transmission (O'Brien et al., 2005). As
pointed out by O’Brien et al., (2005), elk and deer will generally maintain spatial
separation, so interspecies transmission of M bovis more likely occurs through the
contamination of feed and bait piles known to be visited by both deer and elk in the area.
The likely occurrence of indirect disease transmission between white-tailed deer and elk

supports other evidence (lesion distribution and historical practice of winter feeding) that

 

' The bovine TB “core” area is deﬁned as the area where the four comers of Montmorency, Alpena, Oscoda
and Alcona counties meet and includes Michigan Department of Natural Resources deer management unit
number 452.

27

indirect transmission of M bovis through contaminated feed, also contributes to bovine
TB transmission among white-tailed deer and between deer and cattle.

The role of supplemental feeding of wild white-tailed deer, and the inﬂuence of
the practice on the apparent prevalence of bovine TB in Michigan deer, was investigated
by characterizing the association between the risk of M bovis infection and supplemental
feeding practices in the same geographic area (Miller et al., 2003). Although potentially
biased by a high non-response rate among landowners in the region, the data collected
suggest that supplemental feeding practices associated with an increasing risk for bovine
TB in deer were generally indicators of large-scale feeding practices (number of deer fed,
quantity of fruits and vegetables provided, quantity of grain provided and the spreading
of grain). These feeding sites congregate deer and will facilitate both direct and indirect
transmission of M bovis. The provision of larger quantities of feed, however, would also
result in the availability of feed at a single location over a longer period of time;
circumstances that would enhance the likelihood of indirect transmission of M bovis via
the contamination and subsequent ingestion/inhalation of M bovis in feed.
1.2.3.2 Observational Studies of CaLle

Factors associated with the occurrence of bovine TB in cattle on farms in
northeast Michigan were assessed by performing a matched case-control study of 68
farms (17 “cases” farms and 51 “controls”) (Kaneene et al., 2002b). The relationship
between a farm’s TB status and farm management practices and environmental factors
were assessed with multivariable logistic regression analysis. Factors associated with a
reduced risk of TB were the percentage of open land surrounding the farms and the deer

exclusion index (a combination of factors relating to exclusion of deer from cattle

28

housing areas). Univariate analyses suggested an association between an increased risk
of TB and the prevalence of TB in wild deer in the surrounding area, the number of other
TB farms in the same TRS (square-mile township, range, section block), and the presence
of ponds or creeks in cattle housing areas, although these factors did not maintain their
signiﬁcance when adjusted for confounding in the multivariable model.

The results of the case-control study described above contribute to our
understanding of the potential role of indirect transmission in the epidemiology of bovine
TB in northeast Michigan. The identiﬁcation of “TB farms in the same TRS” as a risk
factor for disease reminds us that fence-line contact and local “sharing” of cattle not
necessarily identiﬁed as a “cattle purchase” may contribute to cattle-to-cattle M bovis
transmission in the region. The other factors identiﬁed as signiﬁcant indicate that
opporttmities for indirect transmission of M bovis between deer and cattle increase the
likelihood of a TB positive farm. The protective “deer exclusion index” indicates that
cattle not contained in housing, but presumably with access to open pastures and adjacent
wood lots where deer are present, are more likely to become infected with TB. These
data suggest that opportunities for overlap in infected deer and cattle feeding/ grazing and
watering areas enhances interspecies M bovis transmission. Given the lack of observed
close contact between cattle and deer, indirect transmission through a contaminated
environment is the most likely route of disease transmission.
1.2.3.3 Observational Studies of Other Wildlife

The presence of a wildlife reservoir or maintenance host of M bovis other than
the white-tailed deer has been investigated by surveying for the presence of M bovis in

free-ranging wildlife (Bruning-Fann et al., 2001; Bruning-Fann et al., 1998; Payeur et al.,

29

2001; Schrrritt et al., 2002) and performing experimental disease transmission studies
with some of these species (Butler et al., 2001; Diegel et al., 2002; Fitzgerald et al., 2003;
Palmer et al., 2002c). The same strain of M bovis found in cattle and white-tailed deer in
Michigan has also been detected in elk, black bear (Ursus americanus), bobcat (Felis
ruﬁts), coyote (Canis lantrans), opossum (Didelphis virginiana), raccoon (Procyon
later), and red fox ( Vulpes vulpes) in the bovine TB endemic area of the State (Payeur et
al., 2001). The route of disease transmission in the furbearers is thought to be via
ingestion of M bovis from an infected deer carcass during natural scavenging events
(Bruning—Fann et al., 2001; Bruning-Fann et al., 1998).

The scarcity of gross and histologic lesions in bovine TB positive ﬁrrbearers,
coupled with data from experimental inoculation studies, suggest that none of these
species would likely serve as effective maintenance hosts or reservoir of M bovis.
Additionally, there is little evidence that they would act as a source of environmental
contamination with M bovis. The detection of M bovis in Michigan elk does, however,
raise concerns about the potential for the establishment of M bovis in this population

(O'Brien et al., 2006) and therefore its potential role in indirect transmission of M bovis.

1.3 CONCLUSIONS

The prevalence of bovine TB in deer varies across the landscape of the endemic
region of Michigan as do the environmental conditions and farm management practices
that would facilitate or discourage the indirect transmission of M bovis among or
between livestock and wildlife species. The published literature on the epidemiology of

bovine TB in other systems around the world contains reports that conﬁrm the feasibility,

3O

but do not necessarily emphasize the role, of indirect transmission of bovine TB among
or between wildlife and livestock.

Experimental and observational epidemiologic studies, with a focus on the recent
discovery of bovine TB in white-tailed deer and cattle in Michigan, have investigated
various aspects of disease dynamics and the potential role of indirect transmission of M
bovis in the epidemiology of bovine TB in Michigan. Lacking in the investigative efforts
in this area are more intensive environmental sampling in locations where bovine TB
transmission is known to have occurred, and the determination of the length of time M
bovis remains in the environment as a potential source of infection for cattle and/or
wildlife.

The epidemiology of bovine TB in every geographical location of its occurrence is
complex (Monis et al., 1994). A comprehensive study of the epidemiology of bovine TB
in Michigan requires investigating the role of indirect transmission in the dynamics of
disease in both domestic livestock and wildlife populations. Understanding the persistence
of M bovis in the environment, and its potential role as a source of infection for susceptible
hosts, is essential in the development of epidemiologically appropriate control and
management policies. Informed decisions regarding the feeding and baiting of wildlife,
repopulating cattle farms, designing surveillance programs, and assigning regional bovine
TB accreditation zones can only be made with an improved understanding of the role of

indirect transmission in the epidemiology of bovine TB in Michigan.

31

Chapter 2
OPTIMIZING THE ISOLATION OF M YCOBA CTERI UM BOWS FROM
ENVIRONMENTAL SAMPLES

2.0 ABSTRACT
Objective: To optimize methods for detecting Mycobacterium bovis in samples collected
from the environment by reducing culture contamination rates and increasing the
minimum detection level of the process.
m: A series of experimental inoculation studies designed to compare two techniques
for processing environmental samples for M bovis isolation by bacterial culture.
Sample Population: M bovis inoculated substrates included hay, soil and water. CB-18
and NaOH-based methods were used to process the inoculated samples for isolation of M
bovis by mycobacterial culture.
Procedure: M bovis recovery and culture contamination rates were compared in samples
processed with CB-l 8 and NaOH sample processing methods. The minimum detection
level of M bovis in feed, soil and water was determined for the CB-18 based method.

Results: CB-18 sample processing methods markedly reduced culture contamination

 

rates. The minimum detection level in the absence of contamination was similar for
samples processed with the CB-18 and NaOH-based methods. The minimum detection
level was 120 CFU per processed soil, water or hay sample.

Conclusions and Clinical Relevaﬂe: The development of a more efﬁcient and effective
method for examining samples ﬁ'om the environment for the presence of M bovis is an

important step in the endeavor to characterize the persistence of M bovis in the

32

environment and the potential role of its persistence in the indirect transmission of bovine

tuberculosis among and between susceptible species.

2.1 INTRODUCTION

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become
established in free-ranging wildlife in northeast lower Michigan (O'Brien et al., 2005).
White-tailed deer have been identiﬁed as the current primary reservoir host for TB in the
region and they are the presumed source of M bovis infection in cattle in over 50% of the
herds identiﬁed as TB-positive in Michigan (O'Brien et al., 2002). Understanding the
role of indirect transmission of M bovis in the epidemiology of bovine TB is of particular
importance in Michigan where disease transmission between deer and cattle is thought to
occur in the absence of observations of close contact between the species (DeLiberto et
al., 2004). The ability to detect and quantify the presence of viable Mycobacterium bovis
in the environment is an essential ﬁrst step in understanding the role of indirect
transmission in the epidemiology of bovine TB. Information regarding the persistence of
M bovis in the environment is essential for the improvement of protocols for cattle farm
bio-security, the maintenance of appropriate restrictions on feeding and baiting free-
ranging white-tailed deer and other wildlife, and for informing human health and safety
regulations regarding the potential public health hazards of bovine TB.

The challenges associated with the detection of M bovis in the environment fall
into three major categories. First, environmental samples (soil, feed, fecal material,
pond/stream water, etc.) contain large numbers of saprophytic bacteria, molds and other

potentially infectious organisms. These other organisms interfere with the isolation of

33

mycobacteria by overgrowing and out-competing the mycobacteria during the bacterial
culture process. A decontamination step is, therefore, essential. The decontamination
process not only eliminates saprophytic organisms but also reduces the viability of
mycobacteria in the specimen and, therefore, interferes with the sensitivity of detection of
mycobacteria by bacterial culture methods (Kent and Kubica, 1985; Yajko et al., 1995).

Secondly, evidence suggests that the shedding of M bovis ﬁ'om both infected deer
(Palmer et al., 2001) and infected cattle (Goodchild and Clifton-Hadley, 2001; Neill et
al., 1988) is intermittent. Intermittent or low level shedding of M bovis coupled with the
relatively small size—when compared with the scale of the potentially contaminated
environment—of sample that can be collected and processed for mycobacterial culture
results in a greatly reduced chance of collecting a contaminated substrate in the
environment.

Thirdly, the properties of M bovis make it particularly difﬁcult to process and
culture. Like other mycobacteria, M bovis has a tendency to clump and form cords so it
is often not evenly distributed in a processed sample. Its thick, waxy cell wall makes it
buoyant and reduces the success of centriﬁrgation methods aimed at concentrating the
organism in the sample. In addition, M bovis requires 6-8 weeks for growth on solid
media, prolonging the time the specimen must be maintained at optimal conditions (37°
C, moist and free of other microbial contamination).

A few attempts have been made to isolate M bovis from environmental substrates
in areas identiﬁed as sites of natural transmission of bovine TB in Michigan including a
captive white-tailed deer facility (Palmer et al., 2000) and one of the ﬁrst cattle farms

identiﬁed as TB-positive in the current outbreak of M bovis in Michigan (Kaneene,

34

personal communication). Environmental samples (feed, soil, water and fecal material)
collected from these sites were all negative for M bovis. It has been difﬁcult to
determine whether the lack of positive results is due the limited persistence of M bovis in
the environment or the challenges associated with detection of M bovis in environmental
substrates. Studies performed in indoor bio-safety level III pen facilities have been
designed to examine M bovis transmission among deer (Pahner et al., 2004b; Palmer et
al., 2001) and between deer and cattle (Palmer et al., 2004a). These studies have yielded
positive M bovis cultures from environmental samples collected ﬁ'om the pen ﬂoors or
feeding areas. The M bovis found in the environment is thought to have played a role in
the indirect transmission of TB under these experimental conditions.

Epidemiologic studies of bovine TB in other regions have also failed in efforts to
isolate viable M bovis from environmental samples collected under natural disease
transmission conditions (Pillai et al., 2000). Researchers interested in the persistence of
M bovis in the environment have turned to experimental inoculation studies and an
assessment of the conditions that support or inhibit M bovis survival (Dufﬁeld and
Young, 1985; Jackson et al., 1995; Little et al., 1982; Maddock, 1933, , 1934, , 1936;
Tanner and Michel, 1999; Williams and Hoy, 1930; Wray, 1975). A recent study has
described the successful identiﬁcation of M bovis DNA in the environment under natural
disease transmission conditions (Young et al., 2005) but this does not address the
epidemiologically important question of the persistence of viable and infectious M bovis
in the environment.

The standard procedure for processing samples for mycobacterial isolation

involves the mixing and grinding of the substrates, settling and the removal of the

35

supernatant for further processing, a NaOH-based decontamination step, and
centrifugation to concentrate any mycobacteria in the sample before inoculating culture
media for mycobacterial isolation (Payeur et al., 2001). This standard procedure was
followed for processing the environmental samples collected ﬁom a large enclosure
holding white-tailed deer infected with M bovis (Palmer et al., 2000).

A new method for processing respiratory specimens for mycobacterial detection
with C gg-Carboxypropylbetaine was published in 1998 (Thornton et al., 1998a). The Cu;-
Carboxypropylbetaine is a zwitterionic detergent. It replaces NaOH as the primary
decontamination agent and its addition to processed samples is thought to decrease
surface tension and counteract the natural buoyancy of mycobacteria and facilitate the
more efﬁcient collection of the bacilli (Thornton et al., 1998a).

The CB-18 method was initially reported to signiﬁcantly increase the
mycobacterial detection sensitivity of both culture and acid-fast smear methods, but it
also resulted in an increase in the rate of non-mycobacterial contamination of liquid
cultures (Thornton et al., 1998a). A sediment resuspension buffer with lecithin, to reduce
toxicity, and a mixture of lytic enzymes (lysosyme, zymolyase, Cytophaga and
T richoderma extracts) to reduce contamination was, therefore, added to the CB-18
sample processing protocol (Thornton et al., 1998c).

A follow-up study by Thornton, et al, 1998b, produced results that suggest that M
bovis BCG is particularly sensitive to NaOH, and that processing with this method may
generate a large number of false-negative culture results (Thornton et al., 1998b). The
effect of CB-18 replacement of NaOH as the primary decontamination agent on the

recovery rates of 5 M tuberculosis complex isolates and 4 non-M tuberculosis

36

mycobacterial isolates was studied. The recovery rate of mycobacteria were signiﬁcantly
higher in those specimens processed with CB-l8 as compared to NaOH across isolates,
but the difference in recovery rates (1.6% to 2.3 % for NaOH, and 70.9% to 84.4% for
CB-18) was markedly higher for the M bovis BCG isolate when compared to other M
tuberculosis complex bacteria, and this may be true for all M bovis isolates.

An improved protocol for processing samples collected from the environment for
mycobacterial culture is necessary for the further investigation of the role of M bovis
persistence in the environment and indirect transmission in the epidemiology of bovine
TB in Michigan. The CB-18 sample processing method has resulted in improved
mycobacterial detection when applied to various specimens in a number of different
settings (Comejo et al., 1998; Laserson et al., 2005; Manterola et al., 2003; Ozbek et al.,
2003; Padilla et al., 2005; Thornton et al., 1999; Thornton et al., 1998a). Experimental
M bovis inoculation studies were, therefore, designed to create and evaluate a method for
processing environmental samples collected from areas of suspected bovine TB
transmission in Michigan and elsewhere. The speciﬁc hypothesis tested is that the CB—18
method signiﬁcantly reduces the rate of contamination of M bovis inoculated
environmental substrates and reduces the probability of false-negative results associated
with the standard NaOH-based method. To test the stated hypothesis, M bovis recovery
and culture contamination rates were compared in samples processed with both CB-18
and NaOH. The minimum detection level of M bovis in feed, soil and water was

determined for the CB-18 based method.

37

2.2 MATERIALS AND METHODS
2.2.1 Facility, culture, media, and growth conditions

All Mycobacterium bovis experimental inoculation studies were performed in the
mobile bio-safety level HI (BL3) laboratory on the campus of Michigan State University
(MSU). M bovis was obtained from a frozen culture of M bovis originally isolated in
2001 ﬁom a 5-year-old Charolais cow from northeast Michigan with gross and
microscopic lesions consistent with bovine TB. One ml of the previously frozen culture
was added to 10 ml of Middlebrook 7H9 Broth with Middlebrook ADC Enrichment for
cultivation of mycobacteria (Becton-Dickenson, Cockeysville, Maryland 21030, USA).
Multiple 10 ml vials of M bovis inoculated 7H9 Broth were incubated at 37° C for 21 to
30 days or until the cell density reached 1 X 108 to 5 X 108 colony forming units
(CFU)/ml. The culture was then distributed into 1.5 ml portions, used immediately or
frozen at -80° C.
2.2.2 Environmental substrates

Grass hay, water and soil were selected as environmental substrates for M bovis
inoculation, sample processing and isolation experiments. Grass hay was collected from
the feed storage area of the MSU Large Animal Veterinary Teaching Hospital in East
Lansing, Michigan. Soil was collected ﬁom the Baker Woodlot (Rachana Raj endra
Neotropical Bird Sanctuary) located in the south central section of the MSU campus.
Water was collected from the large pond at the center of the Baker Woodlot and from the

Red Cedar River at the Farm Lane Bridge on the MSU campus.

