Mycobacterium Avium Ssp. Paratuberculosis Infection Research Articles

Seroprevalence and contributing factors of transboundary animal diseases in sheep and goats: a study in Peninsular Malaysia.

Diseases caused by small ruminant lentiviruses, Mycobacterium avium ssp. paratuberculosis (MAP), Schmallenberg virus, and peste des petits ruminants virus (PPR) is globally recognised as serious threats to the ruminant industry due to their potential to spread rapidly across boundaries. Despite their global distribution and negative impacts on ruminant production, there is a gap in knowledge of the current trends in their epidemiology among sheep and goat populations in Peninsular Malaysia. This study was therefore designed to fill the gap of knowledge concerning the seroprevalence and contributing factors of CAEV, paratuberculosis, SBV, and PPRV among small ruminants from selected flocks in Selangor, Negeri Sembilan, and Pahang states in Peninsular Malaysia. A cross-sectional study design was used to collect animal data and blood samples for serological assays simultaneously. The ID Screen (ID.VET, France) indirect ELISA screening tests were used to detect serum antibodies directed against CAEV/MVV (VISNAS Ver 0922), paratuberculosis (PARAS Ver 0516), SBV (SBVC Ver 1114) and PPRV (PPRC Ver 0821). There was 45.4% (95% CI = 40.74-50.74), 6.8% (95% CI = 4.66-9.69), 27.8% (95% CI = 23.35-32.77), and 2.6% (95% CI = 1.11-0.51) true seroprevalence for CAEV, paratuberculosis, SBV, and PPR, respectively. Geographical location and species were the risk factors for CAEV and paratuberculosis, while the management system and age of small ruminants were the risk factors for SBV. The present study is the first to document a large-scale seroprevalence of MAP and PPR infection among sheep and goat flocks in Peninsular Malaysia. The presence of PPRV and MAP antibodies among small ruminant flocks is signalling current or previous exposure to the pathogens or cross reactions with similar antigens. This finding further suggests the potential for future outbreaks of these devastating diseases among sheep and goats in Malaysia. The high seroprevalence of CAEV and SBV among small ruminants indicates high levels of exposure to the viruses in the environment, which is a potential threat to production.

Mapping Crohn's Disease Pathogenesis with Mycobacterium paratuberculosis: A Hijacking by a Stealth Pathogen.

Mycobacterium avium ssp. paratuberculosis (MAP) has been implicated in the development of Crohn's disease (CD) for over a century. Similarities have been noted between the (histo)pathological presentation of MAP in ruminants, termed Johne's disease (JD), and appearances in humans with CD. Analyses of disease presentation and pathology suggest a multi-step process occurs that consists of MAP infection, dysbiosis of the gut microbiome, and dietary influences. Each step has a role in the disease development and requires a better understanding to implementing combination therapies, such as antibiotics, vaccination, faecal microbiota transplants (FMT) and dietary plans. To optimise responses, each must be tailored directly to the activity of MAP, otherwise therapies are open to interpretation without microbiological evidence that the organism is present and has been influenced. Microscopy and histopathology enables studies of the mycobacterium in situ and how the associated disease processes manifest in the patient e.g., granulomas, fissuring, etc. The challenge for researchers has been to prove the relationship between MAP and CD with available laboratory tests and methodologies, such as polymerase chain reaction (PCR), MAP-associated DNA sequences and bacteriological culture investigations. These have, so far, been inconclusive in revealing the relationship of MAP in patients with CD. Improved and accurate methods of detection will add to evidence for an infectious aetiology of CD. Specifically, if the bacterial pathogen can be isolated, identified and cultivated, then causal relationships to disease can be confirmed, especially if it is present in human gut tissue. This review discusses how MAP may cause the inflammation seen in CD by relating its known pathogenesis in cattle, and from examples of other mycobacterial infections in humans, and how this would impact upon the difficulties with diagnostic tests for the organism.

Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn's disease.

1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. Aphage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment.

Human antibodies against Mycobacterium avium ssp. paratuberculosis combined with cytokine levels for the diagnosis and selection of Crohn's disease patients for anti-mycobacterial therapy-A pilot study.

Increasing evidence links a worldwide bacterial infection of cattle and other animal species by Mycobacterium avium ssp. paratuberculosis (MAP) to Crohn's disease (CD). A large, FDA phase 2/3 controlled clinical trial of combination antimycobacterial antibiotic therapy for CD has been completed, and the report describing the trial is pending publication. The identification of MAP infection in CD patients will become increasingly important. Thus, it is desirable to develop MAP-based tests that accurately predict which CD patients have a MAP infection. A prospective, case-control laboratory test study of 199 subjects (61 CD patients and 138 non-CD controls) was performed using a panel of MAP antigens, including Hsp65, PknG, PtpA, CL1, and MAP IDEXX, which were measured under blind conditions in the plasma of the 199 subjects. Results showed that compared to any individual MAP antigen, combinations of antigens showed improved CD classification performance. For the Hsp65 antigen, the sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), correct classification (CC), and area under the curve (AUC) were 59.02%, 58.70%, 38.71%, 76.42%, 59.3% and 0.606, respectively. For the best combination of MAP antibodies (Hsp65 and PknG), the SEN, SPE, PPV, NPV, CC, and AUC were 59.02%, 60.87%, 40.00%, 77.06%, 60.30%, and 0.631, respectively. Further improvement of the CD classification performance was achieved by combining IFN-γ, IL-8, and IL-17 cytokines with antibodies against MAP antigens, yielding SEN, SPE, PPV, NPV, CC, and AUC of 62.3%, 62.32%, 42.22%, 78.9%, 62.31% and 0.708, respectively. Thus, combinations of antibodies against MAP antigens and cytokine levels yield better CD diagnostic predictive performance than any individual antibodies against MAP antigens.

Economic premiums associated with Mycobacterium avium ssp. paratuberculosis-negative replacement purchases in major dairy-producing regions

Johne's disease, or paratuberculosis, is an infectious disorder of the intestines that can affect domestic and wild ruminants that is caused by an infection with Mycobacterium avium ssp. paratuberculosis (MAP). Although the economic losses due to Johne's disease in dairy cattle herds and the benefits and costs of various potential control practices have been estimated before, little is known about the economic value of purchasing MAP-negative dairy replacements in major dairy-producing regions. This study used Markov Chain Monte Carlo simulation techniques to compare 2 sets of MAP-negative and MAP-positive herds across a comprehensive selection of regions: herds purchasing MAP-negative replacement animals and herds purchasing replacement animals with unknown MAP infection status. The economic benefits per MAP-negative replacement purchased were then estimated over a 10-yr horizon, and the additional value of MAP-negative replacements when compared with unknown status replacements were calculated as a percentage premium of the average aggregated dairy replacement price in each region. An average benefit of US$76 per MAP-negative replacement purchase was estimated in major dairy-producing regions, equivalent to a premium of 13%, with higher premiums in regions characterized by below-average replacement prices and on-average farm-gate prices. It was also estimated that the greatest benefits from MAP-negative replacement purchases are associated with MAP-negative herds that successfully remain uninfected.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

Detection of Paratuberculosis in Dairy Herds by Analyzing the Scent of Feces, Alveolar Gas, and Stable Air.

