Mycobacterium Avium Subspecies Paratuberculosis Infection Research Articles

Mycobacterium avium subspecies paratuberculosis (MAP) infection, and its impact on gut microbiome of individuals with multiple sclerosis

The microbial ecology of Mycobacterium avium subspecies paratuberculosis infections (MAP) within the context of Multiple Sclerosis (MS) is largely an unexplored topic in the literature. Thus, we have characterized the compositional and predicted functional differences of the gut microbiome between MS patients with MAP (MAP+) and without (MAP−) infection. This was done in the context of exposome differences (through self-reported filled questionnaires), principally in anthropometric and sociodemographic patterns to gain an understanding of the gut microbiome dynamics. 16S rRNA microbiome profiling of faecal samples (n = 69) was performed for four groups, which differed by disease and MAP infection: healthy cohort (HC) MAP−; HC MAP+ ; MS MAP−; and MS MAP+ . Using a dynamic strategy, with MAP infection and time of sampling as occupancy models, we have recovered the core microbiome for both HC and MS individuals. Additional application of neutral modeling suggests key genera that are under selection pressure by the hosts. These include members of the phyla Actinobacteriota, Bacteroidota, and Firmicutes. As several subjects provided multiple samples, a Quasi Conditional Association Test that incorporates paired-nature of samples found major differences in Archaea. To consolidate treatment groups, confounders, microbiome, and the disease outcome parameters, a mediation analysis is performed for MS cohort. This highlighted certain genera i.e., Sutterella, Akkermansia, Bacteriodes, Gastranaerophilales, Alistipes, Balutia, Faecalibacterium, Lachnospiraceae, Anaerostipes, Ruminococcaceae, Eggerthellaceae and Clostridia-UCG-014 having mediatory effect using disease duration as an outcome and MAP infection as a treatment group. Our analyses indicate that the gut microbiome may be an important target for dietary and lifestyle intervention in MS patients with and without MAP infection.

Mycobacterium paratuberculosis: A HERV Turn-On for Autoimmunity, Neurodegeneration, and Cancer?

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that, over millions of years, became integrated into the human genome. While normally inactive, environmental stimuli such as infections have contributed to the transcriptional reactivation of HERV-promoting pathological conditions, including the development of autoimmunity, neurodegenerative disease and cancer. What infections trigger HERV activation? Mycobacterium avium subspecies paratuberculosis (MAP) is a pluripotent driver of human disease. Aside from granulomatous diseases, Crohn's disease, sarcoidosis and Blau syndrome, MAP is associated with autoimmune disease: type one diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA) and autoimmune thyroiditis. MAP is also associated with Alzheimer's disease (AD) and Parkinson's disease (PD). Autoimmune diabetes, MS and RA are the diseases with the strongest MAP/HERV association. There are several other diseases associated with HERV activation, including diseases whose epidemiology and/or pathology would prompt speculation for a causal role of MAP. These include non-solar uveal melanoma, colon cancer, glioblastoma and amyotrophic lateral sclerosis (ALS). This article further points to MAP infection as a contributor to autoimmunity, neurodegenerative disease and cancer via the un-silencing of HERV. We examine the link between the ever-increasing number of MAP-associated diseases and the MAP/HERV intersection with these diverse medical conditions, and propose treatment opportunities based upon this association.

IFN-γ and IL-10 Immunosensor with Vertically Aligned Carbon Nanotube Interdigitated Electrodes toward Pen-Side Cattle Paratuberculosis Monitoring.

Highly sensitive vertically aligned carbon nanotube arrays (VANTAs) interdigitated electrode (IDE) arrays are developed for electrochemical biosensing of two cytokines (i.e., interleukin-10 (IL-10) and interferon-gamma (IFN-γ)) that are useful for early detection Johne's disease (Bovine Paratuberculosis) in cattle. The high aspect ratio VANTA-IDEs (50-60µm in height) are grown through a chemical vapor deposition process from an iron (Fe) catalyst that is lithographically patterned on a silicon wafer with equal finger width and inter-finger spacing of 25µm. After functionalization with distinct antibodies the VANTA-IDEs are capable of selective detection of both IL-10 and IFN-γ within an actual biological matrix (i.e., diluted bovine implant supernatant) over concentration ranges of 0.1 to 30 pgmL-1 (limit of detection - LOD: 0.0911 pgmL-1) and 50-500 pgmL-1 (LOD: 24.17 pgmL-1), respectively with a response time of <35 min. Results demonstrate important initial steps for rapid, pen-side identification of cattle with stage-I Mycobacterium avium subspecies paratuberculosis infection before physical symptoms of Johne's disease are present. Such a rapid pen-side diagnostic test can be used on cattle at an auction or before they are introduced to a herd to ensure the larger population does not become infected with Johne's disease.

