Introduction

The family of bacteria known as Mycobacteriaceae contains a single genus, Mycobacterium a group of acid-fast bacteria known for their lipid-rich cell enve-lopes. Within this genus exists the Mycobacterium avium complex (MAC), which contains 28 serovars of two species: Mycobacterium avium and Mycobacterium intracellulare. Mycobacterium avium subsp. paratuber-culosis (MAP) belongs to this complex. Several strains of MAP have been identified, and enzyme restriction fragment length polymorphism (RFLP) methods have been designed to identify them (Manning 2001). MAP is the etiological agent of Johne’s disease, a chronic, inflammatory gastrointestinal disease of domestic and non-domestic ruminants and some non-ruminants that is characterized by chronic diarrhea, emaciation, decreased milk production, and often leads to death. It has been known to affect primates, but is most preva-lent in herds of dairy cattle (Manning 2001; Chacon et al. 2004; Clarke 1997). MAP was first described in

1895 (Johne and Frothinghan 1895), but it was not until 1912 that the requirements for Koch’s postulates were successfully met by F. W. Twort when he was able to culture MAP in the laboratory and transmit the infec-tion to healthy cattle (Twort and Ingram 1912).

The purpose of this review is to highlight the dif-ferences that separate MAP from other pathogenic mycobacteria and to examine and compare what is known about the role of MAP in the pathology of both Johne’s and Crohn’s diseases and its potential role in other autoimmune diseases such as type 1 diabetes. This review will also discuss the economic implications of MAP prevalence and Johne’s disease on the U.S. dairy industry, the current measures in place for the control of MAP at the herd level, and the prevalence with which dairy farmers practice them and discuss the future directions of MAP research. In addition, this review will examine current knowledge about Crohn’s disease and type 1 diabetes and future research needs for these diseases.

Critical Reviews in Microbiology, 2011, 1–16, Early Online

Address for Correspondence: Steven C. Ricke, Food Science Department, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704. Tel.: 479-575-4678, E-mail sricke@uark.edu

R E V I E W A R T I C L E

Current perspectives on Mycobacterium avium subsp. paratuberculosis, Johne’s disease, and Crohn’s disease: a Review

Ken Over, Philip G. Crandall, Corliss A. O’Bryan, Steven C. Ricke

Center for Food Safety and Food Science Department, University of Arkansas, 2650 N. Young Ave., Fayetteville, AR 72704

AbstractMycobacterium avium subsp. paratuberculosis (MAP) causes the disease of cattle, Johne’s. The economic impact of this disease includes early culling of infected cattle, reduced milk yield, and weight loss of cattle sold for slaughter. There is a possible link between MAP and Crohn’s disease, a human inflammatory bowel disease. MAP is also a potential human food borne pathogen because it survives current pasteurization treatments. We review the current knowledge of MAP, Johne’s disease and Crohn’s disease and note direc-tions for future work with this organism including rapid and economical detection, effective management plans and preventative measures.

Keywords:Johnes, Crohns, Mycobacterim avium susp. paratuberculosis, epidemiology

(Received 21 May 2010; revised 05 October 2010; accepted 13 October 2010)

ISSN 1040-841X print/ISSN 1549-7828 online © 2011 Informa Healthcare USA, Inc.DOI: 10.3109/1040841X.2010.532480 http://www.informahealthcare.com/mcb

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Genus Mycobacterium

The members of the genus Mycobacterium are acid-fast, aerobic microorganisms that include more than 100 species, several of which are important animal and human pathogens that can be differentiated from envi-ronmental species by their slow growth in comparison to environmental mycobacteria (Chacon et al. 2004). The first mycobacteria to be described were Mycobacterium tuberculosis, originally called Bacterium tuberculosis (Zopf 1885) the causative agent of human tuberculosis and Mycobacterium leprae, originally named Bacillus leprae (Hansen 1880) the causative agent for leprosy or Hansen’s disease. In 1896, Lehmann and Neumann (1899) proposed the genus Mycobacterium and included M. tuberculosis and M. leprae. Differentiation of mem-bers of the genus Mycobacterium from other closely related genera such as Nocardia, Rhodococcus, and Corynebacterium was originally based on 1) acid-alcohol fastness stains, 2) the presence of mycolic acids in the cell wall, and 3) a molecular predominance of 61 to 71 mol% of guanine + cytosine in the genome (Shinnick and Good 1994). Important human pathogenic mycobacte-ria include M. leprae, M. tuberculosis, and M. ulcerans, which are the causative agents of leprosy, tuberculosis, and Buruli ulcers, respectively (Cosma et al. 2003). In addition, M. avium subsp. avium, M. chelonae, M. for-tuitum, and M. kansasii can cause opportunistic human infections in patients with chronic respiratory infections and in immunocompromised individuals, particularly AIDS patients (Inderlied et al. 1993; Brennan and Nikaido 1995).

Important animal pathogenic mycobacteria include M. avium subsp. avium, M. bovis, and M. avium subsp. paratuberculsis, which are the causative agents of avian tuberculosis, bovine tuberculosis, and Johne’s dis-ease, respectively. M. avium subsp. paratuberculosis belongs to the M. avium complex, a group which also includes M. avium subsp. avium, M, intracellulare, and M. silvaticum.

Mycobacterium avium Complex

The Mycobacterium avium complex (MAC) has been subdivided into three subspecies: M. avium subsp. avium, M. avium subsp. paratuberculosis (MAP), and M. avium subsp. silvaticum (Chacon et al. 2004) based on DNA-DNA hybridization studies and other genotypic and phenotypic tests (Hurley et al. 1988; Saxegaard et al. 1988; Thorel et al. 1990; Yoshimura and Graham 1988). M. avium subsp. avium and subsp. silvaticum, are not able to cause disease in healthy hosts, but can in immuno-compromised hosts. MAP can be differentiated from M. avium and M. silvaticum by its very slow growth rate (8 to 24 weeks for visible colony formation) as compared to fast

growing Mycobacteria which form visible colonies in just 7 days. MAP also has a dependence upon the siderophore mycobactin J, an iron-chelating cell wall component, for growth in primary cultures (Chacon et al. 2004).

Many strains of MAP are such slow growers, taking months to years to form colonies, that it is nearly impos-sible to isolate MAP in cultures free of other microorgan-isms (Bull et al. 2003a). Thus, it is necessary to look at molecular methods for isolation and identification. In mixed culture, PCR methods are most desirable because genes and sequences specific to the bacteria of interest can be quickly identified. Mycobacterial interspersed repetitive units (MIRU) are mini-satellite sequences of between 46 and 101 base pairs in length found in the mycobacterial genome. Originally, MIRU were used as a method to specifically identify M. tuberculosis. More recently, MIRUs have been identified in both M. avium subsp. avium and MAP, and have been found to differ between the two subspecies in the number of tandem repeat motifs present in six of the MIRU loci discovered (Bull et al. 2003b), making MIRU typing a valuable tool for the identification of MAP. PCR-based detection of MIRU loci 1 and 4 of these six differentiated MIRU is a potentially reliable means of distinguishing MAP from other members of the MAC.