Paratuberculosis is an important disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Early detection is crucial for successful infection control, but available diagnostic tests are still dissatisfying. Methods allowing a rapid, economic, and reliable identification of animals or herds affected by MAP are urgently required. This explorative study evaluated the potential of volatile organic compounds (VOCs) to discriminate between cattle with and without MAP infections. Headspaces above fecal samples and alveolar fractions of exhaled breath of 77 cows from eight farms with defined MAP status were analyzed in addition to stable air samples. VOCs were identified by GC–MS and quantified against reference substances. To discriminate MAP-positive from MAP-negative samples, VOC feature selection and random forest classification were performed. Classification models, generated for each biological specimen, were evaluated using repeated cross-validation. The robustness of the results was tested by predicting samples of two different sampling days. For MAP classification, the different biological matrices emitted diagnostically relevant VOCs of a unique but partly overlapping pattern (fecal headspace: 19, alveolar gas: 11, stable air: 4–5). Chemically, relevant compounds belonged to hydrocarbons, ketones, alcohols, furans, and aldehydes. Comparing the different biological specimens, VOC analysis in fecal headspace proved to be most reproducible, discriminatory, and highly predictive.

Identification of Long Non-coding RNA Isolated From Naturally Infected Macrophages and Associated With Bovine Johne's Disease in Canadian Holstein Using a Combination of Neural Networks and Logistic Regression

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic enteritis in most ruminants. The pathogen MAP causes Johne's disease (JD), a chronic, incurable, wasting disease. Weight loss, diarrhea, and a gradual drop in milk production characterize the disease's clinical phase, culminating in death. Several studies have characterized long non-coding RNA (lncRNA) in bovine tissues, and a previous study characterizes (lncRNA) in macrophages infected with MAP in vitro. In this study, we aim to characterize the lncRNA in macrophages from cows naturally infected with MAP. From 15 herds, feces and blood samples were collected for each cow older than 24 months, twice yearly over 3–5 years. Paired samples were analyzed by fecal PCR and blood ELISA. We used RNA-seq data to study lncRNA in macrophages from 33 JD(+) and 33 JD(–) dairy cows. We performed RNA-seq analysis using the “new Tuxedo” suite. We characterized lncRNA using logistic regression and multilayered neural networks and used DESeq2 for differential expression analysis and Panther and Reactome classification systems for gene ontology (GO) analysis. The study identified 13,301 lncRNA, 605 of which were novel lncRNA. We found seven genes close to differentially expressed lncRNA, including CCDC174, ERI1, FZD1, TWSG1, ZBTB38, ZNF814, and ZSCAN4. None of the genes associated with susceptibility to JD have been cited in the literature. LncRNA target genes were significantly enriched for biological process GO terms involved in immunity and nucleic acid regulation. These include the MyD88 pathway (TLR5), GO:0043312 (neutrophil degranulation), GO:0002446 (neutrophil-mediated immunity), and GO:0042119 (neutrophil activation). These results identified lncRNA with potential roles in host immunity and potential candidate genes and pathways through which lncRNA might function in response to MAP infection.

Survey of Candidate Single-Nucleotide Polymorphisms in SLC11A1, TLR4, NOD2, PGLYRP1, and IFNγ in Ankole Longhorn Cattle in Central Region of Uganda to Determine Their Role in Mycobacterium avium Subspecies paratuberculosis Infection Outcome.

