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

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

Evaluation of ELISA in raw milk for detection of antibodies to Mycobacterium avium subsp. paratuberculosis in dairy herd.

Paratuberculosis (PTBC) is a chronic intestinal disease of animals caused by Mycobacterium avium paratuberculosis (MAP). MAP infection is diagnosed through indirect tests based on the immune response. The aims of this study were to compare the performance of two milk ELISA for the diagnosis of PTBC and to assess the bulk tank milk (BTM) MAP exposure in dairy cattle in Argentina. A total of 357 fecal, serum, and milk samples were collected. The fecal samples were processed by culture for MAP isolation, while both, serum and milk samples were used for the detection of antibodies by two different ELISA tests, "in-house" and commercial kit. MAP was isolated in 3.9% of fecal samples. For milk ELISA, poor concordances were obtained. Optimized cut-off points were calculated. The highest sensitivity and specificity values (64% and 80% respectively) were obtained with the combination of MAP isolation and commercial milk ELISA. The results indicate that the combination of different techniques to identify of dairy cattle infected with MAP increases the efficiency of diagnosis. In addition, BTM  samples (n=98) were evaluated to determine herd status using the commercial kit during two seasons, identifying 33.3% of positive samples in autumn and 35.4% in spring.

The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation.

Johne's disease (JD) is a chronic enteric infection of dairy cattle worldwide. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of JD, is fastidious often requiring eight to sixteen weeks to produce colonies in culture-a major hurdle in the diagnosis and therefore in implementation of optimal JD control measures. A significant gap in knowledge is the comprehensive understanding of the metabolic networks deployed by MAP to regulate iron both in-vitro and in-vivo. The genome of MAP carries MAP3773c, a putative metal regulator, which is absent in all other mycobacteria. The role of MAP3773c in intracellular iron regulation is poorly understood. In the current study, a field isolate (K-10) and an in-frame MAP3773c deletion mutant (ΔMAP3773c) derived from K-10, were exposed to iron starvation for 5, 30, 60, and 90 min and RNA-Seq was performed. A comparison of transcriptional profiles between K-10 and ΔMAP3773c showed 425 differentially expressed genes (DEGs) at 30 min time post-iron restriction. Functional analysis of DEGs in ΔMAP3773c revealed that pantothenate (Pan) biosynthesis, polysaccharide biosynthesis and sugar metabolism genes were downregulated at 30 min post-iron starvation whereas ATP-binding cassette (ABC) type metal transporters, putative siderophore biosynthesis, PPE and PE family genes were upregulated. Pathway analysis revealed that the MAP3773c knockout has an impairment in Pan and Coenzyme A (CoA) biosynthesis pathways suggesting that the absence of those pathways likely affect overall metabolic processes and cellular functions, which have consequences on MAP survival and pathogenesis.

Epidemiological insights into paratuberculosis in camels in Saudi Arabia: Bayesian estimation of true prevalence and identification of risk factors.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a significant concern in the camel population of Saudi Arabia. This study aimed to provide epidemiological insights into the disease by estimating the true prevalence in camels in the Eastern Province and Riyadh, using a Bayesian estimation framework, and exploring the associated risk factors through a frequentist approach. A total of 1200 camel blood samples were collected and analyzed using an indirect ELISA method. The true herd-level prevalence was estimated at 0.7 (95% probability interval: 0.57 to 0.81), and the mean expected true animal-level prevalence was 0.17 (0.14 to 0.20). Risk factors associated with Map seropositivity were identified, including sex, breed, raising system, and production type. Females, single breed camels, and nomadic raising systems were found to have lower odds of seropositivity, while camels used for racing and show had significantly higher odds. The study's Bayesian approach, adjusting for the imperfect accuracy of MAP tests, provides a nuanced understanding of the disease's prevalence in the region. The integration of true prevalence estimates with risk factor analysis offers a comprehensive framework that can guide future policies and strategies in the fight against paratuberculosis in Saudi Arabia. The findings emphasize the importance of targeted control measures, underscoring the urgent need for interventions in Saudi Arabia's camel population. By understanding the true disease prevalence and its associated risk factors, we can enhance disease management strategies, offering valuable insights for future control and eradication efforts in the region.