38

Samples of soil and hay were stored in 5-gallon capacity black plastic bags and
water was stored in clear 1 liter plastic bottles. Once collected all environmental
substrates were stored at 4° C with no exposure to light.

Sets of environmental substrates for the M bovis inoculation experiments were
prepared as follows. Soil samples were divided into 2 gm portions and placed in 3 X 7
inch Whirl-Pak® bags. Hay samples were chopped with scissors and 2 gm portions were
placed in sterilized Ball® pint regular mason jars. Water was divided into 7.5 m1
portions and place in 50 ml conical centrifuge tubes.

Approximately half of the bulk samples of soil, hay and water were autoclaved for
2 hours at 121° C and 20 psi before they were apportioned into the experimental samples
sets described above. The autoclaved environmental substrates were used to set up the

“pre-sterilized” environmental substrates used in the M bovis inoculation studies.

2.2.3 Inoculation Experiment 1 (CB18 vs. NaOH Processing)

This experiment included 8 soil samples (4 sterilized; 4 non-sterilized), 8 hay
samples (4 sterilized; 4 non-sterilized), and 8 water samples (4 sterilized; 4 non-
sterilized). Two ml of thawed M bovis liquid culture were added to each sample. Four
replicates of each sample type, 2 sterilized before M bovis inoculation and 2 not
sterilized before inoculation, were processed with CB-l8. The remaining 12 samples

were processed with NaOH.

39

2.2.4 Inoculation Experiment 2 (C318 vs. NaOH Processing at Different M bovis

Concentrations)

This experiment included 16 soil samples (8 sterilized; 8 non-sterilized), 16 hay
samples (8 sterilized; 8 non-sterilized), and 16 water samples (8 sterilized; 8 non-
sterilized). The 48 samples were divided into 8 sets of 2 samples (1 sterilized; 1 non-
sterilized) of each substrate. The sample sets were inoculated with either 2 ml of M
bovis liquid culture, a 1:10 dilution, a 1:100 or a 1:1000 dilution. The M bovis liquid
culture was diluted with Middlebrook 7H9 Broth. The inoculated samples were divided
into two equivalent sets and processed with either the CB-18 or NaOH. Samples of the
M bovis liquid culture and 3 dilutions were inoculated onto plates with modiﬁed
Middlebrook 7Hll Agar (Diagnostic Center for Population and Animal Health
(DCPAH), Lansing, MI, USA) and Selective 7H11 agar (Becton-Dickinson) to monitor

for M bovis growth.

2.2.5 Inoculation Experiment 3 (CB18 vs. NaOH Processing 4 Different Known M
bovis Concentrations)

This experiment included 16 soil samples (8 sterilized; 8 non-sterilized), 16 hay
samples (8 sterilized; 8 non-sterilized), and 16 water samples (8 sterilized; 8 non-
sterilized). The 48 samples were divided into 8 sets of 2 samples (1 sterilized; 1 non-
sterilized) of each substrate. The sample sets were inoculated with either Dilution l (1 ml
of M bovis liquid culture + 50 ml of 7H9), Dilution 2 (1 ml of Dilution 1 + 50 ml of

7H9), Dilution 3 (1 ml of Dilution 2 + 50 m1 of 7H9) or Dilution 4 (1 ml of Dilution 3 +

40

50 ml of 7H9). Four (Dilution 1-4) inoculated sample sets were processed with CB-18
and the remaining 4 sample sets were processed with NaOH. Exactly 100 p1 of the 4
dilutions of M bovis were inoculated onto plates with modiﬁed Middlebrook 7H11 Agar
(DCPAH) and Selective 7Hll agar (Becton-Dickinson) to monitor for M bovis growth

and to determine the M bovis concentration (CFU/ml) of each dilution.

2.2.6 Inoculation Experiment 4 (CB18 Processing, with and without Liquifaction,
of Samples with 2 Different Known M bovis Concentrations Stored for
Varying Times Under Different Conditions)

This experiment included 32 soil samples (16 sterilized; l6 non-sterilized), 32 hay
samples (16 sterilized; 16 non-sterilized), and 32 water samples (16 sterilized; 16 non-
sterilized). The 96 samples were divided into 4 sample sets. Each set contained 4
samples (2 sterilized; 2 non-sterilized) of each substrate that were inoculated with 1 ml of
Dilution l (5 ml of M bovis liquid culture + 75 ml of 7H9) and 4 samples (2 sterilized; 2
non-sterilized) of each substrate inoculated with 1 ml of Dilution 2 (1 ml of M bovis
liquid culture + 75 ml of 7H9) of M bovis liquid culture. One sample set was processed
for mycobacterial isolation at time 0 (on the same day as the inoculation), one sample set
was held for 72 hours at room temperature before processing, one sample set was held for
72 hours at 4° C before processing and one sample set was stored for 18 days at ~20 ° C
before processing. All samples were processed with the CB-18 method. The samples
were further divided into 1 set treated with liquifaction solution and another set in which

water was substituted for liquifaction in the sample processing procedure.

41

Samples of the 2 M bovis dilutions were stored alongside the inoculated
environmental substrates. Exactly 100 pl of the 2 dilutions of M bovis were inoculated
onto plates with modiﬁed Middlebrook 7H11 Agar (DCPAH) and Selective 7H11 agar
(Becton-Dickinson) to monitor for M bovis growth and to determine the M bovis
concentration (CFU/ml) of each dilution at time 0, after 72 hours at room temperature,

after 72 hours at 4° C and after 19 days at -20 ° C

2.2.7 Inoculation Experiment 5 (CB18 Processing of Samples with 2 Different
Known M bovis Concentrations Stored for Varying Times Under Different
Conditions)

Inoculation experiment 5 was a repeat of inoculation experiment 4 with '/2 the
number of samples per set (12). Each set contained 2 samples (1 sterilized; 1 non-
sterilized) of each substrate that were inoculated with 1 ml of Dilution l (2.5 ml of M
bovis liquid culture + 37.5 ml of 7H9) and 2 samples (1 sterilized; 1 non-sterilized) of
each substrate that were inoculated with 1 ml of Dilution 2 (0.5 ml of M bovis liquid
culture + 39.5 ml of 7H9) of M bovis liquid culture. One sample set was processed for
mycobacterial isolation at time 0 (on the same day as the inoculation), one sample set was
stored for 72 hours at room before processing, one sample set was stored for 72 hours at

4° C before processing and one sample set was stored for 16 days at —20 ° C before

processing. All samples were processed with the CB-18 method. Exactly 100 pl of the 2
dilutions of M bovis were inoculated onto plates with modiﬁed Middlebrook 7H11 Agar
(DCPAH) and Selective 7H1] agar (Becton-Dickinson) to monitor for M bovis growth

and to determine the M bovis concentration (CFU/ml) of each dilution. In addition,

42

exact amounts (100 pl) of the processed samples were inoculated onto mycobacterial
culture plates to facilitate more precise determination of M bovis recovery rates and

minimum detection levels.

2.2.8 CB-18 Sample Processing
The CB-lST'“I TB Culture Kit with Lytic DeconTM H (Integrated Research
Technology, LLC, Quest Diagnostics Inc., Baltimore, MD) was used to process M bovis
inoculated environmental substrates in the “CB-18” sample sets of the experimental
studies. The sample processing procedure used was an adaptation of the protocol for
processing tissue specimens outlined in the ofﬁcial CB-18T" TB Culture Kit with Lytic
DeconT" H instruction booklet (Technology). The solutions and reagents necessary for the
CB-18"“l processing were made up according to the procedures outlined in the instruction
booklet. These included the following: 1) Decontamination Solution (20X Tris-citrate
Buffer, CB-18T" Stock, N-acetyl-L-cysteine or NALC and water); 2) 2X-Resuspension
Solution (lOX-Enzyme Stock-Trichoderma harzianum extract, lysozyrne and Lysobacter
extract and NALC); and 3) NALC-Citrate Liquefaction Solution trisodium citrate
dehydrate and NALC).
Environmental substrate specimens were processed in the following 10 steps:
1. Pulverize/Homogenize Sample
a. Add 5 ml liquefaction solution (all samples)
b. Add 7 .5 ml sterile water to soil and hay samples
c. Mix

i. Water Samples: Vortex on high for 30 seconds

43

ii. Soil Samples: Place in Stomacher® 80 (laboratory blender) on
medium for 30 seconds

iii. Hay Samples: Secure blade unit head and gasket on jar, invert
and blend with household blender on high for 30 seconds

. Place specimens upright and allow to settle for 30 minutes.

. Remove top 5 ml of ﬂuid for each specimen and transfer to 50 ml conical tube

containing 10 ml of Decontamination Solution.

. Vortex well and incubate at 37° C for 75 minutes.

. Add sterile water to 50 ml mark on each tube and mix.

. Centrifuge specimens at 3,000 g for 20 minutes.

. Decant pellet-containing tubes completely; use a pipette to remove all but 1-3
ml of liquid from samples without a visible pellet. Resuspend the pellet by
vortexing in supernatant backwash.

. Add 1 m1 of sterile water and mix. Remove 0.5 m1 of sample and transfer to
1.5 ml labeled cryo tube and freeze at —80° C.

. Add 1 ml of 2X Resuspension solution to each sample, mix and incubate for

45 rrrinutes at 37° C.

10. Remove samples ﬁom incubator and prepare to inoculate on mycobacteria

isolation media.

44

2.2.9 NaOH Sample Processing

An adaptation of the standard NaOH-based procedure for processing tissue
samples for mycobacteria isolation (Payeur et al., 2001) was used to process the M bovis
inoculated environmental substrates in the “NaOH” sample sets of the experimental
studies. Brieﬂy: An equal volume of phenol red nutrient broth (Becton-Dickinson) was
added to each sample. Sample containers were capped and/or sealed and the contents
were thoroughly mixed. Ten ml of the mixed sample suspension was transferred with a
large oriﬁce pipette to a 50 ml screw-cap conical centrifuge tube containing 5 ml of 0.5 N
NaOH. The samples remained in the NaOH for 20-30 minutes. Drops of 6.0 N HCl were
added to the sample suspension until the rrrixture turned yellow and then the suspension
color was brought back to pale pink with 1.0 N NaOH. The neutralized sample
suspension was then centrifuged at 6,000g (4540 rpm) for 20 minutes. The pellet and
approximately 85% of the overlaying ﬂuid was decanted. The sample sediment was

resuspended in the residual ﬂuid and used to inoculate the M bovis isolation media.

2.2.10 Mycobacterium bovis Isolation and Identification

A range of media, solid and liquid culture systems were used to isolate
Mycobacterium bovis in these experiments. For initial experiments (M bovis inoculation
experiments 1-3) both samples processed with NaOH and those processed with CB-18
were inoculated onto solid media slants containing modiﬁed Middlebrook 7H11 agar
(Becton-Dickinson) with sodium pyruvate (DCPAH), modiﬁed Middlebrook 7H10
(Becton—Dickinson) with sodium pyruvate (DCPAH), Lowenstein Jensen and Mitchison

7H11 Selective agar (Remel, Lenexa, Kansas 66215, USA). Sample sediments were also

45

inoculated into Bactec 12B liquid media (Becton-Dickinson) supplemented with the
antimicrobial PANTAT” (Becton-Dickenson) and erythromycin. In later experiments (M
bovis inoculatoin experiments 4-5) plates containing modiﬁed Middlebrook 7H11 agar
(Becton-Dickinson) with sodium pyruvate (DCPAH) and 7H11 Selective plates (Becton-
Dickinson) were used exclusively.

Solid media slants and plates were incubated at 37°C for 8-12 wk and examined
weekly for colony formation. Inoculated Bactec 12B vials were placed in a BACTEC
460TB system and monitored daily for indications of mycobacterial growth.

Positive mycobacterial cultures and colonies on solid media were subjected to an
acid-fast smear analysis to conﬁrm the presence of acid-fast bacteria using standard
protocols for slide preparation, staining and examination (Kent and Kubica, 1985).
Random samples were identiﬁed to species group, Mycbocaterium tuberculosis complex,

using a genetic probe (AccuProbes, Gen-Probe, San Diego, California 92121, USA).

2.2.11 Recording of Contamination and Calculation of M bovis Recovery Rates and
Minimum Detection Levels
The presence of contamination with non-mycobacteria in liquid and solid media,
on slants and plates, was recorded for every processed sample. The presence of
contamination on solid agar plates was further characterized according to the percent of
total area of the plate affected. “None” (corresponding to 0% of the plate), “some”
(corresponding to 5 25% of the plate), “yes” (corresponding to >25% and 5 50% of the

plate) or “very” (corresponding to >50% of the plate).

46

An M bovis growth curve experiment (concentration of CFU recorded over time)
was used to predict the concentrations of M bovis dilutions used in the inoculation
experiments. This was, however, always conﬁrmed by quantitative culture at the time of
sample inoculation. The conﬁrmed concentration of M bovis dilutions were compared to
the mycobacterial culture results of the processed specimens to determine the minimum
number of M bovis CFUs necessary per sample to detect their presence with
mycobacterial culture. The mycobacterial culture results of the inoculated pre-
sterilization substrates were used to compare sample processing recovery rates in the

absence of contamination.

2.2.12 Data Analysis

Descriptive statistics were generated in Excel (Microsoft Ofﬁce XP Professional).
Comparisons of contamination rates in the CB-l 8 and NaOH-processed samples were
conducted with SAS software (SAS version 9.0, Cary NC: SAS Institute, Inc., 2003)
using the Cochran-Mantel-Haenszel Chi-square or 2-tailed Fisher’s exact test where
appropriate. Odds ratios with 95% conﬁdence intervals that did not contain 1 were

considered signiﬁcant.

47

2.3 RESULTS
2.3.1 Comparison of CB-l8 and NaOH-based Processing of Environmental

Substrates for M bovis Isolation

The results of M bovis inoculation experiments 1 — 3 are presented in tables 2.1,
2.2 and 2.3. Environmental substrates (hay, soil and water) are signiﬁcantly less likely
to be contaminated if processed for mycobacterial culture with the CB-18 method as
compared to the NaOH-based method. The odds of contamination in NaOH processed
M bovis inoculated environmental substrates that were not pre-sterilized was 11 times
that of the same substrates processed with the CB-18 method [OR=11.0; 95% CI. (3.2,
37 .9)]. The actual concentration of M bovis used in dilutions 1-4 in inoculation
experiment 3 was 100 fold lower than intended. Relevant data on rates of contamination
are presented, however, M bovis detection in dilutions 2-4 would not be expected
irrespective of sample processing method.

The presence of contamination resulted in the inability to detect M bovis and the
reporting of false negative results. There was a negative correlation between the number
of contaminated samples and the number of positive results. CB-18 processing
outperformed NaOH processing methods for M bovis detection in the inoculated
environmental substrates that were not pre-sterilized because a greater number of false
negative results were produced by the samples processed with the NaOH based method.

The substrate that was most likely to produce contamination, regardless of sample

processing method, was soil.

48

2.3.2 Effect of Sample Storage Time and Conditions on M bovis Recovery and

Contamination Rates with CB-18 Processing

The results of M bovis inoculation experiments 4-5 are presented in Tables 2.4.1,
2.4.2 and 2.5. The least contamination and least number of false negative results were
found in the pre-sterilized environmental substrates processed with CB-18 immediately
after inoculation (time 0). The number of contaminated samples and the number of false
negative results was highest for inoculated substrates held at room temperature for 72 or
24 hours before processing. The storage of samples at 4° C and ~20° C before CB-18
processing resulted in an intermediate number of contaminated samples and false
negative results. Samples held at -20° C in inoculation experiment 5 were all
contaminated during sample processing. M bovis isolation and contamination data were
not available for these samples.

False negative M bovis culture results in experiments 4 and 5 are associated with
the presence of contamination. Colony count data (not presented in the tables) suggests

that storage at room temperature and 4° C does not have an affect on the numbers of

viable M bovis bacilli, however, testing of M bovis stored at —20° C indicated a 5 fold
reduction in recoverable M bovis after a single freeze-thaw cycle.

The substitution of water for liquefaction in the CB-18 sample processing
procedure (inoculation experiment 4) did not affect sample contamination or M bovis

detection.

49

2.3.3 Minimum Detection Levels

In the absence of contamination, samples processed with CB-18 and those
processed with NaOH performed equally in the detection of M bovis via mycobacterial
culture. Both the NaOH and CB-18 sample processing methods, regardless of the
substrate type, decreased the number of viable M bovis detectable by mycobacterial
culture. In inoculation experiment 2 approximately 120 CFUs of M bovis were delivered
to each substrate. Both CB-18 and NaOH-processed samples consistently detect M bovis
at this original concentration. Colony counts on plates inoculated with exactly 100 pl of
processed specimens originally inoculated with Dilution 1 (120 CFU) yield 1-2 CFU.
The ﬁnal volume of processed specimen was approximate 1 ml, indicating a 80%-90%
loss of M bovis bacilli during processing. The minimum detection level of M bovis in

the volume of environmental substrates processed in this series of experiments was 120

CFUs.

