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We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne's disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity., (Copyright © 2024 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, or paratuberculosis (PTB) in ruminants, besides having zoonotic potential. It possibly changes the gut microbiome, but no conclusive data are available yet. This study aimed at investigating the influence of MAP on the faecal microbiome of cattle naturally infected with PTB. In a follow up period of 10 months, PTB status was investigated in a herd of dairy cattle with history of clinical cases. Each animal was tested for MAP infection using serum and milk ELISA for MAP anti-bodies and IS900 real-time PCR and recombinase polymerase amplification assays for MAP DNA in the faeces and milk monthly for 4 successive months, then a last one after 6 months. The faecal samples were subjected to 16S rDNA metagenomic analysis using Oxford Nanopore Sequencing Technology. The microbial content was compared between animal groups based on MAP positivity rate and production status. All animals were MAP positive by one or more tests, but two animals were consistently negative for MAP DNA in the faeces. In all animals, the phyla firmicutes and bacteroidetes were highly enriched with a small contribution of proteobacteria, and increased abundance of the families Oscillospiraceae, Planococcaceae, and Streptococcacaceae was noted. Animals with high MAP positivity rate showed comparable faecal microbial content, although MAP faecal positivity had no significant effect (p > 0.05) on the microbiome. Generally, richness and evenness indices decreased with increasing positivity rate. A significantly different microbial content was found between dry cows and heifers (p < 0.05). Particularly, Oscillospiraceae and Rikenellaceae were enriched in heifers, while Planococcaceae and Streptococcaceae were overrepresented in dry cows. Furthermore, abundance of 72 genera was significantly different between these two groups (p < 0.05). Changes in faecal microbiome composition were notably associated with increasing MAP shedding in the faeces. The present findings suggest a combined influence of the production status and MAP on the cattle faecal microbiome. This possibly correlates with the fate of the infection, the concern in disease control, again remains for further investigations., (© 2024. The Author(s).)

Johne's disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10 -6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Introduction: As a contagious and chronic disease in the livestock industry, Paratuberculosis is a significant threat to dairy herds' genetic and economic resources. Due to intensive breeding and high production of dairy cattle, the incidence and prevalence are higher. Developing non-destructive diagnostic methods for the early detection and identification of healthy animals is paramount for breeding programs. Conventional methods are almost entirely destructive, have low accuracy, lack precision, and are time-consuming. Near-infrared spectroscopy (NIRS) and aquaphotomics can detect changes in biofluids and thus have the potential to diagnose disease. This study aimed to investigate the diagnostic ability of NIRS and aquaphotomics for Paratuberculosis in dairy cattle., Methods: Blood plasma from dairy cattle was collected in the NIR range (1,300 nm to 1,600 nm) 60 days before and 100 days to 200 days after calving in two groups, positive and negative, using the same consecutive enzyme-linked immunosorbent assay test results three times as a reference test., Results: NIRS and aquaphotomics methods invite 100% accuracy, sensitivity, and specificity to detect Paratuberculosis using data mining by unsupervised method, Principal Component Analysis, and supervised methods: Soft Independent Modeling of Class Analogiest, Linear Discriminant Analysis, Quadratic Discriminant Analysis, Partial Least Square-Discriminant Analysis, and Support Vector Machine models., Discussion: The current study found that monitoring blood plasma with NIR spectra provides an opportunity to analyze antibody levels indirectly via changes in water spectral patterns caused by complex physiological changes, such as the amount of antibodies related to Paratuberculosis by aquagram., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Behdad, Pakdel and Massudi.)

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis ( Map ) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis . While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map .IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis . This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map . This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid., Competing Interests: The authors declare no conflict of interest.