Mycobacterium avium subsp. paratuberculosis (MAP)

Thermal resistance

Mycobacterium avium subsp. paratuberculosis (MAP) has been found to be more resistant to heat treatments than other mycobacteria including M. avium, M. chelonae, M. phlei, M. scrofulaceum, and M. xenopi (Schulze-Robbecke and Buchholtz 1992). Studies examining the thermal tol-erance and effectiveness of pasteurization conditions on the destruction of MAP have detected the organism after typical thermal treatments including low-temperature holding at 63 °C for 30 min, and high temperature-short time (HTST) at 72 °C for 15 sec (Sung and Collins 1998; Millar et al. 1996). Chiodini and Hermon-Taylor (1993) concluded that MAP strains were able to survive HTST thermal processing with survival rate ranges of 3% to 5% and 24.8% to 31.4% for strains isolated from bovine and human tissues, respectively. Grant et al. (1996) con-structed thermal death curves for MAP and determined that when present in large numbers, the pasteurization process was not sufficient to ensure the complete destruc-tion of the organism. A general consensus is that the pres-ence of MAP in concentrations greater than 104 CFU/mL in milk samples may not be completely destroyed by cur-rently practiced pasteurization processes including HTST and low temperature batch holding (Meylan et al. 1996;

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Current perspectives on Mycobacterium avium subsp. paratuberculosis 3

Grant et al. 2001). Grant et al. (2002) found that 1.8% of commercially pasteurized samples in the UK were cul-ture positive for MAP. In the U.S., Ellingson et al. (2005) surveyed 702 samples of pasteurized milk purchased at retail for the presence of MAP and found that 2.8% of the samples were culture positive. The methods for deter-mining resistance of MAP to pasteurization processes are made difficult by the slow growth rate for new isolates (8 to 24 weeks for visible colony formation) and difficulties in culturing the organism. Another problem is presented by the hydrophobic nature of its cell wall, which can lead to clumping and congregation of the organism on the surface of liquids such as milk, and on the walls of tubes (Lund et al. 2002). These both lead to difficulties in detec-tion and quantitation.

Detection and quantitation

The primary methods of detection and quantitation of MAP, as well as the advantages and disadvantages of each are summarized in Table 1. Detection methods based on culturing MAP are very difficult due to the extremely slow growth of the organism on initial isolation (Harris and Barletta 2001). The thick cell wall, along with the inability to produce mycobactin has been suggested as the reason for the unusually long generation time of MAP (Rowe and Grant 2006). Bovine strains may take from 12 to 24 weeks of incubation on initial isolation before visible colonies emerge in culture, while human and sheep isolates may require many months. Many human strains still have not been isolated in pure culture (Bull et al. 2003a). A simple method for identification of MAP utilizes its mycobactin J dependency. It was determined that MAP required an “essential substance” to grow, which was eventually iso-lated and designated mycobactin J (Francis et al. 1953). In early work with mycobacteria it was found that most would grow on laboratory media, except for MAP (Snow 1970). Mycobactin J is used in identification of MAP by culturing samples in media with and without the supple-ment; if a particular Mycobacterium can grow only on the media supplemented with mycobactin J, it is considered to be MAP (Snow 1970).

Tests using serological responses to recombinant MAP antigens have also been used to verify the presence of MAP in sera of Crohn’s patients (El-Zaatari 1999) and to detect MAP in infected animal feces, sera, and milk (Sugden et al. 1987; Sweeney et al. 1994; Jark et al. 1997; Stabel et al. 2002; Singh et al. 2007b; Clark Jr et al. 2008; Pinedo et al. 2008). The primary serological tests include complement fixation (CF), agar gel immunodiffusion (AGID), and enzyme-linked immunosorbent assay (ELISA) (Yakes et al. 2008). Mycobacterial LAM has many immunomodulatory functions and is capable of eliciting an antigen response from its host (Chatterjee 1998). An ELISA to detect MAP was designed using the

lipoarabinomannan (LAM) antigen detected by Sugden et al. (1987) and has been used for the rapid detection of MAP infection in feces, flesh, and milk of infected cat-tle and sheep (Sweeney et al. 1992; Sweeney et al. 1994; Sugden 1989), with results for the ELISA test available for examination within 1 to 2 hours after initial prepara-tion of microtiter plates. Although these tests are rapid in comparison with culturing techniques, they suffer from lack of sensitivity (20 to 60% with results varying by antigen and laboratory methods) and often cannot detect MAP in subclinically infected cattle (Sockett et al. 1992; Clarke et al. 1996; Sergeant et al. 2002; Collins et al. 2005). A direct comparison of single PCR and ELISA reveals that ELISA is more specific, but PCR is more sensitive in detection of MAP from fecal samples (Clark et al. 2008). Recently a sandwich immunoassay was developed to rapidly detect low levels of MAP (Yakes et al. 2008). The method utilized an immobilized layer of monoclonal antibodies specific to a MAP surface protein and extrinsic Raman labels (ERLs) designed to bind to the antibody-captured proteins and emit surface-enhanced Raman scattering (SERS). The researchers have tested it success-fully in a whole milk matrix.

Green et al. (1989) characterized a novel insertion element unique to MAP they termed IS900. A subse-quent PCR method to detect IS900 was created and has since been used as a means to detect the presence MAP (Millar et al. 1996; McFadden et al. 1987). The specificity of IS900 to MAP was called into question when IS900-like sequences were found in other mycobacteria of the MAC, requiring the use of restriction endonuclease analysis to discern false positives (Cousins et al. 1999; Naser et al. 1999; Englund et al. 2002). More recent screenings for specific targets using the sequenced MAP genome have discovered four sequences without homologues in other members of the MAC; ISMap02 is present in six copies (Stabel and Bannantine 2005), ISMav2 is present in three copies (Li et al. 2005), and F57 and Hsp X both occur as single copies (Tasara and Stephan 2005). These gene tar-gets may not be as sensitive due to the limited number of copies present in the MAP genome compared to IS900, but are more MAP-specific and likely will generate fewer false-positives. Real-time PCR detection of MAP, because it affords the ability to determine the initial concentra-tion of target sequence, is more accurate when the target sequence is present in only one copy per genome. Hsp X is a heat-shock protein that has been found to be neces-sary for intra-host survival of M. tuberculosis (Geluk et al. 2007), but the functions of the F57 and ISMav02 gene sequences are as yet unknown.