2.3.4 Mycobacterial Culture Media

The use of Bactec 12B liquid media (Becton-Dickinson) resulted in high rates of
contamination in samples processed with NaOH and CB-18 and the culture media was
not used in inoculation experiments 4~5. The least contamination was recorded on plates
containing modiﬁed Middlebrook 7H1] agar (Becton-Dickinson) with sodium pyruvate
(DCPAH) and 7Hll Selective plates (Becton-Dickinson). The quantiﬁcation of
contamination and the differentiation and identiﬁcation of individual M bovis colonies

was difﬁcult on slants.

50

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56

2.4 DISCUSSION

The detection of Mycobacterium bovis in substrates collected from the
environment under natural conditions of bovine TB transmission will always be a
challenge. The low level and intermittent shedding of M. bovis by infected animals (Neill
et al., 1988; Palmer et al., 1999), and the limits on the volume of substrate that can be
collected and processed for mycobacterial detection, reduce the probability that M. bovis
will be present in a sample collected for testing from a large potentially contaminated
landscape. The detection of M. bovis in a contaminated environmental substrate can,
however, be improved by the development of sample processing and bacterial culturing
methods that limit the growth of non-mycobacterial species and improve the recovery of
M. bovis.

The standard method for processing veterinary samples, primarily tissues, for
mycobacterial detection by culture utilizes NaOH in its decontamination step (Payeur et
al., 2001). This method has been applied to the environmental samples collected in the
ﬁeld under natural TB transmission conditions in Michigan (Palmer et al., 2000) and it
has been applied to environmental samples collected as part of bovine TB experimental
transmission studies with the Michigan TB strain of M. bovis performed with penned deer
in an indoor bio-safety level II facility (Palmer et al., 2002a, , 2004a, 2004b; Palmer et
al., 1999; Palmer et al., 2001). Although M. bovis was detected in a subset of the
environmental samples collected during the experimental bovine TB transmission studies

performed indoors in penned animals, the authors have noted that an improved and more

57

sensitive method for M. bovis detection may be necessary to accurately assess
environmental contamination (Palmer et al., 2004a).

The CB-18TM TB Culture Kit with Lytic Decon H (Integrated Research
Technology, LLC) is a commercially available set of reagents and instructions for
processing specimens for the detection of mycobacteria by culture. The kit contains C13-
Carboxypropylbetaine (CB-l 8), a zwitterionic detergent that replaces NaOH in the
decontamination step and is also thought to decrease surface tension and counteract the
natural buoyancy of mycobacteria and facilitate the more efﬁcient collection of the bacilli
(Thornton et al., 1998a). The kit also contains the components of a resuspension buffer
with lecithin and a mixture of lytic enzymes (lysosyme, zymolyase, Cytophaga and
Trichoderma extracts). The resuspension buffer is added to the sample sediment before
the inoculation of mycobacteria isolation media to reduce contamination (Thornton et al.,
1998c).

The performance of the standard NaOH-based sample processing method and the
CB-18 protocol for processing environmental substrates (hay, water and soil) were
compared. The performance of the two methods was assessed by processing replicate
samples of environmental substrates experimentally “contaminated” or inoculated with
M. bovis. In each study, one of the sample replicates processed by both methods was
sterilized before M. bovis inoculation to remove the effect of competition from
saprophytic organisms in the environmental substrates.

Data from the ﬁrst set of experiments (Table 2.1 — 2.3) clearly indicate that the
CB-18 method reduced the number of samples contaminated with non-mycobacteria as

well as the level of contamination in individual samples. The number of contaminated

58

samples was negatively correlated with the number of positive M. bovis culture results.
When contamination was examined by analyzing the results of the processed
environmental substrates that were not sterilized before M. bovis inoculation, the NaOH
processed samples were found to have 11 times the odds of being contaminated. This
level of contamination led to the reporting of false negative results in 13% of the samples
processed with the NaOH method vs. 3% of the samples processed with the CB-18
method.

A comparison of the NaOH and CB-18 methods for processing environmental
samples did not reveal a difference in the minimum detection level of these two methods
for isolation of M. bovis by culture. Both sample processing methods reduced the
number of M. bovis bacilli, or CFU, by 80% to 90%. The minimum detection level for
both methods of sample processing was approximately 100 CFU/sample. The data from
these comparative studies did not reveal the increased sensitivity of M. bovis to NaOH
reported by Thornton et a1 (Thornton et al., 1998b), however, the quantitative culturing
methods used in these studies may not have been sensitive enough to detect a difference.

The ﬁnal two M. bovis experimental inoculation studies evaluated the effect of
sample storage conditions on the recovery of M. bovis from samples processed with CB-
18 and Lytic Decon II (Table 2.4-2.5). Recovery rates, based on the number of positive
samples, were highest for those samples processed immediately. Storage of the samples
at room temperature led to an increase in contamination and the reporting of false
negative results. When compared to storage at room temperature, refrigerating the
samples at 4° C or freezing the samples at —20° C reduced contamination of samples and

therefore the reporting of false negative results. A separate analysis of the effect of the

59

freeze (-20° C) and thaw cycle on the viability of M. bovis stored in liquid culture media
indicated a 5-fold reduction in the number of viable M. bovis bacilli. This indicates that
the minimum detection level of M. bovis in samples with lower amounts of M. bovis than
those used in these experiments would be increased if the specimens were subjected to a
ﬁeeze-thaw cycle.

The ﬁnal two studies also reveal the variability of results when using sample
substrates collected from the ﬁeld for experimental inoculation studies and the particular
difﬁculty of producing a non-contaminated result ﬁ'om soil samples. Some degree of
contamination was encountered when processing the samples with the CB-18 method at
all time points and under all storage conditions. The unpredictability of the range of
saprophytic organisms encountered in environmental samples necessitates the use of a
range of selective media when attempting to isolate mycobacterial species. A
commercially available Selective 7Hll agar plate (Becton-Dickenson) speciﬁcally
designed to isolate pathogenic bacteria and inhibit the growth of other bacteria and
molds, produced the “cleanest” M. bovis cultures ﬁ'om environmental samples. If
additional efforts are made to keep the agar plates and M. bovis cultures hydrated, the
plates allow for the visual identiﬁcation and selection of M. bovis colonies for further
testing in the event that a sample is contaminated with a mix of bacteria and mold.
Despite the difﬁculties with contamination encountered when using culture as the means
of detecting mycobacteria in the environment, it is the only routine way to assess the
viability and potential infectivity of M. bovis present in environmental substrates. The
application of the CB-l8 method to the processing of environmental samples does

increase the sensitivity of mycobacterial culture by decreasing the contamination that

60

often results in the reporting of false negative results. The CB-18 sample processing
method coupled with the use of selective culture media and special attention applied to
the identiﬁcation of M. bovis colonies in mixed cultures should improve the efﬁciency
and success of efforts to identify and characterize the persistence of M. bovis in the

environment.

2.4.1 Conclusions

Optimized methods for processing environmental samples, and isolating viable M.
bovis with bacterial culture, include the following: 1) Rigorous mixing of the substrate; 2)
Use of CB-18TM TB Culture Kit with Lytic Decon“ II (Intergrated Research Technology,
LLC); and 3) Isolation with Selective 7H11 agar plates (Becton-Dickenson). This
method reduced contamination and improved the sensitivity of M. bovis detection in

environmental substrates with bacterial culture.

61

Chapter 3

AN INVESTIGATION OF BOVINE TUBERCULOSIS TRANSMISSION SITES
(CATTLE FARMS AND WILDLIFE AREAS) IN MICHIGAN, USA

3.0 ABSTRACT
Objective: To culture Mycobacterium bovis from environmental substrates collected
from bovine tuberculosis transmission sites (Michigan cattle farms with conﬁrmed M
bovis infection or Michigan townships with the highest recorded apparent prevalence of
M. bovis in white-tailed deer) in an attempt to document the persistence of M. bovis in the
environment.
D_esjgg: Cross-Sectional Study
Sample Population: 13 cattle farms and 5 wildlife sites within townships with a high
apparent prevalence of bovine TB in white-tailed deer.
Procedure: Bovine tuberculosis (TB) positive cattle farms were those farms with a
culture conﬁrmed case of M. bovis infection identiﬁed through the State and Federal
bovine TB surveillance program. Wildlife sites were all within the 5 Michigan townships
with the highest apparent prevalence of bovine TB in white-tailed deer. The sites
sampled were either, the capture locations of M. bovis-infected small mammals, or areas
of known white-tailed deer congregation. Environmental samples were collected from
the sites and processed for mycobacterial culture.

Results: None of the samples collected were positive for M. bovis.

 

Conclusions and Clinical Relevance: White-tailed deer incursion on cattle farms, wildlife

feeding, and livestock management practices in Michigan create potential opportunities

62

for M. bovis contamination of environmental substrates by infected hosts. Agent, host
and landscape level factors decrease the probability of detecting M. bovis in the

environment through environmental sampling and conventional mycobacterial culture.

3.1 INTRODUCTION

The State of Michigan lost its United States Departrnent of Agriculture
designation as “Free from Tuberculosis (TB)” in 2000. This reversal of the State’s TB
Free status, originally achieved in 1979, was the result of the detection of bovine TB in
Michigan cattle in 1998, and conﬁrmation of the establishment of bovine TB in free-
ranging white-tailed deer (Odocoileus virginianus) in northeast lower Michigan in 1995
(Schmitt et al., 1997). Thirty-seven farms with Mycobacterium bovis infected cattle have
been detected in Michigan since intensive surveillance for TB in livestock was reinitiated
in 1998 (Judge, 2006).

The occurrence of bovine TB in cattle and white-tailed deer in Michigan has a
similar temporal and spatial distribution (Schmitt et al., 2002). In addition, DNA
ﬁngerprinting techniques have revealed that cattle and deer are infected with an identical
strain of M bovis (Whipple et al., 1999c). Evidence supports interspecies transmission of
M. bovis ﬁom the wildlife reservoir (white-tailed deer) to cattle but the exact mechanism
and route of disease transmission is unknown. White-tailed deer are the presumed source
of M. bovis infection in cattle in over 50% of the herds identiﬁed as TB positive in
Michigan (O'Brien et al., 2002). Disease transmission in these instances is thought to
occur in the absence of close contact between cattle and deer, since nose-to-nose contact

between the two species is rarely observed (DeLiberto et al., 2004).

63

Bovine TB in Michigan’s white-tailed deer population has been characterized as
having an endemic focus within the ﬁve-country area of Presque Isle, Montmorency,
Alpena, Oscoda and Alcona counties, where 97% of the TB positive deer have been
found (Hickling, 2002; O'Brien et al., 2002; O'Brien et al., 2006). The detection of
bovine tuberculosis in Michigan cattle has been concentrated in the same area of
Michigan. This region of the State, encompassing the ﬁve counties that make up the
endemic focus of bovine TB in deer in addition to Antrim, Charlevoix, Cheboygan,
Crawford, Emmet, Otsego, and portions of Iosco and Ogemaw counties have been
designated as “infected” with bovine tuberculosis and classiﬁed as “modiﬁed accredited”
under the guidelines of the Federal Bovine Tuberculosis Eradication Uniform Methods
and Rules (USDA, 2004).

This study was designed to investigate potential sites of bovine TB
transmission in an effort to detect M. bovis in enviromnental substrates. Potential sites of
bovine TB transmission were deﬁned as Michigan cattle farms with conﬁrmed M. bovis
infection or Michigan townships with the highest recorded apparent prevalence of M.
bovis in white-tailed deer. Bovine TB positive cattle farms were those farms with a
culture conﬁrmed case of M. bovis infection identiﬁed through the State and Federal
bovine TB surveillance program. Wildlife sites were all within the 5 Michigan townships
with the highest apparent prevalence of bovine TB in white—tailed deer. The sites
sampled were either, the capture locations of M. bovis-infected small mammals, or areas
of known white-tailed deer congregation. Speciﬁc substrates targeted for collection
within the sites were selected based on environmental and farm management factors

identiﬁed as risk factors for bovine TB infection on Michigan cattle farms (Kaneene et

al., 2002b) and previous evaluations of the practice of supplemental feeding of white-
tailed deer (Hickling, 2002; Miller et al., 2003; Schmitt et al., 1997).

The overall goal of this study was to apply targeted sampling and an optimized
technique for processing environmental samples for M. bovis detection to characterize the
persistence of M. bovis in the environment and the role of indirect transmission of M.
bovis in the epidemiology of bovine TB in Michigan. The hypothesis being tested is that
M. bovis can be detected in the environment where it can survive for sufﬁcient lengths of

time to serve as a source of infection for cattle and/or wild deer.

3.2 MATERIALS AND METHODS
3.2.1 Study design

All cattle farms in Michigan identiﬁed as bovine TB positive between June 2002
and September 2004 were included in the study. Two additional cattle farms, one
identiﬁed as bovine TB positive in 2000, and the other identiﬁed in 2001, were added to

the sample. Five wildlife areas were also investigated.

3.2.2 Identification of bovine tuberculosis transmission sites

Data generated by on-going bovine tuberculosis surveillance efforts in domestic
livestock and wildlife in Michigan were used to identify speciﬁc sites of potential bovine
TB transmission.
3.2.2.1 Cattle Farms

Cattle farms identiﬁed as bovine TB positive (conﬁrmation of the presence of

cattle infected with M. bovis on the premises) were presumed to be sites of bovine TB

65

transmission. The farms were identiﬁed with the assistance of the Michigan Department
of Agriculture (MDA). The farm owners were contacted and permission to conduct an
in-person interview and perform a ﬁeld investigation on their property was sought. The
approved protocol for farm identiﬁcation is presented in Appendix 1. The survey used
was reviewed and approved by the Michigan State University Committee on Research
Involving Human Subjects.

All of the farms investigated were located in the region of the State of Michigan
designated as “infected” with bovine tuberculosis and classiﬁed as “modiﬁed accredited”
under the guidelines of the Federal Bovine Tuberculosis Eradication Uniform Methods
and Rules (USDA, 2004). The “modiﬁed accredited” zone encompasses Alcona, Alpena,
Antrim, Charlevoix, Cheboygan, Crawford, Emmet, Montmorency, Oscoda, Otsego, and
Presque Isle counties, and those portions of Iosco and Ogemaw counties that are north of
the southernmost boundaries of the Huron National Forest and the Au Sable State Forest
(Figure 3.1).
3.2.2.2 Wildlife Areas

Presumed sites of bovine TB transmission among wildlife species were selected
in two ways. The 1996-2002 Michigan Department of Natural Resources (MDNR)
database of bovine TB surveillance in white-tailed deer in the 4-county TB “core area”
(Montmorency, Alpena, Oscoda and Alcona counties) was used to identify individual
townships with the highest apparent prevalence of bovine TB positive white-tailed deer.
Opportunities to accompany MDNR biologists during their yearly surveys of the white-
tailed deer population in these high prevalence townships were used to select sites for

environmental sampling as described below in section 3.2.3.

66

The trap locations of small mammals identiﬁed as TB positive in a study
performed by the United States Department of Agriculture (USDA), Animal Plant Health
Inspection Service (APHIS), Wildlife Services (WS), were used as the second method to
identify sites of presumed TB transmission among wildlife. The sites were identiﬁed
with the assistance of USDA/APHIS/W S personnel. Permission to access the sites was
requested from landowners if the trap site was on private land.

Two of the areas were selected based on their location within the range of
townships with the highest apparent prevalence of bovine TB in white-tailed deer from
1996-2001, and three of the areas were selected based on the reported trap location of a
small mammal with culture-conﬁrmed M bovis infection.

Five wildlife areas were selected for further investigation and environmental
sampling. They were all located in the region designated as the “core” of the endemic
area of bovine TB currently affecting white-tailed deer (O'Brien et al., 2002). The “core”
is deﬁned by the administrative boundaries of the Michigan Department of Natural
Resources Deer Management Unit number 452 (Figure 3.2).

The wildlife areas selected for sampling were primarily forested habitat on private
land. Samples were collected from ﬁve of the townships with the highest apparent
prevalence of bovine TB in white-tailed deer surveyed from 1995-2002. Three of the
areas selected within the high apparent prevalence townships were also selected based on
a history of trapping a bovine TB positive small mammal in the immediate vicinity.
Speciﬁc locations for substrate collection within the wildlife areas selected for sampling

included locations with evidence of deer feeding activity, areas of open water with

67

evidence of wildlife congregation along the shores, deer and wildlife fecal material and

soil and vegetation in the immediate surroundings.

