effect of genetic variation in the mhc class ii drb region on resistance and susceptibility to...

doi: 10.1111/j.1744-313X.2012.01092.x

Effect of genetic variation in the MHC Class II DRB region on resistance

and susceptibility to Johne’s disease in endangered Indian Jamunapari

goats

P. K. Singh*, S. V. Singh*, M. K. Singh*, V. K. Saxena†, P. Horin‡,§, A. V. Singh* andJ. S. Sohal–

Summary

The pathogenesis of Johne’s disease (JD), caused byMycobacterium avium subsp. paratuberculosis (MAP), iscomplex and has not been completely understood yet. Inthe present study, we analysed the polymorphism in theexon-2 of the caprine major histocompatibility complex(MHC) Class II DRB region and its association withresistance or susceptibility to JD. A total of 203 Jamuna-pari goats, which is an Indian endangered breed highlysusceptible to JD, kept at a single farm were studied. Onthe basis of clinical signs, microscopic examination,faecal culture, ELISA and diagnostic PCR, 60 and 143goats were classified as resistant and susceptible to JD,respectively. PCR-based restriction fragment length poly-morphism (PCR-RFLP) with two enzymes, PstI and TaqI,was used to assess variation in the DRB gene(s) in all 203goats studied. Two di-allelic single nucleotide polymor-phisms (SNPs), here referred as ‘P’ and ‘T’, were tested. Ineach of them, three genotypes were found in the groupanalysed. The minimum allele frequencies (MAFs) were0.233 and 0.486 for the P and T SNPs, respectively.Statistically significant associations between alleles, indi-vidual genotypes and composed genotypes of both SNPswere found. The frequency of p and t alleles, of individualpp and tt and of composed pptt genotypes were signif-icantly higher (Pcorr < 0.001) in the ‘resistant’ group ascompared to the ‘susceptible’ group, while the P and T

* Central Institute for Research on Goats, Makhdoom, Mathura, UP,

India, † Central Avian Research Institute, Izatnagar, UP, India,

‡ Institute of Animal Genetics, University of Veterinary and

Pharmaceutical Sciences, Brno, Czech Republic, § CEITEC-VFU,

University of Veterinary and Pharmaceutical Sciences, Brno, Czech

Republic, – Canadian Food Inspection Agency, St. Hyacinthe, Que-

bec, Canada

Received 4 August 2011; revised 28 November 2011; accepted 4

January 2012

Correspondence: Pravin Singh, CIRG, Makhdoom, PO-Farah,

Mathura, Uttar Pradesh 281122, India. Tel: +91 565 2763260,

Extn 205; Fax: +91 565 2763246;

E-mails: [email protected]; [email protected]

ª 2012 Blackwell Publishing Ltd

International Journal of Immunogenetics, 2012, 00, 1–7

alleles were associated with susceptibility (Pcorr < 0.001).In heterozygous genotypes, susceptibility was dominantover resistance. The effects of both SNP on resistance andsusceptibility were comparable and composed heterozy-gous genotypes showed intermediate levels of suscepti-bility in terms of the odds ratio and P-values calculated.

Introduction

Johne’s disease (JD) is a chronic infectious disease pri-marily of ruminants caused by Mycobacterium aviumsubspecies paratuberculosis (MAP). MAP infectionleads to granulomatous enteritis, persistent diarrhoea,progressive wasting and death eventually. Associatedlosses because of reduced milk production and prema-ture culling combine to make JD one of the most eco-nomically important infectious diseases. JD causeshuge economic losses by reducing productivity indomestic ruminants worldwide. It has been estimatedthat 68.0% of US dairy herds are infected with JD,costing $200 million to $1.5 billion per year to dairyindustry (National Animal Health Monitoring System,1997). In India, JD is highly prevalent and endemic(Singh et al., 2007a). A controversial but developinglink between MAP and human Crohn’s disease sug-gests that this pathogen may also be an importantfood safety concern (Chiodini & Rossiter, 1996; Her-mon-Taylor et al., 2000). Johne’s disease control pro-gramme is severely hampered because of the lack of100% sensitive and specific diagnostics, ability todiagnose early infection and effective vaccine for JD.There is surprisingly little information on the effect ofhost genetics on paratuberculosis. Recently, some stud-ies have provided indication of genetic factors involvedin resistance to paratuberculosis (Koets et al., 2000;Mortensen et al., 2004; Estonba et al., 2005). Associ-ations of alleles of the MHC class II, NRAMP 1, TLR 2and NOD 2 ⁄ CARD 15 genes with susceptibility to JDwere reported (Horin et al., 1998; Reddacliff et al.,2005; Pinedo et al., 2009; Koets et al., 2010).