A wide range of different molecular approaches have been applied to detect MAP in a variety of sample types such as milk, blood, feces, and excised tissue. Molecular methods used to detect MAP in milk include single PCR, nested PCR, and real-time PCR. These methods are

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Tabl

e 1.

Det

ecti

on a

nd

Qu

anti

tati

on m

eth

ods.

Det

ecti

on m

eth

odB

asis

of t

est

Ch

arac

teri

stic

s of

met

hod

Ad

van

tage

sD

isad

van

tage

s

Cu

ltu

re:

Id

enti

fica

tion

of

mor

ph

olog

ical

feat

ure

s an

d

grow

th r

equ

irem

ents

Req

uir

es: d

econ

tam

inat

ion

of

sam

ple

s u

sin

g ch

emic

als

or

anti

bio

tics

, cen

trif

uga

tion

to

con

cen

trat

e M

AP

cel

ls

Sim

ple

, in

exp

ensi

veE

xtre

mel

y sl

ow g

row

th o

f MA

P,

pot

enti

al o

verg

row

th if

con

tam

i-n

ated

sam

ple

Sero

logi

cal:

ELI

SA. C

FT,

AG

IDU

tiliz

es s

pec

ific

anti

gen

s

(hom

emad

e or

co

mm

erci

al)

for

MA

P

anti

bod

ies

No

spec

ial t

reat

men

t of

sam

ple

s re

qu

ired

.H

igh

sp

ecifi

city

, hig

h th

rou

ghp

ut,

in

exp

ensi

ve, r

apid

Pos

sib

le fo

r in

fect

ed a

nim

als

to

shed

MA

P in

milk

wit

hou

t pro

du

c-in

g M

AP

an

tib

odie

s, p

oten

tial

cr

oss-

reac

tivi

ty b

etw

een

an

tib

odie

s (f

alse

pos

itiv

e), l

ow s

ensi

tivi

ty

San

dw

ich

im

mu

noa

ssay

Mon

oclo

nal

MA

P

anti

bod

ies

are

imm

obili

zed

on

an

tige

n la

yer

and

bou

nd

to

ext

rin

sic

R

aman

lab

els

(ER

Ls)

Mol

ecu

lar

(P

CR

):Si

ngl

e P

CR

Det

ecti

on o

f IS9

00, f

57,

Hsp

X, o

r IS

Mav

02R

equ

ires

MA

P c

ell

con

cen

trat

ion

by

trad

itio

nal

ce

ntr

ifu

gati

on o

r m

ore

re

cen

tly

imm

un

omag

net

ic

sep

arat

ion

(IM

S). I

MS

in

volv

es is

olat

ing

MA

P th

rou

gh

inte

ract

ion

of t

he

targ

et c

ells

w

ith

an

tib

odie

s fi

xed

on

m

agn

etic

par

ticl

es th

at c

an

then

be

sep

arat

ed fr

om a

m

atri

x u

sin

g a

mag

net

ic fi

eld

.

Rap

id c

omp

ared

to c

ult

uri

ng

m

eth

ods

of s

low

gro

win

g

myc

obac

teri

a, h

igh

sen

siti

vity

Can

not

dis

tin

guis

h v

iab

le a

nd

non

-vi

able

cel

ls, d

ifficu

lt to

cor

rela

te

to in

itia

l con

cen

trat

ion

of t

arge

t se

qu

ence

N

este

d P

CR

Det

ecti

on o

f IS9

00, f

57,

Hsp

X, o

r IS

Mav

02

R

T-P

CR

Det

ecti

on o

f IS9

00, f

57, H

spX

, or

ISM

av02

Use

s fl

uor

esce

nt-

la

bel

ed p

rob

e ov

er m

ult

iple

si

ngl

e-P

CR

cyc

les

to m

onit

or

amou

nt o

f flu

ores

cen

ce d

uri

ng

ex

pon

enti

al in

crea

se

in P

CR

pro

du

ct.

In a

dd

itio

n to

ad

van

tage

s of

oth

er

PC

R m

eth

ods,

pro

vid

es a

ccu

rate

co

rrel

atio

n w

ith

init

ial t

arge

t co

nce

ntr

atio

n. A

lso,

aff

ord

s ab

ility

to

det

ect v

iab

le c

ells

if R

NA

bas

ed.

Exp

ensi

ve

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necessarily preceded by concentration and separation (either physical or immunological) steps (Slana et al. 2008). Single PCR method targets the IS900 insertion element and has been used to detect MAP (Singh et al. 2007b; Giese and Ahrens 2000; Pillai and Jayarao 2002; Jayarao et al. 2004).

Tests designed to detect MAP in retail and simulated pasteurization testing include culturing and using PCR methods designed to detect IS900 to confirm identifica-tion of survivors (Pearce et al. 2001; Grant et al. 1996; Ayele et al. 2005). The nested PCR method uses two sets of primers used in sequential PCR amplifications to ensure the product from the second PCR has no unwanted con-tamination products. This method also targets the IS900 insertion sequence and has been employed in several studies (Stabel et al. 2002; Pinedo et al. 2008; Corti and Stephan 2004; Gao et al. 2002). The IS900 insertion ele-ment is present in 17 copies in the MAP genome. This abundance of copies increases the sensitivity of PCR-based detection methods. ISMap02 is present in 6 cop-ies and has been evaluated for efficacy as an alternative target sequence for nested and RT-PCR detection, as well as conventional PCR methods by Stabel and Bannantine (2005). They found no significant differences between sensitivity and specificity of ISMap02 when compared to IS900. Due to the possibility of detecting IS900-like sequences from non-MAP mycobacteria without careful control in the lab, the use of ISMap02 as a target sequence for identification may be useful.

Nested PCR is now more commonly being replaced by Real-Time PCR (Slana et al. 2008). Real-Time PCR (also known as Quantitative Real-Time PCR) utilizes fluores-cent labeled probes to monitor amplicon production in Real-Time after each run of the thermocycler, as opposed to after the endpoint has been reached. This method provides a quantitative analysis because it is possible to estimate the original amount of target template based on the rate of increase in PCR product. To achieve reli-able quantitative information, target gene sequences with fewer copies for increased quantitative accuracy are needed; therefore, F57, Hsp X, or ISMav2 are more suitable (Slana et al. 2008). In a direct comparison of Real-Time PCR and nested PCR and culturing methods, Real-time PCR exhibited a sensitivity of 92 to 96% and a specificity of 92% (Fang et al. 2002). The primary draw-back to RT-PCR is the cost of the equipment.