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease (JD) in a wide range of domestic and wild ruminants. Single-nucleotide polymorphisms (SNPs) in several genes including solute-like carrier 11A1 (SLC11A1), interferon gamma (IFNγ), Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain 2 gene (NOD2), and bovine peptidoglycan recognition protein 1 (PGLYRP1) have been implicated in influencing the infection outcome of MAP in cattle. We have carried out a survey in a population of Ankole cattle from three districts in the central region of Uganda including Isingiro, Lyantonde, and Rakai to determine the role played by several SNPs on the above genes in the infection outcome of local cattle in Uganda. Nine hundred fifty-five heads of cattle obtained from 93 herds were tested using ELISA. Thirty-five ELISA-positive cattle and 35 negative herd mates from a total of 955 cattle tested for MAP were genotyped using iPLEX MassARRAY genotyping systems to detect the presence of a total of 13 SNPS in five different genes (SLC11A1, IFNγ, TLR4, NOD2, and PGLYRP1). The cow-level prevalence of MAP infection in Ankole Longhorn cattle in the three districts was 3.98% (35/955), while the herd-level prevalence was 27.9% and within-herd prevalence was 12 ± 1.5% (95% CI = 9.1–14.8%). The genotypes and allele frequencies of the MAP-positive cattle were compared with those of their ELISA-negative herd mates to determine the significance of the polymorphisms. The results showed that SNPs rs109915208, rs110514940, and rs110905610 on SLC11A1, c.480G>A and c.625C>A on PGLYRP1, and c.2021C>T on TLR4 were monomorphic in both seropositive and seronegative cattle and therefore had no influence on the infection outcome. The remaining SNPs studied in the five genes [SLC11A1: rs109614179; TLR4: rs29017188 (c.226G>C), c.2021C>T; NOD2: rs110536091, rs111009394; PGLYRP1: c.102G>C, c.480G>A, c.625C>A; IFNγ: rs110853455] were polymorphic, but their allele and genotype frequencies did not show any significant difference between the seropositive and seronegative cattle. No significant difference was observed for any haplotype at the gene level.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacterium avium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® – a commercial MAP bacterin – we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer’s patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Test positivity for Maedi–Visna virus and Mycobacterium avium ssp. paratuberculosis in Sarda ewes: Effects on milk composition and coagulation traits and heritability estimates for susceptibility

Maedi-Visna virus (MVV) and Mycobacterium avium ssp. paratuberculosis (MAP) are two pathogens that cause chronic, production-limiting diseases in dairy sheep. Although they are present worldwide, there are no detailed reports on their actual effects on milk traits in the literature. This study was designed to investigate the effects of test positivity to MVV and MAP on ovine milk yield, composition and coagulation properties, and curd-firming over time (CFt) variables in clinically healthy animals at the field level. The additive genetic variation and heritabilities of MVV and MAP positivity were also estimated. Milk samples were collected from 1,079 Sarda sheep kept on 23 farms, and pedigree information was obtained from the flock book. Milk yield was also recorded on the sampling date. Positivity for MVV and MAP was determined from milk samples using indirect ELISA test kits. Milk composition traits were measured by spectroscopy, milk coagulation properties were measured with a Formagraph (Foss Italia, Padua, Italy), and CFt traits were calculated using the data from the Formagraph diagram. The effects of MVV and MAP positivity on milk traits were determined through a set of mixed linear models, which took into account various sources of variation, such as days in milk, parity, and flock effects, and included the effects (positive or negative) of the 2 pathogens. A Bayesian threshold sire model with sire relationship was used to estimate genetic variation and heritability. The overall animal prevalence of MVV-positive ewes was 43.6%; on only 1 farm of the 23 tested were all sampled ewes negative. An overall animal prevalence of 10.6% was recorded for MAP, with 4 farms at 0%. Positivity for MVV significantly affected the logarithmic score of the bacterial count, curd firmness after 30 min and 45 min, and the curd-firming instant rate constant. We found significant effects of MAP infection on milk composition, pH, and rennet coagulation time. The mean of the posterior distributions of heritability estimates on the liability scale was 0.15 for MAP and 0.07 for MVV. Our results demonstrate that only a few traits are negatively affected by MVV and MAP positivity, and that there is exploitable genetic variation in MVV and MAP susceptibility in dairy sheep.

