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    A comparative study between milk- and serum-based antibody detection assays for Johne's disease in New Zealand dairy cattle
    (Elsevier B.V., 2025-08-27) Venkatesh KM; Lopez-Villalobos N; Gupta SK; Udy GB; Laven R; Chiu S-J; Bugde P; Furuya Y; Dukkipati VSR
    Dairy cattle are affected by Johne's disease. It is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Suboptimal diagnostic tests add more to the productivity loss resulting from this disease. Agreement between and within different commercial kits is crucial in the decision-making process of disease surveillance programmes. This study compared two ELISAs, that is, Johne's disease commercial antibody detection kits (A and B), using milk and serum samples from New Zealand dairy cattle. These results were also compared with a subset of faecal PCR results. Five scenarios were considered for the comparison of ELISA tests. The point estimates of kappa coefficients (k) between the serum (0.84–0.94) assays were higher than the milk assays (0.59–0.82). The point estimates of kappa coefficients between serum and milk ELISA outcomes were higher for kit B (k = 0.79–0.86) than for kit A (k = 0.55–0.79). The point estimates of kappa coefficients between the ELISA and faecal PCR outcomes varied between 0.43 and 0.74. ELISA tests had point estimates of sensitivity ranging from 0.67 to 0.88 and specificity from 0.62 to 0.93, relative to the faecal PCR test. Results suggest that serum provides a better choice of sample type when both commercial kits A and B are used for Johne's disease surveillance of dairy cattle in New Zealand. Milk assays can be cost-effective to diagnose MAP-positive animals; kit B can be best suited for New Zealand conditions, provided the repeatability of the results is validated.
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    Comparative Genomics of Mycobacterium avium Subspecies Paratuberculosis Sheep Strains
    (Frontiers Media S.A., 2021-02-15) Mizzi R; Timms VJ; Price-Carter ML; Gautam M; Whittington R; Heuer C; Biggs PJ; Plain KM; Salgado M
    Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations (n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.
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    The development of polyester bead-based particulate subunit vaccine against Johne's disease : a thesis presented in partial fulfillment of the requirements of the degree of Master of Science in Microbiology, Massey University, Palmerston North, Institute of Fundamental Science
    (Massey University, 2015) Sitthirit, Panan
    Johne’s disease is the intestinal infection in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). The disease is economically important in the dairy industry as infection of the calves or mature cattle can result in death. Current vaccination as a part of disease control is not only partially protective against MAP but also interferes with current diagnostic test for bovine tuberculosis. Therefore, more effective and defined vaccines are needed. In this study, vaccine candidates were developed by bioengineering Escherichia coli to produce polyhydroxyalkanoate (PHA) beads displaying selected vaccine candidate antigens as fusion proteins. The selected antigens were the MAP 85 antigen complex (Ag85A and Ag85B), Superoxide dismutase (SOD) and a recombinant fusion protein 74F, however, only the antigenpresenting beads with truncated Ag85A, Ag85B and SOD were successfully produced and purified. The fusion protein comprising the respective antigens was identified and confirmed to be associated with PHA beads. The PHA beads were partially purified for future characterisations such as binding of antigen specific antibodies on PHA beads in vitro and immunological properties in animal models
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    Study of an exported protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Copland, Susan Maree
    Johne's disease is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (M. ptb) from the MAIS complex (M. avium, M. ptb, M. intracellulare and M. scrofulaceum). The lack of specific and sensitive diagnostic tests often leads to M. ptb infected animals being diagnosed with bovine tuberculosis, a member of the MTB complex (M. tb, M. bovis, M. bovis BCG, M africanum, M. microti and M. canetti). Secreted proteins from pathogenic mycobacteria have been found to be important for the development of protective immunity, namely a cell mediated immune response (CMI). The development of reliable differential diagnostic tests will require the use of species-specific secreted protein antigens and the CMI response. Due to the taxonomic distance between the MAIS and MTB complexes our hypothesis was that the M. ptb genome may encode for secreted proteins that are absent from members of the MTB complex. If such proteins can stimulate an immune response they may be suitable for use as antigens in a differential diagnostic test for Johne's disease. To this end, the secreted protein library clone pJEMIl-M ptb281 was examined and its insert found to contain the 5' region of the hypothetical M.ptb281 ORF fused in frame with phoA. The entire ORF was determined using M. avium and M. ptb database sequences then cloned into E. coli and mycobacterial expression systems. These systems incorporate 6x histidine (His6) affinity tags into recombinant proteins allowing them to be semi-purified by Ni-NTA affinity chromatography. Semi-purified recombinant proteins tested positive by western blot analysis to highly specific anti-His6-tag antibodies. Amino acid sequencing to confirm the identity of these recombinant proteins and screening for their ability to stimulate an immune response were prevented by time constraints. Homologs to M. ptb281 were absent from M. tb, M. bovis and M. bovis BCG but present in the MAIS complex, making this protein unsuitable for use as an antigen to differentiate between MAIS complex species in a diagnostic test. M. ptb281 homologs found in the genomes of two members of the Acetomycetes order corresponded to hypothetical proteins predicted by computer software programs trained to identify genes, which may indicate that the hypothetical M. ptb281 ORF may encode a functional protein.
