Differential role of M cells in enteroid infection by Mycobacterium avium subsp. paratuberculosis and Salmonella enterica serovar Typhimurium.

Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-κB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2-positive M cells.

Interferon-gamma producing CD4+ T cells quantified by flow cytometry as early markers for Mycobacterium avium ssp. paratuberculosis infection in cattle.

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.

Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT) MAC-T cells were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.

Analysis of long non-coding RNA expression profile of bovine monocyte-macrophage infected by Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. As a potential zoonotic pathogen, MAP also seriously threatens human health and social security. At present, long non-coding RNA (lncRNA) has attracted wide attention as an useful biomarker in various diseases. Therefore, our study analyzed the lncRNA expression profiles and lncRNA-mRNA regulatory network of MAP infected bovine monocytes-macrophages and uninfected bovine cells by high-throughput sequencing. A total of 4641 differentially expressed lncRNAs genes were identified, including 3111 up-regulated genes and 1530 down-regulated genes. In addition, lncRNA-mRNA interaction analysis was performed to predict the target genes of lncRNA. Among them, after MAP infection, 86 lncRNAs targeted to mRNA, of which only 6 genes were significantly different. The results of Gene Ontology (GO) enrichment analysis showed that the differentially expressed genes significantly enriched in functional groups were related to immune regulation. Multiple signal pathways including NF-κB, NOD-like receptor, Cytokine-cytokine receptor, Toll-like receptor signaling pathway, Chemokine signaling pathway, and other important biochemical, metabolic and signal transduction pathways were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In this study, analysis of macrophage transcriptomes in response to MAP infection is expected to provide key information to deeply understand role of the pathogen in initiating an inappropriate and persistent infection in susceptible hosts and molecular mechanisms that might underlie the early phases of paratuberculosis.

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.

Therapeutic management of Mycobacterium avium subspecies paratuberculosis infection with complete resolution of symptoms and disease in a patient with advanced inflammatory bowel syndrome.

BACKGROUND: A 26-year-old male had a history of frequent bowel movements, mushy stool with mucus and loss of 25 kg body weight in 6 months was diagnosed as a case of inflammatory bowel disease (IBD). The patient did not respond to routine and standard treatment for IBD. His condition was steadily deteriorating, and he was in a very precarious state when he reported to us. METHODS: Upon laboratory investigation by using IS900 specific PCR [which is specific for Mycobacterium avium subspecies paratuberculosis (MAP)], the blood and stool samples were found negative. However, the presence of low titer MAP-antibodies by indigenous ELISA were found followed by detection of the typical acid-fast MAP bacilli (with 3 + or 4 + grade) microscopically. The MAP stool culture was positive after 6 months incubation. The biotyping by IS1311 specific polymerase chain reaction restriction enzyme (PCR-RE) confirmed infection with 'Indian Bison Type Genotype', a dominant biotype infecting the domestic livestock population of India. Standard anti-MAP therapy was initiated under supervision of the treating physician. The drug of choice in prescribed treatment regimen included Isoniazid (5 mg/kg), Rifampicin (10 mg/kg), Ethambutol (15-25 mg/kg) once a day for 24 weeks and Clarithromycin (250 mg)/Levofloxacin (250 mg) twice a day for 6 weeks. RESULTS: Following treatment, the patient started improving progressively with reduction in bowel movement frequency and gained body weight with an enhanced appetite propensity. Upon follow-up of the patient after 1 year of treatment, stool-microscopy and stool-culture were found negative for MAP. Till the recent past, the patient was further monitored for disease relapse, if any. CONCLUSIONS: This patient has experienced a complete resolution of IBD using a combination of anti-MAP antibiotics. The initial detection of heavy shedding of acid-fast MAP bacilli and typical colony morphology with its characterization obtained from culturing of stool sample indicated the infection of MAP. Interestingly, the present case is one more example of the linkage of demonstrable MAP infection treated with anti-MAP therapy in the presence and then absence of disease in the human host.

Who infects whom?-Reconstructing infection chains of Mycobacterium avium ssp. paratuberculosis in an endemically infected dairy herd by use of genomic data.