In recent years, research on the major histocompati-bility complex (MHC) as candidate genes of disease

1

2 P. K. Singh et al.

resistance and susceptibility has become a major focusin animal breeding. MHC is a cluster of closely linkedgenes (usually inherited as a haplotype) havingimmunological and non-immunological functions, andis present in all vertebrates ranging from cartilaginousfish to mammals. The prime function of the MHC is tocode for specialized antigen-presenting glycoproteins,known as histocompatibility molecules, or MHC mole-cules. These molecules bind processed peptide antigensand present them to T-lymphocytes, thereby triggeringantigen-specific immune responses. RFLPs studies dem-onstrated that caprine MHC I and II genes are highlypolymorphic (Cameron et al., 1990). The DRB genesare the most polymorphic among the MHC genes(Andersson & Rask, 1988). Besides the major role indisease resistance (Longenecker & Gallatin, 1978;Schierman & Collins, 1987; Kaufman & Venugopal,1998; Reddacliff et al., 2005; Sayers et al., 2005; Liet al., 2010), MHC polymorphisms have also beenreported to be associated with certain production(Gautschi et al., 1986; Jung et al., 1989; Sheikh et al.,2006) and reproduction traits (Philipsen & Kristensen,1985; Renord & Vaiman, 1989) in various speciessuggesting potential utility as candidate gene marker.

The Jamunapari breed of goats is an importantdual-purpose (milk and meat) breed. However, itspopulation declined very fast over the last two dec-ades, and now, it is considered as an endangeredbreed. An in situ conservation and selective breedingprogramme has been launched in 1984 at CentralInstitute for Research on Goats (CIRG), Makhdoomfarm of Jamunapari goats. This breed is very suscepti-ble to JD (Singh et al., 2009a). Goat herds maintainedin the CIRG, Makhdoom, were shown to be endemicfor JD (Kumar et al., 2007, 2008; Singh et al.,2007a). Despite extensive culling of positive goats andother management practices over the last 25 years, theMAP infection could not be eliminated from this herd.

Two single nucleotide polymorphisms (SNPs) in theDRB region (detected by PCR-RFLP method usingPstI and TaqI restriction endonuclease) were describedby Amills et al. (1995), leading to amino acid substi-tutions in the antigen binding site of the caprine MHCmolecules. Therefore, investigations of possible rela-tionships between these critical SNPs with resistanceor susceptibility to JD are of potential importance.

Therefore, a study on genetic variation in the MHCclass II DRB region and its possible associations withresistance or susceptibility to JD in endangeredJamunapari breed of goats was undertaken.

Materials and methods

Experimental animals and sample collection

A farm of Jamunapari goats located in the CIRG,Makhdoom, India, with a history of endemic MAPinfection resulting in significant losses caused by JDwas selected for the study. As pedigree information was

not available, goats were randomly selected for the pur-pose of this study. As far as it was possible to assess,experimental goats were unrelated. Initially, a total of354 goats were screened for the detection of MAPinfection by ELISA, faecal microscopy, faecal cultureand diagnostic PCR on peripheral blood. Peripheralblood samples from all 354 goats were collected in twoaliquots: 4 mL blood with anticoagulant (for the DNAisolation and PCR) and another 1 mL blood withoutanticoagulant (for harvesting the serum for ELISA test).About 2 g of faecal samples was also collected fromeach goat for the detection of MAP by microscopicexamination of faecal smears and by culture method.

Classification of resistant and susceptible goats

Goats positive for diagnostic PCR, ELISA, microscopicexamination of faecal smears and culture of faecalsamples on Herrold’s egg yolk (HEY) medium wereconsidered as ‘susceptible’ (the ‘S’ group), whereasgoats negative in all diagnostic tests performed wereconsidered as ‘resistant’ (the ‘R’ group). Only thosegoats complied with this resistant ⁄ susceptible criteriawere studied further, whereas others having differencesin results of all four tests were excluded from the fur-ther study. Sex and age ratio of goats (included instudy) in both ‘S’ and ‘R’ groups were also analysed.The diagnostic tests were performed as follows.

