 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the
thesis entitled

SHEDDING OF MYCOBACTERIUM AVIUM SUBSP. ‘
PARATUBERCULOSIS IN NATURALLY EXPOSED DAIRY
CALVES AND ASSOCIATED RISK FACTORS

presented by T

Michael William Bolton DVM

has been accepted towards fulﬁllment
of the requirements for the

MS degree in Large Animal Clinical Sciences

{Epidemiology}

 

V Major Pr fessor's Signature
sz 2/ Z 0 7

Date

MSU is an Afﬁrmative Action/Equal Opportunity Employer

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

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K:IProjIAcc&Pres/ClRC/DateOue.indd

 

SHEDDING OF MYCOBACTERIUM AVIUM SUBSP.
PARATUBERCULOSIS IN NATURALLY EXPOSED DAIRY CALVES AND
ASSOCIATED RISK FACTORS

By

Michael William Bolton DVM

A THESIS

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

MASTER OF SCIENCE
LARGE ANIMAL CLINICAL SCIENCES (EPIDEMIOLOGY)

2009

ABSTRACT

SHEDDING OF MYCOBACTERIUM AVIUM SUBSP.
PARATUBERCULOSIS IN NATURALLY EXPOSED DAIRY CALVES AND
ASSOCIATED RISK FACTORS

By

Michael William Bolton DVM

With the recent development of liquid culture techniques, and associated
higher sensitivity, we conducted a study to detect shedding of Mycobacterium avium
subsp. paratuberculosis (MAP), the causative agent of J ohne’s disease (ID), in
naturally exposed dairy calves from eight Michigan herds. We charted age
distribution, MAP status of the dam, MAP prevalence in the herd, sensitivity of two
sizes of pooled fecal samples as well as concurrently procuring an ELISA blood
sample. We were able to detect MAP in calves with the preponderance of positive
animals falling within the seven to fourteen month age group. A higher percentage of
infected calves were from positive dams and herds with higher MAP prevalence.
There was no apparent association between fecal culture results and ELISA results and
fecal pools of ﬁve animals showed signiﬁcantly higher sensitivity than pools of ten. In
a separate case study we demonstrated the potential danger in retaining a clinically
normal, MAP shedding, cow in a low prevalence herd. The take home message, from
the sum of the components in this study, is that close attention has to be paid to the
young animal and all risk factors must be considered and controlled to

comprehensively manage J ohne’s disease in an infected dairy herd.

DEDICATION

This work is in memory of my Mom, Jo Bolton. It is a cuhnination of a process
she started with a gift of money, to attend the ﬁrst Dairy Certiﬁcate Program at

Michigan State in 1992. That has resulted in many opportunities.

It is also in honor of my Dad, Bill Bolton, a consummate learner and the most
unselﬁsh man I have ever met. He has reveled in each of the post- graduate degrees

obtained by every one of his seven children.

A multitude of thanks goes to each of my children, Molly, Zach, and Ann.

They each have had a hand in the preparation of some portion of this document.

Finally, and above all, my sincerest gratitude goes to my wife Ruthy, for all her

patience, encouragement and love.

ACKNOWLEDGEMENTS

Having completed this thesis after a four year project and extensive class work,
I now have a great deal of appreciation and respect for those who have traveled this
path. It has also become apparent to me that it takes a “community” to complete a task

such as this and I have many to thank.

First on the list is my committee, led by my major professor, the worldly and
family oriented Dr. John Kaneene. It was his unorthodox call to not only allow, but to
encourage — with patience and signiﬁcant remuneration — a long-standing dairy
practitioner to tackle this research endeavor. My gratitude also goes to Dr. Dan
Grooms who provided much of the project guidance, structure of the thesis, solid
leadership, and great friendship throughout this process. Committee member Dr.
Whitney Mauer was invaluable in the preparation of this document, her tireless efforts
along with her encouragement, patience, and friendship allowed this work to see the

light of day.

For ﬁnancial support, thanks go to the United States Department of Agriculture
(J ohne’s disease Demonstration Project), Michigan Department of Agriculture, and the

Center for Comparative Epidemiology at Michigan State University.

Special thanks to Dr. Roxanne Pillars and Dr. Joe Woltanski. who both seemed

to always have a spare hour to help collect calf fecal samples.

My appreciation goes to the folks at the Diagnostic Center for Pepulation and

Animal Health at Michigan State University, especially Joe Hattey and Dr. Steve

Bolin who made the diagnostic efforts on nearly 2,000 fecal samples possible. Special
mention must be given to Dr. Nora Bello who took a second look at the stats, and

Stephanie who rapidly helped compile this document.

On a personal note, I applaud the efforts of my partners in practice, especially
Dr. Steve Edwards, as well as Dr. Peter Blinkilde and Dr. Randy Carpenter, as they
encouraged me to “step out” and do this, and helped me manage my part time status at

the clinic.

Finally, a public thanks to Dr. Jim Lloyd who noticed this graduate position
and put me on to it. Jim also was responsible for nominating me to the board of
directors of the American Association of Bovine Practitioners for which I am grateful.
He is always selﬂessly surveying the landscape for possibilities for other people. I

could not have a ﬁner ﬁ-iend.

I dedicated this manuscript to my entire family who truly deserving it.

Thanks to you all, and Cheers!

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ viii
Introduction .................................................................................... 1
Chapter 1 ........................................................................................ 6

Risk Factors Associated with the Transmission and Detection of Mycobacterium
avium subspecies paratuberculosis in Young Dairy Cattle: A Review

Introduction ............................................................................ 7
General Description of MAP and JD .............................................. 8
Diagnostic Procedures (Direct and Indirect Testing Methods) ..................... 12
Risk Factors: Individual Cow Level Risks and Herd Level Risks ............ 18
Herd Control ......................................................................... 22
Summary .............................................................................. 27
References ............................................................................ 30
Chapter 2 ........................................................................................ 39

Detection of Mycobacterium avium subspecies paratuberculosis in naturally exposed
dairy calves, relationship to dam status, and other risk factors

Objectives ............................................................................. 40
Abbreviations ......................................................................... 42
Introduction ........................................................................... 43
Materials and Methods .............................................................. 44
Results ................................................................................. 48
Discussion .............................................................................. 49
References ............................................................................ 56
Chapter 3 ....................................................................................... 60

Use of Pooled Fecal Cultures to detect Mycobacterium avium subsp. paratuberculosis
in Naturally Exposed Dairy Calves: Comparison of Relative Sensitivity and
Speciﬁcity of Pools of ﬁve compared to Pools of ten

Abstract ................................................................................ 61
Introduction .......................................................................... 62
Materials and Methods ............................................................. 65
Results ................................................................................ 68
Discussion ............................................................................ 71
References ............................................................................. 73
Chapter 4 ....................................................................................... 77

Potential for Environmental Transmission of Mycobacterium avium subspecies
paratuberculosis From Non-Shedding Dairy Cows to Their Calves: A Case Report
Abstract ............................................................................... 78

vi

Problem Statement .................................................................. 80

Materials and Methods ............................................................. 81
Results ................................................................................ 82
Discussion ............................................................................ 85
References ........................................................................... 87
Overall Summary ............................................................................. 88

vii

LIST OF TABLES

Table 2.1: Mycobacterium avian subspecies paratuberculosis calf fecal culture test
results, positive calves and MAP shedding level, positive calves and herd prevalence,
and ELISA test results by calf age group distribution: Michigan 2005-2007 ...... 54

Table 2.2 —Myc0bacterium avium subspecies paratuberculosis fecal culture results of
calves in relation to serum ELISA and fecal culture status of their dam: Michigan
2005 — 2007 ................................................................................. 55

Table 2.3—Mycobacterium avium subspecies paratuberculosis serum ELISA results of
calves in relation to serum ELISA and fecal culture status of their dam: Michigan
2005 —2007 ................................................................................. 55

Table 2.4—Comparisons between age groups of fecal culture positive calves shedding
Mycobacterium avium subspecies paratuberculosis: Michigan 2005—2007 .......... 56

Table 2.5—Multivariable mixed logistic regression of risk factors associated with
Mycobacterium avium subspecies paratuberculosis positive calf fecal cultures:
Michigan 2005 —2007 ......................................................................... 56

Table 3.1—Distribution and fecal culture results for Mycobacterium avium subspecies
paratuberculosis of individual samples across the four age groups as well as combined
age groups of older compared to younger in pools containing ﬁve individual samples
and pools containing ten individual samples per pool. Michigan 2005 — 2007 68

Table 3.2—Distribution of pools containing ﬁve calves compared to pools containing
ten across individual age groups as well as younger compared to older calves and their
subsequent MAP culture status. Michigan 2005 — 2007 .................................. 69

Table 3.3 - Mycobacterium avium subspecies paratuberculosis fecal culture results
when comparing pools looking at false negatives and false positives distributed by age
group. Michigan 2005 — 2007 ............................................................... 7O

viii

Table 3.4 This is 3 2X2 table illustrating relative sensitivity, speciﬁcity, and positive
predictive value comparing pools of ten with pools of ﬁve in detecting Mycobacterium
avium subsp. paratuberculosis in relationship to individual fecal samples from dairy
calves across all age groups. Michigan 2005-2007 ........................................ 71

Table 4.1 - Presence of Mycobacterium avium subspecies paratuberculosz‘s (MAP) by
site swabbed on seven dairy cows and their respective serum ELISA and fecal culture
status: Michigan 2005 ........................................................................ 83

Table 4.2 - Timeline illustrating the presence of dairy cows (seven swabbed for MAP,
two known MAP fecal culture positive) in the “close-up” pen relative to the test date
(> enter pen, X exit pen, ---weeks in pen): Michigan 2005 .............................. 84

INTRODUCTION

J ohne’s disease (JD) caused by Mycobacterium avium subsp. paratuberculosis
(MAP) is a chronic, infectious, inﬂammatory enteric disease of both domestic and
non-domestic ruminants. Although JD is over a century old, described in 1895 in
Northern Germany and characterized soon after (Twort 1910) as an acid-fast bacillus,
it is of increasing international importance in the cattle industry. Very prevalent in the
United States, JD is associated with reduced milk production and economic loss (Ott
1999). There is also mounting evidence of a public health risk associating this
bacteria to Crohn’s disease (N aser 2004). Although widely variable prevalence
ﬁgures have been published (Adaska 2003; Hirst 2004; J ohnson-Ifearulundu 1999;
Pillars 2009) it is safe to assume that MAP is present in about half of the US dairy
herds and a signiﬁcant number of beef herds as well. It is widely accepted that calves
are often infected before the age of six months (Sweeney 1996) but MAP is a slow-
growing bacteria and development of clinical signs may take 2-5 years (Harris 2001).
Fecal culture has been an ineffective method to detect low bacterial shedders (Kim
2002). Due to reports of increased sensitivity with the recently developed
TREK®ESPII liquid culture system (Stitch 2004) we designed a study to determine if

we could detect fecal shedding in naturally infected dairy calves.

HYPOTHESES TESTED

l.

Fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) can
be detected in calves using liquid culture and this may not be equally
distributed across age groups.

An association exists between ELISA test results and fecal culture results of
these calves.

J ohne’s ELISA test status of the dam is a signiﬁcant risk factor to her
offspring.

There is an optimal number of animals contained in a pool of fecal samples
when utilizing this method to access MAP shedding in a group of calves.
There may be a risk associated with retaining a clinically normal, heavy

shedding cow in a dairy herd with low prevalence of J ohne’s disease.

OBJECTIVES

To test hypothesis one:

Prepare and run fecal cultures using TREK®ESPII liquid culture system on

individual calves of four age groups from eight dairy herds. Collect samples at

approximately four month intervals for a two year period. The test data from the dam

and the herd prevalence is already established.

To test hypothesis two:

Compare positive and negative fecal culture samples of these calves with their

ELISA test results to determine if an association exists between them.

To test hypothesis three:

Compare culture or ELISA status of dam with fecal status of various aged
calves.
To test hypothesis four:

a) Pool fecal samples (ﬁve to ten fecal samples in a pool) and determine ability to
detect one positive individual within each of the two pool sizes.

b) Conversely, test that positive pools contain at least one positive sample and
negative pools do not, determining relative sensitivity (Se), speciﬁcity (Sp),
and positive predictive value (PPV) of pools; compare to the MAP shedding
status of the calves that comprise the pool.

To test hypothesis five:
Describe a case study whereby a positive heavy MAP shedder is juxtaposed to
a group of animals ready to calve. Culture teat ends and other areas that a calf is likely

to nuzzle soon after birth for presence of MAP.

OVERVIEW

Chapter 1 is a targeted literature review of J ohne’s disease focusing on early
transmission and detection, utilizing liquid culture methods. Exploration of
environmental sampling and sample pooling as well as discussion of various risk
factors, management strategies to mitigate these risk factors will be the focus of this
literature review. A limited look at various national control programs and the

possibility of zoonotic potential will also be explored.

Chapter 2 addresses hypotheses one, two, and three and is a description of a
two year prospective, longitudinal, multiple cross-sectional study looking at detection
of MAP in calves. Also assessed is if MAP status of the dam is a risk factor to the
fecal culture results of the calf. Thirdly, we explored correlation between a calf fecal
culture and its ELISA test results.

Chapter 3 addresses hypothesis four comparing pooled fecal samples of two
sizes (ﬁve and ten samples per pool) looking at comparative sensitivities.

Chapter 4 assesses hypothesis ﬁve, a case study to illustrate the potential risk

of retaining one clinically normal heavy fecal shedder in a dairy herd.

REFERENCES

Adaska J M, Anderson RJ. Sero prevalence of Johne’s disease infection in dairy cattle
in California, USA. Prev. Vet Med. 2003;3;255-261

Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary
Medicine. Clin. Micro. Rev. 2001 ;l4(3), 489-512

Hirst HL, Garry FB, Morley PS, Salman MD, Dinsmore RP, Wagner BA, McSweeny
KD, Goodell GM. Seroprevalence of Mycobacterium avium subsp. paratuberculosis
infection in dairy cows in Colorado and herd-level risk factors for seropositivity. J.
Am. Vet. Med. Assoc. 2004;225(1), 97-101

J ohnson-Ifearulundu YJ, Kaneene JB, Lloyd J W, 1999. Herd — level economic
analysis of the impact of paratuberculosis on dairy herds. J. Am. Vet. Med. Assoc.
214(6), 822-825.

Kim SG, Shin SJ, Jacobson RH, Miller LJ, Harpending PR, Stehman SM, Rossiter
CA, Lein DA. Development and application of quantitative polymerase chain reaction
assay based on the ABI 7700 system (Taq Man) for detection and quantiﬁcation of

Mycobacterium avium subsp. paratuberculosis. J. Vet. Diagn. Invest. 2002;14(2), 126-
131

Naser, SE, Ghobrial, G, Romero, C, Valentine JF. Culture of Mycobacterium avium
subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet
2004;364,1039—1044

Ott SL, Wells SJ, Wagner BA. Herd- level economic losses associated with Johne’s
disease on US. dairy operations. Prev. Vet. Med. 1999;40, 179-192

Pillars RB, Grooms DL, Woltanski J A, Blair E. Prevalence of Michigan dairy herds
infected with Mycobacterium avium subspecies paratuberculosis as determined by

environmental sampling. Preventive Veterinary Medicine Volume 90, Issues 3-4, 1
August 2009, Pages 223—232

Stich RW, Byrum B, Love B, Theus N, Barber L, Shulaw WP. Evaluation of an
automated system for non-radiometric detection of Mycobacterium avium
paratuberculosis in bovine feces. J. Microbiol. Methods 2004;56(2), 267-275

Sweeney RW, 1996. Transmission of paratuberculosis. Vet. Clin. North Am. Food
Anim. Pract. 1996;12(2), 305-312

Twort FW. A Method for Isolating and Growing the Lepra bacillus of Man.
Proceedings of the Royal Society of London. Series B. 1910;83(562), 156-158

CILAPTER 1

RISK FACTORS ASSOCIATED WITH THE TRANSMISSION
AND DETECTION OF MYCOBACTERIUM AVIUM SUBSP.
PARATUBERCULOSIS IN YOUNG DAIRY CATTLE: A REVIEW

1. INTRODUCTION

Given the breadth of literature pertaining to Johne’s disease (JD) and the
causative bacteria, Mycobacterium avium subsp. paratuberculosis (MAP), this review
targets literature pertinent to the diagnosis of and risk factors related to JD
transmission in cattle. In particular, research pertaining to subjects necessary to either
understand the pathogenesis of JD, MAP diagnostic testing and risk factors related to
management and control of JD on dairy farms is also reviewed. Study of this disease is
important because of its’ devastating ﬁnancial impact of nearly one quarter of a billion
dollars annually to the US dairy industry (Garry 1998). Furthermore there is interest
in this disease beyond the agricultural sector due to potential zoonotic implications
(N aser 2004). Understanding risk factors of this complicated disease will assist in the
formulation of speciﬁc control programs that will be more effective in limiting or

extinguishing the impact of JD.

A. SEARCH METHODS

The electronic reference library at Michigan State University was used for
obtaining peer-reviewed, relevant articles. These included, but were not restricted to,
Science Direct, Pub Med, Medline, CAB abstracts which were used to scan research
using key words of interest (e.g., J ohne’s, calves, immunology, detection, testing
methods, environmental sampling, management, risk factors, control, zoonosis and
others). Articles were restricted to those from refereed journals, peer-reviewed
proceedings, and governmental data including the National Animal Health Monitoring

Systems (N AHMS) resources that were relevant to this review.

