Mycobacterium Avium Subspecies Paratuberculosis Infection Research Articles

MAP template controlling Crohn’s disease?

Crohn’s disease is stated to be an immune-mediated disease initiated by Mycobacterium avium subspecies paratuberculosis (MAP) infection within a period of host immunological vulnerability and induced by the widespread prevalence of that organism in the human food chain. For over two decades, steroids and biologics have, move often than not, been able to induce temporary remissions, but not cures. This thesis examines how the only two, very divergent, therapeutic approaches that have produced cures achieved this positive outcome.

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

Assessment of lipid profile and acute phase protein in Mycobacterium avium subspecies paratuberculosis infected and healthy goats

Present study is based on 24 goats that were located in goat herds endemically infected with Mycobacterium avium subspecies paratuberculosis (MAP) infection. Objective of the study was to access the variations in the lipid profile and acute phase proteins in the serum samples driven from non-infected (negative and physically healthy) and infected (positive and physically weak) goats with MAP infection, cause of incurable Johne’s disease (JD) in domestic livestock. Infected goats had significantly higher cholesterol and albumin levels and significantly ‘reduced level’ of high density lipoprotein (HDL) and ‘reduced level’ of the density of lipoproteins (LDL) in comparison to non-infected goats. Lipid profile and acute phase proteins could be further explored for their significance in pathogenesis and diagnosis of JD in domestic livestock including goats.

23 Differential microRNA expression in jejunal tissue and jejunal lymph nodes following naturally occurring Mycobacterium avium subspecies paratuberculosis infection in Holstein cows

Abstract Mycobacterium avium subsp. Paratuberculosis (MAP) is the causal agent of Johne’s disease (JD), a chronic intestinal disease affecting ruminants worldwide. This study investigated miRNA expression in jejunal intestine (JE) and jejunal lymph nodes (JELN), and the potential regulatory roles of miRNAs during JD progression. JE and JELN tissues were collected from 5 MAP positive (JD subclinical stage) Holstein cows and 5 MAP negative cows. Following miRNA sequencing, bioinformatic processing with a standard pipeline and functional analysis with ClueGo, 272 and 333 miRNAs were identified in JE and JELN, respectively. Compared with MAP negative cows, 13 and 71 miRNAs were differently expressed (DE) (P < 0.05) in MAP infected JE and JELN, respectively. The most up-regulated and down-regulated miRNAs were bta-miR-485 (fold change = 6.18) and bta-miR-451 (fold change = -6.81), and bta-miR-331-5p (fold change = 35.56) and bta-miR-2285bk (fold change = -61.25) in JE and JELN, respectively. In JE, some DE miRNAs (miR-147 and miR-199a-5p) have been associated with glucose metabolism while several DE miRNAs in JELN have associations with immune response to disease (e.g. miR-146a and miR-146b have been associated with bovine mastitis and miR331-5p and miR-184 are important for human cancers). Target genes of JELN DE miRNAs were enriched (P < 0.05) for more gene ontology terms (n = 180) and KEGG pathways (n = 123) as compared with JE (n = 90 and 25, respectively). Furthermore, JELN DE miRNAs were uniquely enriched in several immune related pathways including immune response regulating/activating signal transduction, T-cell/B-cell receptor signaling, Toll-like receptor signaling, TNF signaling pathways, etc., suggesting potential regulatory roles for JELN DE miRNAs in the host immune response to JD. In addition, interactions between DE miRNAs and DE mRNAs (expressed in the same samples) demonstrated important functions for miR-2284/2285 family members in JELN response to MAP infection. In conclusion, these results suggest site specific regulatory roles for miRNAs during MAP infection.

Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy

Quantification of Mycobacterium avium subspecies paratuberculosis (MAP) during in vitro infection experiments is challenging due to limitations of currently utilised methods, such as colony counting. Here we describe quantifying MAP infection of bovine macrophages (Mφ) using confocal microscopy. Bovine monocyte derived macrophages were infected with MAP at a high or low dose and the number of intracellular bacteria calculated at 2 h post infection using confocal microscopy. Bacteria within simultaneously infected Mφ were quantified by colony counting in order to compare confocal microscopy results with results obtained by an established method. Confocal microscopy provided a robust alternative quantification method that allowed for assessment of the infection at the individual Mφ level. This demonstrated that MAP infection was not homogeneous, and that there were higher numbers of both infected Mφ and intracellular bacteria and bacterial aggregates at the high dose compared to the low dose, potentially impacting the Mφ response to infection. Confocal microscopy can therefore provide a level of detail regarding the infection unobtainable by other quantification methods.