Recent evidence of circulation of multiple strains within herds and mixed infections of cows marks the beginning of a rethink of our knowledge on Mycobacterium avium ssp. paratuberculosis (MAP) epidemiology. Strain typing opens new ways to investigate MAP transmission. This work presents a method for reconstructing infection chains in a setting of endemic Johne's disease on a well-managed dairy farm. By linking genomic data with demographic field data, strain-specific differences in spreading patterns could be quantified for a densely sampled dairy herd. Mixed infections of dairy cows with MAP are common, and some strains spread more successfully. Infected cows remain susceptible for co-infections with other MAP genotypes. The model suggested that cows acquired infection from 1-4 other cows and spread infection to 0-17 individuals. Reconstructed infection chains supported the hypothesis that high shedding animals that started to shed at an early age and showed a progressive infection pattern represented a greater risk for spreading MAP. Transmission of more than one genotype between animals was recorded. In this farm with a good MAP control management program, adult-to-adult contact was proposed as the most important transmission route to explain the reconstructed networks. For each isolate, at least one more likely ancestor could be inferred. Our study results help to capture underlying transmission processes and to understand the challenges of tracing MAP spread within a herd. Only the combination of precise longitudinal field data and bacterial strain type information made it possible to trace infection in such detail.

Limitations in the implementation of control measures for bovine paratuberculosis in infected Swiss dairy and beef herds.

Various measures have been advocated for the control of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis, MAP) in different countries. Farmers' compliance has been reported to be variable depending on disease prevalence and incentives to participate in control programs. After the prevalence of MAP shedding and risk factors for within-herd spread of MAP were assessed in 17 Swiss cattle herds (10 dairy and 7 beef), general and herd-specific recommendations were given to the farmers to reduce MAP transmission within the herd. Participation in the study and implementation of control measures were voluntary, no financial incentives were provided for the realization of control measures. After a 3-year period of monitored observation including biannual farm visits and discussion of the situation, the implementation of the recommended control measures and their effect on prevalence of MAP shedding were evaluated. Implementation of recommended general and farm-specific control measures was only partially realized. Neither the number of animals tested positive (before or during the study) nor the farmers' knowledge about paratuberculosis were significantly associated with their compliance for the implementation of management changes. The apparent within-herd prevalence remained constant despite limited implementation of control measures, and no particular group of control measures was found to be associated with changes in prevalence. Farmers' compliance for the implementation of control measures to reduce the impact of Johne's disease in infected farms was very limited under Swiss farming conditions in the frame of voluntary participation in a research project. These results indicate that the losses associated with paratuberculosis in Swiss dairy and beef operations are not estimated to be high enough by the farmers to justify important efforts for control measures, and that incentives may be necessary to achieve efficient implementation of such measures.

Discerning novel drug targets for treating Mycobacterium avium ss. paratuberculosis-associated autoimmune disorders: an in silico approach.

Mycobacterium avium subspecies paratuberculosis (MAP) exhibits 'molecular mimicry' with the human host resulting in several autoimmune diseases such as multiple sclerosis, type 1 diabetes mellitus (T1DM), Hashimoto's thyroiditis, Crohn's disease (CD), etc. The conventional therapy for autoimmune diseases includes immunosuppressants or immunomodulators that treat the symptoms rather than the etiology and/or causative mechanism(s). Eliminating MAP-the etiopathological agent might be a better strategy to treat MAP-associated autoimmune diseases. In this case study, we conducted a systematic in silico analysis to identify the metabolic chokepoints of MAP's mimicry proteins and their interacting partners. The probable inhibitors of chokepoint proteins were identified using DrugBank. DrugBank molecules were stringently screened and molecular interactions were analyzed by molecular docking and 'off-target' binding. Thus, we identified 18 metabolic chokepoints of MAP mimicry proteins and 13 DrugBank molecules that could inhibit three chokepoint proteins viz. katG, rpoB and narH. On the basis of molecular interaction between drug and target proteins finally eight DrugBank molecules, viz. DB00609, DB00951, DB00615, DB01220, DB08638, DB08226, DB08266 and DB07349 were selected and are proposed for treatment of three MAP-associated autoimmune diseases namely, T1DM, CD and multiple sclerosis. Because these molecules are either approved by the Food and Drug Administration or these are experimental drugs that can be easily incorporated in clinical studies or tested in vitro. The proposed strategy may be used to repurpose drugs to treat autoimmune diseases induced by other pathogens.