Network analysis of gut microbial communities reveal key genera for a multiple sclerosis cohort with Mycobacterium avium subspecies paratuberculosis infection

BackgroundIn gut ecosystems, there is a complex interplay of biotic and abiotic interactions that decide the overall fitness of an individual. Divulging the microbe-microbe and microbe-host interactions may lead to better strategies in disease management, as microbes rarely act in isolation. Network inference for microbial communities is often a challenging task limited by both analytical assumptions as well as experimental approaches. Even after the network topologies are obtained, identification of important nodes within the context of underlying disease aetiology remains a convoluted task. We therefore present a network perspective on complex interactions in gut microbial profiles of individuals who have multiple sclerosis with and without Mycobacterium avium subspecies paratuberculosis (MAP) infection. Our exposé is guided by recent advancements in network-wide statistical measures that identify the keystone nodes. We have utilised several centrality measures, including a recently published metric, Integrated View of Influence (IVI), that is robust against biases.ResultsThe ecological networks were generated on microbial abundance data (n = 69 samples) utilising 16 S rRNA amplification. Using SPIEC-EASI, a sparse inverse covariance estimation approach, we have obtained networks separately for MAP positive (+), MAP negative (-) and healthy controls (as a baseline). Using IVI metric, we identified top 20 keystone nodes and regressed them against covariates of interest using a generalised linear latent variable model. Our analyses suggest Eisenbergiella to be of pivotal importance in MS irrespective of MAP infection. For MAP + cohort, Pyarmidobacter, and Peptoclostridium were predominately the most influential genera, also hinting at an infection model similar to those observed in Inflammatory Bowel Diseases (IBDs). In MAP- cohort, on the other hand, Coprostanoligenes group was the most influential genera that reduces cholesterol and supports the intestinal barrier.ConclusionsThe identification of keystone nodes, their co-occurrences, and associations with the exposome (meta data) advances our understanding of biological interactions through which MAP infection shapes the microbiome in MS individuals, suggesting the link to the inflammatory process of IBDs. The associations presented in this study may lead to development of improved diagnostics and effective vaccines for the management of the disease.

Chitosan-alginate/R8 ternary polyelectrolyte complex as an oral protein-based vaccine candidate induce effective mucosal immune responses

Vaccination is the most effective method for preventing infectious diseases. Oral vaccinations have attracted much attention due to the ability to boost intestinal and systemic immunity. The focus of this study was to develop a poly (lactide-co-glycolide) acid (PLGA)-based ternary polyelectrolyte complex (PEC) with chitosan, sodium alginate, and transmembrane peptides R8 for the delivery of antigen proteins. In this study, the antigen protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis (MAP) antigens HBHA, Ag85B, and Bfra, was combined with R8 to generate self-assembled conjugates. The results showed that PEC presented a cross-linked reticular structure to protect the encapsulated proteins in the simulated gastric fluid. Then, the nanocomposite separated into individual nanoparticles after entering the simulated intestinal fluid. The ternary PEC with R8 promoted the in vivo uptake of antigens by intestinal lymphoid tissue. Moreover, the ternary PEC administered orally to mice promoted the secretion of specific antibodies and intestinal mucosal IgA. In addition, in the mouse models of MAP infection, the ternary PEC enhanced splenic T cell responses, thus reducing bacterial load and liver pathology score. These results suggested that this ternary electrolyte complex could be a promising delivery platform for oral subunit vaccine candidates, not limited to MAP infection.

First Genotyping of Mycobacterium avium Subspecies Paratuberculosis from Large Ruminant Population at Satna District in Madhya Pradesh in India to Bison Type

Background: Paratuberculosis or Johne’s disease in large ruminants is caused due to Mycobacterium avium subspecies paratuberculosis. The bacterial disease in large ruminants is characterized by chronic diarrhea, weakness and emaciation. There are no reports on Paratuberculosis in this area’s large ruminant population and its genotyping. Methods: A total of 76 samples (26 fecal and 50 milk) of large ruminants from Satna district in Madhya Pradesh were investigated (microscopical; for the presence of acid-fast organisms and molecular methods; for gene-specific amplification) for the presence of Mycobacterium avium subspecies paratuberculosis infection. Result: Microscopical examinations of fecal samples revealed 11.54% positivity and milk samples 14% positivity of Mycobacterium avium subspecies paratuberculosis infection. A single fecal and 2 milk samples were found positive in the IS900 polymerase chain reaction test (413 bp product). Specific amplification of 608 bp IS1311 product followed by restriction endonuclease analysis proved the genotype of Mycobacterium avium subspecies paratuberculosis as Bison type.

