The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.

No description available.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

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.

La paratuberculose est une maladie entérique incurable causée par la bactérie Mycobacterium avium subsp. paratuberculosis (MAP) endémique dans la plupart des régions du monde. Cette affection ciblant les ruminants est responsable d’importantes pertes économiques dans les productions animales et son contrôle est particulièrement difficile. L’ingestion de matières contaminées (fèces, lait et colostrum) est considérée comme la voie principale de transmission. La situation épidémiologique de la paratuberculose dans les troupeaux ovins du Québec demeure inconnue. Ce projet visait à évaluer la prévalence de MAP et à caractériser les facteurs de risque associés aux statuts positifs des ovins du Québec. Au total, 2942 brebis provenant de 70 fermes ovines, dont 10 productions laitières et 60 productions de boucherie, ont été échantillonnées dans le cadre de ce projet entre février 2020 et mars 2022. La prévalence réelle des moutons positifs dans la province a été estimée à 8.3% (IC 95% 4.7-14.1%) alors que la prévalence apparente de troupeaux positifs a été estimée à 29.9% (IC 95% 25.7-34.1%). L’analyse multivariée des facteurs de risque a permis d’identifier la présence de signes cliniques comme facteur de risque à un statut positif individuel (RC = 3.0; IC 95% 1.3-7.4) et l’utilisation d’un logiciel de régie pour les données de santé des animaux comme un facteur protecteur à un statut de troupeau positif (RC = 0.1; IC 95% 0.0-0.6). Les résultats de cette étude permettront l’exploration de mesures de contrôle de l’infection dans les troupeaux ovins de la province. / Paratuberculosis is an incurable enteric disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), which is endemic in most parts of the world. This disease affecting ruminants is responsible for significant economic losses in livestock production and is particularly difficult to control. Ingestion of contaminated material (e.g., faeces, milk, colostrum) is considered the main route of transmission of MAP. The epidemiological situation of paratuberculosis in Quebec sheep flocks remains unknown. Hence, the purpose of this project was to evaluate the prevalence of MAP as well as to characterize the risk factors associated with positive status within Quebec sheep. A total of 2942 ewes from 70 sheep farms, including 10 dairy and 60 meat farms, were sampled between March 2020 and March 2022. The true prevalence of positive sheep in the province was estimated at 8.3% (IC 95% 4.7-14.1%) while the apparent prevalence of positive flocks was estimated at 29.9% (95% IC 25.7-34.1%). Multivariable analysis of risk factors identified the presence of clinical signs as risk factor for a positive ewe status (OR = 3.0; 95% CI 1.3-7.4) and the use of animal health data management software as a protective factor for positive flock status (OR = 0.1; 95% CI 0.0-0.6). The results of this study will enable the implementation of MAP-tailored infection control measures in the province's sheep flocks.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and has recently been linked to Crohn's disease in humans. To generate an effective vaccine against MAP, it is necessary to identify MAP antigens that trigger protective immunity. Unfortunately, not much is known about MAP proteins despite decades of research. We have previously shown that a 4.8 kb insert from MAP will produce a 16 kDa recombinant protein when expressed in Escherichia coli and 19 kDa recombinant protein when expressed in M smegmatis ( smeg 19K). The difference of 3 kDa in size of these expressed proteins may be related to posttranslational modificatjons that occur in Mycobacterium species. We hypothesized that smeg19K is a lipoglycoprotein since blast analysis revealed approximately 76 % amino acid identity between the MAP 19 kDa protein and a known lipoglycoprotein, the 19 kDa protein of M tuberculosis. This prediction was confirmed following positive staining of smeg19K with Sudan Black 4B, a postelectrophoresis dye used to stain for lipids. Smeg 19K has also stained positively for glycosylation with the lectin concavalin A, a highly specific stain for mannose residues. As expected, treatment with tunicamycin (an antibiotic known to inhibit N-glycosylation) and treatment with deglycosylation assay (non-specific for mannose ), showed no reduction in size of 19 kDa glycolipoproteins. Since covalent modification of proteins with acyl or glycosyl moieties alter immunogenicity and/or pathogenicity, the study here provides foundation for future experiments regarding the antigenicity of MAP 19 kDa lipoglycoprotein and its role in disease pathogenicity.

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

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

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