3.2.3 Sampling site and substrate selection

Following the identiﬁcation of a potential TB transmission site, the area was
investigated to identify speciﬁc locations for substrate collection within the identiﬁed site
that could be contaminated with M. bovis and facilitate the indirect transmission of
bovine TB among or between cattle and wildlife.
3.2.3.1 Cattle Farms

Data generated through the administration of a structured questionnaire and
observations recorded during a walk through of the farm were used to identify speciﬁc
sites for sample collection on TB positive cattle farms. An in-person interview was
conducted with the individual most familiar with the management of cattle and the
general practices on the farm. Part I of the structured questionnaire (Appendix 2) was
used to record farm characteristics including the number, species and type of livestock
and other domestic animals present on the farm, the size and location of the farm relative
to other livestock operations, and the general production cycle on the farm. Speciﬁc
questions about water sources, feeding practices, livestock containment (housing and
fencing), feed storage and cattle movement were also included.

Part H of the structured questionnaire (Appendix 2) was used to gather speciﬁc
information on reported wildlife incursions on the farm. The questions were designed to

identify the sites and patterns of white-tailed deer incursions on the farm, however,

68

information on the presence and observed behavior of other wildlife on the farm was also
recorded.

Following the interview, a map of the farm was drawn with the assistance of the
lamdowner. The structures on the premises, locations of pastures, crop ﬁelds, woodlots,
open water, feed storage and any cattle or wildlife feeding sites were recorded. The
location of cattle on the farm and any seasonal movement patterns were noted, and all
locations of observed wildlife incursions were recorded.

A farm walk through was performed to identify and conﬁrm the locations
indicated on the map. Any deviations from the information recorded on the map were
noted. Observations of wildlife incursion, including the presence of wildlife feces,

footprints, trails and bedding areas, were recorded.

A ﬁnal list of approximately 20 sampling locations was produced based on the
results of the questionnaire and farm walk through. Targeted sites included those with
evidence of animal concentration (mixed and single species), feed and water sites with
open access to livestock and wildlife, the location of the infected cattle if known, and
sites of wildlife incursion including pastures and woodlots. Substrates from the speciﬁed
locations selected for sampling included feed (hay, grain and silage), pasture grass, soil,
fecal material, bedding and water. Figure 3.3 depicts three sites selected for sampling on
bovine TB positive farms.
3.2.3.2 Wildlife Areﬁ

The locations for environmental sampling within the wildlife areas selected as
potential bovine TB transmission sites were identiﬁed with the assistance of MDNR and

USDA/APHIS/W S biologists. Deeryards (naturally sheltered areas used by deer during

69

severe winters with signiﬁcant snow fall), deer feeding sites and adjacent areas of open
water were selected within the townships identiﬁed as having a high prevalence of bovine
TB. The landscape surrounding the trap location of small mammals identiﬁed as TB
positive was surveyed to identify speciﬁc substrates for sampling. Substrates selected for
sampling from both white-tailed deer and small mammal sites included fecal material,
soil, vegetation and water. Figure 3.4 depicts three sites selected for sampling in the

wildlife bovine TB transmission areas.

3.2.4 Sample collection

Approximately 500 grams of substrate was collected ﬁom each of the sampling
locations identiﬁed. Latex gloves or a cleaned and betadine-disinfected shovel were used
to collect each sample to prevent cross contamination. Water samples were collected in
0.5 liter sterile plastic bottles and capped. All other substrates were placed in large
capacity Whirl-PakT" bags and sealed. The sample containers were labeled with a unique
identiﬁcation number. Additional data corresponding to the sample identiﬁcation
number and a description of the sampling site were collected and recorded on the ﬁeld
data sheets (Appendix 3). Recorded data included a description of the sample collected, a
description and the GPS coordinates of the sampling collection location, and a digital
photograph of the sampling site.

The samples were stored in an insulated cooler surrounded with cold packs. They
were transported by vehicle to the Bio-safety Level III laboratory at Michigan State
University within 8 hours of collection. The samples were stored at 4° C for 12 hours

before processing.

70

3.2.5 Sample Processing

All samples were processed for mycobacterial culture with the CB-18T" TB
Culture Kit with Lytic DeconTM 11 (Integrated Research Technology, LLC, Quest
Diagnostics Inc., Baltimore, MD). Collected substrates were thoroughly mixed and
approximately 5 gm of the solid substrates and 7 .5 ml of water were transferred for
further processing. Soil and fecal samples were placed in 3 X 7 inch Whirl- PakT“ bags.
Feed and vegetation samples were chopped with scissors when necessary and placed in
sterilized Ball® pint regular mason jars. Water samples were transferred to 50 ml conical
centrifuge tubes.

Sterile water (7.5 ml) and 5 m1 of Liquefaction Solution (trisodium citrate
dehydrate and N-acetyl-L-cysteine or NALC) were added to the solid substrates.
Samples were then pulverized and homogenized by placing the Whirl-Pak® bags in a
Stomacher® 80 laboratory blender for 30 seconds, and securing a blade unit and gasket
on the jars, inverting and blending them for 30 seconds on high with a household blender.
Five ml of Liquifaction Solution were added to the water samples and they were mixed
on high for 30 seconds with a vortex machine.

The samples were placed upright and allowed to settle for 30 minutes. The top 5
m1 of ﬂuid from each sample was removed and transferred to a 50 m1 conical tube

containing 10 ml of Decontamination Solution (20X Tris-citrate Buffer, CB-18T" Stock,
or NALC and water). Samples were mixed with a vortex machine and incubated at 37° C

for 75 minutes. Sterile water was added to the 50 ml mark on each tube, mixed and

centrifuged at 3,000 g for 20 minutes. Pellet-containing tubes were decanted completely.

71

A pipette was used to remove all but 1-3 ml of liquid from samples without a visible
pellet. The pellet was resuspended in the supernatant backwash. One ml of sterile water
was added and mixed. A 0.5 ml sample was transferred to a 2.0 m1 labeled cryogenic vial
and frozen at —80° C. One ml of 2X Resuspension Solution ( 10X-Enzyme Stock-
Trichoderma harzianum extract, lysozyme and Lysobacter extract and NALC) was added

to each sample and they were incubated for 45 minutes at 37 ° C.

3.2.6 Mycobacterial culture and isolation

CB-18 processed samples were inoculated onto solid media slants and plates
containing modiﬁed Middlebrook 7H11 agar (Becton-Dickinson) with sodium pyruvate
(Diagnostic Center for Population and Animal Health, Lansing, MI, USA) and 7Hll
Selective plates (Becton-Dickinson). Solid media slants and plates were incubated at
37°C for 8-12 weeks and examined weekly for colony formation. Positive
mycobacterial cultures and colonies on solid media were subjected to an acid-fast smear
analysis to conﬁrm the presence of acid-fast bacteria using standard protocols for slide
preparation, staining and examination (Kent and Kubica, 1985). Acid-fast positive
isolates were identiﬁed to the Mycbocaterium tuberculosis complex species group using a
genetic probe (AccuProbes, Gen-Probe, San Diego, California 92121, USA).
Biochemical tests and high performance liquid chromatography was performed by the
Michigan Department of Community Health (MDCH) Tuberculosis/Mycology laboratory
to speciate non-M. tuberculsosis complex mycobacteria or to differentiate between

Mycobacterium bovis and other members of the M. tuberculosis complex.

72

3.2.7 Statistical analysis

Excel spreadsheets and statistical functions (AVERAGE, AVEDEV, MEDIAN,
MAX, MIN) were used to generate simple descriptive statistics characterizing the bovine
tuberculosis transmission sites and the speciﬁc locations identiﬁed as sites of potential
environmental contamination with Mycobacterium bovis (Excel, Microsoft Ofﬁce XP
Professional). Excel spreadsheets were also used to record mycobacterial culture results
and summarize the data on the isolation of acid-fast bacteria and the presence or absence
of contamination. Comparisons between mycobacterial culture result, substrate type and
presence or absence of contamination were made using the Cochran-Mantel-Haenszel test
for n—way cross tabulation tables and the SAS soﬁware program (Proc Freq;

Tables/CMH, SAS version 9.0, Cary NC: SAS Institute, Inc.).

3.3 RESULTS
3.3.1 Identification of potential sites of bovine tuberculosis transmission

Between June 2002 and September 2004, 12 cattle farms in Michigan were
declared bovine tuberculosis positive by State (Michigan Department of Agriculture) and
Federal (U SDA/APHISN eterinary Services) animal health ofﬁcials. DNA ﬁngerprinting
of the M. bovis isolates associated with bovine tuberculosis (TB) on all of these farms
was conﬁrmed as the Michigan M. bovis strain, ﬁrst characterized in the state in 1999
(Judge, 2006; Whipple et al., 1999c). Access to collect environmental samples

potentially contaminated with M. bovis was granted for 11 of the 12 farms. Two

73

additional cattle farms, one identiﬁed as bovine TB positive in 2000, and the other
identiﬁed in 2001, were also investigated at the request of the farm owners.

Farm investigations were scheduled within an average of two months of the
ofﬁcially recorded bovine TB positive date for each of the farms identiﬁed between June
2002 and September 2004 (Table 3.1). One farm was investigated 10 days before the
ofﬁcial TB positive date and the remaining 10 farms were investigated after their
ofﬁcially recorded TB positive date. The average time between environmental sampling
and the ofﬁcial TB positive date was 56.18 days (average deviation=27 .47; median=55,
max=107; min=10). The two farms identiﬁed in 2000 and 2001 were investigated 929
days, and 612 days respectively, after the ofﬁcially recorded TB positive date for each
farm. The cattle ﬁom 9 of the 10 farms identiﬁed as TB positive between 2002 and 2004
were depopulated. The investigation and sampling of the TB positive farms was
accomplished before the cattle were depopulated on ﬁve farms and after the cattle were
depopulated for the remaining 4 farms (Table 3.1). The average time between farm
sampling and cattle depopulation was 32.67 days (average deviation=11.56; median=31;

max=55; min=7).

3.3.2 Cattle Farms: General characteristics, farm management practices and
description of potential sites of M. bovis contamination
Of the thirteen bovine tuberculosis cattle farms investigated, four were dairy
operations, seven were beef cow/calf operations, one was a small beef feeder operation,
and one was a combination cow/calf and feeder operation. The number of adult cattle on

the farms ranged ﬁ'om 14 to 239 and averaged 74. The average size of the farm

74

properties was 251 acres. Only 1 farm reported fence line contact with another cattle
farm. Approximately 1/2 of farms that were identiﬁed as bovine TB positive between June
2000 and September 2004 purchased 100% of their cattle ﬁom outside sources. The
majority of the farms did not raise any other kind of livestock, with the exception of
chickens, and all but one reported the presence of pet dogs and cats on the farm (Table
3.2).

Full farm investigations (interviews with farm owners or primarily cattle
managers and farm walk-through) were performed on the ﬁrst 12 farms sampled.
USDA/APHIS/V S personnel collected environmental samples from the 13th farm, but
only very general farm characteristics were recorded. Although 10 of the 12 farms had
cattle housing (barn, feedlot, or barn yard) facilities, cattle spent more than 50% of their
time outside on 9 of the 12 farms (Table 3.3). The three farms on which cattle spent
more than 50% of their time inside were dairy herds. Only 1 farm, a dairy, never fed
cows outside. Cattle on all of the farms had access to water sources outside and on 10 of
the farms cattle had access to surface water (ponds, streams or water ways). Hay was
provided to cattle in a feeder on 4 of the farms but it was also provided on the ground on
2 of these. Ten of the 12 farms provided bay to their cattle outside on the ground. A
summary of the cattle management practices on each of the farms is summarized in Table
3.3.

An examination of feed storage and general fencing practices on the farms
revealed that hay (round bales) were stored outside and unprotected on 8 of the 12 farms.
Round bales of hay were stored in the ﬁelds, along fencerows or in the woods on 4 of the

farms. Five of the farms used some barbed wire and 9 of the farms used electric fencing.

75

Two of the farms had “deer proof” fencing around their hay that had been installed
through a ’USAID/APHIS/WS program after their cattle were identiﬁed as bovine TB
positive. A summary of the fencing and feed storage practices is provided in Table 3.4.

All of the farmers interviewed reported observing deer on their property. All but one of
the 12 farms reported observing deer in their cattle pastures and all 12 farms reported
observing deer feeding on either their pastures or crop ﬁelds. Deer were not observed
drinking water from outdoor tanks but 5 of the farms reported observing evidence of deer
using the same open water sources accessible to cattle on the farm. Six of the 12 farms
reported observing deer feeding on harvested hay intended for cattle and all but 1 farm
reported the presence of deer attractants (orchards or cedar swamps) on or adjacent to
their property (Table 3.5). Producers on all farms surveyed reported observations of
wildlife other than deer. The species observed included raccoons (Procyon lotor),
opossum (Didelphis virginiana), fox (Vulpes vulpes), coyote (Cam's Iantrans), turkeys
(Meleagﬁs gallopavo), skunks (inephitis mephitis), bobcats (F elis ruﬁls), bears (Ursus

americanus) and porcupine (Erethizon dorsatum).

3.3.3 Cattle farms (samples collected)

A total of 409 samples were collected ﬁom 13 farms. Approximately 20 sites
were selected for sampling on each farm. One sample was collected per site for the ﬁrst
10 farms. An effort was made to intensify sampling on the ﬁnal two farms and more
samples were collected per site. A total of 140 samples were collected from farm 112

and a total of 52 samples were collected from farm 113 (Table 3.6).

76

3.3.4 Wildlife areas (samples collected)

A total of 97 samples were collected ﬁ'om 5 wildlife areas. Approximately 20
locations were selected for sampling in each wildlife area and approximately 1 sample
was collected per site. An attempt was made to distribute the sampling across substrate

type (Table 3.7).

3.3.5 Mycobacterium bovis culture results

None of the samples collected from the bovine TB positive farms were positive
for Mycobacterium bovis based on mycobacterial culture. A number of acid-fast
organisms were isolated but further testing revealed that none of these were members of
the M. tuberculosis complex (Table 3.6). Acid-fast organisms were most commonly
isolated from soil, vegetation, manure and cattle feed samples but an association between
the isolation of acid-fast bacteria and sample type was not found (Mantel-Haenszel X2 =
0.04; degree freedom = 1; p-value = 0.84). Samples processed from wildlife areas
produced similar results. Acid-fast bacteria were isolated from samples of soil and
vegetation but no signiﬁcant associations between substrate type and acid-fast bacterial
isolation were found. The isolates of the non~M. tuberculosis complex acid-fast bacteria
that could be identiﬁed to species included M. fortuitum, M avium, M. fortuitum-
chelonae, and Mycobacterium sp. Group IV.

The prevalence of contamination (overgrth of the cultures with mold and non-
mycobacteria) was high. Twenty-seven percent of the samples collected from bovine TB
positive farms (Table 3.6) and 11% of the samples collected from wildlife areas (Table

3.7) were contaminated. Soil samples and substrates mixed with manure were most

77

likely to be contaminated but no signiﬁcant association between sample substrate and the
presence of contamination was found (Mantel-Haenszel X2 = 0.01; degree freedom = l;

p-value = 0.92)

78

Figure 3.1 Map of Michigan indicating the Bovine Tuberculosis State Status
Designations: 1) Modiﬁed Accredited (Infected Zone), and 2) Modiﬁed
Accredited Advanced (Disease Free Zone). Source: Michigan Department
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Table 3.6 Mycobacterial culture results of environmental substrates collected ﬁ'om
bovine tuberculosis positive farms.

 

 

 

TOTAL AFB + (%) Contamination + (%)
Pasture & Vegetation 79 13 (16%) 15 (19%)
Soil 75 13 (17%) 32 (43%)
Open Water 78 2 (3%) 12 (30%)
Hay 50 5 (10%) 10 (20%)
Cattle Feed 10 3 (30%) 2 (20%)
Barn Water 4 O (0%) 0 (0%)
Bedding 22 0 (0%) 9 (41%)
Manure 40 1 (3%) 12 (30%)
Manure Mix 23 6 (26%) 12 (52%)
Deer Feces 10 0 (0%) 4 (40%)
Wildlife Feces 17 l (6%) 3 (18%)
Bear Hair 1 0 (0%) 0 (0%)

409 44 (11%) 111 (27%)

Table 3.7 Mycobacterial culture results of environmental substrates collected from
potential wildlife bovine tuberculosis transmission sites.

