Paratuberculosis Infection Research Articles

Preliminary exploration of mRNA, lncRNA, and miRNA expressions in the bovine jejunum unveils novel aspects of Mycobacterium avium subspecies paratuberculosis infections

BackgroundParatuberculosis (Johne’s disease, JD) is a chronic and enteric disease in a range of ruminants, often caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, leading to substantial economic losses worldwide. Yet, the molecular underpinning of paratuberculosis remains elusive. Here we performed RNA sequencing (RNA-seq) and small RNA sequencing (sRNA-seq) of the jejunum tissues from the Holstein cows with three distinct statuses of paratuberculosis, i.e., healthy, subclinical, and clinical to screen potential genes, lncRNAs, and miRNAs associated with the resistance or susceptibility to MAP infection and build ceRNA regulatory networks via miRNAs.ResultsWe applied whole transcriptome sequencing analysis to examine the jejunum tissue in nine Holstein cows. Starting with 19,994 expressed genes, 13,529 lncRNAs, and 735 miRNAs, we screened out differentially expressed genes (DEGs), lncRNAs, and miRNAs of three comparison groups, i.e., clinical vs. healthy, subclinical vs. healthy, and clinical vs. subclinical, subsequently identifying ceRNA pairs. Ultimately, we detected 76, 74, and 24 DEGs, 19, 39, and 10 lncRNAs, as well as 28, 61, and 20 miRNAs across the three comparison groups, respectively. Through integrating these DEGs with functional annotation, previously reported QTLs, and GWAS results, we proposed eight genes (LYZ, LYZ1, BOLA-DQB, BOLA-DQA1, TAP, CATD, VNN1, and PPARG), six lncRNAs, 48 miRNAs, and 107 ceRNA pairs implying their potential associations with susceptibility to MAP infection.ConclusionThe present study provided a global view of the dynamics in transcriptomes of the bovine jejunum tissues in terms of JD status. Our results demonstrated that not only mRNAs but also lncRNAs and miRNAs played important roles in regulating MAP infection in dairy cattle. This study provided a valuable resource for understanding the molecular basis of JD, potentially contributing to the genetic improvement of JD resistance in dairy cattle.

Bayesian estimation of diagnostic accuracy of fecal smears, fecal PCR and serum ELISA for detecting Mycobacterium avium subsp. paratuberculosis infections in four domestic ruminant species in Saudi Arabia.

Paratuberculosis, a chronic wasting disease affecting domestic and wild ruminants worldwide, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Various diagnostic tests exist for detecting MAP infection; however, none of them possess perfect accuracy to be qualified as a reference standard test, particularly due to their notably low sensitivity. Therefore, we used Bayesian latent class models (BLCMs) to estimate diagnostic accuracy of fecal smears (FS), fecal PCR and serum ELISA for detecting MAP infections in sheep, goats, cattle, and camels older than 2 years in Saudi Arabia. Data from a cross-sectional study conducted in the Eastern Province of Saudi Arabia on 31 different farms with a history of MAP infection were analyzed. Fecal and blood samples from all animals older than 2 years in each farm were collected, resulting in a total of 220 sheep, 123 goats, 66 cattle, and 240 camels sampled. FS and IS900-PCR were performed on fecal samples to detect acid-fast bacilli and MAP DNA, respectively. The IDEXX ELISA kit was used to detect MAP antibodies in serum samples. For each ruminant species population, a BLCM was fitted to obtain posterior estimates [medians and 95 % Bayesian credible intervals (95 % BCI)] for sensitivity (Se) and specificity (Sp) of the three tests. We assumed FS and PCR to be conditionally dependent on the true animal MAP status. Prior distributions for test accuracy were used if available. FS had the highest Se among all tests and across all species with median values around 80 % in sheep, goats and camels, and near 50 % in cattle. Median Sp estimates of ELISA and PCR were higher than 90 % for all species. FS yielded the lowest Sp of the study when applied in camels, sheep, and goats. Using the prevalence observed in this study, median positive predictive value (PPV) was higher for PCR and ELISA than FS for camels, sheep, and goats. In cattle, PPV of all tests was similar with median estimates > 95 %. In camels, sheep, and goats, median negative predicative value (NPV) of all tests were > 60 %. The lowest median NPV for all tests were observed in cattle (<30 %). Our results suggest that ELISA is a suitable option to identify MAP infected animals in farms with previous history of MAP in the Eastern region of Saudi Arabia.

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.

A review on Paratuberculosis (John’s disease) current diagnostic approaches and future prospects

The objective of the present study is to review the current diagnostic tools used for detection of paratuberculosis infection in animals. It also showed the sensitivity and specificity of each test as well as the difficulty of fecal culture as gold standard technique for paratuberculosis infection diagnosis. It also reviewed the limitation of commercial Elisa tests as they cannot be used for diagnosis of animals at early stages of infection. Lateral flow test has been recently developed. We suggest it could be used as rapid, accurate useful tool for diagnosis of MAP infection.