Pathological Investigation of Double–Stranded DNA Breaks and DNA Oxidation in Natural Infection with Mycobacterium avium subspecies paratuberculosis in Goats

Paratuberculosis, created by Mycobacterium avium subspecies paratuberculosis (MAP), manifests as a chronic affliction marked by persistent diarrhoea and granulomatous enteritis, pervasive in both domestic and global wild ruminants. In this investigation, DNA disruption in lesioned tissues of goat as natural infecte with MAP was pathologically assessed. Accordingly, goats manifesting symptoms suggestive to paratuberculosis, including pronounced emaciation and continual episodic diarrhoea, were subjected to an ELISA diagnostic procedure to ascertain the presence of MAP. This diagnostic approach confirmed the presence of the infectious agent in 20 patients. These patients were subsequently euthanized, and tissue samples from intestinal and regional lenf nods. It were subjected to Hematoxylin and Eosin (HE) staining for histopathological investigatıon, Ziehl Neelsen (ZN) staining to identify acid–fast mycobacteria, γ–H2AX to discern disruptions in double stranded DNA, and 8–Ohdg to detect DNA oxidation by immunohistochemical (IHC) method. Gross anatomical observation serous adipose atrophy, augmented dimensions of mesenterial lymphatic nodes, mucosal hypertrophy and non–retractable mucosal undulations. Histological assessment highlighted epithelial cellular degeneration, an abundance of epithelioid macrophages, lymphocytes, plasmocytes, infiltrating in mucosa. Acid–fast entities, discernible through ZN staining, appeared as luminescent red conglomerates in intestinal and mesenterial tissue. The immunohistochemical analyses evinced positive results for both γ–H2AX and 8–Ohdg across all sampled tissues. Intriguingly, this investigation presented the inaugural global evidence of γ–H2AX and 8–Ohdg expression in a natural MAP infection, demonstrating that this pathological agent precipitates DNA degradation and oxidation, thereby augmenting comprehension of the disease’s pathogenesis.

The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line

BackgroundJohne’s disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne’s disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne’s disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne’s disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved.ResultsImmune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways.ConclusionsIdentifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.

Evaluation of Immune Exhaustion and Co-Inhibitory Receptor Expression in Mycobacterium avium Subspecies paratuberculosis (MAP) Seropositive Diarrhoeic Bovines.

Mycobacterium avium subspecies paratuberculosis (MAP) infection leads to chronic, persistent granulomatous enteritis, causing prolonged diarrhoea and emaciation. The disease is managed using medications such as antibiotics, live vaccines, mycobacteriophage therapies and other treatments; however, a notable proportion of affected animals do not show improvement with this approach. We hypothesise that immunoinhibitory receptors TIM-3 (T cell immunoglobulin mucin protein-3) and PD-1 (Programmed death receptor 1) may be upregulated on Peripheral blood mononuclear cells (PBMCs) of MAP-seropositive bovines, potentially contributing to immune exhaustion. Samples (blood and faeces) were collected from 32 diarrhoeic bovines suspected of MAP infection; eight apparently healthy buffaloes from the dairy farm at Hisar, Haryana and from 14 cows (suffering from chronic diarrhoea, weakness and emaciation) housed in stray cattle shed. MAP infection was estimated using indigenous ELISA (i-ELISA), faecal IS900 PCR, culture and acid-fast staining. TIM-3 and PD-1 gene expression on PBMCs were determined using qRT-PCR. TIM3 expression was relatively higher (~400-fold, 330-fold, 112-fold, 65-fold and 16-fold) in 5 chronically diarrhoeic PBMCs samples (MAP-seropositive), and higher PD-1 expression (around ~7-fold, 1.75-fold, 2.5-fold, 7.6-fold) was recorded in 4 diarrhoeic MAP-seropositive animals, compared to apparently healthy and other MAP-seronegative diarrhoeic animals. High co-expression of TIM-3 and PD-1 levels was also recorded in chronically diarrhoeic, emaciated stray cattle. Understanding immune responses in field conditions might aid in the therapeutic management of paratuberculosis.