Aims: This study aimed to identify specific genomic targets for the detection and strain typing of Map and analyse their sensitivity and specificity, and detect Map directly from faeces., Methods and Results: A comparative genomics approach was used to identify specific genomic targets for the detection and strain typing of Map. A Map specific qPCR using the primer pair 7132 that targets a DNA segregation ATPase protein was able to detect all strains of Map and is more sensitive than the current Johne's disease PCR assays with a sensitivity of 0.0002 fg µl-1. A strain specific qPCR using the Atsa primer pair that targets the arylsulfase gene was able to differentiate between Type S and Type C strains of Map and was more sensitive than the IS1311 PCR and REA with a sensitivity of 40 fg µl-1 and was specific for Type S Map. Both assays successfully detected Map directly from faeces., Conclusion: This study developed and validated two genomics informed qPCR assays, 7132B Map and Atsa Type S and found both assays to be highly specific and sensitive for the detection of Map from culture and directly from faeces. This is the first time that a probe-based qPCR has been designed and developed for Map strain typing, which will greatly improve the response time during outbreak investigations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Johne's disease is an infectious enteric disease caused by Mycobacterium avium subspecies paratuberculosi s (MAP) affecting ruminant species worldwide. In Project 1, an independent performance comparison ring trail was conducted between three different commercial MAP quantitative polymerase chain reaction (qPCR) assay services (B, C, and D) currently marketed in Great Britain by three separate laboratories against each other and against a fourth assay (A) not available commercially in Great Britain. A total of 205 individual ovine and bovine samples from five farms were analyzed to give 41 sets of pooled results (pool size five) from each laboratory according to their specific protocols. The numbers of positive pools for assays A-D were 18, 12, 11, and 1 (43.9%, 29.2%, 26.8%, and 2.4%), respectively. Assessment of interrater reliability produced a Fleiss' kappa coefficient of 0.15, indicating very poor overall agreement between the four laboratories. Laboratories A-D diagnosed 4, 3, 2, and 1 flocks at the farm level, respectively, as MAP positive. In Project 2, 38 pooled ovine samples from 10 flocks were analyzed to compare the performance of laboratories A and B. The numbers of positive results for laboratories A and B were 24 (63.1%) and 17 (44.7%), respectively (Cohen's kappa 0.54), indicating that laboratory A was more sensitive than B in line with results from Project 1. Variation between laboratories offering MAP qPCR assays is a significant concern, and further work is warranted to validate and standardize the performance of assays between laboratories for both ovine and bovine samples.IMPORTANCEOur study reports the findings of an inter-laboratory ring trial comparing the performance of four different quantitative polymerase chain reaction (qPCR) assay services for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle and sheep. MAP is the causative agent of Johne's disease (also known as paratuberculosis), a significant production-limiting disease in livestock populations with a worldwide distribution. The content of this paper is significant and novel as it is the first to highlight the marked variation between the diagnostic sensitivity and reproducibility of the three principal commercial laboratories offering MAP qPCR diagnostic and screening services in Great Britain. The low sensitivity and high variability between the laboratories are of great concern and relevance to veterinary practitioners and livestock producers., Competing Interests: The authors declare no conflict of interest.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mizzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this contribution, a novel, low-cost, high throughput, and ultra-selective electrochemical DNA nanobiosensor was developed for accurate on-site detection of Mycobacterium avium subspecies paratuberculosis (MAP) in real media for practical diagnosis of Johne's disease (JD). The method was designed based on the immobilization of graphene oxide and chitosan biopolymer on the surface of a glassy carbon electrode, modified by electrochemical immobilization of graphene oxide and chitosan biopolymer, followed by activation of biopolymer via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy succinimide (EDC/NHS) coupling system. Afterward, the commercial probe DNA (ssDNA) was stabilized on the activated electrode surface to prepare an ultra-selective ssDNA-stabilized nanobiosensor for MAP sensing called "ssDNA-stabilized GO-CH-EDC/NHS-modified electrode". Several characterization methods distinguished the bioelectrode. The DNA hybridization between the nanobiosensor and target DNA was confirmed by cyclic voltammetry and differential pulse voltammetry. "At optimal experimental conditions, the nanobiosensor showed a linear range of 1.0 × 10 -15 -1.0 × 10 -12  mol L -1 , a detection limit as low as 1.53 × 10 -13  mol L -1 , and a repeatability with a relative standard deviation (%RSD) of 4.7%. The reproducibility was also appropriate, with a %RSD of about 10%. It was used to diagnose MAP in real samples with highly accurate results. Therefore, the developed nanobiosensor can be used for clinical diagnosis of MAP., (© 2024. The Author(s).)

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS 900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS 900 PCR assay with a commercial ISMap 02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS 900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS 900 PCR assay. Among culture-positive samples before incubation, the IS 900 PCR assay yielded significantly more positive results than the ISMap 02 PCR assay; however, among culture-negative samples, the IS 900 PCR assay yielded positive results both before and after incubation. The ISMap 02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS 900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap 02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: To date genomic studies on Map have concentrated on Type C strains with only a few Type S strains included for comparison. In this study the entire pan-genome of 261 Map genomes (205 Type C, 52 Type S and 4 Type B) and 7 Mycobacterium avium complex (Mac) genomes were analysed to identify genomic similarities and differences between the strains and provide more insight into the evolutionary relationship within this Mycobacterial species., Results: Our analysis of the core genome of all the Map isolates identified two distinct lineages, Type S and Type C Map that is consistent with previous phylogenetic studies of Map. Pan-genome analysis revealed that Map has a larger accessory genome than Mycobacterium avium subsp. avium (Maa) and Type C Map has a larger accessory genome than Type S Map. In addition, we found large rearrangements within Type S strains of Map and little to none in Type C and Type B strains. There were 50 core genes identified that were unique to Type S Map and there were no unique core genes identified between Type B and Type C Map strains. In Type C Map we identified an additional CE10 CAZyme class which was identified as an alpha/beta hydrolase and an additional polyketide and non-ribosomal peptide synthetase cluster. Consistent with previous analysis no plasmids and only incomplete prophages were identified in the genomes of Map. There were 45 hypothetical CRISPR elements identified with no associated cas genes., Conclusion: This is the most comprehensive comparison of the genomic content of Map isolates to date and included the closing of eight Map genomes. The analysis revealed that there is greater variation in gene synteny within Type S strains when compared to Type C indicating that the Type C Map strain emerged after Type S. Further analysis of Type C and Type B genomes revealed that they are structurally similar with little to no genetic variation and that Type B Map may be a distinct clade within Type C Map and not a different strain type of Map. The evolutionary lineage of Maa and Map was confirmed as emerging after M. hominissuis., (© 2023. The Author(s).)