2. GENERAL DESCRIPTION OF (MAP) AND JOHNE’S DISEASE (JD)
A. BACTERIA

The organism that causes the infection that can ultimately lead to J ohne’s
disease (JD) is Mycobacterium avium subsp. paratuberculosis (MAP). It is an acid
fast, obligate intracellular, aerobic bacillus that needs the host to multiply, (Krieg
1986) but may be viable in the environment for a minimum of one year under proper
conditions. Although it is quite hardy, it is susceptible to desiccation in the presence
of sunlight (Whittington 2004). Commonly, the dark moist conditions found on

farms allow organism stability for an extended period (Whittington 2005).

B. IMMUNOLOGY

A fundamental understanding of the immunological challenges that MAP
presents to the host will provide the foundation on which to place the topics of
importance, such as risk assessment, testing strategies, environmental and
management challenges, and control programs.

MAP is an intracellular bacteria that initiates a cell mediated T Helper (TH)
Type I response (Bannantine 2008) by the calf, as it is picked up by M-cells in the gut.
The gram positive aerobe is transported from intestinal villi to the endothelium and
engulfed by macrophages. This causes a delayed T type IV hypersensitivity reaction
(Bannantine 2008). Suppression of pro-inﬂammatory cytokines and subsequent
expression of anti-inﬂammatory cytokines as the disease progresses (Stabel 2006) is a
key to the organisms’ survival in the host. As TH 2 responses are usually triggered by

extracellular challenges such as parasites, there is an ineffective response to the

intracellular MAP. This transition from the cytotoxic TH 1 response to an ineffective
TH 2 response is observed at the onset of clinical signs. The source of the cells that
turn off the TH 1 cytokines and inhibit cell-mediated-immunity (CMI) response is
thought to occur within the mediastinal lymph nodes draining the infection site
(Coussens 2004). This transition to an ineffective TH 2 response allowing progression
of JD is not well understood. Additionally, because this does not always happen, not
all exposures are pathologic. The recent use of multicolor ﬂow cytometry (K00 2004)
and access to the sequenced (Catanho 2006) genome of these bacteria will allow
characterization of these immunologic transformations from start to ﬁnish at the

cellular level improving our understanding of JD.

C. TRANSMISSION

Fecal-oral transmission of MAP is the primary mode for perpetuation of JD on
a dairy operation. However, in utero transference is a signiﬁcant risk to the fetus,
especially in dams that are in advanced stages of the disease (Whittington 2009).
Other common sources of calf infection at birth include vaginal contamination during
the birthing process, teat contamination following birth, feeding of MAP positive
colostrums from the dam, and a contaminated calving environment.

In utero transmission becomes a critical issue when other post natal
transmission factors are eliminated (Whittington 2009). In the attempt to identify at-
risk animals, it has been shown retrospectively that the MAP shedding status of the
dam is a major risk factor associated with transmission of JD to her offspring (Aly

2005) due both to congenital transmission as well as periparturient exposure to MAP.

In this situation, removing the infected dam from the herd is the only effective control
method. Additionally, recent reports have shown that infected cows housed next to
naive calves can infect these calves and subsequent shedding from these calves can
horizontally infect other na‘r've calves (van Roermund 2007).

Pooled milk from high somatic cell count (indicating intramarnmary
inﬂammation) cows or feeding bulk tank milk has not been shown to be a signiﬁcant
risk factor for JD transmission to calves, but feeding pooled colostrums was,
according to a recent Danish study (Nielsen 2008). The same study showed that the
practice of feeding colostrum replacer to calves born to positive cows and feeding
colostrum from negative dams only seemed to provide mitigation.

Mycobacterium avium subsp. paratuberculosis is nearly ubiquitous on infected
dairies. Common cow areas such as alleys and lagoons are the most prevalent areas for
this organism to be isolated. Environmental sampling has become a relatively accurate
and easy method of detecting positive herds (Lombard 2006; Pillars 2009). These
high trafﬁc areas contaminated with MAP are high risk areas for transmission to the
unexposed calf. A phenomenon that has been long observed anecdotally is the
clustering of cows with JD within a herd which would indicate that several calves in a
row, a year or two prior, were infected nearly simultaneously, and could be explained

by environmental transmission.

10

D. CLINICAL PICTURE

The MAP bacteria have worldwide distribution and causes granulomatous
enteritis in exotic and domestic ruminants. This bacterium is also harbored in some
non-ruminants such as the fox and the badger. There are typically four stages of the
infection if allowed to run its course; 1) asymptomatic or “silent”, 2) subclinical, 3)
manifestation of clinical disease, and, if not culled, 4) advanced clinical disease
(Whitlock 1996).

The calf, and animals up to two years of age, may harbor “silent” infection.
These animals are asymptomatic, and exhibit only occasional shedding (Bolton 2005).
There are no observed clinical signs and no cost-effective conﬁrmatory tests at this
time to diagnose JD infection at this stage, even though these animals may be
shedding MAP at a low level and be a risk to their herd mates (van Roermund 2007).
As the disease progresses to the subclinical phase there may be minor weight loss and
increased fecal shedding, especially as the animal approaches the clinical phase
(Tiwari 2006).

Early in the clinical portion of JD, vital signs may remain normal, including
appetite, though intermittent diarrhea develops and weight loss occurs. Many animals
are culled before reaching the clinical stage due to production losses (Abbas 1983). If
allowed to progress, the disease enters the advanced clinical stage which is
characterized by constant diarrhea, weakness, mandibular edema, extreme weight loss,
recumbence and death. During the clinical and advanced clinical phases, the infectious
potential of the animal intensiﬁes due to increased shedding of the organism (Manning

2001) in both feces and milk. The common clinical outcome of this severe

ll

granulomatous enteritis, Johne’s disease (JD), is easy to visualize with its chronic
diarrhea and associated cachexia (Waters 1999). This disease usually is non-
responsive to treatment and the results are often fatal or result in culling the animal

from the herd for poor performance.

3. DIAGNOSTIC PROCEDURES (DIRECT AND INDIRECT TESTING
METHODS)

The value of effective early testing may not only lie in the prediction of
outcome of individual animals but also may have the added value of reducing the
amount of bacteria in the herd, thus decreasing environmental bacterial load and
transmission of this pathogen.

Due to the complexity of the immune response, latency of the disease, and
variability of isolation from fecal shedding, (Visser 1999; Kalis 1999) it has been
difﬁcult to develop a highly effective test for detecting a MAP infected animal,

especially early in the infection process.

A. DIRECT TESTS

Diagnostic tests that detect the presence of the actual MAP organism, such as
fecal culture, are direct tests. As it is imperative to identify and cull infected cows, as
well as monitor documented management practices and purchasing strategies to
control the presence of this disease on the farm, (Whittington 2001) Sensitivity (Se)
and Speciﬁcity (Sp) are both important qualities of these tests.

The fecal culture using Herrold’s Egg Yolk medium (HEYM) has been

considered for many years to be the “Gold” standard for direct testing. This egg yolk

12

emulsion is used to provide a source of iron for MAP and for neutralization of toxins
within the sample (Merkal 1974). While this test has high Sp the Sc of this method is
estimated at only 38% - 50%. Therefore, it has limitations in early detection of MAP
when bacterial shedding is the lowest and Se is of the greatest value (Whitlock 2000).
Another drawback of the HEYM fecal culture is the length of time required (up to 16

weeks) to demonstrate the organism (Harris 2005). The strength of the recently

developed TREK®ESPIIa liquid culture system is that the time to positive (TTP) is

reduced from 16 weeks with HEYM to a maximum of 42 days. This method monitors
0; consumption by MAP organisms, if present, by sensing the negative pressure in the
culture vial via a computerized system. Unlike the HEYM test where colony forming
units (CFU) are measured, this test uses TTP as a proxy for the level of MAP
shedding. The lower the TTP, the heavier the bacterial shedding (Williams-Bouyer

2000)

Two other liquid culture systems used widely are the MGIT 960 systemb and

the BACTEC 460c radiometric system. These systems, with differences in sample

handling and preparation, also measure changes in ion levels or pressure changes
resulting from shifts in Oz and C02 concentration that occur with growth of the MAP
bacillus. With these liquid culture systems the Se is at least equal to HEYM when
bacteria are in high concentrations (Sweeney 2007) and they seem to have a slight
advantage in Se with subclinical, low shedding animals, with no sacriﬁce in Sp (Kim

2004). In either case, a critical advantage of liquid culture is a 70% reduction of time

13

needed to produce the test results with at least equivalent sensitivity and speciﬁcity
parameters, which is important for the management of positive animals within a herd.
The most recent and one of the most sensitive of the direct tests is an enhanced
polymerase chain reaction (PCR) test (Bogli-Stuber 2005). This test reﬂects
improvements of sensitivity (Se 52%) and (Se 59%) over serum enzyme-linked
immunosorbent assay (ELISA) and bacterial culture tests, respectively, without
sacriﬁcing speciﬁcity (Sp 99% - same as the other two methods) (Scott 2007). Unlike
direct PCR performed on the fecal sample itself or contents of the culture vial (used in
liquid culture), where the test is less sensitive due to contamination, this newer PCR
technique utilizes a common extraction and enhancement procedure, which helps to
eliminate sample contaminants. This process begins by incubating the fecal samples
with magnetic beads coated with rabbit origin polyclonal antibodies (Khare 2004).
This is followed by washing, lysing, and precipitation to extract the DNA from the
samples. The DNA precipitate is then re-dissolved and 18900 primers are utilized in a
real time PCR. Use of this magnetic separation technique to pull in the immuno-
captured bacteria has increased the diagnostic Se from less than fecal culture (Collins
1993) to approaching that or higher than fecal culture (Cook 2007). The current
challenge with newer diagnostic methods and disease management methods that will
have to be rectiﬁed is that, as Se increases and the time to obtain results decreases, the
price of diagnostic tests also has increased, because newer technologies are expensive.
A balance has to be reached as the tests seems to allow for earlier diagnosis of MAP
infection, which is critical in the management of J ohne’s disease as there are no

effective treatments.

14

B. INDIRECT TESTS

Indirect testing for MAP infection measures an immune response to an antigen
in contrast to direct testing which detects the presence of the organism. To have a
valid indirect test, there has to be a known relationship between the immune response
and the presence and shedding of the organism.

The serum ELISA (enzyme linked immunosorbent assay) Idexx Herdchek is a

popular test for MAP due to its price (approximately $6/test - 2009), (DCPAH —
MSU)e quick results reporting, and, most-importantly, its Se. This test uses optical

density (OD) to measure IgG antibodies that are capable of binding with MAP,
reporting the results as an OD reading. Intra-laboratory variation is corrected by
subtracting the OD from a mean negative control value and recording the difference,
the OD corrected (ODc). Animals are considered positive when the mean OD minus
the plate negative control is greater than 0.100 as recommended by the manufacturer
and as reported in other studies (Alvarez 2009). It has been shown that the mean time
it took for an ELISA positive cow to commence detectable shedding and be identiﬁed
via fecal culture was about nine months after being tested, so ELISA antibodies are
generally present prior to shedding of the MAP organism. Therefore, serum ELISA
was determined to be a good test when used to predict future MAP shedders (N ielson
2008)

A milk ELISA test has been developed in Europe, utilizing the Prionics
Parachekf test and adapted by Antel Biog in the US. This is a non-invasive test, since

the milk of cows is gathered and routinely tested for other components at regular

15

intervals. This allows for screening large numbers of cows. It has been validated by
the United State Department of Agriculture (USDA) and is commercially available in
the US at this time. In several studies it has been shown to perform equally as well as
the serum ELISA when compared to the HEYM fecal culture (Hendrick 2005; Collins
2005; Lombard 2006). This test is performed on the individual milk sample and is
often associated with monthly milk sampling done by the Dairy Herd Improvement

Association (DHIA) which is available to all the DHIA’s members.

C. DEVELOPMENT OF FUTURE TESTS AND TESTING STRATEGIES

A new version of a serum ELISA test using formaldehyde and soniﬁcation
(SELISA) holds promise as its Se is higher than traditional ELISA tests without
sacriﬁcing Sp (Speer 2006).

In the direct testing arena, use of protein arrays of MAP in a ninety six-well
system may be the new frontier. This array may allow us to not only identify
organisms but also identify characteristics of the infective organism that may aid in
assessing virulence and predicting immune response by host. Another advantage of
protein arrays is the lack of any cross-reactivity between antigens. This utilization of
genomics, molecular testing and complicated immunological assays may hold the
collective key for future testing and control of J ohne’s disease (Bannantine 2008).
Although holding promise, these tests are in the development phase and not available
for general use at this time.

Pooled Samples - An example of a new strategy using existing tests involves

the pooling of individual fecal samples to detect the existence of one or more positive

16

MAP shedders within the pool, which has been used as a management tool for
assessing whether or not MAP is present within a herd (Kalis 2000; Wells 2002).
Pooled fecal sampling for culture has been recommended for some time in Australian
sheep ﬂocks (Whittington 2000). The number of individual animals to incorporate in
a pool has been debated in the literature with some studies determining the most cost
effective pool size is ten individual animals per pool (Tavompanich 2004; van Schaik
2007). However, in studies that dealt with low shedding cattle (Eamens 2008) or
tested herds of various Sizes and prevalence levels, it was indicated that pools
containing no more than ﬁve individual animals were appropriate to maintain adequate
Se (van Schaik 2003). The same study showed that pools of ﬁve containing at least
one low shedding animal had at least a 53% chance of culturing positive for MAP
using standard culture methods. Far more variable results were observed using pools
of ﬁve when using the RT-PCR test (Scott 2007). Pools are a cost effective

management tool to utilize in assessing the presence of MAP in an infected herd.

D. UTILIZATION OF TESTS

Because there is no effective therapy for JD, the ﬁrst requirement to determine
which testing method is the most effective is to understand whether it is a desirable
goal to cull “infectious (shedding)” animals to prevent JD spread or not. Therefore,
for all of these tests, it is important to identify the purpose of the testing strategy to be
selected for management purposes (Nielsen 2006). Furthermore, as tests improve over

time, it will be possible to focus our decision analysis on how to handle the disease

17

rather than being diverted by the uncertainty of test results (Smith RD Slenning BD

2000)

4. RISK FACTORS: INDIVIDUAL COW LEVEL RISKS AND
HERD LEVEL RISKS

Risk of acquiring an infectious dose of MAP resides in whatever surrounds the
calf. As described earlier, a herd can maintain infection by many different pathways.
For this reason many questionnaires attempting to indentify various risks associated
with various management practices on the dairy farm have been developed by
investigators. One such survey initially had several hundred questions and after
conducting multi-variable statistical analysis it was determined that assessment of only
eleven questions resulted in nearly the same accuracy in risk determination (Berghaus
2005)

The risks in this review are divided into two major categories. The ﬁrst
category is individual cow level (inherent) risks, risks that are present in the animal at
a point in time and are intrinsic to the cow. Unlike herd level (exposure) risks, inherent
risks such as darn status, in utero transmission, or variable genetic susceptibility can
only be changed over time, by culling or natural attrition. Herd level risks such as
environment, housing, colostrum management, among others, are also important but

can be altered as quickly as the farm is able to initiate programs to mitigate these risks.

18

A. INDIVIDUAL COW LEVEL (INHERENT) RISKS

It has been demonstrated that transfer of the MAP infection can occur in utero
(Sweeney 1992). A recent meta-analysis illustrates that the likelihood of this transfer
is related to within herd prevalence. In a herd with a 5% prevalence of infection, the
annual incidence of an infected calf at birth is 1%. This is particularly problematic in
herds that have managed other risk factors well. Likewise, about 40% of calves born
to a cow clinically manifesting symptoms of JD will be born carrying MAP
(Whittington 2009). These same high rates of in utero transmission of MAP have
been shown in sub-clinically infected red deer (Cervus elaphus) in New Zealand
(Thompson 2007).

MAP status of the dam has always been assumed to be a risk factor. In a
recent retrospective study it was found that dairy cows with sero-positive dams were
6.6 times as likely to be sero-positive, compared to cows from sero-negative dams
(Aly 2005). A retrospective look at zoo ruminants found nearly the same relationship
between the dam and her offspring (odds ratio [OR] = 6.8 p < 0.01) (Witte 2009).
Although a similar retrospective study in beef herds in Texas did not demonstrate the
same relationship (Osterstock 2008) as in the dairy and zoo animal, there was a clearly
deﬁned familial relationship in sero-positivity. This may be explained by genetic work
that shows susceptibility to MAP infections may be associated with mutations in the
captase recruitment domain (CARD 15) gene. Possessing this allelic variant in a
case-control study resulted in more than a threefold (OR 3.35) increase in likelihood

of infection in beef cows (Pinedo 2009).

19

B. HERD LEVEL (EXPOSURE) RISKS

There are many ways a susceptible young calf can be exposed to MAP and
become infected. Exposure of the young dairy animal to feces laden with MAP is still
the primary and most manageable of the exposure risks. A recent retrospective study
conﬁrmed this and the authors speculated that once a control program has been
implemented, the largest risk factor to calves besides the status of their darn was the
recent calving of an infected and MAP shedding cow in the vicinity of birthing
(Benedictus 2008).

Another important and early risk factor is the calf suckling a contaminated cow
or ingesting colostrum containing MAP. A Danish study showed that calves fed
pooled colostrum were 1.24 times as likely to be ELISA positive as calves fed only
their own dams colostrum, and if allowed to suckle compared to being fed milk
replacer the OR was 2.01 (Nielsen 2008). Previous work has also highlighted this
exposure risk (Streeter 1995) by showing that 8/36 of colostrum samples were positive
for MAP from subclinical fecal shedding cows.