Individual and herd-level milk ELISA test status for Johne's disease in Ireland after correcting for non-disease-associated variables

Antibody-detecting tests for Mycobacterium avium ssp. paratuberculosis (MAP) have low sensitivity and imperfect specificity for detection of infection. Sensitivity increases as the disease progresses. Aside from infection status and stage of disease, several factors affect test performance. These factors have not yet been studied in dairy cows producing lower volumes of milk with higher solids concentration, such as those managed in low-input, pasture-based production systems. Furthermore, the effect of correcting for these associations on individual and herd test status is also unknown. The first objective of this study was to examine the relationship between MAP antibody response in milk and milk yield, somatic cell count (SCC), fat and protein contents, and stage of lactation in dairy cows enrolled in the national Johne's Disease Control Programme (JDCP) in Ireland. The second objective was to examine the effect of correcting the antibody response for these associations on the test status of individual cows and herds, given that individual tests are often used to define a herd's status. Data were extracted for herds in the JDCP from January 2014 to December 2015 inclusive, consisting of 42,657 milk recordings from 18,569 cows across 187 dairy herds. Two linear regression models were constructed to investigate the association between log-transformed MAP sample-to-positive ratio and milk recording data and in primi- and multiparous cows. Days in milk was modeled as a B-spline in each model, and cow and herd were included as random effects. Across both models, natural log-transformed MAP antibody response was negatively associated with milk yield, positively associated with protein and fat production, and had a curvilinear association with log-transformed SCC. The association between MAP antibody response and days in milk varied over the course of the lactation. However, when combined, these variables explained only 5.1% of the variation in the antibody response of the population. After correcting for these associations, 93 multiparous cows and 20 primiparous cows changed category (negative, suspect, or positive). When considered at the herd-test level, out of a total of 531 herd tests, 1 herd changed from negative to positive, and 5 herds changed from positive to negative. This study provides useful information to aid in the interpretation of antibody results for herds testing animals for the presence of MAP infection. At an overall population level, correction of the serological response for non-disease-associated factors has the potential to change the status of only a small number of cows. At the herd level, the proportion of herds changing status was minimal. However, depending on the implications of a herd-level serological diagnosis, consideration should be given to correcting for these non-disease-associated variables within the context of national JD control programs.

Short communication: Occurrence and differentiation of Mycobacterium avium ssp. paratuberculosis (MAP) strains from milk of cows from herd with low prevalence of MAP

Mycobacterium avium ssp. paratuberculosis (MAP) is an important pathogen responsible for the chronic progressive granulomatous enteritis known as paratuberculosis. None of the detection methods of MAP infection based on isolation of the bacterium is 100% sensitive or specific. In this article, we describe the comparison of 2 MAP detection methods: direct isolation of genetic material and culture, in individual and pooled milk samples. The genetic types of MAP detected in the samples were also identified. The study was performed in a herd of 321 cows; apparent herd seroprevalence was 3.43%. Seven of 11 individual milk samples from seropositive cows were positive by culture (and confirmed by PCR), whereas all 11 were positive by direct PCR. Of the 62 milk pools from seronegative animals, 15 were positive by culture (and confirmed by PCR) and 13 were positive by direct PCR. Using multiplex PCR and PCR-restriction enzyme analysis (PCR-REA) methods, C (cattle) and S (sheep)-types of mycobacteria were identified. Most of the genetic material tested belonged to C-type. Detection of the MAP type occurring in an infected herd can help track the source of infection. We suggest using genetic material isolated directly from pooled milk samples for quick diagnosis, identification of MAP type, and tracking of infection, without the need to sequence the entire genome.

Short communication: Risk factors associated with Mycobacterium avium ssp. paratuberculosis introduction into dairy herds in Galicia, northwestern Spain

This study assessed potential risk factors associated with introduction of Mycobacterium avium ssp. paratuberculosis (MAP) into dairy cattle herds in the Galicia region, northwestern Spain. The study was carried out with data collected from 93 dairies enrolled in a voluntary MAP control program. Information on potential risk factors was obtained through personal interviews with the farmers and veterinarians in charge of the control program of each farm. In addition, blood samples were taken annually over 2 years from cows on the farms in the program, and analyzed with a commercial ELISA to detect antibodies to MAP. Fecal samples of all ELISA-positive cows were analyzed using PCR. Based on χ2 test and Fisher's exact test, purchase practices, shared manure truck, shared materials, and visitors per month who contacted animals were found to be significantly associated with farm MAP infection status. Multiple logistic regression indicated that purchase practices and herd size (included as a potential confounder) are the variables that best predict MAP status.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

Transcriptome Profiling of Bovine Macrophages Infected by Mycobacterium avium spp. paratuberculosis Depicts Foam Cell and Innate Immune Tolerance Phenotypes.