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in triggering rheumatoid arthritis (RA) could be a population-specific phenomenon. This study explored the relationship between MAP and RA using serological and molecular techniques; In this case-control study, 239 Iranian participants, including 120 RA patients and 119 controls, were enrolled. The indirect ELISA was designed to diagnose antibodies against MAP3865c125-133 and Zinc transporter 8 (ZnT8) 178-186 . The Nested-Polymerase Chain Reaction (PCR) detected MAP in blood; The frequency of MAP in RA patients and controls was 31.9% and 12.5%, respectively (P = 0.002). The antibodies against MAP3865c125-133 and ZnT8 178-186 were 42.9% and 37% in RA patients and 14.2% and 11.7% in the controls, respectively (P < 0.0001). Interestingly, positive ELISA results in previously diagnosed (PD) RA were more common than newly diagnosed (ND) RA patients (P < 0.05).; The findings showed a higher frequency of MAP and its antibodies in the RA patients than in the controls. This data indicated MAP as one of RA's predisposing factors. Also, this first report implies the high positivity of MAP in Iranian RA patients., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Paratuberculosis is an enteric disease caused by Mycobacterium avium subs. Paratuberculosis (MAP). Quantifying the load of MAP in faeces samples offers the advantage of determining the stage of infection and planning control measures. Currently, detection of MAP in faecal specimens relies on cultural assays and quantitative PCR (qPCR), but both methods have limitations such as prolonged isolation times for cultural assay and the absence of nucleic acid standards for qPCR. Digital PCR (dPCR) represents an advancement over qPCR as it allows direct quantification of nucleic acid in a sample without the need for a standard curve. The present paper reports about the validation process, following ISO 20395:2019 guidelines, of a F57 digital PCR assay for quantifying MAP cells in faecal samples. Based on our validation, the Limit Of Detection (LOD) corresponds to 7.85 10 4 MAP cells/g, and the Limit Of Quantification (LOQ) to 7.85 10 5 MAP cells, with an efficiency of recovery at LOQ estimated about 4.5%. To assess precision, we evaluated the same faecal sample extracted by two different operators at different times. The standard deviation under repeatability conditions (S Repeatability) and intersession variability conditions (S Intermediate) were calculated, resulting in values of 0.43 and 0.26, respectively. Trueness was determined at LOQ and a value ten times higher, yielding percentages of 3.35% and 5.16%, respectively. Linearity showed a R 2 value of 0.998, indicating strong linear correlation. Measurement uncertainty was 26% in absolute value and 3% on a logarithmic base 10 scale. Overall, the assay exhibits good specificity and robustness. Our validation underlines the good performance of the quantification method and allow the laboratory to provide quantitative results of MAP/cells on faecal samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test. Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0-20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown., Competing Interests: Declaration of Competing Interest The authors have no financial or personal conflicts of interest that could have inappropriately influenced the development of this research., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease with no fully understood etiology and cure. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of paratuberculosis, is also isolated from samples from human patients with CD. Paratuberculosis is characterized by persistent diarrhea and progressive weight loss and primarily affects ruminants, which eliminate the agent via feces and milk. The involvement of MAP in the pathogenesis of CD and other intestinal diseases is unclear. Thus, the present study aimed to analyze immunological, socioepidemiological, biochemical, and therapeutic variables that may be related to the occurrence of MAP in blood samples and CD patients. The sampling was random, and the population of origin was the patients from the Bowel Outpatient Clinic of the Alpha Institute of Gastroenterology (IAG), Hospital das Clínicas, Universidade Federal de Minas Gerais (HC-UFMG). Blood samples were collected from 20 patients with CD, eight with ulcerative rectocolitis (UCR), and 10 control patients without inflammatory bowel diseases. Samples were subjected to real-time PCR for detection of MAP DNA, oxidative stress analyses, and socioepidemiological variables. MAP was detected in 10 (26.3%) of the patients, seven (70%) were CD patients, 2 (20%) were URC patients, and one (10%) was a non-IBD patient. MAP was found more frequently among CD patients, but not restricted to CD patients. The presence of MAP in the blood of these patients occurred simultaneously with an inflammatory response with an increase in neutrophils and significant alterations in the production of antioxidant enzymes such as catalase and GST., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis , due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL , genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro . First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 10 9 CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with Δ BacA induced a greater upregulation of proinflammatory cytokines than Δ IcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. Δ BacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with Δ BacA at all time points. The Δ BacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the Δ BacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by Δ BacA is stronger compared to Δ IcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Eshraghisamani, Arrazuria, Luo and De Buck.)