Environmental contamination due to housing conditions whereby cows
shedding the MAP organism are allowed access to the area of the young calf is a
major exposure risk. For example housing periparturient cows with pre-weaned calves
for more than 24 hours can increase JD prevalence in dairy herds (Wells 2000) as can
housing them in crates next to shedding cows (van Roermund 2007). It was also
demonstrated that these infected calves became shedders themselves and were able to

horizontally infect na'r've calves house in the same pen (van Roermund 2007). Another

20

study showed an increased herd prevalence when calves of less than Six weeks of age
were housed with positive cows (Obasanjo 1997). Some additional environmental risk
factors, including the use of an exercise lot for lactating cows, (J ohnson-Ifearulundu
1995) and spreading contaminated manure on pasture (Obasanjo 1997), have been
associated with increased prevalence of MAP on dairy operations. The frequency of
purchasing of animals and their source is an indirect, but very important risk factor
(Wells 2000).

There are other, less common but more regional or farm speciﬁc risks of
acquiring MAP infection. In a Minnesota study looking at wildlife (rabbits and deer) it
was shown that although wildlife prevalence was low (2-4%) the probability of daily
contact between cattle and wildlife was 20%, so wildlife reservoirs should be
considered to be a potential source of MAP transmission (Raizman 2005). Another
area of interest is MAP co-infection with pathogens such as the Bovine Viral Diarrhea
Virus (BVDV) and Bovine Leucosis Virus (BLV). Recent work showed proper
vaccination of calves with BVDV vaccine was associated with fewer MAP sero-
positive cows (Tiwari 2009). Although there are many avenues for this agent to infect
the young animal, many of these risks can be managed to decrease within-herd

prevalence.

21

S. HERD CONTROL
A. CONTROL WITHIN HERD (Management Practices)

Due to the ubiquitous nature of MAP and its presence in a high perCentage of
dairy herds, control of J ohne’s disease and the reduction of within-herd prevalence has
been the focus of the USDA, multi-state J ohne’s disease Demonstration Project. Their
focus has been producer education as well as risk assessment and mitigation. In
Michigan, for example, using slightly different sampling protocols, the percentage of
herds with at least one MAP culture positive animal has decreased from 66% to 48%
in the past ten years (J ohnson-Ifearulundu 1999; Pillars 2009). This may, or may not,
be a result of this educational effort and increased awareness by producer, but seems
that there may be an association.

Even in smaller countries, such as Germany, where the pathogen was ﬁrst
identiﬁed, eradication does not seem to be on the near horizon. The reason is that due
to recent work, MAP is considered an environmental pathogen with reservoirs in a
variety of animals, (Stratmann 2005) thus the German focus continues to be on
decreasing spread of the disease by identifying heavily infected herds with milk
ELISA and removing strong shedders in those herds. All herd control strategies
should strive to decrease new infections (i.e. decrease exposure of the calf to MAP)
with adoption of best management practices as well as to decrease the prevalence of
shedding animals in the herd (T iwari 2006; McKenna 2006). There have been
methodologies developed to identify positive herds by environmental sampling of
targeted areas on the dairy. These areas include the lagoon, alleyways, fresh cow

pens, and other “common adult cow areas.” When culturing these environmental

22

samples, rather than individual cows in the herd, the culture of pooled environmental
samples identiﬁed positive herds with relative sensitivity of 70%

compared to individual animal culture of all samples within pools (Raizman 2005).
This is inexpensive and requires no individual animal handling or restraint. A USDA
study validated this earlier work and found that greater than 70% of herds with either a
positive ELISA or positive fecal sample were identiﬁed by this method (Lombard
2006)

A study completed in California (T avompanich 2008) compared particular
management practices with herd seroprevalence, correcting for other variables. Not
surprisingly, this study found utilizing feeding equipment for manure handling,
exposing young calves (less than Six months of age) to manure of adult cattle,
exposure of cattle to lagoon water, and feeding unsalable milk to calves held the
highest predictive values for MAP infected herds. Management practices that were not
signiﬁcant included the use of individual calving areas, time before separation of calf
from cow at birth and frequency of bedding changes.

There have been protocols installed on dairy operations addressing their
particular risk proﬁle. Some things, such as using separate machines for feeding and
manure handling, or moving calves from infected cows, are intuitively obvious. Other
changes to mitigate risk to young stock are somewhat more intensive and creative. An
example of this is the feeding of unsalable milk after running it through a high-
temperature short-time (HTST) pasteurizer. This became an important risk mitigation
practice after the 2002 NAHMS Dairy Survey showed that almost 90% of US dairy

operations fed unsalable milk to neonates (NAHMS 2002 Dairy Survey). Ancillary

23

beneﬁts were removal of other pathogens such as Salmonella and Mycoplasma which
resulted in a better product to feed calves (Stabel 2004). These are just some of the
tools to reduce exposure to the most susceptible animal on the dairy—the calf.

Two other speciﬁc management measures are available to help reduce the
shedding of MAP in dairy herds. The ﬁrst, vaccination, is only allowed in a few states

for calves <35 days of age. At present there is only one vaccine, Mycopar® (Fort

Dodge)h, licensed in the US. It is a whole-cell killed suspension of MAP suspended in

oil. It is tissue reactive and has some human health risks with accidental injection
(Solvay MSDS Mycobacterium paratuberculosis bacterium—1990). This vaccine
seems to have an impact on shedding reduction but not on overall colonization
(Uzonna 2003). With the advent of genomic unraveling of both the cow and the MAP
pathogen, an entire new foray into vaccine development has begun. Due to less
antigenic diversity within MAP, compared to some other Mycobacterium, (Wu 2006),
a recent vaccine containing recombinant MAP proteins has been developed and
successfully tested (Kathperumal 2008). Although there was occasional colonization
of a single tissue site in vaccinates at <10 colony forming units (CFU), the controls
had several tissues found to be culture positive at >250 CFU.

The second herd level MAP management measure to control shedding, which
has been used in Canada for sometime, is the addition of monensin to the ration.
Monensin use was legalized in US for dairy herds effective January 4, 2006 by FDA
(FDA — 2006

(http://www.fsis.usda.gove/News & Events/Agenda NACMCF Mar2006/index.asp

24

Last Accessed July 31, 2009)). It has been shown to be protective in a murine model
against hepatic granulomas in susceptible mice (Brumbaugh 2004). Two studies
designed to test efﬁcacy of this use for monensin were conducted in Canada. It was
found that herd sero-positivity was reduced with monensin use (Hendrick 2006) but
that it only marginally reduced the level of MAP shedding (Hendrick 2006). The
authors concluded that monensin or any other drug would never be a replacement for

good management practices but could aid in prevention and control of J ohne’S disease.

B. US CONTROL PROGRAM

In the US the Johne’s disease control programs are speciﬁc to each state and
are primarily voluntary. They concentrate on managing risk and reducing within-herd
prevalence over time through testing, culling, and management changes rather than
testing and culling only. To increase national uniformity the US Voluntary Johne’s
Control Program was established. Under this umbrella the Uniform Program Standards
for the Voluntary Bovine J ohne’s Disease Control Program (VBJDCP) were
developed with input from the National Johne’s Working Group, J ohne’s Committee
from US Animal Health Association, state veterinarians, and representatives from the
cattle industry. It was approved by USDA Animal and Plant Health Inspection Service
(APHIS) Veterinary Services, effective as of June 1, 2006. Part (1) focuses on
Education, Part (2) on Assessment of Risk and Management, and Part (3) deals with
Testing and Classiﬁcation. (USDA -— APHIS, 2006
hm)://www.aphis.usda.gov/anima1 health/animal diseases/johnes/downloads/johnes-

umrpdf (Last accessed July 31, 2009))

25

Some of the best evidence shows that control, rather than eradication is a
sensible approach to JD management results from complex statistical modeling, which
has shown that vertical transmission and calf-to-calf transmission will allow
persistence of MAP even in well managed herds with low-prevalence and therefore be
difﬁcult to eradicate (Mitchell 2008). In ﬁghting JD, utilizing all the tools in our
knowledge base will be necessary to control the effects of this pathogen in US dairy
herds. A combination of increased producer knowledge and awareness through
education, as well as efﬁcient use of testing, and decreasing exposure risks to the
young diary animal through better management will result in a decrease of JD

prevalence in the US dairy herd.

C. ZOONOTIC POTENTIAL

No literature review on the topic of MAP would be complete without touching
on the zoonotic potential of this pathogen. First it should be noted that live MAP has
been cultured from retail pasteurized milk in the US. Of the 22 brands of milk tested
in the US, 12 yielded at least one sample of viable MAP (Ellingson 2005) with 20/702
of the total pints tested from three top producing milk states (CA, MN, WI) yielding
viable MAP. Thus, a common source of human exposure has been established, but the
association between the MAP organism and inﬂammatory bowel disease (Crohn’s
disease) is a less clear. A study ﬁnding a disproportionately higher number of MAP
positive blood cultures in people with Inﬂammatory Bowel Syndrome has triggered

many studies with variable results (Naser 2004).

26

A systematic review of the literature in 2008 concluded that evidence of
zoonotic potential is not strong but should not be ignored. The review cited several
conﬂicting studies almost equally split between association and no association of
MAP with Crohn’s disease. They also noted the absence of experimental design
consistency that would be needed to conﬁrm such an association (Waddell 2008).
Thus, while the negative impact of MAP on the US dairy cattle industry has been well
documented, another impetus for reducing the shedding of this organism, in both milk

and the environment, is the zoonotic unknown.

6. SUMMARY

During the past three decades there has been an international effort to reduce
the impact of MAP in dairy herds. It is important that we have a better understanding
of MAP transmission and its immunological processes so that it can be better
diagnosed and prevented. The knowledge of management of risk factors to reduce
transmission of organism is a key element to decreasing herd prevalence. The
improvement in testing techniques as well as reﬁnement in testing strategies have
allowed us to establish a comprehensive framework to assist producers in their effort
to mitigate the effects of this disease. AS test sensitivities improve, the ability to detect
infected animals earlier and to better characterize prevalence through targeted
environmental sampling may allow for more timely intervention opportunities that
were not previously possible. Implementation of these developments should be further
explored as we strive to decrease the prevalence of MAP in the US cattle herd. With

the unlocking of the genome of both the cow and the bacterium, further work in the

27

areas of genetic predisposition of the cow as well as the variable virulence of the
pathogen hold promise in understanding the disease. This may be the key to
developing an efﬁcacious vaccine against J ohne’s disease. Furthermore, the impetus
for future research may lie in the results of intense ongoing investigations into the

zoonotic potential of the MAP organism.

28

FOOTNOTES

3 TREK Diagnostic Systems, Inc.
982 Keynote Circle, Suite 6
Cleveland, OH 44131

USA

bMIGIT 960
Becton-Dickinson Biosciences
1 Becton Drive

Franklin Lakes, NJ

USA

CBACTEC 460
Johnson Laboratories
Towson, MD

USA

dIdexx Laboratories Inc.
Westbrook, Maine
USA

C(DCPAH —MSU) Price Guide — 2007

Diagnostic Center for Population Medicine and Animal Health
Michigan State University

East Lansing, MI 48824

USA

fPrionics Paracheck

Prionics AG
Schlieren
S WI I ZERLAND

gAntel Bio

North Star Cooperative
East Lansing MI 48823
USA

29

REFERENCES

Abbas B, Rieman HP, Hird DW: 1983 Diagnosis of Johne’s disease (paratuberculosis)
in northern California cattle and a note on its economic signiﬁcance. Calif Vet 1983:
8:20-24

Aly SS, Thunnond MC, Evaluation of Mycobacterium avium subsp paratuberculosis
infection of dairy cows attributable to infection status of the dam. J Am Vet Med
Assoc. 2005; Aug 1; 227(3):450-454

Alvarez J, de Juan L, Bezos J, Romero B, Siiez JL, Marques S, Dominguez C, .
Minguez O, Femandez-Mardomingo B, Mateos A, Dominguez L, Aranaz A; Effect of
paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed
infection using interferon-gamma detection assay, Veterinary Microbiology; 1 35, 3-4,

30 March 2009; 389—393

Bannantine JP, Bayles DO, Waters WR, Palmer MV, Stabel JR, Paustian ML, Early
antibody response against Mycobacterium avium subspecies paratuberculosis antigens
in subclinical cattle, Proteome Sci. 2008;Jan 28;6:5

Bannantine JP, Paustian ML, Waters WR, Stabel JR, Palmer MV, Li L, Kapur V;
Proﬁling bovine antibody responses to Mycobacterium avium subsp. paratuberculosis
infection by using protein arrays, Infection and Immunity, 2008 Feb;76(2):739-49.
Epub 2007 Nov 26; PMID 18039835

Benedictus A, Mitchell RM, Linde-Widmann M, Sweeney R, Fyock T, Schukken YH,
Whitlock RH; Transmission parameters of Mycobacterium avium subspecies
paratuberculosis infections in a dairy herd going through a control program; Prev Vet
Med. 2008 Mar l7;83(3-4):215-27. Epub 2007 Sep 14. PMID: 17868937

Berghaus RD, Lombard JE, Gardner IA, Farver TB, Factor analysis of a Johne’s
disease risk assessment questionnaire with evaluation of factor scores and a subset of
original questions as predictors of observed clinical paratuberculosis, Prev Vet Med,

2005 Dec 12;72(3-4):291-309. Epub 2005 Sep 1. PMID: 16139906

Bdgli-Stuber K, Kohler C, Seitert G, Glanemann B, Antogrroli MC, Salman MD,
Wittenbrink MM, Wittwer M, Wassenaar T, J emmi T, Bissig-Choisat B, Detection of
Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time
PCR and culture: A comparison of the two assays; J Appl Microbiol. 2005:99(3):587-
97. PMID 16108801

Bolton MW, Kaneene JB, Grooms DL, Fecal Shedding of Mycobacterium avium
subsp. In calves: implications for infection control and management. Manning, EJB,
Collins MR (Eds); 2005 Proceedings of the Eighth International Colloquium on
Paratuberculosis, 596-600

30

Brumbaugh GW, Simpson RB, Edwards JF, Anders DR, Thomson TD, Susceptibility
of Mycobacterium avium subsp paratuberculosis to monensin sodium or tihnicosin

phosphate in vitro and resulting infectivity in a murine model; Canadian Journal of
Vet Research, 2004, July 68(3): 175-181; PMID 153252541

Catanho M, Mascarenhas D, Degrave W, Miranda AB: GenoMchB: a database for
comparative analysis of mycobacterial genes and genomes; Genet M01 Res 2006 Mar
31 ; 5(1): 1 1 5

Collins DM, Stephens DM, de Lisle GW; Comparison of polymerase chain reaction
tests and fecal culture for detecting Mycobacterium paratuberculosis in bovine feces;
Vet Microbiol, 1993, Sep: 36(3-4):289-99, Erraum in Vet Microbiol, 1995, Mar:
43(4):331. PMID 7794290

Collins MT, Wells SJ, Petrini KR, Collins J E, Schultz RD, Whitlock RH., Evaluation
of ﬁve antibody detection tests for diagnosis of bovine paratuberculosis, Clin Dian
Lab Immunol, 2005, June:12(6):685-92. PMID 15939741

Cook KL, Britt J S, Optimization of methods for detecting Mycobacterium avium
susbp. Paratuberculosis in environmental samples using quantitative, real-time PCR J
Microbiol Methods, 2007, Apr:69(1):154-60. Epub 2006 Dec 30. PMID 17257697

Coussens PM, Verrnan N, Coussens MA, Elftman MD, McNulty AM; Cytokine gene
expression in peripheral blood mononuclear cells and tissues of cattle infected with
Mycobacterium avium subsp. paratuberculosis: evidence for an inherent
proinﬂammatory gene expression pattern. Infect Immun. 2004 Mar;72(3):1409-22.
PMID: 14977946

Eamens GJ, Walker DM, Porter NS, Fell SA, Radiometric pooled fecal culture for the
detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle. Aust.
Vet. J. 2008;86(7);259-265

Ellingson JL, Anderson JL, Koziczkowski JJ, Radcliff RP, Sloan SJ, Allen SE,
Sullivan NM, Detection of viable Mycobacterium avium subsp. paratuberculosis in
retail pasteurized whole milk by two culture methods and PCR, J Food Prat, 2005
May:68(5):996-72, Marshﬁeld Clinic Laboratories, Food Safety Services, Marshﬁeld
Clinic, Marshﬁeld, WI 54449, USA, ellingson.iav@marshﬁeldclinic.org

FDA — 2006
http://www.fsis.usda.gov/News &Events/Agenda NACMCF Mar2006/index.asp
Last accessed July 31, 2009

Garry FB, Wells S; Reconsidering Johne’s disease in dairy cows. Proceedings of the
North American Veterinary Conference; 1998; 12:910-911. NOTE: Meeting held on
January 10-14, 1998, Orlando Florida. NAL Call No: SF605.N672

31

Harris NB, Robbe-Austerman S, Payeur JB, Effect of egg yolk on the detection of
Mycobacterium avium subsp. paratuberculosis using the ESP II liquid culture system;
J Vet Diagn Invest Nov 2005: 17(6):544-560. PMID 16475513

Hendrick SH, Dufﬁeld TF, Leslie KB, Lissemore KD, Archambault M, Bagg R, Dick
P, Kelton DF, Monensin might protect Ontario, Canada dairy cows from
paratuberculosis milk-ELISA positivity, Prev Vet Med, 2006 Oct 17;76(3-4):237-48.
Epub 2006 Jun 19, PMID: 16787675