Alternative splicing of pre-mRNA modulates the immune response in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

Little is known about the role of alternative splicing (AS) in regulating gene expression in Mycobacteria-infected individuals in distinct stages of infection. Pre-mRNA AS consists of the removal of introns and the assembly of exons contained in eukaryotic genes. AS events can influence transcript stability or structure with important physiological consequences. Using RNA-Seq data from peripheral blood (PB) and ileocecal valve (ICV) samples collected from Holstein cattle with focal and diffuse paratuberculosis (PTB)-associated histopathological lesions in gut tissues and without lesions (controls), we detected differential AS profiles between the infected and control groups. Four of the identified AS events were experimentally validated by reverse transcription-digital droplet PCR (RT-ddPCR). AS events in several genes correlated with changes in gene expression. In the ICV of animals with diffuse lesions, for instance, alternatively spliced genes correlated with changes in the expression of genes involved in endocytosis, antigen processing and presentation, complement activation, and several inflammatory and autoimmune diseases in humans. Taken together, our results identified common mechanisms of AS involvement in the pathogenesis of PTB and human diseases and shed light on novel diagnostic and therapeutic interventions to control these diseases.

Diagnostic advancements: Isolating Mycobacterium avium ssp. paratuberculosis and unveiling its molecular identity with nested-PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis, which is currently prevalent in many parts of Iran and produces severe economic loss. It is hence necessary to identify and isolate the animals infected with this bacterium, so this research aimed to isolate MAP from milk and fecal samples of ELISA-positive animals and determine the molecular identity of isolates. After performing ELISA on 3,700 bovine blood samples, 115 samples of milk and feces were taken from ELISA-positive cattle and were cultured on Herald's egg yolk medium with and without mycobactin-J and then the acid-fastness of positive samples was determined using Ziehl-Neelsen staining. The 16S rRNA-PCR test was performed after DNA extraction to determine the molecular identity of isolates. Primers IS6110 and IS901 were employed to ensure that the isolates were not related to members of M. tuberculosis complex and  M. avium, respectively. Primer IS900 was also used to determine the molecular identity of MAP isolates. Also, expression levels of MAP-related genes (IS900, ISMAP02, F57, MAP2191, MAP4027) were evaluated via qPCR. Finally, positive samples were confirmed based on the Nested-PCR. Results showed that a total of 9 isolates were obtained from the culture of 90 ELISA-positive samples. The results revealed that all grown samples were positive for acid-fastness. The 16S rRNA-PCR test revealed the 543 bp band, which confirms the presence of Mycobacterium in the samples. The PCR test with Primer IS900 generated the 398 bp fragment in the first step and the 298 bp fragment in the second step, indicating the presence of MAP in samples. Also, relative expression analysis revealed that MAP-related genes were significantly higher in ELIZA-positive samples than in negative ones. Based on the study findings, it can be concluded that MAP-infected animals can be identified by ELISA. In addition, mycobacterium can be isolated by culturing the samples on appropriate media and then its molecular identity can be determined by using nested-PCR.

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

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

Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

Electron microscopic observation of Mycobacterium avium subsp. paratuberculosis in cattle feces after a novel purification using liquid paraffin.

We developed a novel method for purifying acid-fast bacteria from feces. The method enabled the observation of characteristic clumps of Mycobacterium avium subsp. paratuberculosis (MAP) under electron microscopy by removing contaminants and other bacteria. Further refinement of this method will contribute to efficient and effective MAP detection.

[Prevalence of paratuberculosis in Thuringian sheep and goat flocks]. / Verbreitung der Paratuberkulose in Thüringer Schaf- und Ziegenherden.

OBJECTIVE: In Germany, only few data on the current distribution of paratuberculosis in sheep and goat flocks is available. The present study provides an overview of the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) in 165 Thuringian sheep and goat flocks. Also, the study investigated the association between the MAP status of the flock and herd specific factors as well as the association between the individual measured value of ELISA and animal specific factors like age, body condition, sex, and animal species. MATERIAL AND METHODS: To investigate the prevalence of MAP, serum samples from 2550 sheep and 1171 goats from 165 flocks (flock size 2 to 2879 animals) were serologically examined for MAP antibodies in 2021. Additionally, 1 to 6 environmental faecal samples were collected from every flock depending on the flock size. They were examined for the presence of MAP by using both bacteriological cultivation and a commercially available real-time-PCR. RESULTS: MAP antibodies were detected in 41 sheep (1.6%) and 29 goats (2.5%), which accounts to a detection of MAP antibodies in 20.6% of the 165 flocks (on herd level). The symptoms of paratuberculosis, weight loss with preserved appetite and altered fecal consistency, were observed in only four of the flocks. A positive association was identified between the detection of MAP or MAP-specific antibodies in a flock and flock size, as well as positive association between the measured value in the Elisa (s/p ratio) and the age of the animal. Furthermore, an association between an increasing s/p ratio of the ELISA and a decreasing body condition was found. CONCLUSION AND CLINICAL RELEVANCE: Given what is known about the distribution of paratuberculosis in small ruminants, this disease should always be considered as a possible cause of weight loss and diarrhea. In case of high within-herd prevalence herd-specific control measures should be considered. In serological herd monitoring, animals with poor body condition should preferably be included in the sample, as the probability of being able to identify MAP positive animals is higher here.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Bayesian latent class modelling of true prevalence in animal subgroups with application to bovine paratuberculosis infection.