Natural reservoirs

One characteristic that separates MAP from other mem-bers of the MAC is that it is a host-dependent organ-ism (Olsen et al. 2002). Although it can survive in the environment, MAP requires residence inside an animal macrophage to replicate (Collins 2003). The natural hosts for MAP are wild and domesticated ruminants as

well as some non-ruminants. MAP infections have been reported in badger, bison, camelids, crow, deer, elk, fox, non-human primates, rabbits, rats, swine, weasel, and wood mice (Stehman 1996; Beard et al. 2001; Whittington and Sergeant 2001; Manning et al. 2003; Palmer et al. 2005; Anderson et al. 2007; Kopecna et al. 2006; Pedersen et al. 2008). The widespread occurrence of MAP infec-tion in wild ruminants and non-ruminants raises serious concerns over the possibility of interspecies transmission of MAP infection. The occurrence of MAP in carnivorous and scavenging wildlife is thought to be due to preda-tion on rodents and other species commonly found in structures used to house cattle and other small ruminants (Palmer et al. 2005). This potential exchange between domestic and wild animals brings to mind the question of wild animals serving as a reservoir to otherwise non-infected domestic species (Anderson et al. 2007).

Methods to control MAP have been developed in many countries, and are usually based on testing and culling individual animals (Benedictus et al. 2000; Kennedy and Alworth 2000). These efforts are based on the knowl-edge that MAP is classified as an obligate pathogen and parasite of animals (Thorel et al. 1990), and theoretically removing infected animals would lead to eradication. In fact, MAP can survive for long periods outside the host, persisting and spreading in pastures; the question of how long pastures remain infective was raised as early as 1912 (Penberthy 1912). Survival of MAP in manure has been documented to be more than 250 days (Lovell et al. 1944) and in neutral-pH water for over 17 months (Larsen et al. 1956).

Epidemiology

Infection with MAP is a worldwide problem. The disease is capable of affecting all ruminant livestock, and is not restricted to any particular geographic area. In their recent review, Nielsen and Toft (2009) critically com-pared many research studies across Europe to determine the prevalence of MAP infection in ruminants found on farms. They chose to assess data from studies that reported prevalence data from multiple herds and did not assess data from non-farmed animals such as zoos and game parks. In addition, herds that had been vac-cinated against MAP were also excluded. They concluded that no reliable estimate of MAP prevalence at the herd or animal level could be arrived at when comparing different studies due to a variety of complicating issues such as sampling bias where herds were selected based on suspicion of infection, unclear sampling schemes, and discrepancies between studies from the same country. Their best guess conclusions were that, in Europe, overall within-herd level prevalence of MAP in cattle was greater than 20%, with some countries having as low as 3 to 5%. Incidence of Johne’s disease was reported to be increasing

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in the Netherlands, Finland, Italy and Scotland. In other parts of the European continent, no change in incidence has been noted over recent years. Nielsen and Toft (2009) were unable to interpret the within-herd level prevalence data for sheep and goats. Between-herd prevalence was found to be greater than 50% for cattle and greater than 20% in sheep and goat flocks. The Department for Environment, Food and Rural Affairs (DEFRA) conducted a survey of dairy herds in the United Kingdom conducted testing and administered questionnaires in order to esti-mate the prevalence of Johne’s in the UK and they esti-mated that almost 35% of the herds are infected (DEFRA 2009). Once again, this may not be the true prevalence since participation was voluntary and farmers that had experienced Johne’s were more likely to participate, and there may also have been a geographic bias in the results (DEFRA 2009).

In North America, several studies have attempted to assess the prevalence of MAP infection at both the animal and herd levels for beef and dairy cattle (Table 2). Similar problems have arisen when attempting to compare the results of these studies as those outlined in Nielsen and Toft’s review of European MAP prevalence studies (2009). Sampling methodologies between studies differed with at least one study focusing on herds suspected of having infection and several testing culled cattle. In addition, sampling sizes were different, not all samples were effec-tively screened due to non-interpretable data, and detec-tion methods between studies often differed. Chiodini and van Kruiningen (1986) reported an18% animal-level prevalence in 100 culled cattle sent to a central abattoir in New England states. No herd-level prevalence estimates could be relied on to give a true prevalence because only herds suspected of MAP infection were chosen for analysis. Merkal et al. (1987) tested 7,540 cattle from 32 states and Puerto Rico using culture detection of ileocecal lymph nodes and determined an animal-level prevalence of 1.6%. NcNab et al. (1991) conducted a study in Ontario, Canada of 304 herds containing 14,923 animals (first phase), 400 culled cattle from 3 abattoirs, and 120 herds containing 2,376 animals (second phase from first 304 herds), by feces culture, LAM-ELISA, and feces culture, respectively. In their study they concluded animal-level MAP prevalence for the first phase 304 herds, 3 abattoirs, and second phase 120 herds to be, 6.1%, 5.5%, and 0.1%, respectively. Thorne and Hardin (1997) tested 1,954 cattle from 19 dairy and 68 beef herds by ELISA and determined animal and herd-level prevalence of 8% and 5% and 74% and 40% for dairy and beef herds, respectively. Roussel et al. (2005) evaluated 4,579 beef cattle from 115 herds in Texas by collecting blood and fecal samples from veteri-narians participating in their study and determined MAP infection by use of ELISA. They estimated the animal and herd-level prevalence to be 3% and 43.8%, respectively. The Animal and Plant Health Inspection Service (APHIS)

branch of the United States Department of Agriculture (USDA) reported in 2007 (the most recent reporting year for Johne’s) that as many as 25% of the dairy herds in the United States contain cattle infected with Johne’s. The National Animal Health Monitoring System (NAHMS) 2007 study (Dairy 2007) focused on Johne’s disease and surveyed only 17 states but represented 83% of the dairy cows in the U. S. (NAHMS 2007). Of the farmers surveyed, 23% reported a confirmed case of Johne’s in their herd in the previous 12 month period (NAHMS 2007). Large operations had a higher percentage positive (34%) as compared to small herds (17%) and the Eastern region had a higher percent of positives (24%) as compared to the Western region (13%) (NAHMS 2007). Beef cattle are also subject to infection with Johne’s, although there are few studies of the prevalence of the disease in these herds. One study of beef cattle found that 8% of U.S. beef herds had cattle that were seropositive for Johne’s (Dargatz et al. 2001). Participation in the study was voluntary and 68% of the farmers chose not to have the serological tests performed (Dargatz et al. 2001). Additionally, serological testing may underestimate the true level of infection due to test sensitivity as well as the fact that seroconversion occurs in the later stages of Johne’s. Furthermore, Roussel et al. (2007) found that environmental (non MAP) myco-bacteria were isolated from feces of cattle with positive ELISA tests although no MAP was isolated from the fecal samples.