Hendrick SH, Dufﬁeld TE, Kelton DE, Leslie KE, Lissemore KD, Archambault M.;
Evaluation of enzyme-linked immunosorbent assays performed on milk and serum

samples for detection of paratuberculosis in lactating dairy cows, J Am Vet Med Asoc,
2005, Feb 1:226(3):424-8. PMID 15702694

Johnson-Ifearulundu Y, Kaneene J B. Distribution and environmental risk factors for
paratuberculosis in dairy cattle herds in Michigan. Am J Vet Res. 1999 May;60(5):589-
596

Johnson-Ifearulundu YJ, Kaneene JB, Management-related risk factors for M.
paratuberculosis infection in Michigan, USA, dairy herds, Prev. Vet. Med. 1998 Dec
1; 37(1-4):41-54

Kalis CH, Hesselink J W, Barkema HW, Collins MT. Culture of strategically pooled
bovine fecal samples as a method to screen herds for paratuberculosis, J Vet Diagn
Invest. 2000 Nov; 12(6):547-51. PMH): 11108455

Kalis CH, Hesselink J W, Russchen EW, Barkema HW, Collins MT, Visser IJ, Factors
inﬂuencing the isolation of Mycobacterium avium subsp. paratuberculosis from
bovine fecal samples; J Vet Diagn Invest. 1999 Jul; 11(4):345-51. PMID: 10424651

Khare S, Ficht TA, Santos RL, Romano J, Ficht AR, Zhang S, Grant IR, Libal M,
Hunter D, Adams LG, Rapid and sensitive detection of Mycobacterium avium subsp.
paratuberculosis in bovine milk and feces by a combination of immunomagnetic bead
separation-conventional PCR and real-time PCR. J Clin Microbiol. 2004 Mar;

42(3): 1075-8 1. PMID: 15004056

Kim SG, Kim EH, Lafferty CJ, Miller LJ, Koo HJ, Stehman SM, Shin SJ. Use of
conventional and real-time polymerase chain reaction for conﬁrmation if
Mycobacterium avium subsp. paratuberculosis in a broth- based culture system ESP

II. J. Vet. Diagn. Invest. 2004; 16(5), 448-553

Koo HC, Park YH, Hamilton MJ, Barrington GM, Davies CJ, Kim JB, Dahl JL,
Waters WR, Davis WC; Analysis of the immune response to Mycobacterium avium

subsp. paratuberculosis in experimentally infected calves; Infect Immun. 2004
Dec;72(12):6870-83. PMID: 15557608

32

Krieg NR, Holt JG; 1986, Abortion in sheep ed., vol 1, pp. 663-634. In Bergey ’3
Manual of Systematic Bacteriology, matography.

Kathaperumal K, Kumanan V, McDonough S, Chen LH, Park SU, Moreira MA, Akey
B, Huntley J, Chang CF, Chang YF; Evaluation of immune responses and protective
efﬁcacy in a goat model following immunization with a coctail of recombinant

antigens and a polyprotein of Mycobacterium avium subsp. Paratuberculosis; Vaccine.
2009 Jan 1; 27(1):]23-35. Epub 2008 Oct 26. PMID: 18955101

Lombard J E, Wagner BA, Smith RL, McCluskey BJ, Harris BN, Payeur JB, Garry
FB, Salman MD; Evaluation of enviromnental sampling and culture to determine
Mycobacterium avium subspecies paratuberculosis distribution and herd infection
status on US dairy operations; J Dairy Sci. 2006 Nov;89(l l):4163-71. PMID:
17033002

Lombard J E, Byrem TM, Wagner BA, McCluskey 8]., Comparison of milk and
serum enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium

subspecies paratuberculosis infection in dairy cattle, J Vet Diagn Invest. 2006 Sep;
18(5):448-58. PMID: 17037612

Manning EJ, Collins MT.; Mycobacterium avium subsp. paratuberculosis: pathogen,
pathogenesis and diagnosis; Rev Sci Tech. 2001 Apr; 20(1):]33-50. Review. PMID:
11288509

McDonald WL, Ridge SE, Hope AF, Condron RJ. Evaluation of diagnostic tests for
Johne’s disease in young cattle. Aust. Vet. J. 1999; 77(2), 113-119

McKenna SL, Keefe GP, Tiwari A, VanLeeuwen J, Barkema HW; Johne's disease in
Canada part II: disease impacts, risk factors, and control programs for dairy producers;
Can VetJ. 2006 Nov;47(11):1089-99. Review. PMID: 17147140

Merkal RS, Curran BJ, Growth and metabolic characteristics of Mycobacterium
paratuberculosis; 1974 Appl Microbiol 28: 276-279 PMID: 4859342 PMCID:
PMC186701

Mitchell RM, Whitlock RH, Stehman SM, Benedictus A, Chapagain PP, Grohn YT,
Schukken YH; Simulation modeling to evaluate the persistence of Mycobacterium

avium subsp. paratuberculosis (MAP) on commercial dairy farms in the United States;
Prev Vet Med. 2008 Mar l7;83(3-4):360-80. Epub 2007 Nov 26. PMID: 18022716

Naser, SE, Ghobrial, G, Romero, C, Valentine JF. Culture of Mycobacterium avium

subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet
2004; 364, 1039—1044

33

Nielsen SS, Bjerre H, Toft N.; Colostrum and milk as risk factors for infection with

Mycobacterium avium subspecies paratuberculosis in dairy cattle; J Dairy Sci. 2008
Dec;91(12):4610-5. PMID: 19038936

Nielsen SS, Toft N; Age-speciﬁc characteristics of ELISA and fecal culture for
purpose-speciﬁc testing for paratuberculosis; J Dairy Sci. 2006 Feb; 89(2):569-79.
PMID: 16428626

Obasanjo IO, Grohn YT, Mohammed HO; Farm factors associated with the presence
of Mycobacterium paratuberculosis infection in dairy herds on the New York State
Paratuberculosis Control Program; Prev Vet Med. 1997 Oct;32(3-4):243-51.

Osterstock JB, Fosgate GT, Cohen ND, Derr JN, Roussel AJ; Familial and herd-level
associations with paratuberculosis enzyme-linked immunosorbent assay status in beef
cattle; J Anim Sci. 2008 Aug;86(8):1977-83. Epub 2008 May 9. PMID: 18469058

Pillars RB, Grooms DL, Woltanski JA, Blair E.; Prevalence of Michigan dairy herds
infected with Mycobacterium avium subspecies paratuberculosis as determined by
environmental sampling. Prev Vet Med. 2009; Apr 7; 90(3-4) 1 August 2009, 223-232
[Epub ahead of print]

Pinedo PJ, Buergelt CD, Donovan GA, Melendez P, Morel L, Wu R, Langaee TY,
Rae DO; Association between CARD] 5/NOD2 gene polymorphisms and
paratuberculosis infection in cattle; Vet Micro 2009 Mar 2;]34(3-4):346-52. Epub
2008 Sep 19

Raizman EA, Wells SJ, Jordan PA, DelGiudice GD, Bey RR; Mycobacterium avium
subsp. paratuberculosis from free-ranging deer and rabbits surrounding Minnesota
dairy herds; Can J Vet Res 2005 Jan;69(1):32-8

Raizman EA, Wells SJ, Godden SM, Bey RF, Oakes MJ, Bentley DC, Olsen KB; The
distribution of Mycobacterium avium ssp. paratuberculosis in the environment
surrounding Minnesota dairy farms; J Dairy Sci. 2004 Sep;87(9):2959-66; PMID:
15375057

Scott HM, Fosgate GT, Libal MC, Sneed LW, Erol E, Angulo AB, Jordan ER.

Field testing of an enhanced direct-fecal polymerase chain reaction procedure,
bacterial culture of feces, and a serum enzyme-linked immunosorbent assay for
detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle,

AJVR, 2007 March 68:(3):236-45

Smith RD, Slenning BD; Decision analysis: dealing with uncertainty in diagnostic
testing; Prev Vet Med. 2000 May 30; 45(1-2):139-62. Review

Speer CA, Scott MC, Bannantine JP, Waters WR, Mori Y, Whitlock RH, Eda S; A
novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium

34

subsp. paratuberculosis infections (J ohne's Disease) in cattle; Clin Vaccine Immunol.
2006 May; 13(5):535-40. PMID: 16682472

Stabel JR; Host responses to Mycobacterium avium subsp. paratuberculosis: a
complex arsenal. Anim Health Res Rev 2006 Jun-Dec; 7(1-2):61-70. Review. PMID:
17389054

Stabel JR, Hurd S, Calvente L, Rosenbusch RF; Destruction of Mycobacterium
paratuberculosis, Salmonella spp., and Mycoplasma spp. in raw milk by a commercial
on-fann high-temperature, short-time pasteurizer; J Dairy Sci. 2004 Jul;87(7):2177-
83. PMID: 15328232

Stratmann J, Homuth M, Gerlach GF; Observations on the control and eradication of
paratuberculosis in dairy herds; Dtsch T ierarztl Wochenschr. 2005 Aug; 112(8):304-6.
Review. German. PMID: 16218184

Streeter RN, Hoffsis GF, Bech-Nielsen S, Shulaw WP, Rings DM; Isolation of
Mycobacterium paratuberculosis from colostrum and milk of subclinically infected
cows; Am J Vet Res. 1995 Oct;56(10):1322-4. PMID: 8928949

Sweeney RW, Whitlock RH, Bowersock TL, Pruitt GW; 2007 9th ICP (International
Colloquium on Paratuberculosis) Comparison of Liquid Culture to Solid Media for
Quantiﬁcation of Mycobacterium in Tissue following experimental infection, page 38

Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium paratuberculosis
isolated from fetuses of infected cows not manifesting signs of the disease. Am. J. Vet.
Res. 1992; 53(4), 477-480

Tavompanich S, Johnson WO, Anderson RJ, Gardner IA; Herd characteristics and
management practices associated with seroprevalence of Mycobacterium avium subsp
paratuberculosis infection in dairy herds; Am J Vet Res 2008 Jul;69(7):904-11

Tavomparrich S, Gardner IA, Anderson RJ, Shin S, Whitlock RH, F yock T, Adaska
JM, Walker RL, Hietala SK. Evaluation of microbial culture of pooled fecal samples
for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds. Am
J Vet Res. 2004; Aug; 65(8); 1061-1070

Thompson BR, Clark RG, Mackintosh CG; Intra-uterine transmission of
Mycobacterium avium subsp paratuberculosis in subclinically affected red deer
(Cervus elaphus); N Z Vet J. 2007 Dec;55(6):308-13. PMID: 18059649

Tiwari A, Vanleeuwen JA, Dohoo IR, Keefe GP, Haddad JP, Scott HM, Whiting T.
Risk factors associated with Mycobacterium avium subspecies paratuberculosis
seropositivity in Canadian dairy cows and herds. Prev Vet Med. 2009; Jan 1;
88(1):32-41

35

Tiwari A, VanLeeuwen J A, McKenna SL, Keefe GP, Barkema HW; Johne's disease in
Canada Part I: clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy
herds; Can VetJ. 2006 Sep;47(9):874-82. Review. PMID: 17017652

United States Department of Agriculture, Animal and Plant Health Inspection Service,
APHIS 91-45-016: Uniform Program Standards for the Voluntary Bovine Johne’s
Disease Control Program, Effective June 1, 2006.

http://www.aphis.usda.gov/animal health/animal diseases/iohnes/downloads/iohnes-

umrpdf

United State Department of Agriculture’s National Animal Health Monitoring
System’s (NAHMS), 2002, Dairy Survey, Dr. Jason Lombard or Dr. Brian McCluskey
@ 970-494-7000.

Uzonna J E, Chilton P, Whitlock RH, Habecker PL, Scott P, Sweeney RW; Efﬁcacy of
commercial and ﬁeld-strain Mycobacterium paratuberculosis vaccinations with

recombinant IL-12 in a bovine experimental infection model. Vaccine. 2003 Jul 4;
21(23):3101-9. PMID: 12804836

van Roermund HJ, Bakker D, Willemsen PT, de Jong MC. Horizontal transmission of
Mycobacterium avium subsp. Paratuberculosis in cattle in an experimental setting:
calves can transmit the infection to other calves, Vet Microbiol. 2007 Jun 21; 122(3-
4):270-279

van Schaik G, Pradenas F M, Mella N A, Kruze V J; Diagnostic validity and costs of
pooled fecal samples and individual blood or fecal samples to determine the cow- and
herd-status for Mycobacterium avium subsp. Paratuberculosis: Prev Vet Med. 2007
Nov 15;82(1-2):159-65. Epub 2007 Jun 26. PMID: 17597241

van Schaik G, Stehman SM, Schukken YH, Rossiter CR, Shin SJ; Pooled fecal culture
sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and
prevalence; J Vet Diagn Invest. 2003 May; 15(3):233-41. PMID: 12735345

Visser I, 1999. Reproducibility of a Fecal Culture Method for Mycobacterium avium
subsp. paratuberculosis. In Manning, EJB, Collins MT (EDS) Proceedings of the
Sixth International Colloquium on Paratuberculosis, page 512

Waddell LA, Rajic A, Sargeant J, Harris J, Amezcua R, Downey L, Read S, McEwen
SA; The zoonotic potential of Mycobacterium avium spp. paratuberculosis: a
systematic review; Can J Public Health, Mar-Apr 2008, 99(2): 45-55

Waters WR, Stabel JR, Sacco RE, Harp JA, Pesch BA, Wannemuehler MJ: Antigen-

Speciﬁc B-cell unresponsiveness induced by chronic Mycobacterium avium subsp.
Paratuberculosis infection of Cattle; Infection and Immun 1999 Apr;67(4):1593-8

36

Wells SJ, Whitlock RH, Lindeman CJ, Fyock T; Evaluation of bacteriologic culture of
pooled fecal samples for detection of Mycobacterium paratuberculosis; Am J Vet Res.
2002 Aug; 63(8):1207-11. PMID: 12171178

Wells SJ, Wagner BA; Herd-level risk factors for infection with Mycobacterium
paratuberculosis in US dairies and association between familiarity of the herd manager
with the disease or prior diagnosis of the disease in that herd and use of preventive
measures; J Am Vet Med Assoc. 2000 May 1;216(9): 1450-7. PMID: 10800519

Whitlock HR, Buergelt C. Preclinical and clinical manifestations of paratuberculosis
(including pathology), Vet. Clin. N. Am. Anim. Pract. 1996; 12:345-356

Whitlock RH, Wells SJ, Sweeney RW, Van Tiem J. ELISA and fecal culture for
paratuberculosis (J ohne’s disease): sensitivity and speciﬁcity of each method. Vet.
Microbiol. 2000; 77 (3-4), 387-398

Whittington RJ, Windsor PA; In utero infection of cattle with Mycobacterium avium
subsp. paratuberculosis: a critical review and meta-analysis; Vet J. 2009 Jan;
179(1):60-9.

Whittington RJ, Marsh IB, Reddacliff LA; Survival of Mycobacterium avium subsp.
paratuberculosis in dam water and sediment; Appl Environ Microbiol. 2005 Sep;
71(9):5304-8. PMID: 16151118

Whittington RJ, Marshall DJ, Nicholls PJ, Marsh IB, Reddacliff LA Survival and
dormancy of Mycobacterium avium subsp. paratuberculosis in the environment. Appl
Environ Microbiol. 2004; May; 70(5):2989-3004

Whittington RJ, Sergeant-ES; Progress towards understanding the spread, detection
and control of Mycobacterium avium subsp paratuberculosis in animal populations;
Aust VetJ. 2001 Apr; 79(4):267-78. Review. PMID: 11349414

Whittington RJ, Fell S, Walker D, McAllister S, Marsh 1, Sergeant E, Taragel CA,
Marshall DJ, Links IJ; Use of pooled fecal culture for sensitive and economic
detection of mycobacterium avium subsp. paratuberculosis infection in ﬂocks of

sheep; J Clin Microbiol. 2000 Jul; 38(7):2550-6. PMID: 10878042

Williams-Bouyer N, Yorke R, Lee HI, Woods GL; Comparison of the BACTEC
MGIT 960 and ESP culture system II for grth and detection of mycobacteria; J Clin
Microbiol. 2000 Nov; 38(11):4167-70. PMID: 11060085

Witte CL, Hungerford LL, Rideout BA. Association between Mycobacterium avium

subsp. Paratuberculosis infection among offspring and their dams in non-domestic
ruminant species housed in a zoo. J Vet Diagn Invest. 2009 Jan; 21(1):40-47

37

Wu CW, Glasner J, Collins M, Naser S, Talaat AM; Whole-genome plasticity among
Mycobacterium avium subspecies: insights from comparative genomic hybridizations,
J Bacterial, 2006, Jan:188(2):711-23. PMID 16385061

38

CHAPTER 2

DETECTION OF M Y COBA TERI UM A WUM SUBSPECIES
PARA TUBERCULOSIS IN NATURALLY EXPOSED DAIRY
CALVES, RELATIONSHIP TO DAM STATUS, AND OTHER
RISK FACTORS

39

Objectives—Determine 1) if fecal shedding of Mycobacterium avium subspecies
paratuberculosis (MAP) can be detected in naturally exposed dairy calves, 2) if there
is an association between fecal shedding of MAP in calves and their ELISA test status,
and 3) if a relationship exists between MAP ELISA test positive cows and fecal
shedding in their offspring.
Design—28 month-longitudinal study.
Sample Population-Heifer calves from eight dairy herds in Michigan participating in
the Michigan Johne’s Disease Control Demonstration Project.
Procedures—Fecal and blood samples were obtained from calves at 4-month
intervals for 28 months. Liquid culture was used on fecal samples and serum ELISA
testing on blood samples. Multivariable mixed logistic regression was utilized to
evaluate the relationship between herd and dam risk factors and the MAP test status of
calves.
Results—A total of 27/1088 (2.51 %) calves were MAP fecal test positive. A total of
26/ 1036 calves (2.50%) were MAP ELISA test positive. Positive serum ELISA
samples from calves showed no Signiﬁcant association with their concomitant fecal
status, (r2 = 0.16, p< 0.0001). Calves born to ELISA positive dams were 11.5 times
more likely to become a fecal shedder of MAP than calves born to ELISA negative
dams (Odds Ratio = 11.5 [95% CI: 4.7 — 28.2]; p<0.0001).