Inflammatory Th17 responses to infection with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle and their potential role in development of Johne’s disease

Chronic intestinal inflammation typically associated with late stage Johne’s disease (JD) in cattle occurs despite a lack of significant expression of the typical proinflammatory cytokines IFNγ and TNFα derived from Th1- like T cells. In contrast, these cytokines appear to be relatively abundant during early infections with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of JD in cattle. The roles of non-classical immune responses, such as those associated with Th17 cells, in response to MAP infection and development of clinical JD are less clear. In this review, we examine literature suggesting that Mycobacterial infections, including Mycobacterium tuberculosis, Mycobacterium bovis, and MAP, are all associated with expression of Th17 promoting cytokines (IL-23, IL-22, IL-17a). We discuss the possibility that Th17 associated cytokines, particularly IL-23, may act as contributing factors in development and maintenance of inflammation characteristic of clinical JD. An as yet relatively unexplored source of chronic inflammation due to over expression of IL-1α and IL-1β is also presented. We further discuss the fact that, as with the typical Th1-like cytokines IFNγ and TNFα , IL-17a is not significantly expressed in CD4+ T cells from cows with clinical JD, possibly due to T cell exhaustion. Finally, we present the notion that the Th17 driving cytokine IL-23 expressed by infected macrophages and associated epithelial cells may contribute to chronic inflammation during later stages of JD.

Mycobacterium avium sp. paratuberculosis (MAP) induces IL-17a production in bovine peripheral blood mononuclear cells (PBMCs) and enhances IL-23R expression in-vivo and in-vitro

Johne’s disease (JD) is a chronic inflammatory gastrointestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of JD is difficult largely due to insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines. Correlates of protection are lacking in model systems of JD and the sources of inflammation due to JD are not well characterized. Commonly studied immune responses, such as the Th1/Th2 paradigm, do not adequately explain host responses to MAP. A potential role for non-classical immune responses to MAP, such as that mediated by Th17 cells, has been suggested. Indeed, MAP antigens induce mRNAs encoding the cytokines IL-23 and IL-17a in bovine peripheral blood mononuclear cells (PBMCs). IL-23 and IL-17a production have both been associated with Th17-like immune responses. Th17 cells are also defined by surface expression of the IL-23 receptor (IL-23R). To determine the relative prevalence of potential Th17 cells in PBMCs from MAP test positive and MAP test negative cows, PBMCs were isolated and analyzed by immunostaining and flow cytometry. Fresh PBMCs from MAP test positive cows (n = 12) contained a significantly higher proportion of IL-23R positive cells in populations of CD4+, CD8+, and Yδ + T cells than in cells from MAP test negative cows (n = 12; p < 0.05). Treatment with MAP antigens increased the percentage of all T cell subsets with surface expression of IL-23R when compared to untreated (n = 12; p < 0.05) cells. ELISA results for IL-17a secretion revealed a higher concentration of IL-17a secreted from PBMCs treated with MAP antigen (n = 20) than from PBMCs not treated with MAP antigens (n = 20) (p < 0.001), regardless of the JD test status of source cows. Also, we observed a moderate negative correlation between JD diagnostic scores for JD + cows and plasma IL-17a concentration (n = 42; r = −0.437; p-value < 0.004). Plasma with low and mid JD- scores (n = 31; n = 9; 0.1 ≤ X < 0.3) had significantly more IL-17a when compared to plasma with high JD- scores (n = 10; 0.3 ≤ X < 0.46; p-values < 0.05). Similarly, plasma with low JD + score values (0.55 ≤ X < 1.0; n = 9) had significantly more IL-17a when compared to plasma with high JD + score values (X ≥ 2.0; n = 21; p < 0.05). Overall, plasma from JD + cows (0.55 < X ≤ 2.86; n = 41) had significantly less IL-17a than plasma from JD- cows (0 < X ≤ 0.46; n = 70). Our data suggests that Th17-like cells may indeed play a role in early immune responses to MAP infection and development or control of JD.