The prevalence of MAP in cattle, goats, and sheep has been studied in Australia, Europe, India, and the Middle East. Singh et al. (2008) tested the serum samples of 1,425 bovine (buffaloes and cattle) in Northern India and deter-mined the sero-prevalence to be 28.6% in buffaloes and 29.8% in cattle. Al-Majali et al. (2008) using a commercial ELISA kit reported individual and flock level prevalence of 21% and 50%, respectively, for sheep and 18.1% and 45.8%, respectively for goats in Southern Jordan. Using univariable analysis they assessed the primary risk fac-tors to be flock mixing and addition of new animals to the flocks (p ≤ 0.05). Singh et al. (2007a) assessed the prevalence of MAP, using ELISA, in 2 month old kids from 3 goat breeds in India and determined the animal level prevalence to be 8.5%. Sheep flock prevalence in Australia has been estimated to be 6–10% in New South Wales and 2.4–4.4% Australia-wide (Sergeant and Baldock 2002). A more recent study conducted in Portugal (Coelho et al. 2008) used PCR screening of pooled samples (5 animals per pool) to estimate ovine MAP infection. Animals were separated into 2 categories, apparently healthy and sus-pected of infection. They found 20.7% of pooled samples from apparently healthy animals were MAP positive, while 16.7% of pooled samples from animals suspected of being infected were MAP positive. The individual ranges of MAP infection in this study ranged from 6.4% to 15.4%.

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Current perspectives on Mycobacterium avium subsp. paratuberculosis 7

Tabl

e 2.

Pre

vale

nce

of M

AP

infe

ctio

n in

Nor

th A

mer

ica.

Reg

ion

Year

An

imal

Sam

plin

g m

eth

odD

etec

tion

m

eth

odH

erd

-lev

el

Pre

vale

nce

An

imal

-lev

el

pre

vale

nce

No.

cat

tle

No.

her

ds

Cav

eats

Sou

rce

New

E

ngl

and

1983

-198

4C

attl

eR

and

omly

ch

osen

fr

om a

bat

toir

eve

ry

2 to

3 w

eeks

Cu

ltu

re25

her

ds

fo

un

d

pos

itiv

e*

18%

100

Un

know

n*O

nly

her

ds

sus-

pec

ted

of b

ein

g

MA

P p

osit

ive

w

ere

test

ed

Ch

iod

ini e

t al

(198

6)

Mis

sou

ri19

93-1

994

Cat

tle

Ran

dom

sam

plin

g

of c

attl

e sp

ecim

ens

su

bm

itte

d fo

r B

ruce

llosi

s te

stin

g

ELI

SAD

airy

: 74%

Dai

ry:

8% ±

3%

1954

Dai

ry: 1

9

Thor

ne

and

H

ard

in (

1996

)

Bee

f: 4

0%B

eef:

5%

± 2

%

Bee

f: 6

8

On

tari

o19

86-1

989

Cat

tle

Sam

ple

s w

ere

take

n

from

cu

lled

cow

s

sen

t to

3 ab

atto

irs

an

d fr

om 3

04 d

airy

h

erd

s, fu

rth

er

inve

stig

atio

n a

t d

airy

farm

s

Isol

atio

n

from

fece

sN

D6.

1%14

,923

304

*Nin

etee

n

per

cen

t of 2

,943

fe

cal c

ult

ure

s

wer

e n

on-

inte

rpre

tab

le

du

e to

ov

ergr

owth

on

p

late

s

NcN

ab e

t al

(199

1)

LA

M-E

LISA

5.5%

400

3 ab

atto

irs

Isol

atio

n

from

fece

s0.

1%2,

376

120

From

32

st

ates

an

d

Pu

erto

Ric

o

1983

-198

4C

attl

eIl

eoce

cal l

ymp

h

nod

es o

f cu

lled

ca

ttle

* fr

om U

SDA

-in

spec

ted

ab

atto

irs

Cu

ltu

reN

D1.

6%7,

540

Un

know

n*

On

ly c

ulle

d

catt

le te

sted

Mer

kal e

t al.

(198

7)

Texa

s20

00-2

001

Cat

tle

(B

eef)

Blo

od a

nd

feca

l sa

mp

les

from

p

arti

cip

atin

g ve

teri

nar

ian

s

ELI

SA43

.8%

3%4,

579

115

R

ouss

el e

t al

(200

5)

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8 Ken Over et al.

Johne’s Disease

Etiology

Johne’s disease, a slow-developing, chronic wasting disease affecting all domestic and non-domestic rumi-nants and some non-ruminants is caused by MAP. The disease, also known as paratuberculosis, is characterized by regional lymphangitis and lymphadenitis and chronic granulomatous enterocolitis. Large lesions, segmental or diffuse, are usually seen in the intestines and mesenteric lymph nodes (Olsen et al. 2002). The clinical signs of paratuberculosis are chronic weight loss with diarrhea, decrease in milk yield, and eventually death (Clarke 1997; Begg and Whittington 2007). Granulomatous lesions are present that vary in severity from patchy to severe and are typically more pronounced in the distal ileum, ending before the ileocecal valve (Buergelt et al. 1978), and are essentially aggregations of macrophages and lymphocytes at varying developmental stages. Granulomatous lesions have also been associated with the ileocecal lymph nodes (Chacon et al. 2004). The advanced stages of the ileal infection result in a reduced ability to absorb protein, which is the primary cause for weight loss (Clarke 1997).

Upon ingestion of MAP through feces, milk, or colos-trum, or the intrauterine transmission of the infection to the unborn fetus, the pathogen transverses the mucosal epithelium, is subjected to phagocytosis and persists within subepithelial macrophages (Momotani et al. 1988). It is believed MAP enters the intestinal mucosa by way of specialized M cells in the epithelium lining the dome areas of Peyer’s patches (Momotani et al. 1988; Sigurdardottir et al. 2001). It is thought that macrophages may act as vehicles in the dissemination of MAP from infected sites (Valentin-Weigand and Goethe 1999). In the subclinical stage of paratuberculosis, macrophages contain a relatively low number of MAP, but as the disease progresses MAP are able to convert macrophages into favorable environments for replication and the organ-ism can exist in large numbers (Clarke 1997). Zurbrick and Czuprynski (1987) infected macrophages with 10 cells of MAP and found that after 2 h there could be as many as 500 MAP present in each macrophage. In the later, advanced stage of the disease, infected epithelial cells are sloughed into the intestine and passed with the fecal discharge. The number of bacteria in the feces of clinically diseased cattle can exceed 108 cells per g feces (Chiodini et al. 1984). It has been suggested that this large number of organisms shed in feces could account for the rapid spread of the disease from infected to non-infected animals in the same herd (Cocito et al. 1994). It is estimated that only 10–15% of infected animals develop clinical disease. Clinical symptoms usually occur 2–4 years after infection (Woo and Czuprynski 2008). This

long incubation period means that even in a herd that has a high prevalence of infected animals, clinical cases will occur only sporadically and animals may actually be culled for other reasons and never recognized as Johne’s cases. Whitlock and Buergelt (1996) suggested that for every clinically confirmed case of Johne’s disease there were another 25 infected animals.