The objective of this study was to report the occurrence of Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats, via description of their clinical presentation, histopathological findings, and molecular identification of the infectious agent. Screening was performed using IS900 real-time PCR (qPCR) in milk samples from 179 properties in the semiarid of Northeast region of Brazil. Pooled milk samples from all lactating goats from processing plants were submitted to molecular diagnosis. One property had a positive result at qPCR. The production unit which had the positive sample for MAP was located, and an on-site visit to this property was performed to collect individual milk samples, seven of which tested MAP positive by IS900 qPCR. With permission from the owner, two goats (Animal 1 was positive and Animal 2 was negative on first qPCR for MAP) were acquired and euthanized. Animals 1 and 2 had milk and portions of the duodenum, ileum, colon, and mesenteric lymph nodes positive at qPCR for MAP. Animal 1 also had MAP DNA detected in part of the jejunum and cecum. In animal 2, the ileocecal valve tested positive. MAP was not detected in the blood or feces of either animal; however; it was confirmed for the association of clinical findings, histopathology, and qPCR. The gene IS900 from the positive samples were sequenced and showed a 99% similarity with MAP. The MAP was identified for the first time in the goat milk and tissues in the semiarid region of Northeast Brazil., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942 ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Amplification of the IS 900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis , which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS 900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS 900 due to 5' deletions at some IS 900 loci. These IS 900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS 900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS 900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS 900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Although Mycobacterium avium subsp. paratuberculosis (MAP) has threatened public health and the livestock industry, the current diagnostic tools (e.g., fecal PCR and enzyme-linked immunosorbent assay [ELISA]) for MAP infection have some limitations, such as inconsistent results due to intermittent bacterial shedding or low sensitivity during the early stage of infection. Therefore, this study aimed to develop a novel biomarker focusing on elucidating the gut microbial signature of MAP-positive ruminants, since the clinical signs of MAP infection are closely related to dysbiosis. 16S rRNA-based gut microbial community analysis revealed both a decrease in microbial diversity and the emergence of several distinct taxa following MAP infection. To determine the discriminant taxa diagnostic of MAP infection, machine learning-based feature selection and predictive model construction were applied to taxon abundance data or their transformed derivatives. The selected taxa, such as Clostridioides (formerly Clostridium) difficile, were used to build models using a support vector machine, linear support vector classification, k -nearest neighbor, and random forest with 10-fold cross-validation. The receiver operating characteristic-area under the curve (ROC-AUC) analysis of the models revealed their high accuracy, up to approximately 96%. Collectively, taxonomic signatures of cattle gut microbiotas according to MAP infection status could be identified by feature selection tools and applied to establish a predictive model for the infection state. IMPORTANCE Due to the limitations, such as intermittent bacterial shedding or poor sensitivity, of the current diagnostic tools for Johne's disease, novel biomarkers are urgently needed to aid control of the disease. Here, we explored the fecal microbiota of Johne's disease-affected cattle and tried to discover distinct microbial characteristics which have the potential to be novel noninvasive biomarkers. Through 16S rRNA sequencing and machine learning approaches, a dozen taxa were selected as taxonomic signatures to discriminate the disease state. In addition, when constructing predictive models using relative abundance data of the corresponding taxa, the models showed high accuracy for classification, even including animals with subclinical infection. Thus, our study suggested novel noninvasive microbiological biomarkers that are robustly expressed regardless of subclinical infection and the applicability of machine learning for diagnosis of Johne's disease.

Mycobacterium avium ssp. paratuberculosis (MAP), causes Johne's disease (JD), or paratuberculosis, a chronic enteritis of ruminants, which in goats is characterized by ileal lesions. The work described here is a case-control association study using the Illumina Caprine SNP50 BeadChip to unravel the genes involved in susceptibility of goats to JD. Goats in herds with a high occurrence of Johne's disease were classified as healthy or infected based on the level of serum antibodies against MAP, and 331 animals were selected for the association study. Goats belonged to the Jonica (157) and Siriana breeds (174). Whole-genome association analysis identified one region suggestive of significance associated with an antibody response to MAP on chromosome 7 (p-value = 1.23 × 10 -5 ). These results provide evidence for genetic loci involved in the antibody response to MAP in goats., (© 2022 Stichting International Foundation for Animal Genetics.)