Modelling transmission and control of Mycobacterium avium subspecies paratuberculosis within Irish dairy herds with compact spring calving.

Paratuberculosis is a chronic bacterial infection of the intestine in cattle caused by Mycobacterium avium subspecies paratuberculosis (Map). To better understand Map transmission in Irish dairy herds, we adapted the French stochastic individual-based epidemiological simulation model to account for seasonal herd demographics. We investigated the probability of Map persistence over time, the within-herd prevalence over time, and the relative importance of transmission pathways, and assessed the relative effectiveness of test-and-cull control strategies. We investigated the impact on model outputs of calf separation from cows (calves grazed on pasture adjacent to cows vs. were completely separated from cows) and test-and-cull. Test-and-cull scenarios consisted of highly test-positive cows culled within 13 or 4 weeks after detection, and calf born to highly test-positive cows kept vs removed. We simulated a typical Irish dairy herd with on average 82 lactating cows, 112 animals in total. Each scenario was iterated 1000 times to adjust variation caused by stochasticity. Map was introduced in the fully naive herd through the purchase of a moderately infectious primiparous cow. Infection was considered to persist when at least one infected animal remained in the herd or when Map was present in the environment. The probability of Map persistence 15 years after introduction ranged between 32.2-42.7 % when calves and cows had contact on pasture, and between 18.9-29.4 % when calves and cows were separated on pasture. The most effective control strategy was to cull highly test-positive cows within four weeks of detection (absolute 10 % lower persistence compared to scenarios without control). Removing the offspring of highly test-positive dams did not affect either Map persistence or within-herd prevalence of Map. Mean prevalence 15 years after Map introduction was highest (63.5 %) when calves and cows had contact on pasture. Mean prevalence was 15 % lower (absolute decrease) when cows were culled within 13 weeks of a high test-positive result, and 28 % lower when culled within 4 weeks. Around calving, the infection rate was high, with calves being infected in utero or via the general indoor environment (most important transmission routes). For the remainder of the year, the incidence rate was relatively low with most calves being infected on pasture when in contact with cows. Testing and culling was an effective control strategy when it was used prior to the calving period to minimize the number of highly infectious cows present when calves were born.

Acid-base variables in acute and chronic form of nontuberculous mycobacterial infection in growing goats experimentally inoculated with Mycobacterium avium subsp. hominissuis or Mycobacterium avium subsp. paratuberculosis.

In current literature, data assessing the acid-base equilibrium in animals and humans during bacterial infection are rare. This study aimed to evaluate acid-base deteriorations in growing goats with experimentally induced NTM (nontuberculous mycobacteria) infections by application of the traditional Henderson-Hasselbalch approach and the strong ion model. NTM-challenged animals were orally inoculated with either Mycobacterium avium subsp. hominissuis (MAH; n = 18) or Mycobacterium avium subsp. paratuberculosis (MAP; n = 48). Twenty-five goats served as non-infected controls. Until 51st week post-inoculation (wpi), blood gas analysis, serum biochemical analysis, and serum electrophoresis were performed on venous blood. Fifty percent (9/18) of goats inoculated with MAH developed acute clinical signs like apathy, fever, and diarrhea. Those animals died or had to be euthanized within 11 weeks post-inoculation. This acute form of NTM-infection was characterized by significantly lower concentrations of sodium, calcium, albumin, and total protein, as well as significantly higher concentrations of gamma globulin, associated with reduced albumin/globulin ratio. Acid-base status indicated alkalosis, but normal base excess and HCO3- concentrations, besides significantly reduced levels of SID (strong ion difference), Atot Alb (total plasma concentration of weak non-volatile acids, based on albumin), Atot TP (Atot based on total protein) and markedly lower SIG (strong ion gap). The remaining fifty percent (9/18) of MAH-infected goats and all goats challenged with MAP survived and presented a more sub-clinical, chronic form of infection mainly characterized by changes in serum protein profiles. With the progression of the disease, concentrations of gamma globulin, and total protein increased while albumin remained lower compared to controls. Consequently, significantly reduced albumin/globulin ratio and lower Atot Alb as well as higher Atot TP were observed. Changes were fully compensated with no effect on blood pH. Only the strong ion variables differentiated alterations in acid-base equilibrium during acute and chronic NTM-infection.