Transmission

MAP strains have been subdivided into two broad groups named Type I (C, typical in cattle infections) and Type II (S, typical of sheep infections), that can be distinguished by detection of the IS1311 insertion sequence (Begg and Whittington 2007). The bacteria most likely enter the host via the fecal oral route and take up residence in the upper gastrointestinal tract, specifically the mucosa-related lymphoid tissue. Originally, paratuberculosis was con-sidered an enteric infection (Sweeney 1995), but there is now evidence that intrauterine transmission of MAP, as well as infection of supramammary lymph nodes occurs in cases of cows with advanced cases of Johne’s disease (Sweeney et al. 1992; Cocito et al. 1994). A meta-analysis presented in a recent review paper suggests that up to 9% of fetuses of subclinically infected cows and 39% of fetuses from clinically infected cows are infected with MAP (Whittington and Windsor 2009). Newborn calves are susceptible to MAP infection due to their immature immune systems, and can be infected through ingestion of contaminated colostrum and milk from infected cows, since they easily absorb macromolecules through the mucosa during their first 24 h after birth, seemingly in order to absorb protective immunoglobulins from colos-trum (Sweeney 1995). It is thought that repeated ingestion and/or gradual growth of the bacteria lead to the spread of MAP infection (Cocito et al. 1994). Paratuberculosis is often spread across herds by introduction of infected stock to healthy herds, but the lateral spread between adjoining pastures is possible (Whittington and Sergeant 2001). Seargent (2005) noted that although older infected cattle were originally thought to contribute the most to transmission through fecal shedding, recent studies have shed light on the significant contribution of infected calves in the spread of MAP through fecal shedding.

Immune response

There is a paradoxical host immune response to MAP infection. In the early stages there is a strong cell- mediated immune (CMI) response demonstrated by intradermal delayed type hypersensitivity (DTH), periph-eral blood lymphocyte gamma interferon response, and proliferation of lymphocytes. In the later, advanced stages there is a strong humoral immune, antibody-mediated response (Stabel and Bannantine 2005). The cellular

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immune response is sufficient in many infected animals to prevent the progression toward a clinically advanced form of the disease (Olsen et al. 2002). The initial CMI response is reminiscent of the hyperreactive, tuberculoid form of human leprosy, while the later anergic form, lack-ing immunity to the antigen, is similar to the later, lepro-matous stage of human leprosy (Cocito et al. 1994). The cellular immune response is typified by the production of cytokines by T lymphocytes. The CD4+ αβ T lymphocytes function by producing the interleukins IL-2, IL-4, IL-5, and IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α (Olsen et al. 2002). IFN-γ is known to be critical in the host resistance to mycobacterial infection, with studies demonstrating increased susceptibility of myco-bacterial infection in IFN-γ-defective hosts (Flynn 1993; Bellamy 2003).

Another T lymphocyte, CD8+ αβ T, has also been noted to be of importance. CD8+ αβ T cells (also known as cytolytic T cells) function in the lysis of infected cells and secrete cytokines IFN-γ (Stenger et al. 1997). The primary immunologically active compounds of MAP include glycopeptidolipids containing monoglycosylated and acylated peptides linked to oligosaccharide residues and LAM (Cocito et al. 1994). In early work with mecha-nisms of protective immunity, the role of host response in the formation of the clinical signs and pathophysiology of MAP infection was investigated. Adams and Czuprynski (1994) noted that LAM elicited the production of tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and IL-6 by bovine monocytes and murine macrophage cell-line RAW 264.7. They further demonstrated that the LAM component of mycobacterial cell walls elicited the production of the cytokines TNF-α and IL-6 in ex vivo blood from healthy cattle (Adams and Czuprynski 1995). These cytokines have been associated with granuloma-tous formations.

Early in the course of Johne’s the CMI can be detected by hypersensitivity skin reactions, which become weaker as the pathology progresses. As the CMI responses fade the serum antibody concentrations rise and can be detected by AGID or ELISA. Diagnosis of preclinical cases can therefore be difficult, which complicates attempts to control the disease.

Control Measures

Current control measures for the spread of MAP infec-tion in dairy herds include removal of calves prior to suckling, removal of infected animals, using separate water sources and pastures for heifers and cows, and the removal of manure from sick animals. Vaccinations against MAP include killed whole-cell-based vaccines and live-attenuated whole-cell-based vaccines avail-able commercially from several sources. It is possible to slow the rate of clinical paratuberculosis with herd

vaccination, but this measure cannot prevent infection (Manning 2001) because no protective immunity is con-ferred. In addition, complications in diagnosing infected animals may arise from the use of vaccinations as part of a control strategy, due to false-positive serological test results (Naugle 2003). For a complete discussion of available MAP vaccines and their relative effectiveness see Rossels and Huygen (2008). Due to the persistence of MAP infection in otherwise symptom-free animals and the extremely long incubation time (2–4 years), total eradication of MAP from a diseased herd would require complete destocking (Whittington and Sergeant 2001). It has been suggested that control of the continuing spread of paratuberculosis would be enhanced by developing a classification of animals and herds based on history of contact with potentially and known-infected cattle (Kennedy and Benedictus 2001). A detailed exposure history such as this would provide the information necessary to restrict the potential spread of the disease from sick to diseased animals. Herd certification pro-grams directed at detailing the risk of infection posed by a potential restocking source could help to limit the spread of the disease (Sternberg and Viske 2003). There is currently no dietary management program designed to inhibit MAP infection or slow the progression of clini-cal symptoms and economic impact of the disease. In the U.S. there are Voluntary Control Program Standards promulgated by APHIS (APHIS 2010). The program calls for testing and removal of positive animals by euthaniz-ing or being immediately sent to slaughter. There are 4 levels a herd can achieve with the probability that the herd is not infected rising with each level from 85% for level 1 to 99% for level 4 (APHIS 2010). Lu et al. (2010) found that test and cull strategies reduce the prevalence of Johne’s and lead to fade out over time, but the time for elimination at the herd level can be as long as 20 years. They suggest that a combination of test and cull and improved management practices to reduce transmis-sion to susceptible calves would result in elimination of Johne’s in herds in a shorter amount of time (Lu et al. 2010). Ridge et al. (2010) analyzed results of the Victoria, Australia Test and Control program and found that after the original program of test and cull was modified to include externally audited calf management require-ments there was a significant reduction of transmission within infected herds. Research determining the effect of various feeding regimens and silages, as well as the effect of MAP-inhibitory feed additives on MAP infection and the progression of the disease should receive attention in future work.

There is evidence of age-related resistance to MAP infection. Older animals can contract MAP infections, but usually require higher doses and longer incubation periods before noticeable symptoms occur (Whitlock and Buergelt 1996). By the age of one year cattle appear to

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have the same resistance to MAP infection as adult cattle (Larsen et al. 1975). Thus the use of older replacement stock may delay MAP infection and time to reach the advanced clinical stage (Sweeney 1995).