 

 

 

TOTAL AFB + (%) Contamination + (%)
Pasture & Vegetation 17 1 (6%) 4 (24%)
Soil 24 2 (8%) 5 (21%)
Open Water 22 0 (0%) O (0%)
Wildlife Feces 5 0 (0%) 1 (20%)
Deer Feces 28 0 (0%) 0 (0%)
Grain 1 O (0%) 1 (100%)
97 3 (3%) 11 (11%)

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3.4 DISCUSSION

Mycobacterium bovis, the causative agent of bovine TB, continues to circulate
among cattle and white-tailed deer in Michigan. Over the two-year span of this study,
ﬁom June 2002 until September 2004, 12 cattle farms in northern Lower Michigan were
identiﬁed as bovine TB positive (Michigan Department of Agriculture). The cattle herds
identiﬁed as bovine TB positive during this period were all in the USDA designated
“Modiﬁed Accredited Zone” (Figure 3.1) where annual whole-herd bovine TB testing is
required (USDA, 2004). The farms identiﬁed, therefore, likely represent new bovine TB
infection and relatively recent transmission events. Similarly, on-going surveillance for
bovine TB in the white-tailed deer population during the same period revealed an
apparent prevalence ﬂuctuating around 2.0% in animals originating in MDNR, Deer
Management Unit # 452, thought to be the endemic focus of bovine TB in white-tailed
deer in the State (Figure 3.2) (O'Brien et al., 2006). A number of the bovine TB positive
white-tailed deer identiﬁed during this routine surveillance were yearlings, indicating
new infection and recent bovine TB transmission events.

Despite evidence of on- going bovine TB transmission in northeast Michigan,
efforts to isolate Mycobacterium bovis ﬁom environmental substrates collected from
bovine TB positive farms and areas of wildlife TB transmission failed. The ﬁeld
investigations did reveal circumstances (location and practice) that would facilitate the
indirect transmission of bovine TB through the contamination of environmental

substrates among and between cattle and white-tailed deer populations.

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3.4.1 Potential sites of M. bovis contamination of the environment
3.4.1.1 Bovine TB posﬂefaﬂs

The general characteristics of the 13 bovine TB positive farms selected for
investigation and environmental sampling in this study were similar to those of the initial
group of bovine TB positive farms identiﬁed between 1998 and 2002 (Schmitt et al.,
2002). The majority of the farms were small beef cattle operations and they were all
located in northeast lower Michigan. Particular cattle management practices that have
been identiﬁed as risk factors associated with tuberculosis on cattle farms in northeast
Michigan in the past (Kaneene et al., 2002b), and those that would facilitate the indirect
transmission of bovine TB ﬁ'om deer to cattle via M. bovis contaminated substrates
included 1) maintenance of cattle outside for more than 50% of the time outside (75% of
farms); 2) feeding cattle outside (92%) and feeding cattle outside exclusively (58%); 3)
watering cattle outside with access to open water (streams, ponds, etc.) (83%); and 4)
feeding cattle hay on the ground (83%).

The practices outlined above are only a bovine TB risk to cattle if infected white-
tailed deer in the area also have access to the hay, pasture and water sources identiﬁed as
cattle feeding and watering sites. Answers to “deer incursion” survey questions indicated
that deer were seen on the premises of 100% of the farms identiﬁed. Ninety-two percent
of respondents observed deer in pastures, 50% observed evidence of deer feeding on hay
intended for cattle, and 42% observed evidence of deer drinking from open water sources
on the farms. Electric (75%) and barbed wire (42%) fencing was used on these cattle

farms but feed was only stored in “deer proof” facilities on 25% of the farms surveyed.

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Dairy cattle operations generally maintained their cattle inside housing more than
50% of the time, fed their cattle inside and used hay feeders, however, cattle had access
to open water and hay was stored outside and unprotected on many of the premises
investigated.
3.4.1.2 Wildlife bovine TB transmission sites

The investigations of wildlife areas were performed in the summer and spring. In
the spring and summer months particular wildlife management practices that would
facilitate the indirect transmission of bovine TB were not observed with the exception of
the identiﬁcation of small ﬁelds in wooded areas planted with alfalfa grass to attract deer
and a limited number of empty deer feeding stations which would likely have been active

in winter.

3.4.2 Detection of M. bovis in the environment: Study related factors

Every attempt was made to investigate cattle farms as soon as they were identiﬁed
as bovine TB positive with the goal of collecting environmental samples from the
premises as close to the bovine TB transmission event as possible. The combination of
State and Federal protocols for identifying a herd as bovine TB positive, and the
arrangements necessary for gaining access to the farm, resulted in an average delay of 56
days between the date the farm was recorded as bovine TB positive and the date that
sampling occurred. For farms that were depopulated, the average time between sampling
and depopulation was 33 days. The circumstances that facilitated the initial bovine TB
transmission event on the farm were likely still in place at the time of environmental

sampling, but the unknown timing of the M. bovis transmission event, and delayed access

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to bovine TB positive cattle farms, is and will continue to be a weakness of ﬁeld

investigations of natural bovine TB transmission events.

3.4.3 Detection of M. bovis in the environment: Agent related factors

Properties of M. bovis contribute to difﬁculties associated with isolating the
organism from environmental substrates. M. bovis is particularly difficult to culture. The
necessity of a bacteriacidal decontamination step, coupled with the cording behavior and
natural buoyancy of M. bovis, reduces the success of mycobacterial culture methods
(Kent and Kubica, 1985; Yajko et al., 1995). The additional presence of large numbers
of saprophytic bacteria, molds and other infectious organisms in environmental samples
further interferes with the sensitivity of detection of M. bovis by bacterial culture.
Attempts were made to improve the success of isolating M. bovis with bacterial culture
methods by processing specimens with CB-18TM TB Culture Kit with Lytic DeconTM H
(Integrated Research Technology, LLC, Quest Diagnostics Inc., Baltimore, MD).
Although M. bovis was not isolated, other mycobacterial species were successfully
identiﬁed from environmental substrates collected suggesting the techniques used were
capable of detecting mycobacterial species ﬁom environmental samples in the presence
of other competing microbes.

Culture contamination, an overgrowth of mold and non-mycobacterial species,
affected our attempts to isolate M. bovis from approximately 25% of the samples
collected. Although no signiﬁcant associations were found between contamination rates
and sample substrate type, the distribution of contamination in samples suggested that

soil, vegetation samples and those mixed With cattle manure were more likely to produce

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contaminated mycobacterial culture results. These data indicate that the sensitivity of
isolation of M. bovis from these particular substrates types may be further reduced.

The challenges associated with detection of M. bovis in environmental substrates
has been cited as a potential cause of failure to isolate M. bovis from environmental
substrates ﬁ'om other areas identiﬁed as sites of natural transmission of bovine TB in
Michigan. These include a captive white-tailed deer facility (Palmer et al., 2000) and one.
of the ﬁrst cattle farms identiﬁed as TB-positive in the current outbreak of M. bovis in
Michigan (Kaneene, personal communication). M. bovis has been isolated from
environmental substrates contaminated in the course of experimental transmission studies
among deer (Palmer et al., 2004b; Palmer et al., 2001) and between deer and cattle
(Pahner et al., 20043), however, even under these “ideal” circumstances, success has

been intermittent.

3.4.4 DetectiOn of M. bovis in the environment: Host related factors

As previously discussed, one of the limitations of opportunistic enviromnental
sampling of bovine TB positive cattle herds and identiﬁed wildlife bovine TB
transmission areas, is that a time lag likely exists between the time at which the M. bovis
shedding animal (cattle or deer) was present on the premises and the time of sample
collection. This time lag is likely exacerbated by intermittent shedding of M. bovis from
both infected deer (Pahner et al., 2001) and infected cattle (Goodchild and Clifton-
Hadley, 2001; Neill et al., 1988). The probability of collecting an environmental sample
from an identiﬁed bovine TB transmission site in the time period the infected animal is

present and at a time when it is shedding M. bovis is probably very low.

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The denning behavior and patterns of movement of other wildlife reservoirs of
bovine TB, primarily brushtail possums (Trichosurus vulpecula) in New Zealand
(Jackson et al., 1995) and European badgers (Meles meles) in Ireland and Great Britain
(Anderson and Trewhella, 1985; Hutchins and Harris, 1997), allow for a closer
approximation of the opportunities for their potential direct and indirect contact with
cattle on bovine TB affected farms. Although somewhat predictable, free-ranging white-
tailed deer have much larger home ranges and their presence on cattle farms is more
transient (Garner, 2001). The behavior of potentially bovine TB infected white-tailed
deer does not allow for a ﬁne level of targeted sampling of environmental substrates for

the detection of M. bovis.

3.4.5 Detection of M. bovis in the environment: Landscape related factors

Efforts to isolate M. bovis in other regions have yielded similar results when
samples were collected under natural disease transmission conditions (Pillai et al., 2000).
Researchers interested in the persistence of M. bovis in the environment have turned to
experimental inoculation studies and an assessment of the conditions that support or
inhibit M. bovis survival (Dufﬁeld and Young, 1985; Jackson et al., 1995; Little et al.,
1982; Maddock, 1933, , 1934, , 1936; Tanner and Michel, 1999; Williams and Hoy,
1930; Wray, 1975). This is due primarily to the difﬁculty of identifying the exact
location of M. bovis contamination over what is often a very large potentially
contaminated site. This study faced the same challenge. Financial and time constraints
limited the number and volume of environmental substrates that could be collected and

processed from sites potentially contaminated with M. bovis. These constraints limited

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the total surface area of both bovine TB positive farms and wildlife areas that could be

sampled effectively.

3.4.6 Persistence of M. bovis in the environment: Overall conclusions

It is generally accepted that M. bovis is transmitted within deer populations, and
within cattle herds, primarily through close contact and direct routes of disease
transmission (Kaneene et al., 2002a; Pahner et al., 1999). Interspecies transmission of
bovine TB between white-tailed deer and cattle, however, likely occurs through indirect
routes of transmission since little evidence of direct contact between the species exists
(DeLiberto et al., 2004). This study, therefore, focused on characterizing the potential for
the indirect transmission of M. bovis through the contamination of environmental
substrates.

This ﬁeld investigation of bovine TB transmission sites conﬁrmed the ﬁndings of
earlier studies that have identiﬁed environmental and cattle farm management practices
(Kaneene et al., 2002b) in northeast Michigan that may facilitate indirect interspecies
transmission of bovine TB between deer and cattle. Investigations of wildlife TB
transmission areas also produced evidence of deer feeding and baiting practices that have
been identiﬁed by other authors as likely contributing to the indirect transmission of
bovine TB among white-tailed deer (Hickling, 2002; Miller et al., 2003; O'Brien et al.,
2006; Schmitt et al., 1997). The failure to isolate M. bovis from environmental substrates
collected ﬁ'om bovine TB positive cattle farms and wildlife areas was likely due to agent,
host and landscape factors that contribute to the difﬁculty of identifying speciﬁc sites of

M. bovis contamination and recovering M. bovis ﬁ'om environmental substrates.

95

Chapter 4

A STUDY OF THE PERSISTENCE OF M YCOBA C TERI UM B0 VIS IN THE

ENVIRONMENT

4.0 ABSTRACT

Objective: To characterize the persistence of the Michigan strain of Mycobacterium
bovis in the environment under natural weather conditions.

Design: Experimental Study

Sample Population: Mycobacterium bovis inoculated environmental substrates (corn,
hay, soil and water) examined for persistence during three seasons and over a 12-month
period.

Procedure: Re-isolation of M. bovis from experimentally inoculated substrates was used
to follow the persistence of viable M. bovis bacteria in soil, hay, water and corn exposed
to natural weather conditions. Environmental factors (humidity, temperature, rainfall,
and levels of solar radiation) were recorded continuously during the experimental
persistence studies. Mean and maximum survival time for M. bovis in each of the
environmental substrates was determined for each of 3 seasons (F all/Winter,
Winter/Spring, Spring/Summer) of experiments. Factors affecting M. bovis persistence
(substrate type, weather conditions, presence of shade, etc.) were studied using survival
analysis and Cox’s proportional hazards regression.

R_e§rrl_t§: The Michigan strain of M. bovis persisted in the environment for up to 88 days

in soil, 58 days in water and hay and 43 days on corn. The time period, or season, in

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which the M. bovis was exposed signiﬁcantly affected its survival. The shortest survival
across all substrate types was seen in the spring/summer season in which average daily
temperatures and levels of solar radiation were highest.

Conclusions and Clinicg Relevm: Viable M. bovis bacteria persists in environmental
substrates (corn, hay soil and water) under natural Michigan weather conditions for up to
6-10 weeks in the cooler fall/winter and winter/spring seasons. Cattle and/or wildlife
with access to environmental substrates contaminated with M. bovis within days to weeks
of the contamination event are at risk of exposure to bovine TB. These ﬁndings
strengthen evidence that suggests that cattle farm bio-security and efforts to eliminate
supplemental feeding of white-tailed deer will decrease the risk of bovine TB

transmission among and between cattle and deer populations.

4.1 INTRODUCTION

An endemic focus of bovine tuberculosis (TB), caused by a single strain of
Mycobacterium bovis, has been identiﬁed in white-tailed deer (Odocoileus virginianus)
in northeast lower Michigan (O'Brien et al., 2006; Schmitt et al., 1997; Whipple et al.,
1999b). Spillover of M. bovis infection from white-tailed deer to cattle is suspected in
the majority of the 33 cattle farms in the same region of the State identiﬁed as bovine TB
positive since intensive surveillance for TB in Michigan livestock was reinitiated in 1998
(Michigan Department of Agriculture; USDA/APHIS/V S). The emergence of a wildlife
reservoir for bovine TB in Michigan, and evidence of disease transmission between
infected ﬂee-ranging white-tailed deer populations and domestic cattle, has forced a

reevaluation of the understanding of the epidemiology of bovine TB in North America.

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Disease transmission between deer and cattle in Michigan is thought to occur in the
absence of close contact between the species (DeLiberto et al., 2004). This has raised
questions about the role of indirect transmission of M. bovis in the epidemiology of
bovine TB and identiﬁed a need to investigate the persistence of M. bovis in the
environment and the potential role of contaminated substrates in the transmission of M.
bovis among and between wildlife and cattle populations.

The persistence of M. bovis in the environment and role of indirect transmission
in the epidemiology of bovine TB in Michigan has been debated since the current TB
epidemic in Michigan was ﬁrst described in 1997 (Schmitt et al., 1997). Bovine TB
infection in white-tailed deer today is likely linked to the large number of cattle infected
with M. bovis in Michigan during the late 1950’s (Frye, 1995), however, the
establishment and persistence of M. bovis in free-ranging white-tailed deer in northeast
Michigan is thought to have been inﬂuenced by the long-term practice of winter feeding
of deer in the region (Schmitt et al., 1997). These piles of feed, set out to attract deer and
improve their productivity and winter survival, are thought to contribute to the
transmission of TB among white-tailed deer by 1) increasing local density and contact
between animals and 2) providing a site for the indirect transmission of TB through
contamination of the feed by infected deer shedding M. bovis in their saliva or nasal
discharges and the subsequent infection of a naive deer by consumption of contaminated
feed (Hickling, 2002; Schmitt et al., 1997). This is supported by evidence that suggests
that speciﬁc supplemental feeding practices, generally indicative of large-scale feeding
operations, are associated with an increasing risk for bovine TB in deer in Michigan

(Miller et al., 2003).

98

The role M. bovis contaminated environmental substrates in the interspecies
transmission of bovine TB between cattle and deer has also been investigated (Fine et al.
2006—chapter 3). Although M. bovis was not identiﬁed from any of the environmental
substrates tested, particular cattle management practices and environmental factors were
identiﬁed that have been shown to be associated with tuberculosis on cattle farms in
northeast Michigan in the past (Kaneene et al., 2002b). These practices are likely
facilitate the indirect transmission of bovine TB ﬁom deer to cattle via M. bovis
contaminated substrates. The factors and practices identiﬁed included the presence of
ponds or open water in cattle areas, maintaining cattle outside more than 50% of the time,
feeding and watering cattle outside and not protecting feed intended for cattle from deer.

Evidence suggests that opportunities for the indirect transmission of M. bovis
between white-tailed deer and cattle exist in northeast Michigan under current cattle and
deer management practices. Data on the persistence of M. bovis on various environmental
substrates and the factors that inﬂuence its survival are essential to the further
understanding of the complexity bovine TB transmission and epidemiology in Michigan.
In addition to contributing to our understanding of bovine TB dynamics in this system,
information regarding the persistence of M. bovis in the environment will support efforts
to improve protocols for cattle farm bio-security and the maintenance of appropriate
restrictions on feeding and baiting free-ranging white-tailed deer and other wildlife.

Experimental studies conducted recently in New Zealand, Australia, South Africa,
Great Britain and Ireland, have shown that M. bovis persists in typical environmental
substrates for varying amounts of time (Dufﬁeld and Young, 1985; Jackson et al., 1995;

Little et al., 1982; Tanner and Michel, 1999; Young et al., 2005).

99

This study was designed to describe the persistence of the Michigan strain of M.
bovis in typical environmental substrates (corn, hay, soil and water) exposed to natural
weather conditions in Michigan. Factors affecting the length of persistence, or survival
time, of M. bovis in the environment were also investigated. The hypothesis being tested is
that M. bovis can survive in environmental substrates for sufﬁcient lengths of time to serve

as a source of infection for cattle and/or wild deer.