Modelling spread and surveillance of Mycobacterium avium subsp. paratuberculosis in the Swedish cattle trade network.

To monitor a state of disease freedom and to ensure a timely detection of new introductions of disease, surveillance programmes need be evaluated prior to implementation. We present a strategy to evaluate surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) using simulated testing of bulk milk in an infectious disease spread model. MAP is a globally distributed, chronic infectious disease with substantial animal health impact. Designing surveillance for this disease poses specific challenges because methods for surveillance evaluation have focused on estimating surveillance system sensitivity and probability of freedom from disease and do not account for spread of disease or complex and changing population structure over long periods. The aims of the study were to 1. define a model that describes the spread of MAP within and between Swedish herds; 2. define a method for simulation of imperfect diagnostic testing in this framework; 3. to compare surveillance strategies to support surveillance design choices. The results illustrate how this approach can be used to identify differences between the probability of detecting disease in the population based on choices of the number of herds sampled and the use of risk-based or random selection of these herds. The approach was also used to assess surveillance to detect introduction of disease and to detect a very low prevalence endemic state. The use of bulk milk sampling was determined to be an effective method to detect MAP in the population with as few as 500 herds tested per year if the herd-level prevalence was 0.2 %. However, detection of point introductions in the population was unlikely in the 13-year simulation period even if as many as 2000 herds were tested per year. Interestingly, the use of a risk-based selection strategy was found to be a disadvantage to detect MAP given the modelled disease dynamics.

Polymorphisms associated to bovine paratuberculosis: investigation of their role in DNA-protein interactions and transcriptional regulation.

Previous studies led to identify SNPs in putative regulatory regions of the SLC11A1 and CARD15 genes with association to paratuberculosis in cattle. Aim of this study was to investigate the role of these mutations at the regulatory level by DNA-protein interaction analyses and transcriptome comparison between wild-type and mutated animals. Gene regions carrying the SNPs of interest were analysed by bioinformatic tools to predict allele-dependent binding sites for transcription factors (TFBS). Putative TFBS were in vitro explored by Electrophoretic Mobility Shift Assays (EMSA). EMSA did not show specific gel shifts for any allele indicating that these SNPs may eventually influence gene transcription without altering TFBS. Whole transcriptome expression analysis was performed on intestinal tissues of wild-type and mutated cattle by RNA-Seq. Differential regulation of five genes involved in innate immune system was detected. Specifically, ULBP3 was down-regulated, while S100A8, S100A12, LOC510860, and IFI27 were up-regulated. In previous studies, ULBP3, S100A8, and S100A12 resulted differentially expressed in cattle affected by paratuberculosis, suggesting a possible implication in the pathogen response. Further investigations are needed to elucidate the functional role of these SNPs and to understand the gene network involved in the interactions between non-coding SNPs and other genome regions.

High prevalence of subclinical paratuberculosis in buffaloes (Bubalus bubalis) in Maranhão, Brazil.

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8 weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.

Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease.

OBJECTIVES: Although controversial, there is increasing concern that Crohn's disease may be a zoonotic infectious disease consequent to a mycobacterial infection. The most plausible candidate is M. avium subspecies paratuberculosis (MAP) that is unequivocally responsible for Johne's disease in ruminants. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Non-identifiable intestine from patients with documented Crohn's disease was assayed according to the manufacturer's instructions and with suggested modifications. Probes were custom designed for MAP and human ß-actin (as the eukaryotic housekeeping gene) from published genomes. RESULTS: Repetitively, false positive signal was observed in our "No-Probe" negative control. Attempts were made to correct this according to the manufacturer's suggestions (by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters). None prevented false positive signal in the "No-Probe" control. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen resected intestine of humans with Crohn's disease.