Diagnostic PCR

Detection of MAP from peripheral blood has been car-ried out as described previously (Singh et al., 2010)using MAP-specific primers (Millar et al., 1995).

ELISA

Anti-MAP antibodies were detected by indigenousELISA kit as per method of Singh et al. (2007b).

Microscopic examination (Ziehl Neelsen’s staining) and culture

on Herrold’s egg yolk (HEY) medium

Both microscopic examination of faecal smear byZiehl Neelsen staining and culture of processed faecalsamples on HEY slants were performed as describedearlier (Kumar et al., 2010).

Study of the MHC class II DRB gene polymorphism

DNA extraction

DNA was extracted from the blood as describedelsewhere (Singh et al., 2010).

Amplification of the second exon 2 caprine DRB sequences

Amplification of the second exon of the MHC class IIDRB gene was carried out as originally described by



DRB polymorphism and Johne’s disease 3

Amills et al. (1995). Briefly, to increase the PCR yieldand specificity, the second exon was amplified innested PCR under high and low stringent conditions.The primers used for the study were as follows:

1 P-I; 5¢-TAT CCC GTC TCT GCA GCA CAT TTC-3¢;2 P-II; 5¢- TCG CCG CTG CAC ACT GAA ACT

CTC -3¢;3 P-III; 5¢- CGT ACC CAG AGT GAG TGA AGT

ATC –T.

The primers used in the first round were primer P-Iand P-III, while a combination of primers P-I and P-IIwas used for the second-round PCR. A total of 25-lLreaction mixture containing 100–200 ng of DNA tem-plate, 10 pM of each primer, 100 mM of each dNTPs,1.5 mM MgCl2, 1 U Taq DNA polymerase, 10X PCRassay buffer was set for amplification both at first- andat second-round PCR. Thermal cycling conditions forthe first round of PCR were as follows: hot start for5 min at 94�C followed by ten cycles of 60 s at 94�C,120 s at 60�C and 60 s at 72�C. Subsequently, 10 lLof the first-round PCR was used as a template in thesecond-round PCR. The reaction mixture for the sec-ond-round PCR was identical to the first round, wherethe thermal cycling profile was 60 s at 94�C, 30 s at65�C and 30 s at 72�C with 25 cycles.

DNA sequencing

Commercial sequencing of the DRB PCR productsobtained was performed by the Centre for GenomicApplication (TCGA), Okhla, New Delhi. The nucleo-tide sequence reads were subjected to global BLAST(http://www.ncbi.nilm.nih.gov/BLAST) for confirmingthe identity of the PCR product.

Detection of restriction fragment length polymorphisms

Similar to Amills et al. (1995), PCR products weredigested with two restriction enzymes, TaqI and PstI(Fermentas, Hanover, MD, USA), based on the manu-facturer’s instructions. Ten microlitres of the PCRproduct was digested for half an hour at 37�C and at65�C in 5 U PstI and 5 U TaqI, respectively. TaqI andPstI digestion products were electrophoresed in 3%Nusieve agarose gels containing ethidium bromide.

Table 1. Comparative evaluation of ELISA, faecal culture, faecal microsco

Jamunapari flock

Tests

Combinations

1 2 3 4 5 6 7

ELISA + ) + ) ) ) +

Culture + ) ) + ) ) +

Microscopy + ) ) ) + ) )Diagnostic PCR + ) ) ) ) + )Total (354) 143 60 36 3 26 30 3



Statistical analysis

Allelic as well as genotypic frequencies of MHC ClassII DRB region were determined by direct counting.Associations between SNP alleles, genotypes and com-posed and resistance ⁄ susceptibility to infection weretested by the chi-square (v2) test. Bonferroni correc-tions for multiple comparisons were used when appro-priate (for three individual genotypes and for ninecomposed genotypes). Besides the P-values obtained,odds ratios (OR) and their 95% confidence intervalswere computed as parameters of associations.