Mycobacterium avium spp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), also known as paratuberculosis, in ruminants. The mechanisms of JD pathogenesis are not fully understood, but it is known that MAP subverts the host immune system by using macrophages as its primary reservoir. MAP infection in macrophages is often studied in healthy cows or experimentally infected calves, but reports on macrophages from naturally infected cows are lacking. In our study, primary monocyte-derived macrophages (MDMs) from cows diagnosed as positive (+) or negative (–) for JD were challenged in vitro with live MAP. Analysis using next-generation RNA sequencing revealed that macrophages from JD(+) cows did not present a definite pattern of response to MAP infection. Interestingly, a considerable number of genes, up to 1436, were differentially expressed in JD(–) macrophages. The signatures of the infection time course of 1, 4, 8, and 24 h revealed differential expression of ARG2, COL1A1, CCL2, CSF3, IL1A, IL6, IL10, PTGS2, PTX3, SOCS3, TNF, and TNFAIP6 among other genes, with major effects on host signaling pathways. While several immune pathways were affected by MAP, other pathways related to hepatic fibrosis/hepatic stellate cell activation, lipid homeostasis, such as LXR/RXR (liver X receptor/retinoid X receptor) activation pathways, and autoimmune diseases (rheumatoid arthritis or atherosclerosis) also responded to the presence of live MAP. Comparison of the profiles of the unchallenged MDMs from JD(+) vs. JD(–) cows showed that 868 genes were differentially expressed, suggesting that these genes were already affected before monocytes differentiated into macrophages. The downregulated genes predominantly modified the general cell metabolism by downregulating amino acid synthesis and affecting cholesterol biosynthesis and other energy production pathways while introducing a pro-fibrotic pattern associated with foam cells. The upregulated genes indicated that lipid homeostasis was already supporting fat storage in uninfected JD(+) MDMs. For JD(+) MDMs, differential gene expression expounds long-term mechanisms established during disease progression of paratuberculosis. Therefore, MAP could further promote disease persistence by influencing long-term macrophage behavior by using both tolerance and fat-storage states. This report contributes to a better understanding of MAP's controls over the immune cell response and mechanisms of MAP survival.

A preliminary study investigating effects of oral monensin sodium in an enteric Mycobacterium avium ssp. paratuberculosis infection model of calves

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, an enteric infection of ruminants that causes significant economic burden for dairy and beef producers. Efforts to control MAP in endemic herds typically focus on herd management practices such as limiting exposure or early culling of infected animals and, occasionally, vaccination. The ionophore monensin sodium may have protective effects against MAP both in vivo and in vitro; however, this has not been thoroughly evaluated experimentally. Using a direct intestinal MAP challenge model, we have observed similarities regarding persistence of MAP in tissues and apparent resilience to infection compared with experimental oral infection or natural disease. Here we sought to investigate the effects of oral monensin supplementation in experimentally MAP-infected calves. We examined the persistence of MAP in the intestinal tissues, MAP-induced intestinal inflammation, fecal MAP shedding, and seroconversion using a commercial serologic assay. Monensin-supplemented MAP-infected calves demonstrated evidence for resilience to MAP infection earlier in this study compared with monensin-free MAP-infected calves. However, statistical modeling did not identify a significant effect of monensin on outcomes of infection, and more work is required to understand how monensin affects early tissue colonization of MAP in calves.

Accuracy of paratuberculosis diagnostic tests in small ruminants: protocol for a systematic review and meta-analysis.