Herd level risk factors for Mycobacterium avium subsp. paratuberculosis infection and clinical incidence in dairy herds in Chile.

The study objective was to identify risk factors associated to: i) the infection by Mycobacterium avium subsp. paratuberculosis (MAP), and ii) paratuberculosis clinical incidence in Chilean dairy herds. A random sample of forty herds with previous history of MAP infection was selected. At herd level, all lactating cows were tested using a commercial ELISA kit. On the sampling date, a questionnaire gathering information on herd demographics, husbandry practices, and biosecurity measures was applied. Additionally, the farm manager/owner was surveyed regarding the number of paratuberculosis compatible clinical cases (CCC) in the last 12 months. Two Bayesian generalized linear mixed effect models were used to evaluate the association between the questionnaire data, and the proportion of truly infected animals (model 1) or the number of CCC (model 2). A total of 4963 animals were sampled with an average apparent prevalence of 6.3 % (95 % confidence interval (4.0-8.0%). All sampled herds presented seropositive animals. Forty eight percent of the herds did not observe any CCC in the last year. Although, among those herd that did report CCC, a median of two cases per year was estimated. Model outputs showed that the proportion of truly infected animals and CCC reporting rates are associated to management practices. Specifically, positive associations were observed for feeding of calves exclusively with milk replacer, and the distance between the milking parlor and the calves' barn. Additionally, CCC reporting rates were higher in farms that recently purchased animals, and where the distance between the milking parlor and the calves' barn was less than 30 m.

Seroprevalence of anti-Mycobacterium avium subsp. paratuberculosis antibodies in female sheep in Tunisia.

Paratuberculosis (Ptb) is a widespread chronic infection caused by Mycobacterium avium subsp. paratuberculosis (Map) that affects both domestic and wild ruminants. Most of the studies focused on cattle while, the prevalence of Ptb in sheep in different regions of the world is not well investigated. This study aimed to address this gap of knowledge by screening adult female sheep for paratuberculosis antibodies in different geographical regions of Tunisia. A total number of 338 female sheep from 15 small to middle-sized, extensively managed sheep farms in six regions across Tunisia were sampled. Animals were clinically examined before blood sampling. Sera were tested for the presence of anti-Mycobacterium avium subsp. paratuberculosis antibodies using a commercial ELISA kit. Six farms out of 15 comprised at least one seropositive animal and 11 female sheep out of 338 tested animals (3.25%; 95% CI = [1.83-5.73]) were seropositive to M. avium subsp. paratuberculosis. The seroprevalence was significantly lower in 5-year-old females (p = .04) and animals that do not graze (p = .02). Due to its huge economic and social impacts, paratuberculosis represents a health problem in Tunisia and several other countries Further investigations are needed to rank sheep Ptb in Tunisia among other diseases and to assess the main risk factors using a larger nation-wide survey.

Mycobacterium avium paratuberculosis infection of calves - The impact of dam infection status.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic condition of dairy cattle, and is endemic in the UK. Lack of understanding of the relative importance of different transmission routes reduces the impact of control scheme recommendations. The long incubation period for Johne's disease makes evaluation of control schemes difficult, and so this long-term cohort study offers a rare and valuable insight into the disease epidemiology. A longitudinal study was carried out following a cohort of 440 UK dairy cows in 6 herds recruited in 2012-2013. Individuals entering the milking herd were routinely monitored for the presence of MAP using quarterly milk ELISA testing. Using a Cox proportional-hazards regression model the relationship between time until first detection of infection and dam MAP status was investigated. We then compared the magnitude of the effect of dam status with that of other risk factors in order to understand its relative importance. Dam status was found to be the only observed factor that was significantly associated with time to an individual testing MAP-positive (p = 0.012). When compared to negative dams, we found a marginally significant effect of having a positive dam at time of calving, that increased the hazard of an individual testing positive by a factor of 2.6 (95% confidence interval: 0.89-7.79, p = 0.081). Further positive associations were found with dams becoming positive after the birth of the subject; a dam seroconverting within 12 months post parturition being associated with a 3.6 fold increase in hazard (95% confidence interval: 1.32-9.77, p = 0.013), and dams seroconverting more than a year after calving increased the hazard by a factor of 2.8 (95% confidence interval: 1.39-5.76, p = 0.004). These results suggest that cows may be transmitting MAP to their offspring at an earlier stage than had previously been thought, and so raise important questions about how this transmission may be occurring. The results of the study may have important practical implications for the management on-farm of the offspring of MAP-positive animals, with the potential to vastly reduce the time required to eliminate this chronic disease.