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Mycobacterium avium sp. paratuberculosis (MAP) is a pathogen, which causes paratuberculosis in animals; it has also been found to be associated with a number of autoimmune disorders in humans. The emergence of drug resistance has also been found in this bacillus during disease management., Objective: The present study's focus was to identify potential therapeutic targets for the therapeutic management of Mycobacterium avium sp. paratuberculosis infection by in silico analysis., Methods: Differentially-expressed genes (DEGs) can be good drug targets, which can be identified from microarray studies. We used gene expression profile GSE43645 to identify differentiallyexpressed genes. An integrated network of upregulated DEGs was constructed with the STRING database and the constructed network was analyzed and visualized by Cytoscape. Clusters in the proteinprotein interaction (PPI) network were identified by the Cytoscape app ClusterViz. MAP proteins predicted in clusters were analyzed for their non-homology with the human proteins, and homologous proteins were excluded. Essential proteins and cellular localization analysis and the physicochemical characteristics prediction were also done. Finally, the druggability of the target proteins and drugs that can block the targets was predicted using the DrugBank database and confirmed by molecular docking. Structural prediction and verification of drug target proteins were also carried out., Results: Two drug targets, MAP&#95;1210 (inhA) and MAP&#95;3961 (aceA), encoding enoyl acyl carrier protein reductase and isocitrate lyase enzymes, respectively, were finally predicted as potential drug targets., Conclusion: Both of these proteins have been predicted as drug targets in other mycobacterial species also, supporting our results. However, further experiments are required to confirm these results., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn's disease. Therefore, control is becoming priority for governments. Control is largely dependent on "Test and Cull" or "Test and Segregate" policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces., Methods and Results: Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples., Conclusion: Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis . The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis , and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Paratuberculosis is a worldwide chronic enteric disease of ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). While MAP has been widely investigated all around the world, little is known about the different strains that circulate in each country. This study describes the genetic diversity of MAP isolates from different bovine and deer herds from Argentina, analyzed by Multiple-Locus Variable number tandem repeat Analysis (MLVA), as well as the phylogenetic relatedness between geographically distant isolates through Whole Genome Sequencing (WGS) and core-genome analysis. A total of 90 MAP isolates were analyzed. The results showed seven different MLVA genotypes, with almost 75% of them belonging to pattern INMV 1, described in all the herds studied. WGS results suggested the presence of a common INMV 1 strain circulating throughout the country. Our results allow confirming the coexistence of different strains in time and space and the mixed infections identified in some animals. These observations suggest the absence of animal monitoring prior to introduction to the herds and the need for a control program in the country. This study represents the first to report WGS of MAP strains in Argentina., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Paratuberculosis is a chronic enteric progressive disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). Despite cultural methods being considered the gold standard for the diagnosis of paratuberculosis, quantitative PCR (qPCR) assays have been developed for this purpose. These assays showed sensitivity and specificity comparable to cultural method but provide more rapid analysis results. Aim of our work was the validation of an IS900-qPCR assay for detection of MAP in faeces according to the OIE guidelines relative to the validation of assays for infectious diseases. The analytical and diagnostic characteristics and the reproducibility of the qPCR method were assessed. The robustness of the assay was evaluated using two extraction methods (silica column and magnetic beads DNA capture) and two qPCR systems (STEPONE™ and CFX96™). According to our validation, the analytical specificity, inclusivity and exclusivity were found to be appropriate for the use of this qPCR assay as a diagnostic test. Specifically, the limit of detection was approximately 100 CFU/g or even less if binomial approaches were used for the determination of the 95 % probability of detection (logit and clog-log models) with sufficient repeatability and reproducibility. Estimation of test accuracy was performed using a Bayesian two latent class model, in various scenarios combining different priors for prevalence and accuracy of the two tests used. All models were run considering three different cut-offs for qPCR. Our validation study underlines the good performance of this IS900-qPCR assay for diagnosis of MAP representing a valid and robust alternative to culture. Moreover, coupled with the semiautomatic magnetic beads DNA extraction method, this assay allows the rapid processing of numerous samples., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The objective of the present study was to evaluate the diagnostic accuracy of the individual fecal culture (IFC), fecal PCR (FPCR), and serum ELISA for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep from four governorates in Egypt, using a latent class model (LCM) fitted within a Bayesian framework. Furthermore, the within-governorate prevalence of MAP infection in sheep was estimated as a secondary objective. Fecal and blood samples were collected from 370 sheep in four Egyptian governorates. Fecal samples were analyzed by IFC and RT-PCR based on ISMav2 gene, while ELISA was performed on serum samples. The sensitivity (Se) and specificity (Sp) of the three diagnostic tests were estimated using a three-tests-four-populations Bayesian LCM to obtain posterior estimates [medians and 95% Bayesian credible intervals (95% BCI)] for each parameter. The median Se estimates (95% BCI) for IFC, FPCR, and serum ELISA were 31.8% (22.8-41.4), 49.7% (31.8-79.9), and 61.2% (39.8-81.4), respectively. The median Sp estimates (95% BCI) for IFC, FPCR, and serum ELISA were 97.7% (96.1-98.9), 97.7% (95.6-99.5), and 98.4% (96.9-99.3), respectively. The median within-governorate paratuberculosis prevalence (95% BCI) was 5.2% (1.1-13.6), 8.4% (2.9-17.7), 9.4% (3.0-20.7), and 18.2% (10.5-29.5) for the Gharbia, Menoufia, Qalyubia, and Kafr El-Sheikh governorates, respectively. In conclusion, at a ratio of the optical density (OD) sample/OD positive control threshold of > 45%, ELISA showed the highest Se among the three tests and comparable Sp to IFC and FPCR. The test ELISA evaluated in this study is an interesting alternative for detecting MAP in sheep due to its higher Se, lower cost, and shorter turnaround laboratory time compared to IFC and FPCR., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 10 7  CFU/mL, 1.68 × 10 6  CFU/mL and 1.68 × 10 5  CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 10 4  CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 10 9  CFU/mL from feces and no less than 10 4  CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 10 5 and 2 × 10 3  CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log 10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 10 11 to 4.14 × 10 4 for IS900 (FAM), 1.27 × 10 11 to 1.27 × 10 4 for IS900 mRNA (Cy5), and 3.68 × 10 10 to 3.68 × 10 4 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 10 4 , 1.27 × 10 4 , and 3.68 × 10 4 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 10 4 and 3.68 × 10 4 copies for IS900 and ModD, respectively., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in animals with zoonotic potential; it has been linked to many chronic diseases in humans, especially gastrointestinal diseases (GID). MAP has been extensively studied in Europe and America, but little reports were published from Africa. Sudan is a unique country with close contact between humans and livestock. Despite such interaction, the one health concept is neglected in dealing with cases of humans with GID. In this study, patients admitted to the reference GID hospital in the Sudan over a period of 8 months were screened for presence of MAP in their faeces or colonic biopsies. A total of 86 patients were recruited for this study, but only 67 were screened for MAP, as 19 did not provide the necessary samples for analysis. Both real-time PCR and culture were used to detect MAP in the collected samples and the microbial diversity in patients´ faecal samples was investigated using 16S rDNA nanopore sequencing. In total, 27 (40.3%) patients were MAP positive: they were 15 males and 12 females, of ages between 21 and 80 years. Logistic regression analysis revealed no statistical significance for all tested variables in MAP positive patients (occupation, gender, contact with animal, milk consumption, chronic disease, etc.). A unique microbiome profile of MAP-positive patients in comparison to MAP-negative was found. These findings suggest that a considerable proportion of the population could be MAP infected or carriers. Therefore, increase awareness at community level is urgently needed to decrease the risk of MAP at human/animal interface. This study represents the first report of MAP in humans in the Sudan; nevertheless, a better view of the situation of MAP in humans in the country requires a larger study including patients with other conditions., Competing Interests: All authors declare that they have no conflict of interest.