4.2 MATERIALS AND METHODS
4.2.1 Culture, media, and growth conditions

A Michigan strain of Mycobacterium bovis was obtained ﬁ'om a frozen culture of
M. bovis originally isolated in 2002 from the retropharyngeal lymph node of a naturally
infected 2-year-old Holstein cow from Michigan. The animal was classiﬁed as a reactor
on a comparative cervical test and had gross and microscopic lesions consistent with
bovine tuberculosis (TB) at necropsy. The frozen M. bovis culture was added to 10 ml
of Middlebrook 7H9 Broth with Middlebrook ADC Enrichment for cultivation of
mycobacteria (Becton-Dickinson, Cockeysville, MD, USA). Multiple 10 ml vials of M.
bovis inoculated 7H9 Broth were incubated at 37° C for 21 to 30 days. A series of
growth curve experiments were used to estimate the concentration (colony forming units
(CFU) of M. bovis per ml) of the liquid cultures. The exact ﬁnal concentration (CFU/ml)
of the M. bovis liquid culture stock was determined by monitoring growth and performing
colony counts on Selective 7Hll agar (Becton-Dickinson) plates inoculated with exactly

100 pl of M. bovis liquid stock, a 1:10, 1:100, 1:1000 and a 1:10000 dilution. Blood and

100

CNA agar plates were inoculated with M. bovis liquid culture stock to monitor for

contamination. Only pure M. bovis cultures were used for inoculation experiments.

4.2.2 Environmental substrates

Substrates selected for testing included grass hay, soil, water and shelled corn.
Grass hay was collected from the feed storage area of the Michigan State University
(MSU) Large Animal Veterinary Teaching Hospital in East Lansing, Michigan. Soil was
collected from the Baker Woodlot (Rachana Raj endra Neotropical Bird Sanctuary)
located in the south central section of the MSU campus. Water was collected ﬁ'om the
large pond at the center of the Baker Woodlot and from the Red Cedar River at the Farm
Lane Bridge on the MSU campus. Shelled corn was purchased in 20-pound bags from a
local feed store. Environmental substrates were stored at 4° C with no exposure to light.

A set of environmental substrates consisted of 4 samples each of grass hay, soil,
water and shelled corn for a total of 16. Ball® Half-Pint Regular Can-or-Freeze Jars
were ﬁlled with 5 gm of hay, 10 gm of soil, 10 gm of corn or 10 m1 of water. Half of the
sample-ﬁlled jars were autoclaved for 2 hours at 121° C and 20 psi to sterilize the
contents. Each sample set of 16 was identiﬁed with uniquely colored tape and a label
denoting the sample type, sample set number, the autoclave status, and shade or non-

shade treatment. The make up of a full sample set is presented in Table 4.1.

4.2.3 Facility (laboratory and outdoor enclosure)

Mycobacterium bovis sample inoculation, sample processing and M. bovis

isolation procedures were all performed in the bio-safety level III (BL3) laboratory in the

101

Diagnostic Center for Population and Animal Health (DCPAH) at Michigan State
University (MSU).

The M. bovis environmental persistence studies were carried out in a structure
erected within the fenced livestock containment facility south of the DCPAH at MSU.
The structure was erected on a concrete slab along the north fence of the livestock
containment facility (Figure 4.1). The structure consisted of an enclosed “cage” 16-ﬁ. X
25-ft. X 8-ﬁ. with a galvanized steel frame covered with 3/8‘h-inch fencing (chain link-
type fence with 3/8th inch holes) on all sides including the top. The bottom rail of the
cage was ﬂush with the concrete slab or buried below the ground surface. Any gaps
below the bottom rail were closed with Z-ﬂ. X 4-ﬁ. wooden beams. A locked door was
built into one side of the enclosure with a minimum clearance with the concrete slab and
doorframes.

The fencing excluded all birds and small mammals. The structure was built to
exclude livestock and/or deer in the unlikely event that they gain entrance to the fenced
containment facility and access to the experimental enclosure was limited to authorized
individuals.

Speciﬁed sets of M. bovis inoculated environmental substrates were placed within
secondary plastic containers on 2 lines of steel tables set up within the enclosure (Figure
4.2). Sample containers on 1 line of steel tables were covered with black shade cloth.
All secondary sample containers were lined with gravel and sand and secured with wire

mesh covers.

102

4.2.4 Environmental monitoring

A WeatherHawkTM weather station, Division of Campbell Scientiﬁc, Inc., was
positioned at the center of the enclosure. The station was powered by a solar panel
charged battery pack. Environmental data collected included rainfall (mm), wind speed
(m/sec), temperature (°C), humidity level (%), and solar radiation (W/mz).
Evapotranspiration, a combination of solar radiation, temperature, wind speed, and
humidity, was also calculated with the WeatherHawkTM Virtual Weather software. The
weather station was programmed to record data at 20-minute intervals, 24 hours a day.
Environmental data was downloaded from the weather station to an Excel (Microsoft
Corporation, Redmond, WA, USA) computer database on a desktop computer using a

wireless system.

4.2.5 Inoculation with M. bovis

Each sample was inoculated with 50,000 CFUs of a clinical strain of
Mycobacterium bovis isolated from a cow in Michigan. Samples were inoculated in the
BL3 laboratory. Sample jars were sealed with a plastic, leak-proof lids and transported to
the outdoor experimental enclosure, located 500 meters ﬁ'om the BL3 laboratory, in

sealed and labeled coolers.
4.2.6 Study design and sampling

The persistence of M. bovis in environmental substrates was evaluated over 4

sampling periods. The ﬁrst sample period spanned 12 months ﬁ'om November 2004 to

103

December 2005. Twelve sets of 16 samples were inoculated with M. bovis as described
above and placed in their assigned positions in the experimental enclosure. One sample
set was collected at the end of the 1st through 12th month of the experiment and processed
for M. bovis isolation. The remaining three sampling periods covered 12 weeks and
began on November 8, 2004, February 4, 2005 and May 20, 2005 (Figure 4.3). At the
start of each sampling period twelve sets of 16 samples were prepared and inoculated
with M. bovis and placed in the environmental sample enclosure. The persistence of M.
bovis over time was detemrined by processing sample sets for mycobacterial culture at

the time of inoculation, every other day and then at weekly intervals for 12 weeks.

4.2.7 Environmental sample processing

All samples were processed for mycobacterial culture with the CB-18T" TB
Culture Kit with Lytic Decon” H (Integrated Research Technology, LLC, Quest
Diagnostics Inc., Baltimore, MD). Collected samples were processed within their
original container (Ball® 1/2 pint regular jar). If necessary sterile water (5-10 ml) was
added to the solid substrates. Samples were pulverized and homogenized by securing a
blade unit and gasket on the jars, inverting and blending them for 30 seconds on high
with a household blender.

The samples were placed upright and allowed to settle for 30 minutes. The top 5
ml of ﬂuid ﬁ'om each sample was removed and transferred to a 50 ml conical tube
containing 10 ml of Decontamination Solution (20X Tris-citrate Buffer, CB-18T“ Stock,
NALC and water). Samples were mixed with a vortex machine and incubated at 37° C

for 75 minutes. Sterile water was added to the 50 ml mark on each tube, mixed and

104

centrifuged at 3,000 g for 20 minutes. Pellet-containing tubes were decanted completely.
A pipette was used to remove all but 1-3 ml of liquid ﬁom samples without a visible
pellet. The pellet was resuspended in the supernatant backwash. One ml of sterile water
was added and mixed. A 0.5 ml sample was transferred to a 2.0 ml labeled cryogenic vial
and ﬁ'ozen at —80° C. One ml of 2X Resuspension Solution (lOX-Enzyme Stock-
Trichoderma harzianum extract, lysozyme and Lysobacter extract and NALC) was added

to each sample and they were incubated for 45 minutes at 37° C.

4.2.8 Mycobacterial culture and isolation

CB-18 processed samples were inoculated onto solid 7Hll Selective plates
(Becton-Dickinson) and incubated at 37 °C for 8-12 weeks and examined weekly for
colony formation. Typical M. bovis colonies were counted and recorded on laboratory
data sheets. Acid-fast smear analysis was performed to conﬁrm the presence of acid-fast
bacteria using standard protocols for slide preparation, staining and examination (Kent
and Kubica, 1985). A subset of the acid-fast positive isolates were conﬁrmed to be
Mycobacterium tuberculosis complex species group using a genetic probe (AccuProbes,

Gen-Probe, San Diego, CA, USA).

4.2.9 Data analysis

Mycobacterial culture results, recorded on laboratory data sheets for each
sampling period, were entered in an Excel spreadsheet database (Excel, Microsoft Ofﬁce
XP Professional, Redmond, WA, USA). Excel data from the weather records and

mycobacterial culture results were imported into the SAS software program (SAS version

105

9.0, SAS Institute, Inc., 2003, Cary, NC, USA) and combined. Imported Excel
spreadsheet data were analyzed using the SAS software program. Summaries of the
weather records for each sampling period were created and descriptive statistics were
generated for the persistence of M. bovis on each sample type for all sampling periods
(SAS Institute, Inc., 2003).

The t-test (Proc TTEST) was used to test for signiﬁcant differences between mean
M. bovis survival time (persistence) in shade/non-shade treated samples and
sterilized/non-sterilized treated substrates. An AN OVA (Proc AN OVA), using the
Bonferroni approach for multiple comparisons, was performed to test for signiﬁcant
differences in the mean survival time (persistence) in sample types and sampling seasons
(fall/winter, winter/ spring and spring/summer). Assessment of associations between the
bovine TB status of experimental samples (positive vs. negative) and covariates (sample
type, shade/non-shade, sterilized/non-sterilized substrate) were conducted using the
Cochran-Mantel-Haenszel Chi-square (Proc FREQ/CMH) or 2-tailed Fisher’s exact test
where appropriate.

Survival analyses to compare the persistence of M. bovis in the environment
between seasons, and to study the effects of environmental factors on M. bovis survival
across the seasons, were conducted in SAS. The time ﬁ'om sample inoculation to the ﬁrst
negative bovine TB culture after the last positive bovine TB culture was used as the
survival, or M. bovis persistence period, for each sample. Parametric (Proc LIFEREG)
procedures, assuming an exponential distribution were used. The Log rank and Wilcoxon
tests were used to compare the survival distributions for M. bovis persistence in the

environment across the 3 seasons tested. The survival function or Kaplan-Meier curves

106

for M. bovis persistence in each of the three seasons and in each of the 4 substrates across
the three seasons were plotted. Cox’s proportional hazards regression procedure (Proc
PHREG) was used to study the effects of the non-weather related covariates (sample
type, shade/non-shade, sterilized/non-sterilized substrate) and season on M. bovis survival
in the environment.

Model selection in Cox regression (Proc PHREG) was used to identify speciﬁc
weather or seasonal factors that inﬂuenced the survival of M. bovis in the environment.
The weather data were summarized as daily means, maxirna and mirrima for rainfall,
wind speed, temperature, humidity, barometer readings, solar radiation and
evapotranspiration. Univariable Cox proportional hazards regression was conducted for
each weather-related risk factor and the likelihood ratio test was used to determine the
inﬂuence of each covariate on the outcome variable. Spearrnan correlation coefﬁcients
(r) were computed to identify potential areas of multicollinearity between the weather
related risk factors.

A multivariable Cox proportional hazards regression model was then developed
(SAS Institute Inc., 2003). The model contained all weathered-related risk factors that
had a univariable model Likelihood ratio statistic (LRS) p-value <0.15. Highly correlated
weather-related risk factors were removed due to redundancy of information and
multicollinierity (e.g. solar radiation and evapotranspiration were both highly correlated
with temperature). Purposeful selection of covariates and a modiﬁed stepwise method of
variable evaluation with the entry criteria set at LRS p-value of 0.15 and the “stay” or
removal criteria set at LRS p-value of 0.20 was used to identify the ﬁnal multivariable

model for M. bovis survival in the environment.

107

 

Table 4.1 The make up of one sample set used in the bovine TB environmental
persistence study

 

 

 

 

 

 

TREATMENT Shade Shade Non-Shade Non-Shade
Sterilized Non- Sterilized Non-Sterilized
Sterilized
Soil 1 1 1 1
Water 1 l 1 1
Hay 1 l l 1
Corn 1 1 l 1

 

 

 

 

 

 

Total = 16 samples

108

Figure 4.1 A diagram of the location of the enclosure used for the bovine TB
environmental persistence study on the campus of Michigan State
University.

 

Experimental
Site

 

 

 

 

 

 

MiChjgan Cattle Barns Cattle Barns . 5
Dept. of

Natural V
Resources Cattle Barns Cattle Barns l-—-]

 

 

 

 

 

 

 

 

 

 

 

.Gat °°°°°° ‘-
e / Gate

 

 

“Deer- , . .
Proof” Dragrrostrc Center for Population

Fence and Animal Health

 

 

 

 

 

 

109

Figure 4.2 Placement of samples and weather station within the bovine TB
environmental persistence study enclosure.

 

  

 

        
  
  
 

 

 

 

 

 

 

 

   
   

 

 

 

 

 

 

 

 

   

Shaded from Sun Not Shaded from Sun
.- 2CAD, 2CND, 2HAD, 2CAL, 2CNL, 2HAL,
2HN D, 2HNL,
ZSAD, 28ND, ZWAD, 2WND ZSAL, zsNL, ZWAL, 2WNL
SET 3 DARK SET 3 LIGHT
SET 4 DARK SET 4 LIGHT
SET 5 DARK SET 5 LIGHT
SET 6 DARK SET 6 LIGHT

 

  

 

 

 

 

 
  
 

SET 7 DARK
SET 8 DARK

 

   

SET 7 LIGHT
SET 8 LIGHT

  

 

 

 

 

 

SET 9 DARK . 9 SET 9 LIGHT
SET 10 DARK , SET 10 LIGHT

 

 

 

 

 

 

SET 11 DARK :5; SET 11 LIGHT
SET 12 DARK SET 12 LIGHT

 

 

 

 

 

 

 

Key: C=com; H=hay; S=soil; W=water; A=autoclaved; N=non-autoclaved; D=dark;
L=light

110

Figure 4.3 Timing of sampling periods: Monthly over 1 year and weekly
over 3 seasons.

 

 

 

Year:
11/8/04 11/9/05
+
“A” Fall/Winter:
1 1/8/04 1/6/05
b
“B” Winter/Spring:
2/4/05 5/3/05
b
“C” Spring/Summer:
5/20/05 8/2/05

 

P

111

4.3 RESULTS
Samples of M. bovis inoculated substrates set up for the twelve-month study were
negative for months three through twelve. Data from the initial two months of the

twelve-month study are represented by the fall/winter season “A”.

4.3.1 Mycobacterial culture

One hundred and ninety-two sample replicates from each of the three sampling
periods (total 576) were processed for M. bovis isolation. Contamination of cultures with
mold and other non-mycobacterial species was detected in 13% of the samples processed
in sampling period fall/winter “A” and winter/spring “B” and 50% of the samples
processed in sampling period spring/summer “C”. Sample substrates that were sterilized
before M. bovis inoculation had signiﬁcantly lower odds of contamination across all
sampling periods (“A”: X2 = 20.28, p < 0.01; “B”: X2 = 10.29, p < 0.01; “C”: X2 = 4.7, p

< 0.05).

4.3.2 Environmental conditions

The coldest sampling period was the fall/winter “A”, winter/spring “B” was
intermediate and spring/summer “C” was the warmest with the greatest amount of
precipitation, highest solar radiation and greatest degree of evapotranspiration (Table
4.2). Weekly average temperatures and weekly average rainfall recorded at a Michigan
Automated Weather Network weather station in the endemic bovine TB area were

identical to the trends recorded at the study site. Average weekly temperature at the

112

northern weather station in the bovine TB endemic region was slightly higher across all

sampling periods.

4.3.3 Persistence of M. bovis in the environment

M. bovis persisted in substrates exposed to environmental conditions for an
average of one month in cool fall/winter and winter/spring conditions and for an average
of 7 days in warmer spring/summer conditions (Table 4.3). Both the time from
inoculation to the last positive M. bovis sample and the time ﬁ'om inoculation to ﬁrst
negative after last positive M. bovis sample are presented. The average and maximum
survival times were lowest in the spring/summer sampling period “C”. The maximum
survival time across all substrate types and sampling seasons was recorded in a soil
sample in the winter/spring “B” period. The soil sample was M. bovis positive at the
ﬁnal sampling point of 88 days. The shortest survival period was recorded in a hay
sample in the spring/summer “C” period. The sample was positive at time 0 but negative
at the 18t sampling point at 3 days. The mean and maximum survival time in each
substrate type in each season is presented in Figure 4.4. The overall mean and maximum
survival time across all substrate types in each season is presented in Figure 4.5.