Long-term outcomes of the Italian Mycobacterium avium subspecies paratuberculosis control programme for dairy cattle.

The considerable epidemiological and economic implications of paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), have placed importance on control efforts aimed at preventing MAP transmission. In this context, Italy issued national guidelines for the control and status certification of MAP in dairy cattle in 2013. We assessed the long-term outcomes of the Italian MAP control programme for 14 dairy farms located in northern Italy by retrospectively reviewing the results of yearly serological tests, presence of clinical cases, MAP faecal shedding in serologically positive animals, farm management and health ranking as indicators of herd health between 2014 and 2021. A significantly higher number of serologically positive animals were observed between 2014 and 2016 than between 2017 and 2021, as well as an improving trend in the paratuberculosis health ranking for nine of the 14 farms. No clinical cases were reported. MAP shedding was detected in 9.4% of serologically positive animals. Discarding colostrum and prioritised culling of seropositive animals assisted by adoption of standardised serological testing were presumed to have a key role in MAP control, despite the reluctance of some farmers to address hygienic issues and improve the separation of calves from adult animals. The small number of farms included in this study and the fact that these were not randomly selected may limit the generalisability of the findings. The Italian paratuberculosis control plan has provided measures to limit the uncontrolled spread of MAP infection within and between herds by promoting animal trading between farms certified as negative or low risk.

Innate and adaptive immune responses in the intestine of camel (Camelus dromedarius) naturally infected with Mycobacterium avium subspecies paratuberculosis.

The spread of John's disease in camel herds (Camelus dromedarius) has been worldwide reported. Despite extensive studies on Mycobacterium avium subspecies paratuberculosis (MAP) infection in camels, the complete pathogenesis and epidemiology of this infection have not been fully exploited. The objective of the study is focusing on the nature of the immune responses, and the types of the recruited cells were studied in the intestine of naturally infected camels employing immunohistochemistry to analyze the expression of CD335, CD103, CD11b, and CD38 markers. Marked expression of some or all of the markers was observed in the ileum, mesenteric, and supramammary lymph nodes of the old infected camels. The expression of CD335, a well-known natural killer (NK) cell marker, was detected in the mesenteric lymph node, while the dendritic cell (DCs) marker, CD103, was markedly expressed in the villi and propria submucosa (PS) of the ileum in old infected camels. CD103 + and CD11b + DCs were detected in the mesenteric lymph nodes of young infected camels. The expression of CD38, a crucial proinflammatory marker, was more noticeable in the peripheral region of the mesenteric lymph node. The expression of these markers in the infected camel intestine was peculiar and is reported for the first time. In summary, the unique expression patterns of CD335, CD103, CD11b, and CD38 markers in naturally infected camel intestines revealed through immunohistochemistry new insights into the immune responses associated with MAP infection. These first-time observations suggest potential roles of innate and adaptive immunity, highlighting specific aspects of MAP immunopathology. Further studies with targeted tools are crucial for a precise understanding of these markers' roles in the infected intestines.

Mycobacterium avium Subspecies paratuberculosis Infection and Immunopathological Changes in Animals: A Review

Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic granulomatous intestinal disease and causative agent of Johne’s disease (JD) in domestic and wild ruminants. This study explores the intricate dynamics of the host immune response in MAP infection and its implications for disease progression and control. Our investigation revealed the pivotal role of interleukin-10 (IL-10) in mediating the communication between the host’s innate and adaptive immunological responses. IL-10 inhibits interferon-gamma (IFN-γ) secretion, altering the balance between Th1 and Th2 responses. In the early stages, a robust Th1 response marked by increased IFN-γ production is observed, but as the disease progresses, a shift towards a humoral Th2 response occurs. Furthermore, we found that IL-10 exerts suppressive effects on macrophage (Mφ) anti-mycobacterial action, leading to increased intracellular survival of mycobacteria. IL-10 also hampers Mφ antimicrobial activity by suppressing the production of essential cytokines such as IL-12 and tumor necrosis factor alpha (TNF-α), crucial for activating natural killer (NK) cells and inducing differentiation of CD4+ T lymphocytes into Th1 effector cells. Importantly, our study sheds light on the intricate interplay between past infections or concurrent active co-infections and their influence on the immune response to unrelated pathogens in the context of paratuberculosis. This review underscores the multifaceted role of IL-10 in the immunopathological manifestation of MAP infection and host immunomodulation. These findings provide valuable insights for both animal and human research in the fight against MAP infection, emphasizing the importance of balancing immune responses and considering the influence of concurrent infections in disease progression.