Results

Susceptible and resistant goats

Of 354 Jamunapari goats maintained in the same herdand at the same place, 59.6, 44.1, 59.9 and 56.8%goats were detected as positive for MAP infectionusing ELISA, faecal culture, faecal microscopy anddiagnostic PCR, respectively. Comparative evaluationof four tests result showed that per cent agreementamong four tests was 57.34%. Of the 354 Jamunparigoats, 29.6% (60) and 70.4% (143) were positive andnegative in all applied tests, respectively (Table 1).Therefore, only 203 Jamunapari goats (77 male and126 female from 6 months to 4 years old) compliedwith the classification criteria defined and were consid-ered as resistant (60) and susceptible (143) for furtherstudy. The percentage of male and female goats in thesusceptible group was 39.86 and 60.14%, and 33.33and 66.67% in the resistant group. A total of 22.37,33.56 and 44.05% goats of susceptible group and28.33, 31.67 and 40% goats of resistant group werein the age group of 6–12 months, 1–2 year and>2 years, respectively.

Amplification of the DRB gene and sequencing

A 285-bp PCR product was always obtained from thegoat genomic DNA samples analysed as also reportedby Amills et al. (1995). Sequencing of two PCR prod-ucts resulted in 229-nucleotide-long, clear and read-able nucleotides. Nucleotide BLAST and multiplealignment of the sequences obtained from Jamunaparigoats with other goats breeds and species confirmed

py and diagnostic PCR for the detection of MAP infection in targeted

8 9 10 11 12 13 14 15 16

) ) + ) + + ) + +

+ ) ) + ) + + + )+ + + ) ) + + ) +

) + ) + + ) + + +

7 17 18 0 10 0 0 0 1


that they are exon 2 DRB sequences. Sequence wassubmitted to GenBank database (Accession numberJF416295). There was a significant sequence similaritywithin the DRB region of caprine, ovine and bovine(93–95% homology) species.

Polymorphisms

The RFLP analysis revealed polymorphic patterns indi-cating the existence of two polymorphic positions(SNPs) within the PCR product obtained. The PstIRFLP revealed the presence of a polymorphic and anon-polymorphic PstI site at 241st and 15th nucleo-tide position of the amplified fragment, respectively.Therefore, three restriction patterns were detected bythe PstI PCR-RFLP. The fragments of the three restric-tion patterns were 15, 226, 44 (the ‘P’ allele) or15 bp, and 270 bp (the ‘p’ alleles). Four fragments,270, 226, 44 and 15 bp, indicated the heterozygousgenotype. Hence, two alleles (P, p) and three geno-types for the P SNP were observed in the Jamunaparigoats (Fig. 1a). The TaqI RFLP revealed the presenceof a polymorphic TaqI site at the nucleotide position122. For the TaqI RFLP, two restriction patterns, onewith 163 and 122 bp (T) and another with 285 bp (t),were found. Thus again, two alleles (T, t) and threegenotypes were identified (Fig. 1b).

DRB polymorphisms and its associations with Johne’s

disease

Genotype frequencies in the two groups are compared,and their associations with infection are summarized inTable 2. The results showed a non-random distributionof P and T genotypes in the resistant and susceptiblegroups (P < 0.001 and 0.007, respectively). Significantdifferences between homozygotes and heterozygotes inSNPs were observed. For the P SNP, heterozygositywas associated with susceptibility, while a heterozygoteadvantage was observed for the T site (Table 3).

Associations with individual nucleotide RFLP sitesare summarized in Table 4. The presence of allele Pwas strongly associated with susceptibility(P < 0.0003); no PP individual was found among resis-tant goats. The allele p was associated with resistanceto infection (P < 0.03). The allele t was associated

270 bp 226 bp

(a)

Figure 1. Restriction profile of amplicons of different samples. (a) By Pst

PP genotype, Lane 3: pp genotype and Lane 4 and 6: Pp genotype. (b) By

genotype, Lane 2,3,4 and 6: Tt genotype and Lane 5: tt genotype.

with resistance (P < 0.05), while only non-significantassociation (P < 0.08) was found for T.

Analysis of composed P and T genotypes showed avery strong association between the genotype ppTtand resistance to infection (Table 5).