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    A study on secreted proteins of Mycobacterium avium subspecies paratuberculosis vaccine strain 316F : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Sriasih, Made
    Doctoral Student Citation: Mycobacterium avium subspecies paratuberculosis strain316F is the organism in the live attenuated vaccine Neoparasec, which has been used to control Johne’s disease in cattle and sheep. Ms Sriasih’s research focussed on the identification of secreted proteins of this organism, which may have application in the development of diagnostic tests for Johne’s disease. Her results have contributed to the general body information concerning the immunogenic proteins of the vaccine strain 316F. The genetic approach undertaken to search for novel secreted proteins has resulted in the identification and characterisation of an immunogenic glutamine binding protein. [Abstract not able to be extracted from thesis.]
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    The epidemiology of Johne's disease in New Zealand dairy herds : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2007) Norton, Solis
    Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, debilitating enteritis of cattle, other domestic livestock and some wildlife species. JD was first identified in the late 1800s and today it is a worldwide problem in dairy cattle. Heavily infected cows have reduced milk production, a higher risk of removal from the herd and low slaughter value. Several countries have implemented national level control strategies. In New Zealand, JD was first reported in 1912 and today the prevalence of infected dairy herds is thought to be high. To improve our understanding of the epidemiology of JD and to evaluate the feasibility of a national control strategy, four studies were conducted. The first study was a questionnaire based case-control study to identify associations between management practices and the occurrence of clinical JD on farms from four regions of New Zealand. The second study was on the effect of sub-clinical JD on milk production and the risk of removal from the herd in four dairy herds over four milking seasons. The effect of misclassification of disease status on productivity estimates was also studied. In the third study diagnostic test result data from the productivity study was combined with a novel Bayesian regression model to estimate performance of the ELISA and faecal culture tests as a function of covariates and utilising repeated tests on individual cows. Finally, results from these three studies were used to adapt an existing JD simulation model, 'JohneSSim', to represent the epidemiological behaviour of JD in New Zealand dairy herds. Control strategies for the disease were simulated and evaluated based on their cost effectiveness. Of the 427 farmers responding to the questionnaire, 47% had suspected clinical cases of JD in their herd in the preceding 5 years. Only 13% of suspected infected herds had an average incidence of greater than 0.5 cases per 100 cow years at risk. The disease was not considered a serious problem by 20% of herd managers who reported the presence of disease in the preceding 5 years. The presence of Jersey cows in the herd and the purchase of bulls had strong positive associations with the presence of clinical JD. Grazing calves in the hospital paddock, larger herds, the purchase of heifers, and the use of induction were also positively associated with JD. In the productivity study the herd-level prevalence of JD by ELISA and/or faecal culture ranged from 4.5% (95% CI 2.6-6.9) to 14.2% (95% CI 9.2-20.6). Daily milksolids production by JD positive cows was 0.8% (95% CI -6.1%-4.5%) less than that of JD negative cows. However in herd D, JD positive cows produced 15.5%, (95% CI 6.75%-24.2%) milksolids less than JD negative herd mates daily. This equates to a loss of 53kg of milksolids/305 day lactation, or NZD 265/lactation, given a price of NZD 5/kg of milksolids. In herd D only, the annual hazard ratio of removal for JD positive cows was significantly increased. It was 4.7 times and 1.4 times higher in cows older than 5 years and younger than 5 years. The results were insensitive to misclassification. Analysis of the diagnostic test data demonstrated the strengths of our Bayesian regression model. While overall estimates of sensitivity and specificity by this method were comparable to estimates by existing methods, it showed a broad trend of increasing sensitivity in higher parity groups and higher sensitivity in early, relative to late, lactation. It also showed that estimates of prevalence may in fact decline with repeated, relative to single, testing. Our novel approach demonstrated trends that could not be shown by existing methods, but could be improved by application to a larger data set. Simulation showed that control strategies for JD based on either test-and-cull, vaccination, breeding for genetic resistance, or removal of offspring from clinically affected cows, were not cost effective for the average infected herd. Improvement of the hygiene associated with calf management provided the greatest reduction in the within-herd prevalence of JD. While JD is present in a high proportion of New Zealand dairy herds, the incidence of clinical cases is usually low, and most farmers consider it to be of little importance. However, JD causes significant losses in productivity in some herds. The disease would probably be best controlled on a herd-by-herd basis, given the limited success of national-scale control programs for JD in other countries. The education of dairy farmers regarding risky management practices, and the offer of a risk assessment to farmers wishing to control the disease, would provide a combination of wide reaching and targeted approaches, of low cost, for JD control. It seems likely that JD will persist in some capacity in the years ahead, but will remain of minor concern next to major animal health issues, such as infertility and mastitis. Clarification of the effect of genetic strain on the virulence of MAP may help explain differences in the effect of the disease between herds. This knowledge could then be used to further improve the efficiency of JD control.