Paratuberculosis is a worldwide infectious disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Various ruminant species can be affected by the disease, and the diagnosis of the disease is challenging in the absence of a gold standard test. The aim of this systematic review protocol is to determine the accuracy of the direct and indirect diagnostic tests for MAP infection with a special focus on sheep and goats.

Reduction of viable Mycobacterium avium ssp. paratuberculosis in slurry subjected to anaerobic digestion in biogas plants

Cattle infected with Mycobacterium avium ssp. paratuberculosis (MAP) shed the bacterium in their feces. This may lead to considerable concentrations of MAP in slurry, which has been postulated to contribute to MAP transmission when this slurry is used as fertilizer. For other bacterial species, anaerobic digestion has been shown to reduce bacterial load and to increase the safety of organic waste. Therefore, the objective of this study was to investigate the effects of anaerobic digestion in biogas plants on MAP survival in slurry from 16 dairy farms with a history of MAP infection. Presence of MAP was determined using MAP culture and a commercial MAP IS900 quantitative PCR (qPCR) applied on untreated slurry samples, slurry samples after primary fermentation, and digestate. Unfermented slurry samples from most enrolled farms tested positive for MAP, via both culture and qPCR. After the fermentation process, MAP could no longer be cultured in most samples, with the exception of 2 samples from farms where high numbers of MAP-shedding cows were kept at the time of sampling. A Bayesian binomial model predicted a probability of 93% for a MAP-negative culture result after fermentation. In most samples, MAP DNA was still detectable when using the IS900 qPCR. The probability of a negative result in qPCR was estimated to be 27%. Results of this study indicate that subjecting MAP-positive slurry to anaerobic digestion in biogas plants leads to a reduction of viable MAP below the detection limit; however, MAP DNA remained detectable. It remains undetermined whether MAP DNA detected in fermentation products is a residue of MAP degradation or belongs to viable MAP below the detection limit or in a dormant state. In conclusion, subjecting MAP-positive slurry to anaerobic mesophilic digestion reduces viable MAP concentration below the detection limit. The use of digestion products as fertilizer on pasture and agricultural soils instead of untreated slurry may therefore reduce the risk of MAP transmission.

Prevalence of Mycobacterium avium ssp. paratuberculosis infections in Canadian dairy herds

Johne's disease is a progressive, chronic disease with inflammation of the small intestine of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Accurately estimating prevalence of MAP infections is important when controlling spread of infection or monitoring effectiveness of control programs. In the absence of a consistent test method used in prevalence studies across Canada, prevalence estimates among regions and programs cannot be compared. The aim of the current study was to estimate and compare prevalence of MAP infection in Western Canada, Ontario, Québec, and the Atlantic provinces, as well as among varying herd sizes and housing types. On 362 dairy farms located in all 10 provinces of Canada, environmental samples were collected and cultured for detection of MAP. For each herd, 1 sample was collected from the lactating cow area and manure storage. An additional environmental sample was collected from the area where breeding-age heifers were housed. Using prior distributions from previous research, diagnostic sensitivity and specificity were calculated to assess the ability of only 2 environmental samples (manure storage and lactating cow area) to identify MAP-positive farms, resulting in a sensitivity and specificity of 38 and 100%, respectively. We found no difference in sensitivity and specificity when including breeding-age heifers environmental samples. Test characteristics were applied to environmental culture results from the 362 participating farms in all 4 regions, resulting in true prevalence estimates of 66% for farms in Western Canada, 54% in Ontario, 24% in Québec, and 47% in Atlantic Canada. Herds housed in tiestalls had lower prevalence than freestall-housed herds, and herds with 101-150 and >151 cows had higher prevalence than herds with ≤100 cows. This was the first time MAP prevalence was determined using 1 detection method, performed in 1 laboratory, and within a single year across Canada, enabling direct comparisons of prevalence among regions, housing types, and herd sizes.