The number of M. bovis positive replicate samples, and the number of M. bovis
colonies recovered per sample, drops off quickly over the ﬁrst 7 to 14 days of exposure to
environmental conditions. The isolation of M. bovis from substrates exposed to

environmental conditions was more intermittent after 14 days and positive samples were

often identiﬁed based on the isolation of less than 5 M. bovis colonies per 100 pl of

113

sample. The percent of M. bovis positive replicates recorded at each sampling point and

the number of colony forming units isolated are displayed in Figures 4.6 and 4.7.

4.3.4 Effects of non-seasonal factors on the persistence of M. bovis in the
environment

The effect of substrate type on the persistence of M. bovis in the environment is
variable. Symbols indicating signiﬁcantly different mean survival times are displayed in
Table 4.3 next to the mean survival period for M. bovis in each of the tested substrates.
In the spring/summer period “C” survival is signiﬁcantly longer in water. In the
fall/winter “A” period survival appears to be signiﬁcantly longer in hay and in the ‘
winter/Spring “B” period survival appears to be signiﬁcantly longer in soil. Survival
probability curves for M. bovis in soil, corn, hay and water across all seasons are
illustrated in Figure 4.8. The curves appear similar and log rank statistics conﬁrm that
the survival curves for the different substrates types are not signiﬁcantly different from
one another (X2 = 5.03, p=0.l7). Among all seasons there is no signiﬁcant difference in
the survival of M. bovis in one sample type versus another.

No signiﬁcant associations were found between the bovine TB status of a sample
and whether or not it was sterilized before M. bovis inoculation. The placement of the
inoculated samples in shade or direct sunlight did not have a statistically Signiﬁcant effect
on M. bovis survival but the mean survival time was longer across all samples and
seasons in those placed under shade. The difference in mean survival time in the shaded
and non-shaded samples approached signiﬁcance in the fall/winter “A” and

spring/summer “C” seasons (Table 4.4).

114

4.3.5 Effects of season on M. bovis persistence in the environment

Figure 4.9 illustrates the survival probability curves of M. bovis organisms
exposed to environmental conditions in fall/winter “A”, winter/spring “B”, and
spring/summer “C”. The log rank statistics were associated with highly signiﬁcant
differences (chi-square = 19.88, p<0.0001) for between season probabilities. An analysis
of maximum likelihood estimates when other covariates (Shade/non-shade, substrate type,
sterilize/non-sterilized substrates and interaction between shade/non-shade and season)
are added to the model makes it clear that it is the season that drives the difference in the

survival probability (Table 4.5).

4.3.6 Effects of weather on M. bovis persistence in the environment across seasons
The Cox’s proportional hazard regression model, used to determine the relative
inﬂuence of various weather related factors that together contribute to seasonal
differences in the environmental persistence of M. bovis, revealed that temperature is the
most inﬂuential factor in M. bovis survival. ”A number of the weather-related factors
recorded throughout the sampling periods were signiﬁcantly associated with the survival
of M. bovis in the environment, however, many of these factors correlated with one
another. The univariable hazard ratios and 95% conﬁdence intervals for the weather
related factors tested are presented in Table 4.6. Although all of these variables were
signiﬁcant at the p < 0.15 level, evapotranspiration and solar radiation were removed due
to redundancy. The ﬁnal multivariable Cox proportional hazard regression model is

presented in Table 4.7.

115

 

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Figure 4.4 The mean and maximum survival of M. bovis in corn, hay, soil and water
exposed to environmental conditions in F all/Winter “A”, Winter/ Spring
“B”, and Spring/Summer “C”. Error bars represent standard deviation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fall/Winter "A"
100
- so
3 60 X oMean
g 40 u- x T x Maximum
‘ f
20 § 5 l
0 Corn Hay Soil Water
Winter/Spring "B"
100
X
80 1
E
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g 40 ! )KMaximum
: l
20 >§ X x
0 Cam Hay SoiL film
Spring/Summer ”C"
100
80
E 60
X OMean
E 40 XMaximum
: 20
o X § ¥ L
.20 Com Hay Soil Water

 

 

 

118

Figure 4.5 The overall mean and maximum survival time (persistence) of M. bovis in
all substrates exposed to natural environmental conditions in F all/Winter
“A”, Winter/Spring “B”, and Spring/Summer “C”. Error bars represent
stande deviation.

 

All Substrate Types

 

 

 

 

 

 

 

 

 

 

 

 

 

100
so X
E 6° >K ..
é 40 I )K oMean
° + xMaximum
3 20 l ’ $
0 ..
.1.
66A,, ((B” 66C”
-20

 

 

 

 

119

Figure 4.6 The percent (n=4 of each substrate) of M. bovis positive replicates of corn,
hay, soil and water at each sampling point in F all/Winter “A”,
Winter/Spring “B”, and Spring/Summer “C”.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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120

Figure 4.7 The average number of M. bovis colony forming units isolated from
processed replicates of corn, hay, soil and water samples over time in
F all/Winter “A”, Winter/Spring “B”, and Spring/Summer “C”.

FALLMIINTER "A"
401 -<\\

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TIME (DAYS)

 

 

 

121

Figure 4.8 Survival probabilities of M. bovis in water, corn, hay and soil across all
seasons tested. Images in this dissertation are presented in color.

Survival
Function

 

 

1.00
—1 {—' Water

‘ 0.715 r] n
1 0.50 L ‘— Hay i

Corn “*

1 0.25 1

. 0.00 l

0 20 40 60 80 100

 

 

 

 

 

 

 

 

 

Soil

 

 

 

 

ci‘_

 

 

 

 

 

Days Post Inoculation

122

Table 4.4 Mean duration in days of M. bovis persistence in the environment on corn,
hay, water and soil samples in season “A”, “B” and “C” in shaded and non-

 

 

shaded conditions
Shade No Shade
Mean (Std. Dev.) Mean (Std. Dev.) t statistic p-value
Fall/Winter “A” 12.79 (12.27) 9.58 (9.38) -1.68 0.10
Winter/Spring “B” 18.40 (20.65) 17.41 (20.48) -O.20 0.84
Spring/Summer “C” 7.18 (12.52) 2.38 (4.03) -1.48 0.15

 

123

Figure 4.9 Survival probabilities of M. bovis exposed to natural environmental
conditions in “A”, “B”, and “C” sampling periods. N=16 samples per

. season. Images in this dissertation are presented in color.
Survrval

Function

 

Min—'1:—
s

 

 

 

 

Fall/Winter “A”

l

0.7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i 0.50
4 Spring/Summer Winter/Spring “B”
0.25 ¢ 60C”
__| O
O
I000 I I 1 l I I
0 20 4O 60 80 100

Days Post Inoculation

124

Table 4.5 Cox’s proportional hazard regression model for persistence of M. bovis over
three different sampling periods (fall/winter “A”, winter/spring “B” and
spring/summer “C”) or seasons

 

 

Risk Factor Parameter Standard Hazard Ratio
Estimate Error (95% Conﬁdence Interval)
Season A -2.13 0.49 0.12 (0.05 — 0.32)
Season B -l.94 0.49 0.14 (0.5 — 0.38)
Season C 0 -- --
Sample Type -0.28 0.16 0.76 (0.55 — 1.05)
Sterilized -0.50 0.35 0.61 (0.31 —- 1.20)
Shade -0.68 0.33 0.51 (0.26 — 1.05)

 

125

Table 4.6 Univariable analysis of the inﬂuence of each weather related variable on the
hazard (survival) of M. bovis in the environmental using the score test in
Cox regression.

 

 

Weather Factor Score p-value
(Average) Chi—Square

Solar Radiation 7.50 0.0062

Humidity 2.46 0.1 165

Temperature 9.85 0.0017

Precipitation 5.44 0.0197

Evapotranspiration 17.19 <.0001

 

Table 4.7 Multivariable Cox proportional hazards model of weather related factors
associated with the survival of M. bovis in the environment.

 

 

Risk Factor Parameter Standard Hazard Ratio
Estimate Error (95% Conﬁdence Interval)
Temperature" 0.06 0.02 1.06 (1.02 — 1.10)

 

* Results of purposeful selection of covariate and modiﬁed stepwise modeling in Cox
regression. Only temperature remains in the model.

126

4.4 DISCUSSION

Mycobacterium bovis is an obligate intracellular pathogen but it has been shown
to survive in the environment, outside a host, for substantial periods of time under
favorable conditions (Dufﬁeld and Young, 1985; Jackson et al., 1995; Little et al., 1982;
Maddock, 1933, , 1934, , 1936; Mitscherlich and Marth, 1984; Tanner and Michel, 1999;
Van Donsel and Larkin, 1977; Williams and Hoy, 1930; Wray, 1975; Young etal., 2005).
Although all of these studies demonstrated the persistence of M. bovis in the
environment, their experimental nature, the use of high bacterial loads, and the variability
of results have generally led to the conclusion that the environmental persistence of M.
bovis does not play a signiﬁcant role in the epidemiology of bovine tuberculosis through
the indirect transmission of the pathogen among or between susceptible species.

The outbreak of bovine tuberculosis (TB) in northeast Michigan, and the
establishment of a wildlife reservoir (white-tailed deer) for M. bovis in the region, has
renewed interest in the characterization of M. bovis persistence in the environment and its
role in the epidemiology of bovine TB in North America. This study clearly
demonstrates that the Michigan strain of M. bovis persists in the environment under
typical Michigan weather conditions. The study replicated the natural conditions under
which M. bovis would be deposited on substrates in the environment and the weather to
which the organisms would be exposed. Since the study site was approximately 200
miles to the south of the endemic bovine TB area in Michigan, basic indicators of
environmental conditions (average weekly temperature and precipitation) were compared
between the two areas by using data from the Michigan Automated Weather Network.

The trends in temperature and precipitation were found to be identical with the

127

temperature in the northern TB endemic area an average of 1-2 °C cooler throughout the
sampling periods. These slightly cooler temperatures in the north could be expected to

support slightly longer persistence of M. bovis in the environment.

4.4.1 The Persistence of M. bovis in the Environment

The general pattern of M. bovis persistence in the environment, across all seasons
and in all substrate types, was an initial decline in the numbers of recoverable bacilli over
a 7 to 14 day period and then the persistence of small numbers of M. bovis bacilli for up
to 4 to 10 weeks. The persistence of the Michigan strain of M. bovis on corn, hay, soil
and water recorded in this natural environmental exposure study conﬁrms the ﬁndings of
laboratory-based studies conducted earlier in which the Michigan strain of M. bovis was
found to persist for up to 12 weeks on feed (hay, corn, sugar beets, apples, carrots and
potatoes) stored at 46° F and 0° F and for shorter periods when stored at 75° F (Whipple
and Palmer, 2000). The similarity between the ﬁndings of the laboratory-based studies
on M. bovis persistence and this one, in which the experimentally infected substrates
were exposed to natural environment conditions, indicates that failure to detect M. bovis
in the environment in known areas of M. bovis transmission is likely associated with the
highly clustered spatial distribution of contaminated substrates under natural conditions
and the inability to pin point the exact location of a M. bovis contaminated substrate for
sampling, and not the inability of M. bovis to survive in the environment in a viable state.

The results of early studies performed in Europe and designed to characterize the
persistence of M. bovis in the environment, have been scrutinized due to the large

numbers of M. bovis bacilli used to experimentally inoculate of substrates (Maddock,

128

1933, 1934, 1936; Williams and Hoy, 1930). In this study the initial bacterial load used
to inoculate the substrates was 50,000 CFU of M. bovis. This amount of M. bovis is more
than the minimum infective oral dose of M. bovis established through experimental
studies with the Michigan strain of M. bovis in both cattle (5,000 CFU) and white-tailed
deer (300 CFU) (Palmer et al., 2002a, 2004a). The 50,000 CFU inoculums used is
thought to be indicative of the amount of M. bovis that could be deposited by a bovine TB
infected and shedding animal. The 50,000 CFU inoculum is smaller than the number of
M. bovis bacilli recorded in the exudates from a lesion in an infected brush-tailed possum
in New Zealand (5 X 108 CFU/ml) (Smith, 1972) but larger than number of bacilli
(approximately 70 CFU) detected in the nasal mucous of an experimentally infected cow
(McCorry et al., 2005). The amount of M. bovis used to inoculate substrates is believed
to be relevant both in terms of the probability of contamination of environmental
substrates through shedding of M. bovis ﬁom an infected animal and the likelihood of
ingestion of M. bovis bacilli present in the environment by a susceptible host. This is
particularly true in the 1St 7 days of exposure of M. bovis to environmental conditions
when the number of CFUs recovered from the experimentally inoculated samples

remained high.

4.4.2 Factors Influencing the Persistence of M. bovis in the Environment

This study was designed to characterize the persistence of M. bovis in a number of
substrates exposed to natural environmental conditions over a 12-month period. A
review of the literature indicated the necessity to supplement the 12-month study with a

number of seasonal experiments designed to capture the effects on the persistence of M.

129

bovis under different weather conditions. The persistence of M. bovis during the
Michigan spring/summer season (May 20th to August 2”“) was signiﬁcantly shorter than
the persistence recorded in the fall/winter season (November 8th to January 6'”) and
winter/spring season (February 4th to May 3rd). The spring/summer season was
associated with higher average daily temperatures, greater intensity of solar radiation and
higher loss of moisture through evapotranspiration. This ﬁndings are in agreement with
those reported elsewhere in which an increase in temperature and a loss of moisture were
found to be associated with a decease in the persistence of M. bovis in the environment
(Dufﬁeld and Young, 1985; Jackson et al., 1995; Tanner and Michel, 1999; Wray, 1975).

Other factors, including substrate type, did not signiﬁcantly affect the pattern of
M. bovis persistence. The detection of M. bovis in a soil sample at 88 days (the ﬁnal
sampling point) in the winter/spring sampling period is in agreement with other studies
that recorded the longest survival of M. bovis in cool, moist soil that presumably protects
the bacilli ﬁom desiccation and provides an organic environment that supports its
persistence (Dufﬁeld and Young, 1985; Maddock, 1933, 1934). Detection of M. bovis in
a water sample at 48 days in the spring/summer sampling period, in which all other
substrate types were negative at 20 days, indicates that even in the presence of high
temperatures and intense solar radiation, viable M. bovis can persist under moist
conditions.

The sterilization of substrates before M. bovis inoculation did not affect the
persistence of M. bovis but it did signiﬁcantly decease the occurrence of contaminated

bacterial cultures. Pre-sterilized substrates were not used exclusively in this study

130

because decreased survival of M. bovis in sterile substrates has been reported (Dufﬁeld
and Young, 1985; Young et al., 2005).

The location of the M bovis inoculated substrates under shade had an effect on
persistence. The mean survival time was longer for samples under shade than those
exposed to direct sunlight. This was true in the fall/winter season and the spring/summer
season but not in the winter/spring season. This apparent lack of a protective effect of
shade during the winter/spring season is likely due to the fact that the cloth used to cover
the “shaded” samples during this sampling period was severely damaged by wind and
removed. It was replaced before the ﬁnal spring/summer sampling period.

Survival analysis was used to evaluate the impact of weather over the three
seasons on the persistence of M. bovis in the enviromnent. Univariate analysis using
Cox’s proportional hazard regression indicated that the survival probability or persistence
of M. bovis was decreased as temperatures increased, solar radiation intensiﬁed and
evapotranspiration (a measure of moisture loss from a system) increased. The effects of
average precipitation and percent humidity lost their signiﬁcance in the presence of
temperature. Since solar radiation and evapotranspiration are both directly related to
temperature, the ﬁnal conclusion was that temperature drives the seasonal effect seen in

M. bovis persistence in the environment.

4.4.3 Implications of the Persistence of M. bovis in the Environment
The evidence that viable M. bovis persists in the Michigan environment under
near natural conditions has signiﬁcant implications on the efforts to control and

eventually eliminate the occurrence of bovine TB in the region. Authors have argued

131

 

when examining other systems of bovine TB in other parts of the world that the
conditions that contribute to M. bovis persistence in the environment also make the
organisms inaccessible to susceptible hosts (Jackson et al., 1995; Morris et al., 1994).
This is not the case in Michigan. The types of substrates tested (soil, water, hay and
corn) are present in and around cattle farms in northeast Michigan. Additionally, white-
tailed deer have access to these substrates on many cattle operations. Although many
feed piles are exposed to sunlight and summer temperatures, there are periods of the year
throughout the region in which low temperatures, cloud cover and the location of feed
and water sources under the cover of a forest canopy or otherwise protected from the
elements, would facilitate the long-term (4-12 weeks) survival of M. bovis bacilli
deposited by a bovine TB infected animal.