Conclusions and Clinical Relevance—Calves born to ELISA positive cows
are at high risk for shedding MAP, thus management of ELISA positive cows is

important. Given the relatively high likelihood of a calf shedding MAP when their

40

dam is ELISA positive, consideration must be given to identifying ELISA positive

dams and housing their calves separately.

41

ABBREVIATIONS

MAP

JD

OR

CI

DCPAH

Mycobacterium avium subspecies paratuberculosis
J ohne’s disease

Odds ratio

Conﬁdence interval

Diagnostic Center for Population and Animal Health

42

Introduction

J ohne’s disease (JD), caused by Mycobacterium avium subspecies
paratuberculosis (MAP), is a chronic granulomatous enteric disease of both domestic
and non-domestic ruminants. It was ﬁrst described in Germany and its etiologic agent
was characterized as an acid fast bacillus by Twort (Twort 1910). Recent studies
placed the percentage of infected herds in Michigan at 64% (J ohnson-Ifearulundu
1999) and 48% (Pillars 2009) respectively. Nationally the published prevalence ﬁgures
are widely variable (Adaska 2003; Hirst 2004). The cost to the US dairy producer is
more than $250 million annually (Ott 1999). Although JD has been recognized for
over a century, it has emerged as a major economic factor in the US dairy industry in
the past three decades.

Calves are most often infected before the age of six months via the ingestion of
MAP contaminated feces, colostrum, or waste milk (Sweeney 1996). Transplacental
infection (Kopecky 1967; Seitz 1989; Sweeney 1992) also occurs. Because MAP is a
slow-growing bacterium, development of clinical signs of JD may take 2-5 years yet
transmission can occur on farms via unapparent carriers (Harris 2001).

Attempts to identify naturally infected calves at a young age have been
unsatisfactory (Ayele 2004; McDonald 1999), presumably due to low levels of
bacterial shedding present in the feces of young animals. Furthermore, immunological
assays looking for antibodies are not as diagnostic with Mycobacteria, as with some
organisms (Bannantine 2008) due to a cell-mediated, rather than hmnoral, response to
infection. Also compromising detection efforts in calves are intermittent shedding of

MAP (Whitlock 1996) and reproducibility deﬁcits within the same fecal sample

43

(Visser 1999). Thus, standard fecal culture has not been effective at detecting low-
level bacterial shedding (Kim 2004) and overall sensitivity may be as low as 33%
(Whitlock 2000).

Early identiﬁcation of calves that may be at risk of MAP infection or that may
be shedding MAP into the environment is important for ensuring that effective JD
prevention and control strategies exist in dairy herds therefore, the purpose of this
study was to determine 1) if fecal shedding of Mycobacterium avium subspecies

paratuberculosis (MAP) can be detected in naturally exposed calves using the

TREK®ESPIIa liquid culture system, 2) if there is an association between fecal

shedding of MAP in the calf and their ELISA test status, and 3) if there is an

association between MAP status of the dam and the MAP status of her calf.

Materials and Methods

Study design and criteria for inclusion— This was a longitudinal study
spanning 28 months. The study population included calves from eight commercial
dairy herds located in Michigan that were enrolled in the Michigan Johne’s Disease
Control Demonstration Program. Herds were enrolled in the JD demonstration project
based on known infection with MAP, herd size, geographic location, and willingness
to cooperate in the study. This study met the Michigan State University guidelines for
animal research administered by the Animal Use Committee and owner’s permission
was received to conduct the study. Ten heifer calves from each of four age groups: 0-3
months, 4-6 months, 7-14 months, and 15-24 months were selected for fecal culture

and serum ELISA testing during each herd visit. Age group composition was based on

distinct housing groups including pre-weaning (hutch calves), post-weaning (small
pens), grth phase (large pens) and breeding group. Calves from fecal or ELISA
positive dams were preferentially targeted for testing and the remainder of each age
cohort were chosen randomly. Throughout the study period, samples were collected at
approximately four-month intervals with a maximum of eight visits over 28 months.

Fecal samples were collected from each calf by digital exculpation using
individual latex gloves and sterile water for lubrication. At least eight grams of fecal
material was collected, labeled, reﬁigerated at 5°C, and submitted to the laboratory
within 1-2 days of collection.

A total of ﬁve milliliters (5 m1) of blood was collected from the jugular vein or
caudal tail vein. Blood samples were labeled, placed on ice, centrifuged within six
hours, and serum submitted to the laboratory within 1-2 days of collection.

Laboratory testing— Both fecal and serum samples were analyzed at the
Diagnostic Center for Population and Animal Health (DCPAH) at Michigan State

University. The DCPAH is an accredited laboratory and has been certiﬁed by USDA

National Veterinary Science Laboratory to conduct TREK®ESPHa liquid fecal culture

for MAP, as well as Parachek®b serum ELISA antibody testing.

The Cornell method (Stabel 1997) was used to prepare fecal samples for

culture. The samples were placed in the TREK®ESPIIa culture system incubator.

Positive and negative controls were used on each batch of forty fecal samples.
Because this is a semi-quantitative test, a positive sample was described as a high

shedder if it took 7—21 days to turn positive, moderate shedder if it took 22-28 days to

45

turn positive and low shedder if it took 29-42 days to turn positive. If a culture was not

positive by day 42 it was stained with acid fast Kenyon stain and the supernatant

tested with ISP900 real time PCRc (Kim 2002). If negative to this follow-up it was

classiﬁed as not shedding (negative). All positive samples from liquid culture were

also conﬁrmed with both acid fast Kenyon stain and IS900 real time PCRC. This

method was also used on the dams on an annual basis to determine their fecal MAP
status.

The blood samples were centrifuged and the serum tested for MAP antibodies

using the Parachek®b ELISA test following the manufacturers recommended
procedures. A corrected optical density (ODC) 21.0 was considered ELISA positive

while an ODc< 1.0 was considered ELISA negative for this study. This same

procedure was also used to assess the dam status on an annual basis.

Risk factors— The primary outcome of interest was the presence, or lack
thereof, of MAP in the calf fecal sample. Additionally, the results of the calf serum
ELISA test were evaluated.

Several risk factors were evaluated to determine their relationship with the
MAP test status of calves. The primary risk factor of interest was the MAP ELISA test
status of the dam. Dam fecal culture status was also evaluated.

Because the age of the calf may be associated with whether or not it is
Shedding MAP (Weber 2005) the relationship between the age group when tested and
the calf MAP status was also evaluated. Age at testing was categorized as follows: 0-3

months, 4—6 months, 7-14 months, and 15-24 months.

46

The relationship between calf MAP status and average herd size was evaluated
by calculating the population size recorded closest to the midpoint of calf sample
collection for each herd. Average herd size was categorized as small (80—130 cows
[three herds]), medium (140—165 cows [two herds]), and large (330—600 cows [three
herds]).

In addition, herd test prevalence of MAP, using serum ELISA, was established
annually. This was evaluated by using the herd MAP prevalence recorded closest to
the midpoint of calf sample collection for each herd. Prevalence was categorized as
low (0.5-5% [three herds]), moderate (6.5-8% [three herds]), and high (10—15 % [two
herds]).

Statistical analysis——Summary statistics were computed to identify

relationships between the outcome (calf MAP test status) and the risk factors of

interest (Proc FREQd). Spearman correlation coefﬁcients were computed to identify

potential collinearity between the risk factors of interest (Proc CORRe). Results were

considered statistically signiﬁcant at p50.05.
Multivariable mixed logistic regression models were created to describe the

association between the outcome (calf fecal MAP) and the risk factors of interest (herd

size, herd prevalence, age group) (Proc GLIMMIXf). The random effect term
incorporated herd and observation visit to ensure the model contained adequate
variance and degrees of freedom relative to the study design. All risk factors with

p<0.05 in the univariable model were considered for inclusion in the multivariable

model. Herd size served as a proxy for undeﬁned management factors as there were no

47

herds that grazed entirely or were totally conﬁned. Herd prevalence was explored but
since it was reﬂected by dam ELISA, it was not included as a variable to avoid model

over-speciﬁcation.

R_esELt§

Calf Fecal Culture—Calf MAP fecal culture test results, positive calves and
MAP shedding level, positive calves and herd prevalence, and ELISA test results by
calf age group distribution were evaluated (Table 2.1). Overall, results showed that
27/ 1088 (2.48%) individual calves cultured positive for MAP. A total of 7 /27
(25.92%) positive calves were positive on two or more serial cultures.

Calf ELISA—The distribution of samples collected and results of the ELISA
testing by age of calf and their darn ELISA test results were evaluated (Table 2.2).
Overall, results showed that 26/ 1038 (2.50%) calves were ELISA positive for MAP
and 20/26 (77.0%) were < 6 months of age; 8/26 (30.77%) ELISA positive calves
tested positive on two or more serial cultures.

Dam MAP Status—There were signiﬁcantly more MAP fecal culture positive
calves born to MAP serum ELISA positive dams and fecal culture positive dams
compared to their test negative cohorts (Table 2.2).

There also were signiﬁcantly more serum ELISA positive calves born to MAP
serum ELISA positive dams and fecal culture positive dams compared to their test
negative cohorts (Table 2.3).

Statistical analysis—With respect to age, there was no signiﬁcant difference

in positive fecal cultures between age group 1 (0-3 months) compared to age group 2

48

(4-6 months), and age group 3 (7-14 months) compared to age group 4 (15-24 months)
(Table 2.4). However, there was a signiﬁcant difference when comparing younger
calves (age groups 1 and 2 combined [0-6 months]) to older calves (age groups 3 and
4 combined [7-24 months]). Furthermore, signiﬁcantly more positive fecal cultures
were found in age group 3 (7-14 months) compared to age group 2 (4-6 months).

No correlation was demonstrated between the fecal culture status of a calf and

their concomitant serum ELISA results (r2 = 0.16; p <.0001).

A multivariable mixed logistic regression model was constructed (Table 2.5).
The exposure variable of interest was dam serum ELISA test status. Calves born to
ELISA positive dams were 11.5 times more likely to become a fecal shedder of MAP
than calves born to ELISA negative dams. Using Least Squares Means it was
determined that the probability of a calf shedding MAP from an ELISA positive dam
was 6.9% while the probability was < 1% (0.0064) when the dam was ELISA negative

(p < .0001).

Discussion

In this study we were able to detect MAP in young dairy calves using

TREK®ESPIIa liquid fecal culture, a relatively new diagnostic technique. One of the

goals of J ohne’s disease management is early detection of MAP. The response variable
of interest was the calf fecal culture status, as this is considered the best indicator of
MAP infection (Stich 2004; Wells 2006). We evaluated four age groups of calves to
determine if there was a group with a higher likelihood of shedding MAP (positive

fecal culture). In our prospective study, in a natural setting, not only did the older

49

animals have signiﬁcantly more fecal culture positives than younger calves but, more
Speciﬁcally, age group 3 (7-14 months) had signiﬁcantly more fecal positives than age
group 2 (4-6 months). Retrospectively, it has been shown that, starting at seven
months of age, there has been detection of MAP shedding, which then wanes before
recurring at two years of age, or older (Weber 2005). This may allow for development
of JD prevention and control strategies, targeting this younger age group, that utilize
fecal culture for early detection of MAP in dairy herds.

The majority (76.9%) of serum ELISA positive calves were less than 6 months
of age. However, there was virtually no correlation between their ELISA and fecal
culture results. Since many of these positive calves were less than two months of age,
most of the ELISA positive samples may be attributed to the presence of matemal
antibodies which have been shown to persist in calves for 200 days (Menanteau-Horta
1985). Additionally, Mycobacteria tend to stimulate more of a cell-mediated, rather
than humoral, response early on in the infection (Bannantine 2008; Kalis 2003). Most
studies report that ELISA test results are a better indicator of infection in the older
animal (over two years of age) (McDonald 1999). Therefore, ELISA results in young
calves are probably not a good indicator of infection at this age (Antongnoli 2007).

Although herd size was not found to be signiﬁcant in this study it was
maintained in the ﬁnal model to account for potential confounding factors. It has been
reported to have a signiﬁcant impact on JD occurrence in another study (Crossley
2005). The difference could be due to the fact that, in the former study, they measured
shedding level differences in adults of various herd sizes, while we were looking at

calves and thus concentrated on number of shedding animals. Also, with n=8, we had

50

fewer comparative herds. Though herd prevalence was not assessed as a risk factor for
calf MAP status in this study, the majority of positive fecal cultures were in calves
from high prevalence herds which does agree with earlier work study (Crossley 2005).

In a natural setting, we found that a calf born to an ELISA positive darn was
11.5 times more likely to become fecal culture positive than a calf born to an ELISA
negative dam. The probability of a calf with an ELISA positive dam becoming fecal
culture positive was 6.8 % compared to only 0.6 % when the dam was ELISA
negative. It has been reported in retrospective studies that the MAP status of the dam
signiﬁcantly increases the risk of positive MAP fecal cultures in the offspring of both
dairy cows (Aly 2005) and non-domestic ruminants from zoos (Witte 2009). This may
be important knowledge in developing a target testing model utilizing this piece of
information while targeting the 7-14 month age group.

In conclusion, results of this study suggest that there is a target age (7-14
months) to start testing for fecal shedding of MAP in the naturally exposed dairy calf.
Additionally, dam ELISA status may be an important predictor of MAP fecal shedding
in the dairy calf. Even if MAP shedding in the young calf does not progress to JD later
in life, it is, at the very least, a potential risk factor for transmission to nearby calves
through environmental contamination (van Roermund 2007). Given the relatively
high likelihood of a calf shedding MAP when their dam is ELISA positive,
consideration must be given to identifying ELISA positive dams and housing their
calves separately.

Further investigation needs to be done using serial testing of 7-14 month old

calves with positive MAP fecal cultures, over time, to follow their production

51

parameters and clinical outcomes, especially in high prevalence herds. Given recent
work with pooled fecal samples, (Tavompanich 2004; Eamens 2008) it may also be
interesting to investigate implementation of a fecal culture pooling strategy in this age
group of calves to obtain an early, more cost effective, assessment of management

changes.