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.

Retrospective Single Nucleotide Polymorphism Analysis of Host Resistance and Susceptibility to Ovine Johne's Disease Using Restored FFPE DNA.

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.

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.

Differentiation of Bovine Tuberculosis and Paratuberculosis Infections with Antemortem Diagnostic Methods

In this study, based on the results of tuberculin skin tests (Bovine and Avian PPD) used in the antemortem diagnosis and differentiation of Bovine Tuberculosis, the animals in the farms with suspected Tuberculosis were serologically examined to diagnose Paratuberculosis infection and fecal bacterioscopy was performed. In addition, it was aimed to obtain data that will contribute to the eradication studies of Bovine Tuberculosis disease by comparing the antemortem diagnostic methods of Bovine Tuberculosis disease, which is endemic in Türkiye and by determining the sensitivity and specificity values of the interferon gamma (IFN-γ) test. In this context, intradermal tuberculin test was applied to 423 cattle with suspected Tuberculosis in a total of 5 dairy cattle farms, one each from Çankırı, Çorum, Ankara, Eskişehir and Konya regions, and this test was determined as the gold standard method and the sensitivity and specificity of the IFN-γ test were determined as 86% and 97%, respectively. For the diagnosis of Paratuberculosis infection, antibody ELISA, fecal bacterioscopy and IFN-γ ELISA were performed on these animals and the prevalence of these tests were 10.4%, 5.44% and 4.96% respectively and 4 (0.95%) of the cattle were positive for each of the diagnostic methods for Map infection. As a result, it was concluded that IFN-γ test, which gives similar results to intradermal tuberculin test results, can also be used in the antemortem diagnosis of Bovine Tuberculosis. Also, in the comparative intradermal tuberculin test for the diagnosis of Tuberculosis infection, avian PPD positive animals were found to play a decisive role in the detection of nonspecific reactions or Paratuberculosis infected animals, supported by other tests used for the diagnosis of Paratuberculosis.

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.

The role of Mce proteins in Mycobacterium avium paratuberculosis infection

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention.

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.

No Evidence of Neutrophil Response Modulation in Goats after Immunization against Paratuberculosis with a Heat-Inactivated Vaccine.

Neutrophils are believed to play a role in the initial stages of paratuberculosis, and it has recently been demonstrated that vaccination can modulate their function via priming or through epigenetic and metabolic reprogramming (training). Modulation of the neutrophil response against Mycobacterium avium subspecies paratuberculosis (Map) through vaccination has been demonstrated in a rabbit model but not in ruminants. Therefore, in the present work, the effect of vaccination on the response of caprine neutrophils against Map was studied. Neutrophils were isolated from non-vaccinated (n = 7) and Gudair®-vaccinated goat kids (n = 7), before vaccination and 30 days post-vaccination. Then, several neutrophil functions were quantified ex vivo: cell-free and anchored neutrophil extracellular trap (NET) release, phagocytosis, and the differential expression of several cytokines and TLR2. The induction of cell-free NETosis and TLR2 expression by Map is reported for the first time. However, vaccination showed no significant effect on any of the functions studied. This suggests that the protection conferred by Gudair® vaccination is based on mechanisms that are independent of the neutrophil function modulation. Further research into the impact of alternative vaccination strategies or the paratuberculosis infection stage on ruminant neutrophil function could provide valuable insights into its role in paratuberculosis.

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.

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.

Large-scale serological survey on Mycobacterium avium subsp. paratuberculosis infection in sheep and goat herds in Sicily, Southern Italy.

Paratuberculosis (PTB) is a worldwide chronic, contagious enteric disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) mainly affecting ruminant species. PTB is a WOAH-listed disease with direct and indirect economic losses in the livestock sector, negative impact on animal welfare and significant public health concerns. In spite of this, MAP prevalence in small ruminants is still unknown and the prevalence appears to be underestimated in many countries. The aim of this study is providing a first large-scale serological survey on MAP infection in small ruminants in Sicily, a region of Southern Italy with the 11.3 and 8.9% Italian national heritage of sheep and goats, respectively. For this purpose, we analyzed a total of 48,643 animals reared in 439 flocks throughout Sicily. MAP seroprevalence was estimated both at herd-level and animal-level within breeds reared in all the nine sampled provinces. Our results revealed a high overall apparent prevalence at herd-level of 71.8% in sheep and 60.8% in goat farms with an animal-level prevalence of 4.5 and 5.1% in sheep and goats, respectively. Significant statistical differences were found between the provinces and within the breeds both in sheep and goats. Our study provides the first large-scale serological survey on PTB infection in small ruminants in Sicily and showed a high prevalence of disease depending to the species, breed and province. This study represents the first step to better understand the MAP epidemiology in a typical Mediterranean breeding context, suggesting the need of in-depth study on the herds risk factors, including the eventual presence of candidate genes for resistance/susceptibility to PTB in native breeds.