Efficacy of dairy on-farm high-temperature, short-time pasteurization of milk on the viability of Mycobacterium avium ssp. paratuberculosis.

Feeding pasteurized milk to suckling calves is a popular practice used increasingly on dairy farms. Waste milk is frequently fed to calves because of its high nutritional value and economic benefits compared to milk replacement products. However, one of the disadvantages of feeding waste milk is the potential for exposure to a high number of bacterial contaminants, which may lead to serious illnesses or infections in calves. One of these contaminants is Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne's disease (paratuberculosis). The transmission and distribution of paratuberculosis in dairy herds occurs mostly through the feeding newborn calves with contaminated colostrum or milk, because this age group is believed to be most susceptible to infection. To reduce the risk of transmission of pathogens, on-farm pasteurization of milk has become increasingly popular. In this study, we analyzed the efficacy of a new commercial high-temperature, short-time pasteurizer (73.5°C for 20 to 25 s) in terms of MAP inactivation under experimental on-farm conditions. The pasteurizer uses a newly developed steam-heating technique, allowing for the pasteurization of the transition milk without clumping. In 3 independent trials, we spiked fresh raw milk samples to a level of 107 or 104 viable MAP cells/mL before pasteurization. We examined the thermal inactivation and viability of MAP using culture and a D29 bacteriophage-based assay. To verify the identity and number of MAP cells, we also performed PCR assays. Pasteurization of the inoculated milk (107 and 104 MAP cells/mL) resulted in a remarkable reduction in viable MAP cells. The mean inactivation rate of MAP ranged from 0.82 to 2.65 log10 plaque-forming units/mL, depending on the initial MAP amount inoculated and the addition of conservative agents to the pasteurized milk. Nevertheless, approximately 103 MAP cells/mL remained viable and could be transferred to calves after high-temperature, short-time pasteurization of milk.

Quantifying transmission of Mycobacterium avium subsp. paratuberculosis among group-housed dairy calves.

Johne's disease (JD) is a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), with control primarily aimed at preventing new infections among calves. The aim of the current study was to quantify calf-to-calf transmission of MAP among penmates in an experimental trial. Newborn Holstein bull calves (n = 32) were allocated into pens of 4, with 2 inoculated (IN) calves and 2 calves that were contact exposed (CE). Calves were group-housed for 3 months, with frequent collection of fecal and blood samples and tissue collection after euthanasia. The basic reproduction ratio (R0) was estimated using a final size (FS) model with a susceptible-infected model, based on INF-γ ELISA and tissue culture followed by qPCR. In addition, the transmission rate parameter (ß) for new shedding events was estimated using a general linearized method (GLM) model with a susceptible-infected-susceptible model based on culture, followed by qPCR, of fecal samples collected during group housing. The R0 was derived for IN and CE calves separately, due to a difference in susceptibility, as well as differences in duration of shedding events. Based on the FS model, interferon-γ results from blood samples resulted in a R 0 IG of 0.90 (0.24, 2.59) and tissue culture resulted in a R 0 T of 1.36 (0.45, 3.94). Based on the GLM model, the R0 for CE calves to begin shedding (R 0 CE ) was 3.24 (1.14, 7.41). We concluded that transmission of MAP infection between penmates occurred and that transmission among calves may be an important cause of persistent MAP infection on dairy farms that is currently uncontrolled for in current JD control programs.