52

Footnotes

aTREK®ESPII

TREK Diagnostic Systems Inc.
Cleveland, OH 44131

USA

bParachek®
Prionics AG
Wagistrasse 27a
CH-8952
Schlieren-Zurich
SWITZERLAND

cIS900 PCR

Applied Biosystems
Foster City, CA 94404
USA

dProc FREQ,
SAS Institute
Cary, NC
USA

eProc CORR
SAS Institute
Cary, NC
USA

fProc GLIMMIX
SAS Institute
Cary, NC

USA

Sources of Funding -

USDA J ohne’s Demonstration project
Michigan Department of Agriculture

Michigan State University — Center for Comparative Epidemiology

53

Table 2.1: Mycobacterium avian subspecies paratuberculosis calf fecal culture test

results, positive calves and MAP shedding level, positive calves and herd prevalence,

and ELISA test results by calf age group distribution: Michigan 2005-2007

 

 

 

 

 

Test Result Age Group Age Group Age Group Age Group ' Total
1 (0-3 2 (4-6 3 (7-14 4 (15-24 # (%)
months) months) months) months)
Fecal Culture
# Tests 424 438 438 437 1737
# ' 2 3 26 10 41(2_4)a
Positive
Tests
# Calves 395 317 181 195 1088
Tested
# 2 2 l 5 8 27(2. 5)b
Positive
Calves
Positive Calves and MAP Shedding Level
Low 2 2 12 7 ' 23
Mod 0 0 1 0 1
erate
High 0 0 2 1 3
Positive Calves and Herd Prevalence
Low 1 0 2 3
Mod 1 1 3 9
erate
High 0 1 10 4 15
ELISA Serum
# Tests 402 418 415 424 1659
# 1 7 9 4 6 36(2'2)a
Positive
Tests
# Calves 378 304 166 190 1038
Tested
# l4 6 2 4 260.5)b
Positive
Calves

 

b
a % positive of total fecal or ELISA tests % positive of total individual calves tested

54

Table 2.2 —Mycobacterium avium subspecies paratuberculosis fecal culture results
of calves in relation to serum ELISA and fecal culture status of their dam: Michigan

2005 — 2007

Dam Status Calves (+) Calves (-) Total % Positive sz

 

ELISA + 17 174 191 8.9

ELISA - 10 887 897 1.1 <.0001
Total 27 1061 1088 2.5

Fecal + 10 125 135 7.4

Fecal - 4 683 687 0.6 <.0001
Total 14 808 822 1.7

 

"‘ X2 p — Chi Square P— value signiﬁcantly different between groups (p< 0.05)

Table 2.3—Mycobacterium avium subspecies paratuberculosis serum ELISA results
of calves in relation to serum ELISA and fecal culture status of their dam: Michigan

2005 —2007

Dam Status Calves (+) Calves (-) Total % Positive sz

 

ELISA + 15 152 167 9.0

ELISA - 11 843 854 1.3 <.0001
Total 26 995 1021 2.6

Fecal + 11 124 135 8.1

Fecal - 7 681 688 1.0 <.0001
Total 18 805 823 2.2

 

* X2 p — Chi Square P— value signiﬁcantly different between groups (p< 0.05)

55

Table 2.4—Comparisons between age groups of fecal culture positive calves

shedding Mycobacterium avium subspecies paratuberculosis:

Michigan 2005—2007

 

Ag Group df F Value Pr > F
Age group 1 vs. age group 2 165 0.01 0.913
Age group 3 vs. age group 4 165 0.25 0.618
Older vs. younger age groups (1&2) vs. (3&4) 165 10.890 0.001
Age group 3 vs. all others 3 vs. (1, 2, & 4) 165 3.130 0.079
Age group 2 vs. age group 3 165 4.38 0.038

 

Table 2.5-Multivariable mixed logistic regression of risk factors associated with

Mycobacterium avium subspecies paratuberculosis positive calf fecal cultures:

Michigan 2005 -2007

 

Risk Factor t-value p valuea dfb ORG 95% Cld

Dam ELISA + 5.33 <.0001 911 11.498 4.680, 28.246
Dam ELISA - - - -

Age group 1(0-3 mos.) - 2.61 0.01 165 0.166 0.043, 0.648
Age group 2 (4-6 mos.) - 2.50 0.0135 165 0.152 0.034, 0.674
Age group 3 (7-14 mos.) - 0.50 0.618 165 0.743 0.230, 2.404
Age group 4 (15-24 mos.) - - - 1.000 -, -
Herd size 1 (80-130 cows) - - - 1.000 -, -

Herd size 2 (140-165 cows) 1.27 0.203 911 0.315 0.053, 1.871
Herd size 3 (330-600 cows) 1.03 0.301 911 2.009 0.535, 7.546

aP-value-level of signiﬁcance for t-value
bdf-degrees of freedom

cOR-Odds Ratio

dCI-95% Conﬁdence Interval

56

REFERENCES

Adaska JM, Anderson R]. Sero prevalence of J ohne’s disease infection in dairy cattle
in California, USA. Prev. Vet Med. 2003; 3; 255-261

Aly SS, Thunnond MC, Evaluation of Mycobacterium avium subsp paratuberculosis
infection of dairy cows attributable to infection status of the dam. J Am Vet Med
Assoc. 2005; Aug 1; 227(3):450-454

Antognoli MC, Hirst HL, Garry FB, Slarnan MD. Immune response to and fecal
shedding of Mycobacterium avium ssp. Paratuberculosis in young dairy calves, and
the association between test results in the calves and the infection status of their dams.
Zoanoses Public Health, 2007; 54(3-4):152-159

Ayele WY, Bartos M, Svastova P, Pavlik 1. Distribution of Mycobacterium avium
subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls.
Vet Microbiol. 2004; 103(3-4), 209-217

Bannantine JP, Bayles DO, Waters WR, Pahner MV, Stabel JR, Paustian ML, Early
antibody response against Mycobacterium avium subspecies paratuberculosis antigens
in subclinical cattle, Proteome Sci. 2008;] an 28;6:5

Crossley BM, Zagrnutt-Vergara FJ, Fyock TL, Whitlock RH, Gardner IA. Fecal
shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows. Vet
Microbial 2005; 107; 257-263

Eamens GJ, Walker DM, Porter NS, Fell SA, Radiometric pooled fecal culture for the
detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle. Aust.
Vet. J. 2008; 86(7); 259-265

Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary
Medicine. Clin. Micro. Rev. 2001; 14(3), 489-512

Hirst HL, Garry FB, Morley PS, Salman MD, Dinsmore RP, Wagner BA, McSweeny
KD, Goodell GM. Seroprevalence of Mycobacterium avium subsp. paratuberculosis

infection in dairy cows in Colorado and herd-level risk factors for seropositivity. J.
Am. Vet. Med. Assoc. 2004; 225(1), 97-101

J ohnson-Ifearulundu YJ, Kaneene JB, Lloyd J W. Herd — level economic analysis of
the impact of paratuberculosis on dairy herds. J. Am. Vet. Med. Assoc. 1999; 214(6),
822-825

Kalis CH, Collins MT, Hesselink JW, Barkema HW. Speciﬁcity of two tests for the
early diagnosis of bovine paratuberculosis based on cell-mediated immunity: the
Johnin Skin test and the gamma interferon assay, Vet Microbiol 2003; Dec 2; 97(1-
2):73-86

57

Kim SG, Kim EH, Lafferty CJ, Miller LJ, Koo HJ, Stehman SM, Shin SJ. Use of
conventional and real-time polymerase chain reaction for conﬁrmation if
Mycobacterium avium subsp. paratuberculosis in a broth- based culture system ESP
II. J. Vet. Diagn. Invest. 2004; 16(5), 448-553

Kim SG, Shin SJ, Jacobson RH, Miller LJ, Harpending PR, Stehman SM, Rossiter
CA, Lein DA. Development and application of quantitative polymerase chain reaction
assay based on the ABI 7700 system (Taq Man) for detection and quantiﬁcation of

Mycobacterium avium subsp. paratuberculosis. J. Vet. Diagn. Invest. 2002; 14(2),
1 26-1 3 1

Kopecky KE, Larsen AB, Merkal RS. Uterine infection in bovine paratuberculosis.
Am. J. Vet. Res. 1967; 28 (125), 1043-1045

McDonald WL, Ridge SE, Hope AF, Condron RJ. Evaluation of diagnostic tests for
Johne’s disease in young cattle. Aust. Vet. J. 1999; 77(2), 113-119

Menanteau-Horta AM, Ames TR, Johnson DW, Meiske J C. Effect of maternal
antibody upon vaccination with infectious bovine rhinotracheitis and bovine virus
diarrhea vaccines; Can J. Comp Med. 1985; Jan; 49(1): 10-14.

Ott SL, Wells SJ, Wagner BA. Herd- level economic losses associated with J ohne’s
disease on US. dairy operations. Prev. Vet. Med. 1999; 40, 179-192

Pillars RB, Grooms DL, Woltanski J A, Blair E. Prevalence of Michigan dairy herds
infected with Mycobacterium avium subspecies paratuberculosis as determined by
environmental sampling. Prev Vet Med. 2009; Apr 7 [Epub ahead of print]

Seitz SE, Heider LE, Huesten WD, Bech—Nielsen S, Rings DM, Spangler L. Bovine
fetal infection with Mycobacterium paratuberculosis. J. Am. Vet. Med. Assoc. 1989;
194(10), 1423-1426

Stabel JR. An improved method for cultivation of Mycobacterium paratuberculosis
from bovine fecal samples and comparison to three other methods. J. Vet. Diagn.
Invest. 1997; 9(4), 375-380

Stich RW, Byrum B, Love B, Theus N, Barber L, Shulaw WP. Evaluation of an
automated system for non-radiometric detection of Mycobacterium avium

paratuberculosis in bovine feces. J. Microbiol. Methods 2004; 56(2), 267-275

Sweeney RW, 1996. Transmission of paratuberculosis. Vet. Clin. North Am. F aad
Anim. Pract. 1996; 12(2), 305-312

58

Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium paratuberculosis
isolated from fetuses of infected cows not manifesting signs of the disease. Am. J. Vet.
Res. 1992; 53(4), 477-480

Tavompanich S, Gardner IA, Anderson RJ, Shin S, Whitlock RH, Fyock T, Adaska
JM, Walker RL, Hietala SK. Evaluation of microbial culture of pooled fecal samples

for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds. Am
J Vet Res. 2004; Aug; 65(8); 1061-1070

Twort F W. A Method for Isolating and Growing the Lepra bacillus of Man.
Proceedings of the Royal Society of London. Series B. 1910; 83(562), 156-158

van Roermund HJ, Bakker D, Willemsen PT, de Jong MC. Horizontal transmission of
Mycobacterium avium subsp. Paratuberculosis in cattle in an experimental setting:
calves can transmit the infection to other calves, Vet Microbiol. 2007 Jun 21; 122(3-
4):270-279

Visser I. Reproducibility of a Fecal Culture Method for Mycobacterium avium subsp.
paratuberculosis. In Manning, EJB, Collins, M.T. (Eds.) Proceedings of the Sixth
International Colloquium on Paratuberculosis, 1999; 512

Weber MF, Kogut J, de Bree J, van Schaik G. Evidence for Mycobacterium subsp.
avium shedding in young stock. Proceedings 8'” International Conference on
Paratuberculosis. Copenhagen, Denmark. Proceedings.2005; 679-689

Wells SJ, Collins MT, Faaberg KS, Wees C, Tavompanich S, Petrini KR, Collins JE,
Cernicchiaro N, Whitlock RH. Clin Vaccine Immunol. 2006; Oct; 13(10):1125-30

Whitlock HR, Buergelt C. Preclinical and clinical manifestations of paratuberculosis
(including pathology), Vet. Clin. N. Am. Anim. Pract. 1996; 12:345-356

Whitlock RH, Wells SJ, Sweeney RW, Van Tiem J. ELISA and fecal culture for
paratuberculosis (J ohne’s disease): sensitivity and speciﬁcity of each method. Vet.
Microbiol. 2000; 77 (3-4), 387-398

Witte CL, Hungerford LL, Rideout BA. Association between Mycobacterium avium

subsp. Paratuberculosis infection among offspring and their dams in non-domestic
ruminant species housed in a zoo. J Vet Diagn Invest. 2009 Jan; 21(1):40-47

59

CHAPTER 3

USE OF POOLED FECAL CULTURES TO DETECT
MYCOBACTERIUM A VIUM SUBSP. PARA TUBERCULOSIS IN
NATURALLY EXPOSED DAIRY CALVES: COMPARISON OF

RELATIVE SENSITIVITY AND SPECIFICITY OF POOLS OF
FIVE AND TEN INDIVIDUAL SAMPLES

60

ABSTRACT

Objectives—This study was conducted using Mycobacterium avium subsp.
Paratuberculosis (MAP) liquid fecal culture to determine if pooled sample of ﬁve or
ten individual fecal samples from dairy calves in a natural ﬁeld setting from eight
known J ohne’s disease infected herds could be used to detect a single positive
individual sample within their respective pool. If so, then to determine if there was any
difference between the sensitivity of pools containing one positive calf sample in a
pool of ﬁve individual samples compared to one positive sample in a pool of ten
individual samples.

Desi n— 28-month longitudinal study.

Sample Population—Heifer calves categorized into four age groups ﬁ'om eight dairy
herds in Michigan participating in the Michigan J ohne’s Disease Control
Demonstration Project.

Procedure— At each herd visit an eight gram fecal sample was collected ﬁ'om ten
individual calves within each of four age groups: 0-3 months, 4-6 months, 7-14
months, and 15-24 months. From each group of individual fecal samples, two pools of
ﬁve and one pool of ten. The pools, as well as the individual fecal samples, were
cultured using the rapid liquid culture (TREK®ESPH) system and positive samples
were conﬁrmed with acid fast staining and IS900 real time PCR. Sensitivity (Se) and
Speciﬁcity (Sp) of the two sizes of pooled samples were calculated and compared to
the individual calf fecal culture result as the gold standard.

Egg—Pools containing ﬁve compared to ten calves were more sensitive (Se) (79%

and 67%, respectively) and had higher positive predictive value (PPV) (52%

61

compared to 18%). These pools represented 2405 individual samples of which 32
were culture positive. No pool contained more than one MAP positive calf sample
and all positive calves were classiﬁed as light shedders. Using Chi Square, test status
results from pools of ten as well as pools of ﬁve showed a relationship with the fecal
status of the individual calves that comprised these pools (p = 0.0353 and p > 0.00001
respectively). Pooling of feces from individual calves for MAP culturing was
ineffective in animals less than seven months of age but showed no difference in
results between age group three (7-14 months) and age group four (15-24 months) in

both pools of ten (p = 0.401) and pools of ﬁve (p = 0.860).

Conclusions and Clinical Relevance Pooling individual fecal samples from dairy
calves naturally exposed to MAP may be used as a tool to determine if a population of
calves is shedding this organism. Pools of ﬁve are more sensitive than pools of ten and
have a higher positive predictive value making it a more optimal pool size. Pooling
was shown to be ineffective in the detection of MAP in calves less than seven months
of age. Pooling may offer the opportunity to utilize samples from calves greater than
Six months old to get an earlier assessment of any management changes instituted to

mitigate MAP transmission.

Introduction
J ohne’s disease (JD), caused by Mycobacterium avium subspecies
paratuberculosis (MAP), is a chronic granulomatous enteric disease of both domestic

and non-domestic ruminants. It was ﬁrst described in Germany and its etiologic agent

62

was characterized as an acid fast bacillus by Twort (Twort 1910). Recent studies
placed the percent of infected herds in Michigan at 64% (J ohnson-Ifearulundu 1999)
and 48% (Pillars 2009) respectively. Nationally the published prevalence ﬁgures are
widely variable (Adaska 2003; Hirst 2004). The cost to the US dairy producer is more
than $250 million annually (Ott 1999). Although JD has been recognized for over a
century, it has emerged as a major economic factor in the US dairy industry in the past
three decades.

Calves are most often infected with MAP before the age of Six months via the
ingestion of MAP feces, colostrum or waste milk contaminated with this organism
(Sweeney 1996). Transplacental infection also occurs (Kopecky 1967; Seitz 1989;
Sweeney 1992). Because MAP is a slow-growing bacterium, development of clinical
signs of JD may take 2-5 years yet transmission can occur on farms via unapparent
carriers (Harris 2001).

Attempts to identify naturally infected calves at a young age using fecal
culture have been unsatisfactory (Ayele 2004; McDonald 1999), presumably due to
low levels of bacterial shedding present in the feces of young animals and intermittent
shedding of MAP (Whitlock 1996). Reasons for this include the fact that standard
fecal culture has not been effective at detecting low-level bacterial shedding (Kim
2004) and overall sensitiVity may be as low as 33% (Whitlock 2000). Immunological
assays looking for antibodies are not as diagnostic with Mycobacteria, as with some
organisms (Bannantine 2008), due to a cell-mediated, rather than humoral, response
infection. Also compromising detection efforts in calves is reproducibility deﬁcits

within the same fecal sample (Visser 1999) with low levels of bacterial shedders.

63

Thus, standard fecal culture has not been effective at detecting this low-level bacterial
shedding (Kim 2004).

Early identiﬁcation of calves that may be at risk of MAP infection or that may
be shedding MAP into the environment is important for ensuring that effective JD
prevention and control strategies exist in dairy herds. An example of a recent strategy
using existing tests involves the pooling of individual fecal samples to detect the
existence of one or more positive adult MAP shedders within the pool, which has been
used as a management tool for assessing whether or not MAP is present within a dairy
herd (Kalis 2000; Wells 2002). Pooled fecal sampling for JD culture has been
recommended for some time in Australian sheep ﬂocks (Whittington 2000).

The number of individual adult cattle to incorporate in a pool has been debated
in the literature. Some studies report that the most cost effective pool size is ten
individual animals per pool (Tavompanich 2004). However, in studies that dealt with
low Shedding cattle (Eamens 2008) or tested herds of various sizes and prevalence
levels were cultured, (van Schaik 2003) pools containing no more than ﬁve individual
animals were appropriate to maintain adequate sensitivity. Pools of ﬁve containing at
least one low Shedding animal had at least a 53% chance of culturing positive for
MAP using standard culture methods (van Schaik 2003). In contrast far more variable
results were observed using pools of ﬁve with the direct qRT(real time)-PCR test
(Scott 2007).

In general it has been shown that, in adult dairy cattle, the culture of pooled
fecal samples may be a cost effective management tool to utilize in assessing the

presences of MAP in an infected herd. The advantage of pooled samples is that more

64

individual animals can be represented per test which may increase overall herd Se of
the test although it will decrease the Sc of detecting a speciﬁc individual (especially
low shedding) animal (van Schaik 2003). Thus it is important to have a strategy for
the use of this method, and an a priori sense of overall prevalence. Although most of
the studies were prospective and done in a natural ﬁeld setting few have utilized liquid
culture diagnostic testing and more focused on the adult animal, although on study
grouped animals by age (Kalis 2000).

The purpose of this study was to determine 1) if pooled fecal samples could be

utilized to detect a single MAP positive animal within the pool using the

TREK®ESPII:1 liquid culture system in a population of naturally exposed dairy calves

2) to determine if there was a difference in the sensitivity (Se) and speciﬁcity (Sp) of
the test when comparing pools of ten to pools of ﬁve and their respective positive
predictive value (PPV) and 3) to determine if age of the dairy calves tested affected

MAP detection in pooled fecal samples.

Materials and Methods

Study design and criteria for inclusion— This was a longitudinal study
Spanning 28 months. The study population included calves from eight commercial
dairy herds located in Michigan that were enrolled in the Michigan J ohne’s Disease
Control Demonstration Program. Herds were enrolled in the JD demonstration project
based on known infection with MAP, herd size, geographic location, and willingness

to cooperate in the study. This study met the Michigan State University guidelines for

65

animal research administered by the Animal Use Committee and owner’s permission
was received to conduct study.