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples., (© 2022. The Author(s).)

Paratuberculosis is a major endemic disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and leads to huge economic loss in the dairy sector worldwide. Alternative splicing (AS) events, playing indispensable regulatory roles in many protein functions and biological pathways, are shown to be associated with complex traits and diseases. In this study, by integrating the RNA sequencing (RNA-seq) data of 24 samples from three tissues (peripheral blood, jejunum and salivary gland) of Holstein cows, we obtained 2,706,541,696 uniquely mapped reads in total that represented 12,870 expressed genes, and detected 4285 differentially expressed genes (DEGs) between MAP-infected and healthy cows (p < 0.05). Of them, 92 differentially expressed splicing factors (DESFs) were included. Further, 119, 150 and 68 differential alternative splicing (DAS) events between MAP-infected and healthy cows were identified in peripheral blood, jejunum and salivary glands, respectively. Of note, six DAS events were highly and significantly correlated with the DESFs (R2 > 0.9; p < 0.01), and their corresponding genes (COPI coat complex subunit gamma 2gene (COPG2), kinesin family member 2C gene (KIF2C), exocyst complex component 7 (EXOC7), Rab9 effector protein with kelch motifs gene (RABEPK), deoxyribonuclease 1 gene (DNASE1) and early endosome antigen 1gene (EEA1)) were significantly enriched in immune response such as vesicle-mediated transport, regulation of acute inflammatory response and tuberculosis through gene ontology (GO) and KEGG analysis. KS test showed that the DAS events in the EXOC7 and KIF2C genes indeed displayed differences between MAP-infected cows and healthy cows. The DAS in EXOC7 might produce a new protein sequence with lack of 23 amino acids, and the DAS in KIF2C induced a stop codon of premature occurrence and resulted in a lack of functional domain. In summary, this study identified the DAS events and corresponding genes related to MAP-infection base on the RNA-seq data from multiple tissues of Holstein cows, providing novel insights into the regulatory mechanisms underpinning paratuberculosis in dairy cattle.