Molecular and Serological Detection of Mycobacterium avium Subspecies Paratuberculosis in Ruminants of Jabalpur Region

Background: Paratuberculosis is a chronic infectious disease of wild and domestic ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). The present study aimed to conduct the molecular and serological detection of MAP in ruminants of Jabalpur region. Methods: A total of 261 faecal, 124 blood and 51 tissue samples were collected from goats, buffaloes and cattle of the region irrespective of their age, sex and breed. ZN staining of faecal and tissue samples was performed as a preliminary screening test followed by anti-MAP antibodies detection in serum samples using indirect ELISA and IS900 PCR analysis of ZN positive faecal and tissue samples. Result: A pattern of acid-fast bacilli shedding in faecal samples of ruminants was obtained based on ZN staining with a majority of +1 shedders. In ELISA, 33.87% of serum samples were found positive for anti-MAP antibodies whereas, 22.50% ZN positive faecal samples and 100% ZN positive tissue samples were found positive in IS900 PCR. Therefore, a conclusion of moderately prevalent MAP infection in ruminants of the Jabalpur region can be drawn.

Immunohistochemical Examination of TNF-α and SAA Expressions in Goats Infected with Mycobacterium avium subspecies paratuberculosis

Paratuberculosis (Johne Disease), caused by Mycobacterium avium subspecies paratuberculosis (MAP) and commonly seen in ruminants, is a contagious disease that causes chronic diarrhea and serious economic losses. In this study, the presence of pro-inflammatory cytokine and acute phase protein expressions in lesional tissues of naturally infected goats with MAP was investigated. For this purpose, paratuberculosis suspected goats complaining of chronic diarrhea and excessive weight loss were examined by ELISA method for Mycobacterium avium subspecies paratuberculosis infection. After sacrificing 20 animals found to be infected with MAP, tissue samples taken from the intestines and lymph nodes were stained with Hematoxylin-Eosin (HE) for histopathological examination and Ziehl-Nelsen (ZN) to show acid-fast mycobacterium. Immunohistochemical staining was performed to examine the expressions of TNF-α as a pro-inflammatory cytokine and SAA as an acute phase protein. Macroscopic examination revealed in serous fat atrophy, mesenteric lymphadenitis, and granulomatous enteritis in the intestines. Histopathological examination revealed atrophy in villi, degeneration of enterocytes, inflammatory cell infiltration consisting mostly of mononuclear leukocytes in the mucosa, and epithelioid macrophage aggregation. Lymphoid hyperplasia and epithelioid histocyst aggregates were found in the mesenterial lymph nodes. In staining with ZN revealed the presence of bright red acid-fast agents in the intestine and mesenteric lymph nodes. In immunohistochemical examination, TNF-α and SAA expressions were observed in all samples. With this study, for the first time, the expressions of TNF-α, a proinflammatory cytokine, and SAA, an acute phase protein, were shown in the lesional tissues of MAP-infected animals, contributing to the immunopathogenesis of the disease.

Cross-reactivity between Mycobacterium avium subspecies paratuberculosis 4027 peptide and Human IRF5 may contribute to Multiple Sclerosis in Iranian patients

BackgroundThe etiology of Multiple sclerosis (MS) is complicated and can be affected by several environmental factors, such as Mycobacterium avium subspecies paratuberculosis (MAP) infection in genetically predisposed individuals. The link between MAP and MS depends on host genetic and epigenetic aspects and population-based features that require further investigation. We aimed to study the possible role of MAP in triggering MS using molecular and serological methods. Materials and methodsThis case-control study examined 200 blood samples (100 MS patients and 100 HCs) to search for the MAP-specific IS900 gene. In addition, ELISA was conducted to determine the humoral response against MAP_402718-32 and its human IRF5424–434 peptide homolog. ResultsThe frequency of MAP detection based on the molecular method in MS patients and HCs was 48 % and 13 %, respectively (p < 0.0001). The presence of antibodies against MAP_402718-32 and IRF5424–434 was 55 % and 65 % in MS patients versus 9 % and 7 % in HCs, respectively (p < 0.0001). A good correlation was observed between MAP_4027 and IRF5 antibodies (r = 0.5782, p < 0.0001), indicating that the same antibodies recognized common peptide epitopes. ConclusionOur research revealed a significant association between MAP and MS, highlighting the possible role of MAP as an important infection trigger factor of MS. It is hypothesized that cross-reactivity between MAP4027 and IRF5 may dysregulate immune homeostasis.