The study of epizootic sera obtained from ruminant animals in complement fixation test (CFT) with the use of Paratuberculous antigen

The paper presents results of the study of epizootic blood sera in the complement fixation test (CFT) with paratuberculous antigen. Blood sera were sampled from the cattle and goats. The antigen was produced from the culture filtrate of Mycobacterium avium subspecies paratuberculosis (MAP) in the laboratory for tuberculosis study. The aim of the present study was to clarify the epizootic situation concerning Johne’s disease among the dairy cattle in different regions of Ukraine. To achieve this aim the blood sera from cattle and goats were collected from farms in different regions of Ukraine. Those sera samples were studied in the complement fixation test with the use of paratuberculous antigen that was produced from the culture filtrate of MAP. The above mentioned blood sera were collected from the cattle that had positive allergic reactions on the use of tuberculin (PPD) for mammals. Those animals belonged to the free from tuberculosis and paratuberulosis milk farms. The study of obtained samples of blood sera was conducted in the accordance with the methodological guidelines “Laboratory diagnostics of paratuberculosis” (shutter. NMR FEFU pr. No. 1, dated December 19, 2014). There were studied 1098 blood sera samples from cattle. In addition to this, investigation was conducted on 24 samples of blood sera from goats. As the result of conducted study it was found that 17 samples of blood sera contained specific antibodies against MAP (serum solution 1:10). These blood sera collected from the cattle belonging to 4 farms in Poltava, Donetsk and Khmelnitsky regions. Along with this it was obtained 9 uncertain results in compliment fixation test that was conducted between paratuberculous antigen (ACF) and blood sera from those 4 farms. The results of monitoring studies indicate that M. avium subsp. paratuberculosis pathogen circulates in studied farms. This can lead to the complication of the epizootic situation regarding paratuberculosis and contribute to the spreading of this pathogen to other free from MAP infection farms. There are no anti-paratuberculosis antibodies in blood serum from goats. It is necessary to conduct annual monitoring serological studies of productive dairy cattle and imported animals in order to clarify and control epizootic situation concerning paratuberculosis on the territory of Ukraine

Is the transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection through milk intended to feed calves an overlooked item in paratuberculosis control programs?

As in many parts of the world, Chile shows a high Mycobacterium avium subsp. paratuberculosis (MAP) infection rate. Evidently, the control recommendations have been inefficient. In the author's opinion, the potential risk of MAP transmission to susceptible calves through milk consumption is largely overlooked. Two observational studies were performed, one to confirm MAP shedding in young stock in a high MAP-infected dairy herd and MAP load in milk intended to feed these calves was estimated. In parallel, in a second study, we estimated the relationship between MAP herd seroprevalence and the cow MAP shedding level as well as the presence of this pathogen in milk used to feed dairy calves. In the first study, 53.7% of cows and 22.5% of calves showed positive culture results. Among all cows tested, 9 (2.19%) animals had a positive serum ELISA, and MAP load in milk reached 106bacteria/mL. In the second study, three seroprevalence categories were established as follows: high (≥ 9%), medium (> 5% and ≤ 9%), and low (≤ 5%). Statistical significant differences among these categories were observed. Animals from the high seroprevalence category shed significantly more MAP than the others. However, in the low category, heavy shedder animals were also observed. Finally, in all study herds, MAP presence in milk intended to feed calves was reported, even from herds without ELISA-positive animals.

Epidemiology and risk factors of Mycobacterium avium subspecies paratuberculosis in semi-extensive dairy sheep and goat farms of Apulia, southern Italy

Paratuberculosis (PTB) is a well-documented chronic and sometimes fatal infection that affects the small intestine of ruminants both in captive and free-ranging living conditions. Although the infection has been detected on small ruminant farms worldwide, epidemiological and risk factor information regarding Mycobacterium avium subspecies paratuberculosis (MAP) infection on semi-extensive sheep and goat farms is generally still scarce, particularly in the Mediterranean area and in Italy. This paper reports the epidemiological findings and risk factors of the infection on semi-extensive sheep and goat farms in Apulia. It particularly focuses on the involvement of biological, structural and management factors, as well as the farmers’ socioeconomic data as predisposing causes of PTB, and investigates their possible connection with the survivability of the infection.The true seroprevalence values of MAP on semi-extensive Apulian sheep and goat farms are reported and the risk factors causing the spread of infection analyzed. Data were collected through a two-year survey over the whole regional area, involving 419 farms, 16,903 sheep and 9369 goats. The epidemiological results showed a true seroprevalence of 66.2% for flocks and of 9.7% at the animal level. Analysis of the risk factors showed that the spread of infection occurs via several concomitant biological, managerial, and farmer-related factors.The survey highlighted the need for urgent and suitable control plans providing guidelines to help Apulian farmers and their veterinarians contain and eliminate the disease, prevent biological risks for animal and humans as well as negative economic effects on the regional livestock sector.