Mycobacterium avium subsp. paratuberculosis (MAP) has been identified as one of the environmental agents that causes multiple sclerosis (MS). The global prevalence of MS has been upsurging over the years; however, efforts to divulge the role of MAP in MS have been limited. As a result, the present study aimed at assessing the odd ratios (ORs) associated MAP with the risk of MS. MAP-related MS data were obtained from 6 databases using the terms 'multiple sclerosis' or 'MS' and 'paratuberculosis' without regard for time or language restrictions following PRISMA standards. A total of 2,538 participants' data from 12 studies presenting anti-MAP antibodies and MAP DNA from 4 studies were fitted in random-effects (RE) and fixed-effects (FE) meta-analytic models. Furthermore, the between-study heterogeneity was measured using I 2 -values with a significant limit set at an I² > 75%. Analytical rigor and publication bias was determined using leave-one-out-analytics, Egger's tests, and p-curve analysis. In the FE and RE models, anti-MAP antibodies data significantly associated MS risk with MAP as 10.71 OR (95%-CI [7.78; 14.74], p-value < 0.0001) and 12.76 OR (95%-CI [8.13; 20.02], p-value < 0.0001) respectively, with an I 2 value of 34.9% (95%-CI [0.0%; 67.2%]; p-value = 0.11). Similarly, the MAP DNA dataset in FE significantly present MS risk due to MAP as 5.53 OR (95%-CI [3.54; 8.66], p-value< 0.0001) while, RE showed 5.27 OR (95%-CI [3.22; 8.60], p = 0.0017), with an I 2 -value = 0.0% (95%-CI [0.0%; 84.7%]; p-value = 0.71). Eggers' test, on the other hand, found publication bias in anti-MAP antibodies data (intercept = 1.61, 95% CI: 0.45 - 2.77, t = 2.72, p = 0.021), but not in MAP DNA dataset (intercept = -5.57, 95% CI: -20.44 - 9.29, t = -0.74, p = 0.54). The robustness of the meta-analyses was demonstrated by all sensitivity analyses. In addition, there is no evidence of p-hacking observed (right-skewness test (P Full < 0.001, P Half <0.001; statistical power ≥ 94% (95%-CI: 72.5%-99%)). In conclusion, the synthesis revealed a strong association between MAP and MS, indicating that MAP is a significant environmental agent that may trigger MS. Thus, early screening of MAP in MS cases may assist in the therapeutic approach to its management/treatment. Therefore, future studies should be tailored towards the role of MAP in the severity of MS phenotypes, as well as address global data gaps and low disease surveillance., (Copyright © 2022. Published by Elsevier B.V.)

Native hosts for the bacterial agent that causes Johne's disease are ruminants, which include cattle, sheep and goats among others. These large animals are often too costly to be used in testing experimental vaccines. In this chapter, we provide detailed methods to use an inexpensive and more manageable animal host, the ferret, to test efficacy and immunogenicity of live-attenuated Mycobacterium avium subspecies paratuberculosis (MAP) mutant strains prior to consideration as vaccine candidates., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. These sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by Mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. We employed ten PCR assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. The most stable genes were RPL13A and HMBS, followed by TFRC and B-ACT. NormFinder analysis provided similar results with B-ACT as the best reference gene, followed by RLP13A and TFRC. This validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qRT-PCR.

Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale., (Copyright © 2021. Published by Elsevier B.V.)

This study aimed to systematically collect and appraise the scientific evidence to answer the research question: What MAP genotypes have been isolated from cattle, sheep, and goats in Latin America and the Caribbean? An electronic search was conducted on three platforms (i.e., OVID®, Web of Science®, SciELO) as well as on the proceedings of the International Colloquium on Paratuberculosis. Inclusion and exclusion criteria were defined a priori and conserved through the systematic process and only articles published in peer-reviewed journals were considered. A total of 26 articles met the definitive inclusion criteria. All were published in English, in 15 different journals, and between 1989 and 2020. The relevant articles reported the use of six different genotyping techniques (i.e., polymerase chain reaction-restriction endonuclease analysis, restriction fragment length polymorphism, type-specific-PCR, mycobacterial interspersed repetitive units-variable number of tandem repeats, multi-locus short sequence repeat, single nucleotide polymorphism) in isolates from seven countries. Genotypes found so far in the region using typing techniques were mainly C type. MIRU-VNTR mostly reported INMV 1, INMV 2, and INMV 11 subtypes, among others. MLSSR reported genotypes from four different countries, reporting nine different subtypes of which 7g-10g-4ggt was the most common for loci 1, 2, and 8, respectively. Regardless the high diversity of techniques used so far to genotype Latin American and Caribbean MAP isolates, the original question of this systematic review has been answered. In addition, a relative genetic similarity between MAP strains recovered from cattle, goats, and sheep unrelatedly of the matrix and geographic origin was identified., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bovine paratuberculosis (PTB) is a chronic inflammatory disease caused by Mycobacterium avium susbp. paratuberculosis (MAP). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) significantly associated with susceptibility to bovine PTB. The main objective of this study was to identify quantitative trait loci (QTLs) associated with MAP infection in Spanish Holstein cows (N = 983) using combinations of diagnostic tests and imputed whole-genome sequence (WGS) data. The infection status of these animals was defined by three diagnostic methods including ELISA for MAP-antibodies detection, and tissue culture and PCR for MAP detection. The 983 cows included in this study were genotyped with the Bovine MD SNP50 Bead Chip, and the corresponding genotypes were imputed to WGS using the 1,000 Bull genomes reference population. In total, 33.77 million SNP variants per animal were identified across the genome. Linear mixed models were used to calculate the heritability (h2) estimates for each diagnostic test and test combinations. Next, we performed a case-control GWAS using the imputed WGS datasets and the phenotypes and combinations of phenotypes with h2 estimates > 0.080. After performing the GWAS, the test combinations that showed SNPs with a significant association (PFDR ≤ 0.05), were the ELISA-tissue PCR-tissue culture, ELISA-tissue culture, and ELISA-tissue PCR. A total of twelve quantitative trait loci (QTLs) highly associated with MAP infection status were identified on the Bos taurus autosomes (BTA) 4, BTA5, BTA11, BTA12, BTA14, BTA23, BTA24, and BTA28, and some of these QTLs were linked to immune-modulating genes. The identified QTLs on BTA23 spanning from 18.81 to 22.95 Mb of the Bos taurus genome overlapped with several QTLs previously found to be associated with PTB susceptibility, bovine tuberculosis susceptibility, and clinical mastitis. The results from this study provide more clues regarding the molecular mechanisms underlying susceptibility to PTB infection in cattle and might be used to develop national genetic evaluations for PTB in Spain., Competing Interests: The authors have declared that no competing interests exist.