First serological diagnosis of Mycobacterium avium subsp. paratuberculosis infection in sheep in the state of Pernambuco, Brazil.

This study aimed to diagnose Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep in the state of Pernambuco, Brazil. A total of 276 blood samples were analyzed using the enzyme-linked immunosorbent assay IDEXX Paratuberculosis Screening kit, and 261 fecal samples were submitted for bacterial culture and polymerase chain reaction tests. An animal-level sero-frequency of 0.72% (n = 2/276) and a farm-level sero-frequency of 20% (n = 2/10) were found. All fecal sample cultures were negative, and molecular analyses were also negative. To the best of our knowledge, this is the first study of MAP infection in sheep in the state of Pernambuco and one of the pioneers in the country. It is an asymptomatic disease that is difficult to diagnose in this species because the susceptibility of sheep to the organism is lower than that of other ruminant species. However, the sero-frequency found reveals that there is MAP exposure in sheep flocks in the region. In addition, serological monitoring can contribute to the observation of the organism's behavior in herds. Our results support the potential risk of MAP infection in sheep in the state of Pernambuco, Brazil.

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.

Microbiological characteristics and antibiotic sensitivity of &lt;i&gt;Mycobacterium avium&lt;/i&gt; subsp. &lt;i&gt;paratuberculosis (MAP)&lt;/i&gt; isolated on the territory of Western Siberia

The results of the studies of microbiological features of cultures of standardized and clinical strains isolated from biological material of the animals on the territory of Western Siberia belonging to Mycobacterium avium subsp. paratuberculosis (MAP) are presented. Microbiological studies of the pathogens consisted of the bacterioscopic method (staining of smears of cultures according to Ziehl – Neelsen) and the culture method (processing of biomaterial by the method of A.P. Alikaeva with subsequent sowing of the obtained sediment on Lowenstein-Jensen and Finn-2 egg nutrient media with mycobactin). In addition, biochemical tests with the cultures isolated from the material were used, including examination of colony growth at 30, 37 and 42 C, on the medium with sodium salicylate, with 5% sodium chloride, nitrate reduction, determination of amidase, catalase, arylsulfatase activity, hydrolysis of Tween-80 and a biological method consisting of intravenous infection with suspensions of standardized and clinical strains of non-linear white mice. The results of the studies showed that the cultures of clinical strains belonged to the 3rd group of mycobacteria according to the Runyon classification and their properties were identical to the standardized strain of M. paratuberculosis, which allows us to attribute them to mycobacteria of paratuberculosis. Antibiotic sensitivity studies of the standardized strain of M. paratuberculosis (Central-Lubinsky) and clinical strains revealed their susceptibility to all the drugs used in the studies. In a biological assay, mice infected with paratuberculosis pathogens had lower body weight than in the control groups. Autopsy revealed enlargement of lungs, spleen and liver, single purulent foci on liver, spleen and mesentery, and the liver was marbled, the mucosa of the small intestine is not changed. The growth intensity of cultures from the biomaterial (lungs, liver, spleen) was 2(++) to 3(+++) to 4(++++) points, the growth intensity of the cultures from the mucosa of the small intestine is 0(+/-) points. Bacterioscopic examination of smears of colony cultures of the studied pathogens isolated from the internal organs of mice of the experimental groups and stained by Ziehl – Neelsen staining showed the presence of single acid-fast granular bacilli arranged in groups or in the form of a "palisade", which is characteristic of the causative agent of paratuberculosis. The biological method of research on laboratory animals revealed the susceptibility of nonlinear white mice to the tested cultures and the possibility of reproducing experimental paratuberculosis infection on them.

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

The 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. This case-control study examined 200 blood samples (100 MS patients and 100HCs) 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. The 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. Our 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.

Vaccination of White-Tailed Deer with Mycobacterium bovis Bacillus Calmette-Guérin (BCG): Effect of Mycobacterium avium ssp. paratuberculosis Infection.

In many parts of the world, bovine tuberculosis eradication efforts are hampered by wildlife reservoirs of Mycobacterium bovis, which serve as a constant source of M. bovis for nearby cattle. The human tuberculosis vaccine, M. bovis BCG has been investigated for use in several wildlife species, including deer. In the US, white-tailed deer in Michigan have been the source of infection for over 82 cattle herds since M. bovis was discovered in free-ranging deer in 1995. The efficacy of BCG may be influenced by many factors, including prior exposure or infection with non-tuberculous mycobacteria, that is, species other than members of the M. tuberculosis complex. M. avium subspecies paratuberculosis (Map) infection is not uncommon in ruminants such as deer. Using natural exposure to Map and experimental infection with M. bovis, we demonstrate that Map infection increased BCG vaccine efficacy as measured by lesion severity scores.