Gene expression profiles during subclinical Mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome

Paratuberculosis in ruminants is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP) however exposure does not predetermine progression to clinical disease. The pathogenesis incorporates a subclinical phase during which MAP is capable of evading host immune responses through adaptation of host cellular immune mechanisms. Presented are results of transcriptomic analysis of Merino sheep experimentally exposed to MAP and repeatedly sampled over the subclinical phase, identifying genes consistently changed over time in comparison to unexposed controls and associated with different disease outcomes. MAP exposed sheep were classified as diseased 45% (n = 9) or resilient 55% (n = 11). Significant gene expression changes were identified in the white blood cells of paucibacillary (n = 116), multibacillary (n = 98) and resilient cohorts (n = 53) compared to controls. Members of several gene families were differentially regulated, including S100 calcium binding, lysozyme function, MHC class I and class II, T cell receptor and transcription factors. The microarray findings were validated by qPCR. These differentially regulated genes are presented as putative biomarkers of MAP exposure, or of the specified disease or resilience outcomes. Further, in silico functional analysis of genes suggests that experimental MAP exposure in Merino sheep results in adaptations to cellular growth, proliferation and lipid metabolism.

Nilotinib: A Tyrosine Kinase Inhibitor Mediates Resistance to Intracellular Mycobacterium Via Regulating Autophagy.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne’s disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population.

Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence of PTB in Indian cattle. A total of 213 cattle, were subjected to multiple diagnostic tests viz. Johnin PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P < 0.01) different in case-control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.

HEMATO-BIOCHEMICAL AND MOLECULAR MARKERS (IS900) OF CATTLE INFECTED WITH JOHNE’S DISEASE IN EGYPT

In Egypt, limited studies were performed to investigate Jhon’s disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP). This study was concerned with diagnosis of MAP in cattle dairy farms using indirect-ELISA technique and amplification of IS900 using PCR technique for confirmation of infection and investigated adequate diagnostic markers for MAP through determining hemato-biochemical profile in cattle dairy farms. Screening of MAP was done by indirect ELISA between the periods from January 2017 to June 2018. A total of 200 cows suffered from persistent diarrhea (n=17) and apparently healthy cows in contact with them (n=183) were used in this study. Positive JD was reported in 62 cases (31%) and negative JD was reported in 138 cases (69%). Out of the positive JD, 17 cases were clinically affected by persistent diarrhea and weight loss (clinical JD), where 45 cases were apparently healthy (sub-clinical JD). Typical colonies of MAP were isolated from the fecal samples of clinically affected cows with positive staining of MAP bacilli. Amplification of IS900 using PCR technique confirmed the presence of MAP infection. Phylogenetic analysis revealed identical similarities with MAP strains in India and Egypt. Hemato-biochemical investigations revealed significant decrease in RBCs, PCV, total protein, globulin and glucose with non-significant change in oxidative stress markers in clinically affected cows. This study is considered one of the limited researches that highlight the role of MAP infection in dairy farms in Egypt.Key words: dairy cattle; ELISA; IS900; Johne’s disease; MAP

Mycobacterium avium subspecies paratuberculosis is able to manipulate host lipid metabolism and accumulate cholesterol within macrophages

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Closely related pathogenic mycobacteria such as M. tuberculosis are capable of altering host lipid metabolism, highlighting the need to explore the role of lipid metabolism contributing to intracellular survival. This study aimed to identify whether MAP is able to manipulate host lipid metabolic pathways and accumulate host cholesterol during early infection. Macrophages were exposed to four different MAP strains and non-pathogenic M. phlei for up to 72 h, with changes to lipid metabolism examined using fluorescent microscopy and gene expression. MAP-infected macrophages displayed strain-dependent differences to intracellular cholesterol levels during early infection, however showed similarly increased intracellular cholesterol at later timepoints. Gene expression revealed that MAP strains similarly activate the host immune response in a conserved manner compared to M. phlei. MAP significantly upregulated host genes associated with lipid efflux and endocytosis. Moreover, lipid biosynthesis genes were differentially regulated in a strain-dependent manner following MAP infection. Collectively, these results demonstrate that MAP manipulates host lipid metabolism during early infection, however the extent of these modulations are strain-dependent. These findings reflect a conserved pathway contributing to intracellular MAP survival.