Crohn’s Disease

Disease characteristics and clinical symptoms

Recognition for the first description of Crohn’s disease was given to Crohn et al. (1952). Crohn’s disease is an inflammatory bowel disease that can affect any por-tion of the gastrointestinal tract from mouth to anus, although most cases occur in the terminal ileum and colon (Hermon-Taylor and Bull 2002). Patients with Crohn’s disease typically suffer from chronic diarrhea, fever, abdominal pain, vomiting, anorexia, weight loss, occasional constipation, and general malaise (Chacon et al. 2004; Chiodini 1989). These patients often live with chronic pain and must make frequent visits to hospitals throughout their lives (Chiodini 1989). Besides the physi-cal impact on lifestyle and quality of life, Crohn’s disease also carries a heavy financial burden for those who have the disease.

Crohn’s disease shares many characteristics of gross pathology and clinical symptoms with Johne’s disease (Chacon et al. 2004). In fact, MAP has been isolated from the biopsy tissue and blood samples of patients suffering from Crohn’s disease (Chiodini 1986). This association has elicited much concern over the potential role of MAP in the etiology of Crohn’s disease. Further, the transmis-sion of MAP through supramammary lymph nodes to the milk of lactating cattle and the ability of MAP to resist thermal treatments raises additional concern over the risk of MAP food borne transmission particularly from the consumption of raw milk by persons with immature or compromised immune systems.

Costs and prevalence

A 2001 study estimated the prevalence of Crohn’s dis-ease cases in North America to be between 400,000 and 600,000 (Loftus et al. 2002). More recently the incidence rate and the prevalence of Crohn’s disease were esti-mated at 3.6 to 15.6 cases per 100,000 persons and 26 to 201 cases per 100,000 persons in North America (Loftus 2004; Kappelman et al. 2007). Silverstein et al. (1999) used a Markov model analysis of a population of Crohn’s patients in Minnesota to estimate the morbidity and healthcare expenses over a 24-year period. They found the median lifetime cost per patient to be $39,906 and the mean lifetime cost per patient to be $125,404. Another study (Feagan et al. 2000) examined the health care and morbidity-related costs of Crohn’s in 607 patients

separated into three groups based on the severity of their condition: group 1 consisted of 117 patients (17%) who required hospitalization, group 2 consisted of 31 patients (5%) who required glucocorticoid or immunosuppressive drug therapy for more than 6 months, and group 3 con-sisted of 459 (76%) who were the remaining patients. They reported the average annual cost to be $12,417. Groups 1, 2, and 3 mean annual costs were $37,135, $10,033, and $6,277, respectively. A study analyzing expenditures for patients with Crohn’s disease from 1999-2005 was conducted and investigators found the average direct expenditures for patients with Crohn’s disease were approximately $19,000 per year (Gibson et al. 2008). The evidence provided by these studies implicates Crohn’s disease as a significant economic burden that affects a substantial proportion of the population. In addition to the financial impact of Crohn’s disease, the effects of the disease on the quality of life of those who suffer from it make this a disease of considerable importance.

Similarities to Johne’s disease

The etiology of Crohn’s disease has not been clearly defined. It appears that several etiological factors such as genetic predisposition, infectious agents or normal flora, abnormal immune function by the host and envi-ronmental factors such as smoking and chemicals may be involved (Chacon et al. 2004). Due to similarities in gross pathology, symptoms, and epidemiology of Johne’s and Crohn’s diseases, concern over the potential role MAP may play in the development of Crohn’s has been expressed. Dalziel (1913) reported both the histopatho-logical and clinical similarities between Crohn’s disease, intestinal tuberculosis and Johne’s disease. Historically, MAP was suspected of being linked to Crohn’s disease but standard culturing and staining techniques failed to detect it. Eventually, Chiodini (1986) was able to isolate cell-wall-deficient cells (spheroplasts) from tissue sam-ples of Crohn’s patients that initially failed to stain in the characteristic acid-fast manner of mycobacteria. After subculturing, the spheroplasts developed cell walls that stained in the traditional acid-fast manner of mycobac-teria. DNA hybridization confirmed these formerly cell-wall-deficient cells to be MAP (Chiodini et al. 1986).

Presence of MAP in Crohn’s patients

Several studies have been conducted that have attempted to determine the prevalence of MAP in Crohn’s disease patients (Green et al. 1989; Sanderson et al. 1992; Lisby et al. 1994; Mishina et al. 1996). Mishina et al. (1996) found MAP in all of 8 Crohn’s patients tested using a RT-PCR method to detect IS900 RNA, but they also found MAP in 2 of 4 controls that were afflicted with ulcerative colitis. MAP has been detected in 7 of 8 patients with

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Crohn’s disease and none in 3 controls by culturing in Mycobacterial Growth Indicator Tubes (MGIT) (Naser et al. 2004). Cell-wall-deficient MAP was isolated from the breast milk of two mothers who had Crohn’s disease, but not from 5 lactating mothers who did not have the disease. These cell-wall-deficient MAP later reverted to the typical bacillary form (Naser et al. 2000). A study by Bull et al. (2003a) detected MAP in 26% of patients with noninflammatory bowel disease (nIBD), and 92% of those with Crohn’s disease using nested PCR and MGIT. They held the opinion that difficulties in lysing the MAP cell and subsequent DNA extraction might have nega-tively impacted the number of MAP-positive diagnoses in Crohn’s disease patients in previous studies. Naser et al. (2004) used both culturing with MGIT and PCR to detect the IS900 insertion sequence of MAP in the blood of Crohn’s patients, those with ulcerative colitis, and those without inflammatory bowel disease. Their results found MAP in 46% of patients with Crohn’s, 44% with ulcera-tive colitis, and 20% of those without inflammatory bowel disease (IBD) using a MAP-specific IS900 detection pro-tocol. Using the MGIT culturing method, they detected MAP in 50% of patients with Crohn’s, 22% with ulcerative colitis, and none without IBD. More recently, Scanu et al. (2007) found MAP in 20 of 23 Crohn’s patients (87%) 2 of the 3 patients who were MAP-negative were being treated with multiple drug therapies that included rifampin and clarithromycin.

Jeyanathan et al. (2007) attempted to directly visualize MAP in tissues of Crohn’s disease patients at two differ-ent treatment centers. They used two different staining methods and found a strong association between myco-bacteria and Crohn’s disease. They determined that MAC organisms were associated with Crohn’s tissue just over half the time as opposed to being rarely found in the control samples. There were limitations to the study, including the fact that the subjects were not matched for age and tissue origin, and the site of tissue varied and was not ileum specific. Although they were able to detect that MAC organisms were present, they were not able to discriminate MAP from other MAC organisms.