Discussion

A major problem for investigations of genetics of dis-ease (paratuberculosis) resistance in animals is the diffi-culty in accurately identifying the susceptible andresistant animals. The phenotypic classification shouldaim to distinguish between genetically different groups.Tissue histopathology and culture are considered asmost specific and sensitive tests for the identification ofactive MAP infection (Reddacliff et al., 2005), but it isa single test not suitable for in vivo diagnostics. Accu-rate diagnosis of active infection of paratuberculosismay be achieved using multiple diagnostic tests (Kumaret al., 2007; Singh et al., 2008a,b). In a previous studyon associations between MHC Class II and susceptibil-ity ⁄ resistance to JD, Reddacliff et al. (2005) used mul-tiple diagnostic tests for this purpose. Here, we usedresults of four laboratory tests and clinical disease asclassification criteria to avoid individual weaknessesreported for each of the diagnostic approaches (Sohalet al., 2007). All goats were kept at the same placeand at the same time, the level of pathogen exposurebeing identical. Age and sex factors may also interferewith susceptibility ⁄ resistant to MAP infection (Hineset al., 2007). Here, the two groups were similar inboth criteria. We thus may conclude that possiblebiases because of variation in breed, level of MAPexposure, sex and age were minimized.

The caprine DRB region molecular variation wasfirst characterized by Amills et al. (1995) and used forstudying another Indian goat breed – Changthangi(Sahar & Othaman, 2006; Sheikh et al., 2006) – usingPstI and TaqI PCR-RFLP. Our results showed the con-served nature of both polymorphic sites among differ-ent goat breeds. They are located in the b1 domain ofthe DR molecule, which is in close contact with anti-gen. Because of its functional importance, the poly-morphism of this MHC region was studied in othermammalian species with similar results (Muggli-Cock-ett & Stone, 1991; Van Eijk et al.,1992; Amills et al.,

122 bp 163 bp 285 bp

(b)

I restriction enzyme-Lane M: 100 bp DNA ladder, Lane 1,2,5 and 7:

Taq I restriction enzyme – Lane M : 100 bp DNA ladder, Lane 1: TT



Table 4. Significant associations of ‘P’ and ‘T’ allele carrier status

with MAP infection in Jamunapari goats

P-value

Associated

with

Odds

ratio

95% confidence

interval

Presence of P 0.0003 Susceptibility 4.839 1.946–12.030

Presence of p 0.03 Resistance Nc Nc

Presence of t 0.05 Resistance 0.454 0.205–1.008

Nc, not calculable because of null frequencies.

Table 2. Distribution of ‘P’ and ‘T’ genotypes in the resistant and

susceptible Jamunapari goats (n = 203)

MHC Class II

DRB genotype

Genotypic frequency

P-value

Susceptible

group (%)*

Resistant

group (%)**

‘P’ genotypes

PP 11 (07.70) 0 (0.0) 0.001

Pp 39 (27.27) 6 (10.0)

pp 93 (65.04) 54 (90.0)

‘T’ genotypes

TT 40 (27.97) 9 (15.0) 0.007

Tt 65 (45.46) 42 (70.0)

tt 38 (26.57) 9 (15.0)

*(n = 143 goats); **(n = 60 goats).

Table 3. Associations between homozygosity in two Caprine MHC

DRB SNPs and MAP infection in Jamunapari goats

Homozygotes ⁄Heterozygotes P-value

Associated

with

Odds

ratio

95%

confidence

interval

‘P’ SNP

PP or pp 0.007 Resistance 0.296 0.118–0.744

Pp Susceptibility

‘T’ SNP

TT or tt 0.002 Susceptibility 2.800 1.472–5.325

Tt Resistance


1995; Aldridge et al.,1998; Sahar & Othaman, 2006;Sheikh et al.,2006).

Amills et al. (1995) and Sheikh et al. (2006) showedthat the nucleotide variation results in changes in aminoacid sequences. The presence of the PstI site is associatedwith the TAC codon (tyrosine), whereas its absence isassociated with the TGT codon (cysteine) at the samenucleotide position. Likewise, TaqI site was associatedwith a TTC codon (phenylalanine), whereas its absence

Table 5. Association between a two-SNP composed genotype and resista

Genotypes

Frequency in

‘Resistant groups’

Frequency in

‘Susceptible group’

ppTt 0.65 (39 ⁄ 60) 0.27 (39 ⁄ 143)

Other

genotypes

0.35 (21 ⁄ 60) 0.73 (104 ⁄ 143)



leads to the TAC codon (tyrosine) in the t allele. As thesechanges are thought to be functionally importance, thestatistical associations with resistance ⁄ susceptibility toJD observed in this study seem to be biologically plausi-ble. A bias related to possible stratification of this specialpopulation and false-positive results cannot be excluded.However, it does not seem to be the case of this study.Besides the functional importance of the variation stud-ied, breeding policy used in this Jamunapari flock avoidsusing limited number of sires whose haplotypes woulddominate the gene pool in subsequent generations.A minimum of 10 sires are used for every breeding sea-son, each of them mating 25 females approximately.The breeders are selected based on breeding value esti-mates for production and fertility traits.