Antimicrobial treatment with the fixed-dose antibiotic combination RHB-104 for Mycobacterium avium subspecies paratuberculosis in Crohn’s disease: pharmacological and clinical implications

ABSTRACTIntroduction: Crohn’s disease (CD) is an inflammatory bowel disease of unknown etiology. However, increasing evidence suggests Mycobacterium avium subspecies paratuberculosis (MAP) as a putative causative agent: 1) MAP is the etiological agent of Johne’s disease, a granulomatous enteritis affecting ruminants, which shares clinical and pathological features with CD; 2) MAP has been detected in tissues and blood samples from CD patients; 3) case reports have documented a favorable therapeutic response to anti-MAP antibiotics.Area covered: This review provides an appraisal of current information on MAP characteristics, diagnostic methodologies and emerging drug treatments. The authors focus on RHB-104, a novel oral formulation containing a fixed-dose combination of clarithromycin, clofazimine and rifabutin, endowed with synergistic inhibitory activity on MAP strains isolated from CD patients.Expert opinion: Based on encouraging in vitro data, RHB-104 has entered recently the clinical phase of its development, and is being investigated in a randomized, placebo-controlled phase III trial aimed at evaluating its efficacy and safety in CD. Provided that the overall clinical development will support the suitability of RHB-104 for inducing disease remission in CD patients with documented MAP infection, this novel antibiotic combination will likely take a relevant position in the therapeutic armamentarium for CD management.

Baseballs, tennis balls, livestock farm manure, the IDH1 mutation, endothelial cell proliferation and hypoxic pseudopalisading (granulomatous) necrosis: Mycobacterium avium subspecies paratuberculosis and the epidemiology, cellular metabolism and histology of diffuse gliomas, including glioblastoma.

An increased rate of diffuse gliomas, including glioblastoma, has been noted in livestock farmers in Western countries. Some researchers have suggested that a zoonotic virus or bacteria present in the livestock animal’s feces or manure may be a possible etiologic factor. Mycobacterium avium subspecies paratuberculosis (MAP), the cause of a chronic enteropathy in domestic livestock and a probable zoonosis, is heavily excreted in an infected animal’s feces or manure, contaminating soil and ground on the animal’s farm. Once excreted in an animal’s feces, MAP lasts indefinitely in a dormant but viable form, and easily spreads outside farms to the surrounding environment. MAP’s presence throughout the soil in countries where MAP infection of domestic livestock is extensive and long-standing may explain the increased rates of glioblastoma in tennis and baseball players who handle balls coated with MAP-contaminated dirt. MAP infection is consistent with glioblastoma’s two defining histopathologic characteristics: endothelial cell proliferation and pseudopalisading necrosis. MAP is a non-tuberculous or atypical mycobacterium, which can cause hypoxic necrotizing granulomas, granulomas that resemble areas of pseudopalisading necrosis. There are known bacterial causes of endothelial cell proliferation. Almost unique amongst intracellular bacteria, MAP’s variant isocitrate dehydrogenase 1 (IDH1) enzyme, a type 2-oxoglutarate ferredoxin oxidoreductase, can use a host cell’s cytosolic α-ketoglutarate in its own Krebs or tricarboxylic acid cycle. MAP’s ability to use a host cell’s α-ketoglutarate may explain the survival advantage of the cytosolic IDH1 enzyme mutation for patients with diffuse gliomas including glioblastoma, astrocytoma, and oligdendroglioma, a mutation that results in a reduced supply of cytosolic α-ketoglutarate. MAP may therefore be one possible infectious cause of glioblastoma and the other histologic categories of diffuse glioma.