Unravelling transmission of Mycobacterium avium subspecies paratuberculosis to dairy calves: results of a lifelong longitudinal study.

Johne's disease (JD) is a chronic disease of ruminants endemic in the UK and other countries and responsible for large economic losses for the dairy sector. JD is caused by Mycobacterium avium subspecies paratuberculosis (MAP), which typically infects calves that remain latently infected during a long period, making early detection of infection challenging. Cow to calf transmission can occur in-utero, via milk/colostrum or faecal-orally. Understanding of the different transmission routes to calves is important in informing control recommendations. Our aim in this longitudinal study was to measure the association between the transmission routes via the dam and the environment on a calf subsequently testing serologically positive for MAP. The study population comprised of 439 UK dairy calves from 6 herds enrolled between 2012 and 2013. These calves were followed up from birth until 2023. At birth individual calf data was captured. During follow-up, individuals entering the milking herd were quarterly tested for the presence of MAP antibodies using milk ELISA. Cox regression models were used to measure the association between exposure from the dam (in-utero and/or colostrum) or from the environment (long time in dirty yard) and time to first detection of MAP infection. An association between calves born to positive dams and probability of having a MAP positivetestresultremainedafter excluding potential MAP transmission via colostrum(Hazardratio:2.24;95% CI:1.14-4.41). Calves unlikely to be infected with MAP via the in-utero or colostrum route, had 3.68 (95% CI: 3.68 1.45-9.33) higher hazard of a positive test result when they stayed longer in a dirty calving area. The effect of the dam infection status on transmission to calves precedes the dam's seroconversion and persists after excluding the potential role of transmission via colostrum. The association betweentime spent in a dirty calving area and probability of a MAP positive test resulthighlights the role of environmental contamination as a source of infection in addition to the dam.

Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle

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.

Serodiagnosis of map infection by latex agglutination test in slaugtered buffaloes of Malwa region (M.P., India)

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of chronic enteritis which is commonly called as Johne’s disease (JD) in animals which is associated with several incurable, auto-immune diseases like Crohn’s disease in human beings. In the current study, 19 serum samples of buffaloes were collected irrespective of their age, sex and breed which were slaughtered at Cantonment board slaughterhouse, Mhow and Nagar Nigam, Indore. These animals were brought from different places of Malwa region of Madhya Pradesh. For detection of anti-MAP antibodies, the latex agglutination test was performed following the standard procedure (Cheong Koo et al., 2004). The present investigation recorded 52.63% MAP infection in slaughtered buffaloes.

Immunopathological mechanisms in the early stage of Mycobacterium avium subsp. paratuberculosis infection via different administration routes in a murine model.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic emaciating disease of ruminants that causes enormous economic losses to the bovine industry, globally. However, there are still remaining clues to be solved in the pathogenesis and diagnosis of the disease. Therefore, an in vivo murine experimental model was tried to understand responses in early stage of MAP infection by oral and intraperitoneal (IP) routes. In the MAP infection size, and weight of spleen and liver were increased in the IP group compared with oral groups. Severe histopathological changes were also observed in the spleen and liver of IP infected mice at 12 weeks post-infection (PI). Acid-fast bacterial burden in the organs was closely related to histopathological lesions. In the cytokine production from splenocytes of MAP-infected mice, higher amounts of in TNF-α, IL-10, and IFN-γ were produced at early stage of IP-infected mice while IL-17 production was different at time and infected groups. This phenomenon may indicate the immune shift from Th1 to Th17 through the time course of MAP infection. Systemic and local responses in the MAP-infection were analyzed by using transcriptomic analysis in the spleens and mesenteric lymph nodes (MLN). Based on the analysis of biological processes at 6 weeks PI in spleen and MLN in each infection group, canonical pathways were analyzed with ingenuity pathway analysis in the immune responses and metabolism especially lipid metabolism. Infected host cells with MAP increased in the production of proinflammatory cytokines and reduced the availability of glucose at early stage of infection (p < 0.05). Also, host cells secreted cholesterol through cholesterol efflux to disturb energy source of MAP. These results reveal immunopathological and metabolic responses in the early stage of MAP infection through the development of a murine model.

Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.