Koch’s postulates and Hill’s criteria

Proof of the causative agent of an infectious disease stud-ies rely on the fundamental principle known as Koch’s Postulates. Briefly, Koch set forth 4 criteria for positively associating a pathogen with an illness, 1) the organ-ism should be present in diseased individuals but not in healthy ones, 2) ability to grow the organism in pure culture, 3) the ability to recreate the disease in a healthy individual using the purified organism, and 4) the organ-ism should be reisolated from the inoculated individual and shown to be identical to the original organism (Koch 1890). This approach is problematic in a chronic disease

such as Crohn’s, since demonstrating association and causality become difficult. Koch himself was forced to abandon the second part of the first postulate when he discovered asymptomatic carriers of cholera (Koch 1893). Indeed, Koch’s postulates have been recognized as being too rigid since not all organisms can be cultured in media, for instance M. leprae (Gupta and Katoch 1995). While some researchers claim that MAP has met Koch’s Postulates for Crohn’s disease (Greenstein 2003), many others do not (Podolsky 2002; El-Zaatari et al. 2001).

Epidemiologists, on the other hand, adhere to Hill’s criteria (Hill 1965). These state that 1) the hypothesized association should make sense biologically, 2) increased exposure should increase risk, 3) the condition can be altered by an experimental regimen 4) the association should be strong, 5) the association should be specific, 6) findings of different groups should be consistent, 7) infec-tion should precede disease. 1) In the context of Crohn’s and MAP the association does make sense biologically as MAP is associated with a chronic bowel inflammatory dis-ease in other hosts such as cattle. 2) Increased exposure should increase risk, but there is not an increased rate of Crohn’s disease in rural populations who should be disproportionately exposed to actively shedding of MAP infected herds (Jones et al. 2006). 3) Members of the MAC, including MAP, are generally resistant to antituberculosis drugs. However, Feller et al. (2010) conclude that a long term treatment with nitroimidazoles or clofazimine often results in remission in Crohn’s patients. 4) The strength of the association between Crohn’s and MAP has been variable in two meta-analyses. A systematic review and meta-analysis by Feller et al. (2007) of 28 case-control studies showed that MAP infection in Crohn’s patients is considerably higher than in the control group, regard-less of the method used to detect the organism or the sample type (i.e., tissue, serum, and other biological preparations). One study found MAP in 20% of granulo-matous tissue samples from Crohn’s patients using PCR amplification and detection of IS900 in a double blinded study (Fidler et al. 1994), while another study concluded 65% infection of Crohn’s patients with MAP by the same PCR method (Sanderson et al. 1992). 5) There have been reports of MAP being associated with ulcerative colitis and sarcoidosis (Bernstein and Shanahan 2008; El-Zaatari et al, 1996). In addition, there is speculation for a role for MAP in the development of type 1 diabetes mellitus (Dow 2006, Rosu et al. 2009), so MAP is known not to be specific for Crohn’s in humans, and 6) previously cited research results indicate variable results with detection of MAP in Crohn’s patients. 7) It is also unknown in humans that exposure to MAP occurs before the development of Crohn’s disease. As of now, Crohn’s disease cannot be definitely linked to MAP organisms, but the association between Crohn’s disease tissue and MAP organisms should not be overlooked. Hill’s criteria are more flexible

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than Koch’s Postulates in that there is not a set number of criteria needed to pass or fail the test for causality. Hill’s criteria are used mostly as guidelines and not all criteria may be of the same importance when studying a particu-lar epidemiological association. Opportunities exist for research into understanding the molecular basis for MAP producing inflammatory bowel disease as compared to other organisms of the MAC that do not.

Treatment strategies

Treatments for Crohn’s disease depend on the specific symptoms being exhibited in the individual and their severity. Often for mild symptoms antidiarrheal medi-cines are given to help alleviate stomach cramps. For mild to moderate severity symptoms treatments include: ami-nosalicylates (sulfasalzine and mesalamine), antibiotics (rifampin, ciprofloxacin, metronidazole), corticosteroids (budesonide and prednisone), and immunomodulators (azathioprine, 6-mercaptopurine, and methotrexate). Severe symptoms are often treated with intravenous corticosteroids or TNF, and in some cases intestinal resection surgery is required (WebMD 2006). Numerous antimycobacterial drugs have been used in the treatment of paratuberculosis in animals and Crohn’s disease in humans. In some cases symptoms were lessened or even abated completely, although these cessations were never maintained and neither disease was ever completely eradicated on the individual or group level (Hermon-Taylor et al. 2000).

Conclusions

MAP is an important animal pathogen that has a major economic impact on the dairy cattle, sheep, and goat industries in the United States and worldwide. Prevention and control of the proliferation of Johne’s disease is made more difficult due to interspecies transmission both from domestic to non-domestic animals, as well as the reverse. Current control and prevention strategies for Johne’s dis-ease focus on removal of infected cattle and separation of calves from infected lactating mothers prior to first suckling. Prevention of the spread of Johne’s disease is exacerbated by the slow development of the disease (2–4 years), the ability of MAP to persist in the soil, and the potential for re-infection from wild animal vectors. The potential control of MAP infection through dietary management needs additional investigation.

MAP has been recovered from tissue and blood samples of patients suffering from Crohn’s disease, a gastrointestinal inflammatory bowel disease, leading to speculation of an etiological role of MAP in Crohn’s disease. There has been no definitive evidence for or against this theory thus far. MAP has been found to be

transmitted through the raw milk of infected cattle and to be resistant to thermal pasteurization treatments; this raises concern over the potential transmission to humans through MAP infected milk.

Future directions of MAP research should focus on the improvement of control and prevention measures of Johne’s disease in domestic animal species. The impact of wild animal vectors on transmission to domestic livestock needs to be better understood, as well as potential control methods for this transmission source. More rapid, eco-nomical, easier, and more accurate detection methods for the identification of subclinically infected animals, as well as a systemized universal means of determining the risk associated with addition of new cattle to existing herds are needed. This would improve the success of within-farm control strategies and provide a means of ensuring the health of newly purchased replacement stock. Research addressing the positive effects of prescribed feeding regi-mens and potential inhibitory feed additives is required in an effort to exhaust the possible means of control of MAP infection and the progression of the disease.

Further investigations into the etiological role of MAP in Crohn’s disease are also needed. Crohn’s disease, in addition to being a potentially debilitating disease that severely affects the quality of life of those who suffer from it, represents a significant financial cost to the United States in the form of treatment and morbidity costs. More research is needed to definitively answer the questions regarding the etiological nature of the disease.

Acknowledgements

Preparation of this review was supported by a grant from the Arkansas Biosciences Institute to authors Crandall and Ricke.

Declaration of interest

The authors report no declarations of interest.

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