The sequence obtained was similar with a caprineDRB sequence (accession number AY496935.1). Ascomplete exon 2 sequences and family data were notavailable, full alleles and ⁄ or haplotypes could not bedetermined and analysed. Further cloning andsequence analysis would better characterize allelesidentified in resistant and susceptible animals. Here,only effects of single SNPs and of composed genotypescould be determined, comparable to the previousreports (Amills et al., 1995; Sheikh et al., 2006). Theeffect of allele ‘P’ on susceptibility was so strong thatit even influenced susceptibility of heterozygotes as agroup. On the other hand, the effects of ‘T’ and ‘t’alleles were relatively weak as compared to the strongassociation with resistance observed for ‘Tt’. Theseresults suggest a heterozygote advantage for this nucle-otide position within the antigen binding site (ABS).Synergic effects of the two SNPs on resistance to infec-tion were also observed. The genotype ‘ppTt’ wasstrongly associated with resistance, where the P-valuewas increased by two orders of magnitude as com-pared to associations determined for individualalleles ⁄ genotypes. These data suggest that like in otherspecies, the outcome of a natural mycobacterial infec-tion can be influenced by the major histocompatibilityregion. Associations of the MHC region with incidenceof ovine Johne’s disease (JD) were reported previouslyby Reddacliff et al. (2005) using microsatellite mark-ers closely associated with MHC region. Recently,Rastislav & Mangesh (2011) reported the associationof an SNP within the same DRB gene with susceptibil-ity to paratuberculosis in cattle.

At the cellular level, down-regulation of MHC ClassI and Class II molecules in bovine macrophages

nce to MAP infection in Jamunapari goats

For eight genotypes

P-value ⁄P corrected

Odds

ratio

95% confidence

interval

5.10)7 ⁄ 4.10)6 0.202 0.106–0.385


infected with live and killed MAP was observed (Weisset al., 2001). MHC class II polymorphisms thus seemto be good candidate markers of resistance ⁄ susceptibil-ity to JD in goats. However, effects of MHC on pro-duction and reproduction traits must be taken intoconsideration. Sheikh et al. (2006) found a significanteffect of OLA-DRB PstI polymorphism on the firstand second year of pashmina production in the IndianChangthangi goat breed.

Although the associations observed seem to be biologi-cally plausible, further population and functional studiesare needed to confirm these relationships and to clarifythe underlying mechanisms before these candidate mark-ers can be considered for marker-assisted selection.

Another important aspect of genetic variation inresistance ⁄ susceptibility of Jamunapari goats is theconservation issue. On the basis of herd prevalence ofMAP (Singh et al., 1990) as well as pathogenicity trialof MAP native strain in goats of different breeds(Singh et al., 2009a), Jamunapari goats were reportedas highly susceptible to MAP infection. Moreover, inour previous study, kids and adult goats from samegoat herd of CIRG, Makhdoom, were screened forMAP septicaemia by diagnostic PCR and a very high(77.5%) level of MAP septicaemia was recorded(Singh et al., 2010). Therefore, high prevalence ofMAP infection in Jamunapari goat herds at CIRG,Makhdoom, is a serious threat for this endangeredindigenous breed. Genotyping of the MAP isolatesrevealed exclusively the ‘Indian Bison Type’ MAPfound in this herd. When studying the host responseto experimental MAP infection, we hypothesized thatthe reason for the high incidence of MAP infection inJamunapari goats is high pathogenicity of the ‘IndianBison Type’ MAP after natural infection (Singh et al.,2009b; Sohal et al., 2009, 2010). In terms of conser-vation genetics, OLA-DRB markers associated withthe risk of infection can be used for monitoring theextent of desirable genetic variation of this endangeredpopulation in a functionally important genetic region.This knowledge is useful not only for a conservationprogramme but also for a programme aiming to elimi-nate MAP infection from the herd. As direct and indi-rect transmission of infection from goats to humanswas observed (Shankar et al., 2010; Singh et al.,2011), this becomes a public health concern.

Acknowledgement

Sincere thanks to Council of Scientific and IndustrialResearch, New Delhi for awarding ‘Senior Researchfellowship’ to P.K.Singh.

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