Genome-wide association study of Mycobacterium avium subspecies Paratuberculosis infection in Chinese Holstein

BackgroundParatuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle.ResultsUsing Illumina Bovine 50 K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50 K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P < 5 × 10− 5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1 Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5 Mb on BTA23, where the TDP2 gene was anchored.ConclusionsIn conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

Low accuracy of Bayesian latent class analysis for estimation of herd-level true prevalence under certain disease characteristics—An analysis using simulated data

Estimation of the true prevalence of infected individuals involves the application of a diagnostic test to a population and adjusting according to test performance, sensitivity and specificity. Bayesian latent class analysis for the estimation of herd and animal-level true prevalence, has become increasingly used in veterinary epidemiology and is particularly useful in incorporating uncertainty and variability into analyses in a flexible framework. However, the approach has not yet been evaluated using simulated data where the true prevalence is known. Furthermore, using this approach, the within-herd true prevalence is often assumed to follow a beta distribution, the parameters of which may be modelled using hyperpriors to incorporate both uncertainty and variability associated with this parameter. Recently however, the authors of the current study highlighted a potential issue with this approach, in particular, with fitting the distributions and a tendency for the resulting distribution to invert and become clustered at zero. Therefore, the objective of the present study was to evaluate commonly specified models using simulated datasets where the herd-level true prevalence was known. The specific purpose was to compare findings from models using hyperpriors to those using a simple beta distribution to model within-herd prevalence. A second objective was to investigate sources of error by varying characteristics of the simulated dataset. Mycobacterium avium subspecies paratuberculosis infection was used as an example for the baseline dataset. Data were simulated for 1000 herds across a range of herd-level true prevalence scenarios, and models were fitted using priors from recently published studies. The results demonstrated poor performance of these latent class models for diseases characterised by poor diagnostic test sensitivity and low within-herd true prevalence. All variations of the model appeared to be sensitive to the prior and tended to overestimate herd-level true prevalence. Estimates were substantially improved in different infection scenarios by increasing test sensitivity and within-herd true prevalence. The results of this study raise questions about the accuracy of published estimates for the herd-level true prevalence of paratuberculosis based on serological testing, using latent class analysis. This study highlights the importance of conducting more rigorous sensitivity analyses than have been carried out in previous analyses published to date.

Comparative diagnosis of Mycobacterium avium subspecies paratuberculosis in the tissues of clinical and subclinical sheep of paratuberculosis endemic farm

Paratuberculosis (Johne’s disease) is the chronic infectious granulomatous enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis. Due to the lack of definite diagnostic tests, the early detection of the disease is difficult. The present study was undertaken to assess comparative efficacy of diagnostic methods routinely used in the identification and confirmation of Mycobacterium avium subspecies paratuberculosis (MAP) in the tissue samples of naturally infected clinical and subclinical sheep. The ileum and mesenteric lymph node (MLN) tissues were collected from 20 paratuberculous sheep from organized farm of Rajasthan. These animals were further classified as paucibacillary (PB) (n = 8) or multibacillary (MB) (n = 12) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN sections from PB and MB and uninfected control groups were tested by indirect immunoperoxidase technique (IPT), Ziehl Neelsen’s (ZN) method, bacterial culture, IS900 PCR and 251 gene PCR. In PB sheep, IPT and ZN were positive in 87.5 and 50% of intestinal sections and 75 and 37.5% of MLN sections, respectively. In MB sheep, IPT and ZN had equal sensitivity. The overall sensitivity of IPT was found to be superior (95%) than ZN method (80%) in demonstration of acid-fast bacteria in intestinal tissues. On the bacterial cultural examination of the intestinal and MLN tissues using Herrold’s egg yolk medium, MAP was isolated in eight (66.6%) MB and in four (50%) PB sheep. Among 20 sheep, IS900 PCR detected 13 (65.0%) and 251 gene PCR detected 16 (80%) sheep as positive for MAP. In the PB sheep, the culture, ZN test and PCR sensitivity was found low in comparison to MB sheep. The sensitivity of IPT in PB sheep was better than other tests in detecting the MAP infection, which underlines its utility in confirmation of subclinical and clinical cases of paratuberculosis in sheep. The sensitivity of 251 gene PCR assay was found better than IS900 gene PCR and has potential to be used as screening test for clinical and subclinical paratuberculosis in sheep flocks.