The elimination of feeding and baiting sites for white-tailed deer and other
wildlife should remain a component of the efforts to reduce bovine TB prevalence in this
population and part of the management recommendations designed to reduce deer-to-deer
bovine TB transmission events. Bovine TB eradication programs designed to eliminate
the occurrence of bovine TB in cattle must consider M. bovis contaminated feed or water
as a possible route for the indirect transmission of bovine TB between infected white-
tailed deer and cattle. Farm bio-security measures focused on the elimination of the
possibilities of cross contamination of feed and water sources should be added to the
current protocols designed to eliminate cattle-to-cattle and the direct transmission of
bovine TB. Speciﬁcally, cattle should be fenced out of open water sources and they
should be provided an alternative source of water. Cattle should not be fed hay on the

ground in the woods or in pasture adjacent to wooded areas and crops ﬁelds known to

132

attract white-tailed deer. The programs designed to encourage the fencing of feed storage
areas should continue but emphasis should also be placed on the fencing and protection

of cattle feeding areas.

4.4.4 Conclusions

These studies on the environmental persistence of M. bovis were designed to
mimic natural conditions in the endemic region of bovine TB transmission in Michigan.
The data clearly indicate that there is a real potential for the indirect transmission of M.
bovis among and between cattle and white-tailed deer populations in Michigan.
Persistence of M. bovis can be expected to be longer in cooler seasons. Practices that
facilitate the cross contamination of substrates by infected and susceptible animals should
be restricted at all times but especially during the cooler seasons of the year.

Failure to isolate M. bovis from environmental substrates collected from areas '
with known bovine TB transmission, is likely due to the inability to pin point the exact
location of environmental contamination for sampling and less to do with the persistence
of the M. bovis bacilli in the environment. Difﬁculties in isolating viable M. bovis from
environmental substrates due to the limitations of sample processing and mycobacterial
culture will also continue to hinder the accurate assessment of the quantity of M. bovis
organisms in the environment. However, this study indicates that the organisms do
persist over a time period that would allow a susceptible animal to become exposed and
infected with M. bovis from an environmental source. Indirect transmission of M. bovis
plays a role in the interspecies transmission of bovine TB and will continue to hinder

programs designed to eliminate the disease if not addressed.

133

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

SUMMARY

This dissertation has examined the persistence of Mycobacterium bovis in the
environment and the potential role of indirect transmission in the epidemiology of bovine
tuberculosis (TB). Special attention has been paid to systems in which a wildlife species
is believed to act as the reservoir of M. bovis infection. Experimental and observational
studies with a focus on the Michigan outbreak of bovine TB in cattle and white-tailed
deer have been discussed with the aim of enhancing our understanding of the role of
indirect transmission in the epidemiology of bovine TB in the region.

One of the primary limitations to assessing the persistence of M. bovis in the
environment was addressed by optimizing a technique for processing environmental
substrates for the detection of M. bovis with mycobacterial culture, the only real testing
option available for determining the viability of M. bovis bacilli in the environment.

Attempts were made to isolate M. bovis, with the optimized techniques, from
environmental substrates collected for cattle farms with a known history of M. bovis
infection. Opportunistic sampling of areas with a high prevalence of M bovis infection
in white-tailed deer was also performed. Though mycobacterial isolation was successful,
none of the isolates were identiﬁed as M. bovis.

To address the question of M. bovis persistence in the environment, given the
limitations of detecting the bacilli under natural conditions, a 12-month experimental

study was designed. Environmental substrates were inoculated with M. bovis and

134

exposed to natural weather conditions. The effect of environmental conditions on the

survival of M. bovis over time was assessed.

CONCLUSIONS

The recorded persistence of M. bovis in the Michigan environment under natural
weather conditions, strongly suggests the potential contribution of indirect means to the
transmission of bovine TB in the region. These data supplement those produced through
experimental M. bovis disease transmission studies that have proven the feasibility of
indirect transmission of M. bovis among and between cattle and white-tailed deer. They
also support the analyses of observational data on M. bovis infection in cattle and white-
tailed deer in Michigan that indicate the importance of indirect transmission in the
interspecies transmission of M. bovis in the region.

Conclusions speciﬁc to this dissertation research include the following:

1. It is possible to improve methods for processing environmental samples for M.
bovis isolation by bacterial culture, however, the necessity of decontaminating
samples limits the sensitivity of M. bovis detection.

2. Mycobacterium bovis is not distributed across the landscape of known sites of
bovine TB transmission and challenges associated with identifying the speciﬁc
point of contamination make detecting M. bovis in the environment under natural
disease transmission extremely unlikely.

3. Under experimental conditions Mycobacterium bovis does persist in the
environment for a long enough period to serve as a source of infection to a

susceptible host.

135

4. Survival of M bovis is longest in the cooler months of the year, in which
persistence of M bovis in a soil sample was recorded at 88 days.

5. Indirect transmission of M bovis is likely to occur if the host and environmental
factors provide an opportunity for substrate contamination by an infected animal

and subsequent contact by a susceptible individual.

RECOMMENDATIONS

Local, State and Federal bovine TB control and eradication policy needs to
consider indirect transmission of M bovis through contaminated environmental substrates
in the creation and implementation of epidemiologically appropriate disease management
plans. In the bovine TB endemic region of Michigan, interspecies transmission of bovine
TB should be considered by both wildlife and livestock health agencies. If this
component of the epidemiology of M bovis transmission in the region is ignored, efforts
to control and eventually eradicate the disease will fail.

Regulations restricting the supplemental feeding and baiting of wildlife should be
upheld and approaches to cattle farm bio-security should be reviewed with the intent of
preventing indirect transmission of M bovis between white-tailed deer and cattle. Cattle
farm repopulation protocols that rely only on time, in the absence of management
changes, to reduce the probably of TB re-infection are inadequate. The current focus on
preventing access of deer to feed intended for cattle is a step in the right direction,
however, that emphasis needs to be carried beyond issues surrounding feed storage to
those surrounding feeding sites and the opportunities for cross contamination of

environmental substrates in and around cattle operations. Speciﬁcally, cattle should be

136

fenced out of open water sources and they should be provided an alternative source of
water. Cattle should not be fed hay on the ground in the woods or in pasture adjacent to
wooded areas and crops ﬁelds known to attract white-tailed deer. The programs designed
to encourage the fencing of feed storage areas should continue but emphasis should also

be placed on the fencing and protection of cattle feeding areas.

137

Appendix A

Bovine Tuberculosis: Identification of Environmental Reservoirs of Mycobacterium
bovis in Michigan

On-Farm Environmental Sampling Protocol

1.

The Michigan Department of Agriculture will inform the owners of bovine
tuberculosis (TB) affected cattle herds of the opportunity to have environmental
testing done on their farm by the research group at Michigan State University.

The names and contact information of interested bovine TB-affected herd owners will
be forwarded to Dr. Amanda Fine, Population Medicine Center, Department of Large
Animal Clinical Sciences, Michigan State University.

Dr. Amanda Fine will contact the TB-affected cattle herd owners by phone and
discuss the project. An appointment for a farm visit will be made at a time that is
convenient for the cattle farm owners.

During the initial farm visit the project will be explained, the herd owners will have
the opportunity to ask any questions and they will be presented with a waiver form to
sign allowing the collection of environmental samples from their farm.

A walk through of the premises will take place with the herd owner's guidance. A
sampling plan will be formed after observations and discussions with the farm
owners. Sites for sampling will be marked on a rough map of the premises.

Approximately 20 samples will be collected from different sites around the farm. It is
expected that these samples will be of feed, water, soil, manure, bedding and pasture
grass.

Dr. Amanda Fine, a veterinarian trained in biological hazards and safety, will collect
all samples.

The samples will be placed in sterile, leak-proof containers or Whirl-Pak bags
ranging in size ﬁ'om 50 ml to 1,000 ml.

The containers will be sealed with their screw top caps and plastic Whirl-Pak bags
will be sealed with additional tape.

10. Smaller containers will be placed in larger plastic bags or containers and sealed.

11. All samples will be placed in a cooler on ice.

12. The samples will be transported by car to the Michigan State and will be delivered

within 12 hours of collection.

138

Appendix B

Bovine Tuberculosis: Identiﬁcation of Environmental Reservoirs of Mycobacterium
bovis in Michigan: Farm Management Survey (Part I)

F arm # Years cattle have been on this farm: years

 

1. Operation type (check all that apply): ~ Dairy ~ Beef cow/calf ~ Beef feedlot

Type of cattle # in herd
Dairy milking herd

 

 

Beef cow-calf pairs

 

Beef feeders

Bulls

 

 

2. Herd Size:

3. How large is your farm? acres total
a) Acres of pasture:
b) Acres of crop ﬁelds (no cattle access):
0) Acres of cattle housing (besides pasture):
d) Acres of homestead (yard, garden, etc):

4. Do you raise any other livestock on your farm?

~ Yes, list:
~ No

 

5. Do you have any pets or barn cats on you farm?

~ Yes, list:
~ No

 

6. Do your cattle have fence line (nose to nose) contact with any livestock you do not own or
manage?

~ Yes

~ No

Notes:

139

7. Please describe the movement of cattle on your farm as they age from birth to adult.

AGE (months)

LOCATION

 

 

 

 

 

8. Please describe the seasonal cycle of the movement of adult cattle on your farm. We are
interested in knowing when and for what period of time cattle are housed in or have access to

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

the following:
Beef Cow/Calf Pairs:
F eedlot/ Fenced Fenced Other
Indoors DryLot Pasture Cropland Woodlot (describe)
Season (%) (%) (%) (%) (%) (%)
Summer
Fall
Winter
Spring
Beef Bulls:
Feedlot/ Fenced Fenced Other
Indoors Dry Lot Pasture Cropland Woodlot (describe)
Season (%) (%) (%) (%) (%) (%)
Summer
Fall
Winter
Spring
Beef Feeders:
Feedlot/ Fenced Fenced Other
Indoors Dry Lot Pasture Cropland Woodlot (describe)
Season (%) (%) (%) (%) (%) (%)
Summer
Fall
Winter
Spring
Notes:

140

8. Seasonal cycle of the movement (cont.)

Dairy Cattle: period of time cattle are housed or have access to the following, in:

Summer

Feedlotl
Indoors Dry Lot
Cattle Group (%) (%)

Pre-weaned
calves

Pasture
(%)

Fenced Fenced Other
Cropland Woodlot (describe)
(%) (%) (%)

 

Weaned calves
& Heifers

 

Lactating dairy
cows

 

Dry cows

 

Bulls

 

Other
(describe):

 

Winter

Feedlotl
Indoors Dry Lot
Cattle Group (%) (%)

Pasture
(%)

Fenced Fenced Other
Cropland Woodlot (describe)
(%) (%) (%)

 

Pre-weaned
calves

 

Weaned calves
& Heifers

 

Lactating dairy
cows

 

Dry cows

 

Bulls

 

Other
(describe):

 

141

8. Seasonal cycle of the movement (cont.)

Dairy Cattle: What housing facilities did this operation use during the last 12 months for
the following (check all that apply):

Hutch Single Multiple
(single Tie Stall or Animal Animal
animal) Freestall Stanchion housing housing

A. Preweaned calves

 

C. Lactating dairy
cows

D. Dry dairy cows

 

E. Bulls
H. Other (describe)

 

 

9. Animals from outside the operation:
a) What percent of your animals were purchased from outside your operation?

Less than 5% 5 to 10% 11 to 25% 26 to 50% More than 50%

~ ~ ~ ~

Notes:

b) What percent of your animals spend any time at a location other than your farm?

Less than 5% 5 to 10% 11 to 25% 26 to 50% More than 50%

~ ~ ~ ~ ~

Notes:

142

9. c) Were any of the following groups of animals brought onto this operation from outside
sources during the last 12 months?

 

 

 

 

 

 

 

Brought onto Percent were brought
operation? onto operation?

A. Preweaned (milk-fed) calves? ~ Yes ~ No %

B. Weaned calves or heifers? ~ Yes ~ No %

C. Dairy cows? ~ Yes ~ No %

D. Bulls? ~ Yes ~ No %

E. Beef cow-calf pairs? ~ Yes ~ No %

F. Feeder cattle? ~ Yes ~ No %

G. Other (describe)? ~ Yes ~ No %

 

Water Sources
10. Where do your livestock drink? (mark all that apply)

~ Inside water tank/trough/individual waters
~ Outside water tank/trough
~ Surface Water (Pond, Lake or Stream)
~ Other, please describe:
Feeding Practices
11. What percent of the following feeds were purchased (rather than grown at your farm):

Forages: %

 

Grains: %

 

143

12. How are your livestock fed? (mark all that apply)

 

 

 

Indoors Outdoors
on Ground In Feeder on Ground In Feeder
Harvested forage, in whole bales: ~ ~ ~ ~
Harvested forage unrolled or ground up: ~ ~ ~ ~
Silage or Haylage: ~ ~ ~ ~
Concentrates (grain or pellets) ~ ~ ~ ~
Supplements (salt and minerals) ~ ~ ~ ~
Feed Storage
13. What type of hay bales do you use? (mark all that apply)
~ Round bales
~ Square bales
~ Other, please describe:
14. How do you store your hay? (mark all that apply)
In a building: ~ Outside: ~ Uncovered
~ Covered
Round bales: ~ Left in ﬁeld Fencing: ~ Fenced
~ Along fence rows ~ Unfenced
Notes:

15. How do you store grain? (mark all that apply)

~ In a grain bin

~ Bagged, indoors
~ Bagged outside
~ On the ground outside, mrcovered/unfenced
~ On the ground outside, covered/unfenced

~ On the ground outside, covered/ fenced

~ Other, please specify:

 

144

16. How do you store your silage/haylage? (mark all that apply)

~ Upright silo

~ Bunk with sides

~ Silage bags

~ Covered pile on ground
~ Uncovered pile on ground
~ Other, please describe:

 

Fencing:
17. What kind of fencing do you use for your livestock enclosures? (please specify units!)

Average fence Length of type of

 

 

 

 

 

 

 

Type of Fence Used? height fence used
Electric ~
High tensile wire ~
Woven wire ~
Barbed wire ~
Board/pipe ~
Building wall (barn, shed, etc.) ~
Other, please describe: ~

 

18. What kind of fencing do you use for your feed storage areas?

Average fence Length of type of

 

 

 

 

 

Type of Fence Used? height fence used
Electric ~
High tensile wire ~
Woven wire ~
Barbed wire ~
Board/pipe ~

 

Building wall (barn, shed, etc.) ~

 

Other, please describe: ~

 

145

Bovine Tuberculosis: Identification of Environmental Reservoirs of Mycobacterium

bovis in Michigan: Wildlife Incursion (Part 2)

19. Deer Incursions

Homestead
Ave. #lday Summer
None

Fall

Winter

SpringL

 

1-5
5-20

 

>20

Cattle Housing
Ave. #lday Summer
None

Fall

Winter

Spring

 

1-5

 

5-20

 

>20

Pasture/Wood Lot (Cattle Access)
Ave. #lday Summer
None

Fall

Winter

Spring

 

l-S

 

5-20

 

>20

 

Cropland (N 0 Cattle Access)
Ave. #lday Summer
None

Fall

Winter

SpﬂnL

 

1-5

 

5-20

 

>20

Feed Storage Areas
Ave. #lday Summer
None
1-5

Fall

Winter

Spring

 

5-20
>20

Other Land (No Cattle Access)
Ave. #ld4ay Summer
None
1-5
5-20
>20

146

Fall

Winter

Spring

20. Have you seen deer or evidence of deer using your livestock water somces?
~ No
~ Yes

21. Ifyes, what was the water source? (mark all that apply)
~ Water tank/trough/individual waterers
~ Sru'face Water (Pond, Stream, Lake)
~ Other

22. Where was the water source located?
~ In barn/shed
~ Barnyard
~ Pasture
~ Wooded lot
~ Other, please specify:

23. Where have you seen deer feeding activity on your farm? (mark all that apply)
~ Inside shed/bam
~ Inside or near calf hutches
~ Outside near shed/barn
~ Within a fenced livestock area
~ Within a fenced feed storage area
~ Outside a fenced area
~ Other, please specify:
~ Have not seen deer feeding activity on my farm

24. What type of feed are deer eating? (mark all that apply)

Forages: ~ Hay
~ Silage:~ Haylage
~ Corn silage

~ Other silage:
Grains: ~ Corn
~ Oats
~ Other grains:
Supplements: ~

25. Describe any other deer feeding activity (i.e. in orchard, landscape trees, etc.) on yoru' farm:

147

26. Are there any cedar swamps, orchards, or croplands that might attract deer onto your
preperty?

~ No
~ Yes Ifyes, please describe these

27. Have you observed any other wildlife species on your farm?

List:

Notes:

148

 

Appendix C

Bovine Tuberculosis: Identification of Environmental Reservoirs of Mycobacterium
bovis in Michigan

On-Farm Environmental Sampling

 

 

 

 

 

 

Data Collection Form

1 Farm #:

Sampling Date:

Sampling Time:

Weather Conditions:

Sample # Type Location Photo # GPS mark Notes
Soil, Description Digital photo Mark of Microclimate
hay, of sample site exact site of (damp, sunny,
water, sampling etc.)

etc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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