Ten heifer calves from each of four age groups: 0-3 months, 4-6 months, 7-14
months, and 15-24 months were selected for fecal culture. Age group composition was
based on distinct housing groups including pre-weaning (hutch calves), post-weaning
(small pens), growth phase (large pens) and breeding group. Calves from fecal or
ELISA positive dams were preferentially targeted for testing and the remainder of
each age cohort were chosen randomly. Throughout the study period, samples were
collected at approximately four-month intervals with a maximum of eight visits over
28 months.

Fecal samples were collected from each calf by digital exculpation using
individual latex gloves and sterile water for lubrication. At least eight grams of fecal -
material was collected and the individual samples from each of the four age groups
were identiﬁed. Two grams from each individual calf sample were used to assemble a
pool of ten and also another two grams were used to make two pools of ﬁve across
each of the four age groups. The pools were complied without knowing a priori the
status of an individual calf. When a pool was complete, a standard tongue depressor
was used to mechanically stir the pool for one minute to homogenize the contents
within the container. From these pools a total of four grams of feces, gathered ﬁ'om
four sites within pool, was placed in a sample vial. The pooled samples as well as the
individual samples from each of the age groups were reﬁigerated at 5°C, and.
submitted simultaneously to the laboratory for MAP culture within one to two days of

collection. Although the majority of pools of ten were comprised of the same

66

individual calves that were in the two pools of ﬁve in a particular age group, there
were pools of ten that stood alone as well as pools of ﬁve tested that were not included
in pools of ten. Thus we did not always directly compare the ability of pools of ten to
pools of ﬁve to detect the same positive individual, although this was the most
common scenario, but rather we compared the difference between pools of ten to
pools of ﬁve to detect a positive MAP sample across a particular age group.

Laboratory testing—Fecal samples were analyzed at the Diagnostic Center
for Population and Animal Health (DCPAH) at Michigan State University. The

DCPAH is an accredited laboratory and has been certiﬁed by USDA National
Veterinary Science Laboratory to conduct TREK®ESPIIa liquid fecal culture for
MAP.

The Cornell method (Stabel JR 1997) was used to prepare fecal samples for
culture. The samples were placed in the TREK®ESPIIa culture system incubator.
Positive and negative controls were used on each batch of 40 fecal samples from
individual calves and their respective pools. Because this is a semi-quantitative test, a
positive sample and/or pool was described as a high shedder (or in the case of a pool,
contained high concentration of MAP) if it took 7-21 days to turn positive, moderate
shedder (moderate concentration of MAP) if it took 22-28 days to turn positive and

low shedder (low concentration of MAP) if it took 29-42 days to turn positive. If a

culture was not positive by day 42, it was stained with acid fast Kenyon stain and the

supernatant tested with ISP900 real time PCRc (Kim 2002). If negative to this follow—

up it was classiﬁed as not shedding (negative). All positive samples, individual animal

67

as well as pool, from liquid culture were also conﬁrmed with both acid fast Kenyon

stain and IS900 real time PCRC.

Statistical Analysis — Descriptive statistics were used, including frequencies,

Mantel- Haenszel Chi-Square table, as well as Fisher’s Exact Test - where appropriate.

Results — During the course of this study 940 individual samples were combined in
pools of ten grouped by age and 1465 individual samples comprised the pools of ﬁve.

Of these individual samples, 32 cultured MAP positive. (Table 3.1)

Table 3.1—Distribution and fecal culture results for Mycobacterium avium subspecies
paratuberculosis of individual samples across the four age groups as well as combined
age groups of older compared to younger in pools containing ﬁve individual samples

and pools containing ten individual samples per pool: Michigan 2005 — 2007

 

 

Pools of Ten Pools of Five

Age Group # Individual # Positive # Individual # Positive

Samples Samples Samples Samples
Age Group 1 240 1 365 1
Age Group 2 270 l 380 2
Younger (Age Group 510 2 745 3
1 and 2)
Age Group 3 210 6 380 9
Age Group 4 220 3 340 9
Older (Age Group 3 430 9 720 18
and 4)
Total 940 1 1 1465 21

 

 

 

Six of 94 pools of ten and 14 of 291 pools of ﬁve were culture positive.

Distribution of the total pools tested and the positive pools is shown in Table 3.2. The

68

distribution of positive pools across age groups for both pools of ten and pools of ﬁve
were similar, (p = 0.827) and (p = 0.887) respectively. There was no difference in
positive MAP test status between age group one and age group two (p :10) and age
group three compared to four (p = 1.0) regardless of pool size. However, there was a
signiﬁcant difference in MAP positive results when comparing pools ﬁ'om age groups
one and two (younger) with age groups three and four (older) across both pool sizes
(p = 0.0143 (pools of ten) and p = 0.002 (pools of ﬁve)), Table 3.2 with nearly equal.
sample distribution between groups across both pool sizes (p = 0.401 and p = 0.860

respectively( (Table 3.2).

Table 3.2—Distribution of pools containing ﬁve calves compared to pools containing
ten calves across individual age groups as well as younger compared to older calves

and their subsequent MAP culture status: Michigan 2005 — 2007

_
Pools of Ten Pools of Five

Age Group # Pools # Positive # Pools # Positive

pools pools

Age Group 1 24 0 71 0
Age Group 2 27 0 76 0
Younger (Age Group 1 and 2) 51 0 147 0
Age Group 3 21 3 76 7
Age Group 4 22 3 68 7
Older (Age Group 3 and 4) 43 6 144 14
Total 94 6 291 14

Although we did not know the individual calf MAP test status prior to

assignment to a pool, the prevalence of MAP detected in the individual samples was

69

low (2.3%) such that any positive pool that happened to contain a MAP positive calf,
contained only one MAP positive calf. Also, all of the individual MAP positive
samples contained in the pools were low shedders. Both of these factors helped
standardize the comparison of positive pools. Furthermore, it was found that there
were a higher proportion of false negative pools in pools containing ten calves when

compared to pools containing ﬁve calves indicating lower Se (Table 3.3).

Table 3.3 - Mycobacterium avium subspecies paratuberculosis fecal culture results
when comparing pools looking at false negatives and false positives distributed by age

group: Michigan 2005 — 2007

 

+ Pools of 10 (-) Pools of + Pools of 5 (-) Pools of 5

 

Age Group with no + 10 with + with no + with + calves)
calves calves calves
Youngera 0 2 0 3
Olderb 1 7 3 7
Total 1 9 3 10

 

3 Age Groups 1 and 3 (0-6 months) b Age Groups 3 and 4 (7-24 months)

The relative Se of the fecal culture from pools containing ten calves was 67%
and the relative Sp was 90% (Table 3.4). The positive predictive value (PPV) was
18%. In comparison, pools containing ﬁve individual calves had a Se of 79%, Sp of

96% and a PPV of 52%.

'70

an

 

Table 3.4 Relative sensitivity, Speciﬁcity, and positive predictive value comparing

pools of ten with pools of ﬁve in detecting Mycobacterium avium subsp.

Paratuberculosis in relationship to individual fecal samples from dairy calves across

all age groups: Michigan 2005-2007

Pools of Ten

Pools of Five

 

 

Positive Negative Positive Negative
Pools Pools Pools Pools
Pools containing 1
positive calf 2 9 11 10

 

Pools containing no
positive calves

   

 

 

1

 

82

 
 

3

 

 
 

 

 

 

 

 

 

Sensitivity (Se) 67% (95% CI 79% (95% CI
21-94%) 56-92%)

Speciﬁcity (Sp) 90% (95% CI 96% (95% CI
89-91%) 95-97%)

Positive Predictive 18% (95% CI 52% (95% CI

Value (PPV) 6-26%) 37-61%)

Mantel-Haenszel

(Yates Corrected) p = 0.036 p > 0.0001

Discussion

Pooling fecal samples in calves for the fecal culture of MAP, using liquid

culture, from targeted age groups may be useful in the overall management of J ohne’s

disease on a dairy. Pooling allows the testing of multiple animals with one test which

signiﬁcantly decreases cost (Kalis 2000; van Schaik 2007). Although detection of

shedding in the individual animal may be decreased by pooling, shown previously in

adult dairy cattle, (van Schaik 2003) overall herd Se may be increased because you use

71

many more animals per test. This increases the odds of detecting MAP in the herd,
especially in a herd with low prevalence (van Schaik 2003). Calves that are infected
tend to be in the early phase of bacterial shedding and shed in low numbers (Weber
2005; Bolton - Chapter 2 of this thesis) so pooling is a tool that increases odds of
MAP detection in a low prevalence population.

In this study, there was more success in detecting a positive animal in a pool of
ﬁve compared to pools of ten. Our positive predictive value of 52% with pools of ﬁve
was similar to a previous report using pools of ﬁve in low shedding adult dairy cows
(van Schaik 2003). In this study, pooling was found to be inefﬁcient in calves less than
seven months of age as zero positive pools were detected with a corresponding low
number of calves actually shedding MAP within these groups. Interestingly, in our
study, there was no difference between the older two age groups (7-14 months of age
and 15-24 moths of age) in Se, Sp, or PPV of pooled cultures. This break point of
seven to nine months before onset of MAP shedding has been shown in a prior study
(van Roermund 2005). However, the sensitivity of pools of ﬁve was greater than pools
of ten.

Given these ﬁndings, it may be sensible to target the age group 7-14 months in
a strategic pooling strategy, using pools of ﬁve to gain an early assessment of the
effectiveness of any management changes in the herd. Early detection of shedding in
this age group and subsequent housing changes may also decrease the shedding threat
of horizontal spread of MAP to young herd mates (van Roermund 2005 However with
a 53% PPV for pools of ﬁve, this would be an inappropriate test for ﬁnding individual

shedders, as pools of three were far better for this application in a study that focused

72

on low prevalence adult cattle (van Schaik 2003). There is a need for future research
in this area to explore the practical applications of these ﬁndings. In lieu of repeating a
comprehensive ﬁeld study such as this, a more prudent approach may be to examine
herds of various prevalence levels, and, utilizing pools of ﬁve in 7-14 month old
calves, sample these animals to determine if a change in prevalence of this age group
within a herd is a predictor of future herd prevalence. This procedure may produce an

“early report card” of the efﬁcacy of prior management changes.

73

REFERENCES

Adaska J M, Anderson RJ. Sero prevalence of J ohne’s disease infection in dairy cattle
in California, USA. Prev. Vet Med. 2003; 3; 255-261

Ayele WY, Bartos M, Svastova P, Pavlik 1. Distribution of Mycobacterium avium
subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls.
Vet Microbiol. 2004; 103(3-4), 209-217

Bannantine JP, Bayles DO, Waters WR, Palmer MV, Stabel JR, Paustian ML, Early
antibody response against Mycobacterium avium subspecies paratuberculosis antigens
in subclinical cattle, Proteome Sci. 2008;J an 28;6:5

Eamens GJ, Walker DM, Porter NS, Fell SA, Radiometric pooled fecal culture for the
detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle. Aust.
Vet. J. 2008; 86(7); 259-265

Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary
Medicine. Clin. Micro. Rev. 2001; 14(3), 489-512

Hirst HL, Garry FB, Morley PS, Salman MD, Dinsmore RP, Wagner BA, McSweeny
KD, Goodell GM. Seroprevalence of Mycobacterium avium subsp. paratuberculosis
infection in dairy cows in Colorado and herd-level risk factors for seropositivity. J.
Am. Vet. Med. Assoc. 2004; 225(1), 97-101

J ohnson-Ifearulundu YJ, Kaneene JB, Lloyd JW. Herd — level economic analysis of
the impact of paratuberculosis on dairy herds. J. Am. Vet. Med. Assoc. 1999; 214(6),
822-825

Kalis CH, Hesselink J W, Barkema HW, Collins MT. Culture of strategically pooled
bovine fecal samples as a method to screen herds for paratuberculosis, J Vet Diagn
Invest. 2000 Nov; 12(6):547-51. PMID: 11108455

Kim SG, Kim EH, Lafferty CJ, Miller LJ, Koo HJ, Stehman SM, Shin SJ. Use of
conventional and real-time polymerase chain reaction for conﬁrmationif
Mycobacterium avium subsp. paratuberculosis in a broth- based culture system ESP
II. J. Vet. Diagn. Invest. 2004; 16(5), 448-553

Kim SG, Shin SJ, Jacobson RH, Miller LJ, Harpending PR, Stehman SM, Rossiter
CA, Lein DA. Development and application of quantitative polymerase chain reaction
assay based on the ABI 7700 system (Taq Man) for detection and quantiﬁcation of
Mycobacterium avium subsp. paratuberculosis. J. Vet. Diagn. Invest. 2002; 14(2),
126- l 3 1

Kopecky KE, Larsen AB, Merkal RS. Uterine infection in bovine paratuberculosis.
Am. J. Vet. Res. 1967; 28 (125), 1043-1045

74

McDonald WL, Ridge SE, Hope AF, Condron RJ. Evaluation of diagnostic tests for
Johne’s disease in young cattle. Aust. Vet. J. 1999; 77(2), 113-119

Ott SL, Wells SJ, Wagner BA. Herd- level economic losses associated with Johne’s
disease on US. dairy operations. Prev. Vet. Med. 1999; 40, 179-192

Pillars RB, Grooms DL, Woltanski J A, Blair E. Prevalence of Michigan dairy herds
infected with Mycobacterium avium subspecies paratuberculosis as determined by
environmental sampling. Prev Vet Med. 2009; Apr 7 [Epub ahead of print]

Scott HM, F osgate GT, Libal MC, Sneed LW, Erol E, Angulo AB, Jordan ER.
Field testing of an enhanced direct-fecal polymerase chain reaction procedure,
bacterial culture of feces, and a serum enzyme-linked immunosorbent assay for

detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle,
AJVR, 2007 March 68:(3):236-45

Seitz SE, Heider LE, Huesten WD, Bech-Nielsen S, Rings DM, Spangler L. Bovine
fetal infection with Mycobacterium paratuberculosis. J. Am. Vet. Med. Assoc. 1989;
194(10), 1423-1426

Stabel JR. An improved method for cultivation of Mycobacterium paratuberculosis
from bovine fecal samples and comparison to three other methods. J. Vet. Diagn.
Invest. 1997; 9(4), 375-380

Sweeney RW, 1996. Transmission of paratuberculosis. Vet. Clin. North Am. Food
Anim. Pract. 1996; 12(2), 305-312

Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium paratuberculosis
isolated from fetuses of infected cows not manifesting signs of the disease. Am. J. Vet.
Res. 1992; 53(4), 477-480

Tavompanich S, Gardner IA, Anderson RJ, Shin S, Whitlock RH, Fyock T, Adaska
J M, Walker RL, Hietala SK. Evaluation of microbial culture of pooled fecal samples

for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds. Am
J Vet Res. 2004; Aug; 65(8); 1061-1070

Twort FW. A Method for Isolating and Growing the Lepra bacillus of Man.
Proceedings of the Royal Society of London. Series B. 1910;83(562), 156-158

van Roermund, HJ W, deJohn MCM, 2005. Horizontal transmission experiment of
paratuberculosis. Proceedings 8th International Conference on Paratuberculosis.
Copenhagen, Denmark. Proceedings release date: September 2006

van Schaik G, Pradenas FM, Mella NA, Kruze VJ; Diagnostic validity and costs of
pooled fecal samples and individual blood or fecal samples to determine the cow- and

75

herd- status for Mycobacterium avium subsp. Paratuberculosis; Prev Vet Med 2007
Nov 15;82(l-2):159-65. Epub 2007 Jan 26. PMID 17597241

van Schaik G, Stehman SM, Schukken YH, Rossiter CR, Shin SJ; Pooled fecal culture
sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and
prevalence; J Vet Diagn Invest. 2003 May;15(3):233—41. PMID: 12735345

Visser I. Reproducibility of a Fecal Culture Method for Mycobacterium avium subsp.
paratuberculosis. In Manning, EJB, Collins, M.T. (Eds) Proceedings of the Sixth
International Colloquium on Paratuberculosis, 1999;512

Weber MF, Kogur J, van Schaik G, 2005. Evidence for Mycobacterium subsp. Avium
shedding in young stock. Proceedings 8th International Conference on
Paratuberculosis. Copenhagen, Denmark. Proceedings release date: September 2006

Wells SJ, Whitlock RH, Lindeman CJ, Fyock T; Evaluation of bacteriologic culture of
pooled fecal samples for detection of Mycobacterium paratuberculosis; Am J Vet Res.
2002 Aug;63(8):1207-11. PMID: 12171178

Whitlock HR, Buergelt C. Preclinical and clinical manifestations of paratuberculosis
(including pathology), Vet. Clin. N. Am. Anim. Pract. 1996;12:345-356

Whitlock RH, Wells SJ, Sweeney RW, Van Tiem J. ELISA and fecal culture for
paratuberculosis (J ohne’s disease): sensitivity and speciﬁcity of each method. Vet.
Microbiol. 2000;77 (3-4), 387-398

Whittington RJ, Fell S, Walker D, McAllister S, Marsh 1, Sergeant E, Taragel CA,
Marshall DJ, Links U; Use of pooled fecal culture for sensitive and economic

detection of mycobacterium avium subsp. paratuberculosis infection in ﬂocks of
sheep; J Clin Microbiol. 2000 Jul;38(7):2550-6. PMID: 10878042

76

CHAPTER 4

POTENTIAL FOR ENVIRONMENTAL TRANSMISSION OF
M Y C OBA C TERI UM A VIUM SUBSPECIES PARA TUBERCULOSIS
FROM NON-SHEDDIN G DAIRY COWS TO THEIR CALVES: A

CASE REPORT

77

ABSTRACT

Objectives—This study was conducted to determine if skin contamination of dairy
cows with Mycobacterium avium subsp. paratuberculosis (MAP) could be detected on
areas that a suckling calf would nuzzle during teat seeking. Fecal and ELISA testing of
cows was also conducted to identify the potential source of MAP contamination.
_De_sigg—A case report.