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    A search for genetic factors influencing immune responses to a killed Mycobacterium avium subspecies paratuberculosis vaccine in Australian fine-wool merino sheep : thesis in fulfilment of the degree of Doctor of Philosophy in Animal Science, Institute of Veterinary, Animal and Biomedical Sciences, College of Sciences, Massey University
    (Massey University, 2007) Dukkipati, Venkata Sayoji Rao
    VSR Dukkipati (2007). A search for genetic factors influencing immune responses to Mycobacterium avium subspecies paratuberculosis. Doctoral thesis, Massey University, Palmerston North, New Zealand. A study was conducted to identify associations between genetic markers and immune responses in Australian fine-wool Merino sheep to a killed Mycobacterium avium subspecies paratuberculosis (Map) vaccine (GudairTM). Blood samples and immune response data (antibody and interferon gamma, IFN-gamma results) were obtained from 934 sheep from a longterm Map vaccination trial undertaken on three independent properties in New South Wales, Australia. Blood samples were genotyped for eight microsatellite markers that included four (DYMS1, OLADRW, OLADRB and SMHCC1) from the Ovar-Mhc region, two each from the SLC11A1 (OVINRA1 and OVINRA2) and IFN-gamma (o(IFN)gamma and OarKP6) gene regions. Vaccination with GudairTM induced strong antibody and IFN-gamma responses as early as two weeks post-vaccination. Between-property differences in magnitude and trend of immune responses, concomitant with season of vaccination and magnitude of natural infection prevalent in individual flocks, were evident. Immune responses in controls on all the three properties remained consistently low, except for slightly elevated IFN-gamma levels at a few time points in controls of properties 2 and 3, concomitant with exposure to natural infection. There were only 2 alleles and 3 genotypes for marker o(IFN)gamma but other loci exhibited extensive polymorphisms, the most occurring at OLADRW which had 42 alleles and 137 genotypes. Heterozygosities varied between 33% (OVINRA2) and 87% (SMHCC1), while polymorphic information contents ranged from 0.31 (o(IFN)gamma) to 0.88 (OLADRW). Genotypes at loci DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)gamma were in Hardy- Weinberg equilibrium (HWE), while those at OarKP6 were in HWE only when rare alleles (<1.0% frequency) were pooled with the closest size class. Departure from HWE, resulting from possible preferential amplification of alleles in heterozygotes, was evident at OLADRW and OVINRA2. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both genotypic and allelic affects. The study revealed several genotypes/alleles at different marker loci to be significantly associated with antibody and IFN-gamma responses to vaccination with GudairTM. However, the majority of those effects were inconsistent across the three properties. Based on significance and consistency in effects across the three properties, five genotypes (two at DYMS1 and one each at OLADRB, SMHCC1 and OVINRA1) and three alleles (one each at DYMS1, OLADRB and o(IFN)gamma) were considered either ‘probable’ or ‘most likely’ to be associated with low IFN-gamma responses, while a genotype at o(IFN)gamma was considered ‘most likely’ to influence high IFN-gamma responses. An allele at OarKP6 was considered ‘probable’ to be associated with low antibody responses to vaccination. Considering the significance of IFN-gamma responses in protection against Map, it is likely that the identified genotype/alleles influencing IFN-gamma responses to vaccination would also influence immune responses to natural Map infections. However, further studies need to be conducted to determine the role of these marker genotypes/alleles in protection against paratuberculosis under natural infection conditions. Key words: paratuberculosis, OJD, Johne’s disease, sheep, immune response, genetic markers, gene polymorphisms, MHC, SLC11A1, IFN-gamma