Background: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility., Methods: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]., Results: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance., Conclusions: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Background: Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease (JD), a chronic enteritis widespread in ruminants, resulting in substantial economic losses, especially to the dairy industry. Understanding the genetic diversity of Map in Australia will assist epidemiological studies for tracking disease transmission and identify subtype characteristics for use in development of improved diagnostic typing methods. Here we investigated the phylogenetic relationships of 351 Map isolates and compared different subtyping methods to assess their suitability for use in diagnostics and accuracy., Results: SNP-based phylogenetic analysis of 228 Australian isolates and 123 publicly available international isolates grouped Type S and Type C strains into two distinct lineages. Type C strains were highly monomorphic with only 20 SNP differences separating them. Type S strains, when aligned separately to the Telford strain, fell into two distinct clades: The first clade contained seven international isolates while the second clade contained one international isolate from Scotland and all 59 Australian isolates. The Australian Type B strain clustered with US bison strains. IS1311 PCR and Restriction Enzyme Analysis (REA) intermittently generated incorrect results when compared to Long Sequence Polymorphism (LSP) analysis, whole genome SNP-based phylogenetic analysis, IS1311 sequence alignment and average nucleotide identity (ANI). These alternative methods generated consistent Map typing results. A published SNP based assay for genotyping Map was found to be unsuitable for differentiating between Australian and international strain types of Map., Conclusion: This is the first phylogenetic analysis of Australian Map isolates. The Type C lineage was highly monomorphic, and the Type S lineage clustered all Australian isolates into one clade with a single Scottish sheep strain. The Australian isolate classified as Type B by IS1311 PCR and REA is likely to be descended from bison and most closely related to US bison strains. Limitations of the current typing methods were identified in this study.

Information on bio-type profile of Mycobacterium avium subspecies paratubeculosis (MAP) in sheep flocks and goat herds of Himalayan region is not reported earlier. The aim of our study was to determine the bio-type of MAP infecting livestock of this region. A total of 71 faecal samples (sheep-57, goats-14) were screened by Ziehl-Neelsen (ZN) staining and IS900 PCR, and then processed for culture on Herrold's egg yolk medium (HEYM) having mycobactin J (MJ). Out of 71 faecal samples, MAP colonies were seen only in four samples (sheep-3 and goat-1). Isolates were confirmed as MAP on the basis of slow growth, acid fastness, MJ dependency, IS900 and IS1311 PCR. All the IS900 and IS1311 PCR positive samples were bio-typed by IS1311 PCR-REA (restriction endonuclease analysis), which confirmed all four isolates as 'bison type.' In IS1311 based phylogeny of MAP isolates by ClustalW method of the MegAlign program of DNASTAR Lasergene software, the four sequences of MAP isolates (NCBI sequence nos. MH988763, MH988765, MH988766 and MH988764) did not show any distinct clustering/grouping pattern. However, these four isolates showed a bit of closeness to the MAP sequences (KC990353.1 and KC990352.1) of 'bison type' isolated from wood bison in Canada. In conclusion, this is the first report on isolation and bio-type profile of MAP infecting sheep and goats of Himalayan region. Study will help in devising prevention and control strategies against spread of MAP infection in livestock population of Himalayan region.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic granulomatous enteropathy in ruminants. Determining the genetic diversity of MAP is necessary to understand the epidemiology and biology of MAP, as well as establishing disease control strategies. In the present study, whole genome-based alignment and comparative analysis were performed using 40 publicly available MAP genomes, including newly sequenced Korean isolates. First, whole genome-based alignment was employed to identify new genomic structures in MAP genomes. Second, the genomic diversity of the MAP population was described by pangenome analysis. A phylogenetic tree based on the core genome and pangenome showed that the MAP was differentiated into two major types (C- and S-type), which was in keeping with the findings of previous studies. However, B-type strains were discriminated from C-type strains. Finally, functional analysis of the pangenome was performed using three virulence factor databases (i.e., PATRIC, VFDB, and Victors) to predict the phenotypic diversity of MAP in terms of pathogenicity. Based on the results of the pangenome analysis, we developed a real-time PCR technique to distinguish among S-, B- and C-type strains. In conclusion, the results of our study suggest that the phenotypic differences between MAP strains can be explained by their genetic polymorphisms. These results may help to elucidate the diversity of MAP, extending from genomic features to phenotypic traits.