Sample Population—Seven adult dairy cows, nearly due to calve and residing in the
“close-up” pen (n=6) and maternity pen (n=1), were selected for testing from a 120
cow dairy herd in Michigan (USA) with a MAP fecal test herd prevalence of 3%.
Procedure—The base of the left front teat and 5cm X 5cm areas of the lateral left
tarsus (hock) and the left lateral brisket posterior to the olecranon were swabbed using
sterile technique. Blood for MAP serum antibody ELISA testing and feces for MAP
culture were also collected. Skin swabs and fecal samples were analyzed using the
rapid liquid culture (TREK®ESPII) system and positive samples were conﬁrmed with
acid fast staining and IS900 real time PCR. Serum MAP antibody levels were
determined using the Parachek® ELISA assay (Prionics).

M— Six of the seven (86%) cows tested had at least one positive skin swab. The
hock (71%) and the udder (57%) were the most common sites where MAP was
isolated. MAP was isolated from the skin of ﬁve of the six (83%) cows residing in the
“close-up” pen, yet each of the 5 was fecal and ELISA test negative. The seventh cow,
residing in the maternity pen was fecal positive (a heavy shedder 100-300cfu), ELISA

positive, and MAP was isolated from all three Skin Sites.

78

Conclusions and Clinical Relevance—MAP was isolated from the skin of dairy
cows despite their negative MAP serum antibody or fecal culture status. This suggests
that 1) environmental contamination with MAP may occur even in low prevalence
dairy herds, 2) the immediate removal of calf from dam at birth is an important
element in prevention of MAP transmission, and 3) removal of MAP fecal positive
cows, especially heavy shedders, from dairy herds may be critical in eliminating an
important source of environmental MAP contamination, and subsequent risk of MAP

transmission to newborn calves.

BACKGROUND

J ohne’s disease (JD), caused by the acid-fast bacteria Mycobacterium avium
subspecies paratuberculosis (MAP), is of international importance in the cattle
industry. Prevalent in the US, it is known to cause reduced production and increased
culling rates on dairy farms, (Ott 1999) and there is mounting evidence of a potential
public health risk (Naser 2004).

This case study is focused on a 120 lactating cow dairy herd which is
participating in the Michigan J ohne’s Disease Control Demonstration Project. In 2002
this herd had a MAP culture positive test prevalence of 12% and Six JD clinical cases.
After the 2005 annual whole herd test, there were 4 MAP test positive cows (MAP
prevalence: approximately 3%) and only one clinical case. This change was attributed
primarily to intense culling and feeding of milk replacer instead of unpasteurized
whole milk to newborn calves. Environmental sampling of this herd also demonstrated

a concomitant reduction in the number of contaminated areas on the farm, with only

79

the manure storage lagoon testing positive in 2005 compared to multiple positive sites
in 2003 (e. g., common alleyways, maternity pen) (Grooms 2003-2007).

With this historical backdrop, and the knowledge that most infections with
MAP occur in the neonate (Hendrick 2005) the purpose of this study was to determine
if MAP could be isolated from areas on the exterior of dairy cows that are commonly
nuzzled in “teat seeking” behavior by the newborn calf (V entorp 1992). The presence
of MAP on these areas could have implications for transmitting MAP to calves.
Understanding this risk will provide additional information to support development of
management strategies for effective J ohne’s disease prevention and control

(Zdanowicz 2004).

PROBLEM STATEMENT

The ﬁrst objective of this study was to determine if skin contamination of dairy
cows with MAP could be detected on areas that a suckling calf would nuzzle during
teat seeking resulting in possible exposure to the pathogen.

The second objective of this study was to determine the MAP fecal culture and
ELISA antibody status of cows to identify potential sources of MAP skin

contamination.

80

MATERIALS AND METHODS

Population Description—The source population for this study was a Michigan
J ohne’s Disease Control Demonstration Project herd of 120 Holstein cows with a
MAP culture positive test prevalence of nearly 12% in 2002 and approximately 3% in
2005 (Grooms 2003-2007). The study population included all cows due to calve
within the three weeks following the test date (based on a 283-day gestation period
and their artiﬁcial insemination date), which occurred on a single herd visit in 2005.
This population was chosen as they present a risk to the newborn calf (Whittington
2009)

On the day of sample collection, Six of the seven cows sampled were housed in
a single row of sand-bedded free stalls adjacent to a maternity pen that was referred to
as the “close-up” pen, our target area. The seventh cow swabbed had recently been
moved to the maternity pen. She had spent the prior four weeks in the “close-up” pen.

Sample Collection—Three areas known to be commonly nuzzled by newborn
calves during teat seeking behavior (Ventorp 1992) were swabbed including the base
of the left front teat, and a 5cm X 5cm area on the lateral left tarsus (hock) and the left
lateral brisket area posterior to the olecranon. Using a scrubbing method described in
an Ohio study (Shulaw 2005), sterile 2in X 2in gauze pads saturated in 0.9% saline
were used. Each area was scrubbed briskly for ten seconds and the gauze pad
deposited in labeled sterile conical tubes. Serum for MAP antibody ELISA testing and
feces for MAP culture were collected from each cow.

T esting—All skin swabs and fecal samples were analyzed at the Diagnostic

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

81

using the rapid liquid culture system; TREK®ESPII, and processing using the Cornell
method (Stabel 1997). All positive samples were conﬁrmed with acid fast staining
and IS900 real time PCR (Kim 2004). Serum MAP antibody levels in each cow were

determined using the Parachek® ELISA assay (Prionics).

RESULTS

Six of the seven (86%) cows tested had at least one MAP positive skin swab
(Table 4.1). Three of the swab positive cows (43%) had more than one positive site.
The hock (71%) and the udder (57%) were the most common Site of MAP isolation.
Each of these six cows was fecal and ELISA test negative. The seventh cow (#7) was
MAP positive on all three swabbed skin sites and was also MAP fecal culture and
serum ELISA positive. She was classiﬁed as a “heavy shedder” based on the days to

positive in the TREK®ESPH culture system (Shin 2001; van Schaik 2003).

82

Table 4.1 - Presence of Mycobacterium avium subspecies paratuberculosis (MAP) by
site swabbed on seven dairy cows and their respective serum ELISA and fecal culture

status: Michigan 2005

C_ov1 Total # Serum Fecal
Number Brisket Hock Udder Sites (+) ELISA Culture
1 + + + 3 - -

2 - - - O - -

3 - + - 1 - -

4 - + + 2 - -

5 - - + 1 - -

6 - + - 1 - -

7 + + + 3 + +++*
Total 3 s g 11/21T

 

*
Cow #7 was characterized as a MAP “Heavy Shedder” on this fecal test.

1.

Total number of positive swab samples from total number of collected samples.

Additionally, during post-test investigation, it was discovered that, of the 3
other fecal culture positive cows in the herd, 2 of them (Cows A and B) had also
recently been housed in the “close-up” pen, calved and vacated the maternity pen
(Table 4.2). Table 4.2 depicts the timeline of cows housed in the “close-up” pen for all
seven of the cows swabbed and the two additional MAP fecal culture positive cows in

the herd.

83

Table 4.2 - Timeline illustrating the presence of dairy cows (seven swabbed for MAP,
two known MAP fecal culture positive) in the “close-up” pen relative to the test date

(> enter pen, X exit pen, ---weeks in pen): Michigan 2005.

TEST DATE

Pretest (Weeks) Posttest (Weeks)

3 2 4
X

 

 

*
MAP ”Heavy Shedder”
Fecal positive cow in herd but not part of swab study.

84

DISCUSSION

In this case study, it was demonstrated that MAP skin contamination of cows
near calving can be common. This may be a signiﬁcant risk factor for MAP
transmission to the newborn calves. Although the two maternity pens evaluated were
relatively small (8ft X 12ft), acceptable maternity management practices were being
used. These included one cow in a matemity pen at a time, frequent cleaning and
bedding of the maternity pen, and removing calf from cow and maternity pen as soon
as possible after birth. There have been many studies describing the importance of
maternity pen management in controlling JD (J ohnson-Ifearulundu 1999; Tiwari
2009), and no reports were identiﬁed that addressed the potential risk to the
environment or to newborn calves of MAP-contaminated animals entering the
maternity area. Isolating MAP on the exterior of these cows, especially areas where
newborn calves suckle, suggests that the potential for MAP transmission to the
newborn calf exists.

Another factor of interest in this study was the impact of a heavy shedding
animal on environmental contamination and subsequent contamination of non-infected
cows. There is evidence that many animals classiﬁed as “heavy shedders” actually
shed at even higher rates than once thought (Whitlock 2005). The concept of a “super
shedder” has been used in characterizing these animals in dairy herds. This “super
shedder” concept has also been modeled in other disease risk assessments, such as E.
coli 0157 (Matthews 2006). These animals can shed a hundred fold more of MAP
than the parameters of a heavy shedder (100-300) but are classiﬁed as heavy shedders

due to limitations in quantifying the standard fecal culture. As cow #7 was a “heavy

85

shedder” of MAP, the potential existed for her to be a “super shedder”. Furthermore,
although MAP is an obligate intracellular pathogen, it is hardy and can live in the
environment for more than a year (Whittington 2004). This highlights the potential
risk to newborn calves for MAP transmission when retaining a heavy shedder of MAP
in a herd.

Most of the high-risk swabbed areas on the cows tested were positive for MAP
contamination. Therefore, there is evidence, through our MAP fecal culture results,
that #7, a heavy shedder, along with her two known fecal positive herd mates, had the
opportunity to contaminate the environment of the fecal culture negative cows, as they
had passed through the same pen only days earlier.

In summary, MAP was isolated from multiple skin sites on cows that were not
themselves shedding MAP. This suggests that 1) a reduced herd test prevalence of
MAP does not free a dairy herd from environmental MAP contamination, 2) that
immediate removal of calf from dam, as suggested by USDA Johne’s group (Uniform
Program Standards for the Voluntary Bovine Johne's Disease Control Program
Effective June 1, 2006), is an important element in prevention of MAP transmission,
and 3) removal of “heavy shedders” may be critical in eliminating an important source
of environmental MAP contamination and subsequent risk of transmission to newborn
calves. Furthermore, attention to the cleanliness of the cows themselves as they enter
the maternity pen may be important. Future prospective studies should be conducted to
examine the efﬁciency of MAP transmission to the calf as a result of contaminated
skin on cows and the subsequent development of clinical Johne’s disease in these

calves as they mature.

86

REFERENCES

Grooms DL, Pillars RB, Bolton MW Collection Data from Michigan J ohne’s Disease
Control Demonstration Project. Michigan State University, East Lansing, Michigan
2003-2007; unpublished

Hendrick, SH, Kelton, DF, Leslie, KE, Lissemore, KD, Archambault, M, Dufﬁeld, TF,
Effect of paratuberculosis on culling, milk production, and milk quality in dairy herds.
JA VMA 2005;227:8, 1302-1308

J ohnson-Ifearulundu Y, Kaneene JB. Distribution and environmental risk factors for
paratuberculosis in dairy cattle herds in Michigan. Am J Vet Res. 1999 May;60(5):589-
596

Kim, SG, Kim, EH, Lafferty, CJ, Miller, LJ, Koo, HJ, Stehman, SM, Shin, SJ. Use of
conventional and real-time polymerase chain reaction for information of
Mycobacterium avium subsp. paratuberculosis in a broth- based ulture system ESP II.
J Vet Diagn Invest 2004;l6(5) 448-553

Matthews L, McKendrick U, Tement H, Gunn GJ, Synge B, Woolhouse ME Super-
Shedding cattle and the transmission dynamics of Escherichia coli 0157. Epidemial
Infect. 2006; Feb;l34(1):131-142

Naser, SE, Ghobrial, G, Romero, C, Valentine JF. Culture of Mycobacterium avium
subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet
2004;364,1039—1044

Ott, SL, Wells, SJ, Wagner, BA, Herd- level economic losses associated with J ohne’s
on US. dairy operations. Prev Vet Med 1999;40, 179-192

Shin SJ, Kim SG, Miller LJ, Harpending PR, Patten VH, Stehman SM, Rossiter CA,
McDonough PL, Lien DH Further evaluation of ESP Culture System 11, for detection
of Mycobacterium avium subsp. Paratuberculosis in bovine clinical samples. AAVLD
2001 Hersey, PA

Shulaw, W. Udder Scrubbing Cows. (Abstract) Proceedings USAHA. 2005;161
Stabel, JR. An improved method for cultivation of Mycobacterium

paratuberculosis from bovine fecal samples and comparison to three methods. J Vet
Diagn Invest 1997;9(4), 375-380

Tiwari A, Vanleeuwen JA, Dohoo IR, Keefe GP, Haddad JP, Scott HM, Whiting T.
Risk factors associated with Mycobacterium avium subspecies paratuberculosis

87

seropositivity in Canadian dairy cows and herds. Prev Vet Med. 2009;J an 1;88(1):32-
41

Uniform Program. Standards for the Voluntary Bovine. J ohne's Disease. Control
Program. Effective June 1, 2006 . Animal Health Inspection Service. APHIS 91—45—
016.
http://www.iohnesdisease.org/Unifonn%20Program%203tandards%20for%20the%20
Voluntarv%20Bovine%20National%20Johne's%20Disease%20Programpdf (Last
Accessed 5/3/2009)

 

van Schaik G, Rossiter CR, Stehman SM, Shin SJ, Schukken YH. Longitudinal study
to investigate variation in results of repeated ELISA and culture of fecal samples for

Mycobacterium avium subsp paratuberculosis in commercial dairy herds. Am J Vet
Res. 2003;Apr;64(4):479-84

Ventorp, M., Michanek, P. The Importance of Udder and Teat Conformation for Teat
Seeking by the Newborn Calf. J. Dairy Sci 1992;75, 262-268

Whitlock, RH, Sweeney, RW, Fyock, TL, Smith, J. MAP Super Shedders: Another
factor in the control of J ohne’s disease. Proceedings of 8th International Colloquium
on Paratuberculosis 2005;164

Whittington RJ. Evidence for age susceptibility of cattle to J ohne's disease. Windsor
PA, Vet J. 2009 Feb 24. [Epub ahead of print] PIVHD: 19246220

Whittington RJ, Marshall DJ, Nicholls PJ, Marsh IB, Reddacliff LA Survival and
dormancy of Mycobacterium avium subsp. paratuberculosis in the environment. Appl
Environ Microbiol. 2004;May;70(5):2989-3004

Zdanowicz, M, Shelford, JA, Tucker CB, Weary, DM, von Keyserlingk, MAG,
Bacterial Populations on Teat Ends of Dairy Cows Housed in Free Stalls and Bedded
with either Sand or Sawdust. J Dairy Sci 2004;87, 1694—1701

88

OVERALL SUMMARY

Nearly one hundred years after Mycobacterium avium subsp. paratuberculosis
(MAP) was characterized as the causative agent of J ohne’s disease in cattle the battle
to control this disease continues world wide. The difﬁculties are many, as MAP
infection is characterized by latent signs, lives up to a year in the environment, resides
in wildlife reservoirs, and is refractory to treatment. Also, to date, there are no
satisfactory vaccines commercially available, and present testing methods are helpful
but continue to need improvement.

However, substantial money and time have been invested in the past three
decades and our understanding of this pathogen has increased many—fold. Researchers
have shown that the calf is most susceptible, can shed bacteria to others, and there may
be some heritability involved in successful transmission. We have shown, with our
work that one of the greatest risks for a dairy calf is the MAP status of her dam. A calf
from a MAP positive dam is eleven times more likely to have a calf that will also shed
MAP. Colostrum and waste milk have to be monitored and the environment kept
clean, as they are also risk factors. Besides early MAP detection in calves greater than
six months of age utilizing liquid culture, we also found that we were able to pool
fecal samples of these low shedding calves and detect a single calf in the pool. Small
pools (pools of ﬁve calves) had higher sensitivity than pools of ten in detecting a
single positive sample. This parallels work that has been done in pooling samples of
low Shedding adult dairy cattle.

A demonstrative case study completed during the course of this research

pointed to the ability that one heavy shedding cow could contaminate the environment

89

in such a fashion as to cause the organism to reside on the teats of several other “non-

infected” cows. This also highlights the need to remove all calves from their dams

quickly after birth, so as to minimize likelihood of suckling. As the zoonotic potential

of MAP continues to be explored by researchers, it is clear that for the health of the

dairy industry and the consuming public, J ohne’s disease needs to be controlled.

Recommendations-

From our work several factors emerged:

1)

2)

3)

4)

The status of the dam should be considered when deciding to retain or cull a
newborn calf. If retaining calf, identiﬁcation as a calf from MAP infected cow
and relegation to separate housing may be indicated.
MAP fecal culture status of the calves may indicate future prevalence of

J ohne’s disease in the herd. More work should be conducted in this area,
especially in calves greater than seven months of age.
Pooling of calf fecal samples should be explored as a less expensive method to
monitor status of calves in a herd.

Indications are that, no matter how low the prevalence of MAP is within a
herd, it is extremely important to remove calf before it has a chance to suckle.
To mitigate this, concentration on pre-calving udder hygiene should be

emphasized.

These are some of the management recommendations that were illustrated or

augmented by this study.

90

   

"'lllllllljlgllllllllllllljllllllllllll“