The prevalence of an infectious disease of animals living in separate groups (e.g. herds) is naturally analyzed using a Bayesian hierarchical latent class model. We propose an extension to this methodology by including subgroup level prevalence measures within the groups of animals. As an application illustrating the merits of our methodology, we reassessed the prevalence of bovine paratuberculosis (PTBC) infection in Hungarian commercial dairy farms. Our aim was to consolidate previous findings using a large amount of recent data and priors based on historical data. To model the subgroup level infection prevalence within animal groups, we considered correlated prevalences following beta distributions derived from independent normally distributed random herd effects. In the application, infection status of herds was handled as latent classes, multiparous and primiparous cows as within-herd subgroups. The novel methodology allows us to estimate both the mean and median conditional within-herd true prevalence (CWHP) related to each animal subgroup as well as other measures characterizing the interrelation of subgroups. The results of the application aligned with the findings of the former PTBC study, while the more recent and considerably larger dataset and the use of historical priors increased the reliability of the results. The STAN and JAGS codes of the application are available in Supplementary material.

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

Objectives: 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. Methods: 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. A phage 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. Results: 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. Conclusion: 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.

Seroprevalence of reproductive and infectious diseases in cattle: the case of Madre de Dios in the Peruvian southeastern tropics.

OBJECTIVE: The objective of this study was to determine the seroprevalence of reproductive and infectious diseases in tropical cattle in the Tambopata and Tahuamanu Provinces in the department of Madre de Dios, Peru. SAMPLE: 156 bovines from 7 cattle farms were sampled. These farms used exclusive grazing for food and natural mating for reproduction and did not have sanitary or vaccination programs. METHODS: The serum of blood samples was subjected to ELISA with commercial kits for the detection of antibodies against Neospora caninum, Mycobacterium avium subsp paratuberculosis (MAP), Leptospira interrogans, pestivirus bovine viral diarrhea virus-1, retrovirus bovine leukemia virus (BLV), orbivirus bluetongue virus (BTV), and herpesvirus bovine herpes virus-1 (BHV). The data were analyzed by means of association tests with χ2 (P < .05) and Spearman rank correlation (P < .05) in the SPSS v.15.0 software (IBM Corp). RESULTS: A low prevalence of antibodies to L interrogans, N caninum, M avium subsp paratuberculosis, bovine viral diarrhea virus-1 was found, but it was high to BTV, BLV, and BHV (100%, 53.85%, and 72.44%, respectively). The presence of BLV and BHV was higher in the Las Piedras District, bovines less than 5 years old, and cattle with breed characteristics of zebu and crossbred (P < .01). In addition, there was a significant correlation between both infections, showing 83.3% of BLV positivity that were also BHV positive (P < .01). CLINICAL RELEVANCE: The high prevalence of antibodies to BTV, BHV, and BLV could be due to livestock management practices, direct contact with infected animals, and variation of the presence of vectors and natural reservoirs in the context of climate change in the tropics.

Inter-laboratory ring trial to compare four quantitative polymerase chain reaction assays employed for detection of Mycobacterium avium subspecies paratuberculosis.

Johne's disease is an infectious enteric disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) affecting ruminant species worldwide. In Project 1, an independent performance comparison ring trail was conducted between three different commercial MAP quantitative polymerase chain reaction (qPCR) assay services (B, C, and D) currently marketed in Great Britain by three separate laboratories against each other and against a fourth assay (A) not available commercially in Great Britain. A total of 205 individual ovine and bovine samples from five farms were analyzed to give 41 sets of pooled results (pool size five) from each laboratory according to their specific protocols. The numbers of positive pools for assays A-D were 18, 12, 11, and 1 (43.9%, 29.2%, 26.8%, and 2.4%), respectively. Assessment of interrater reliability produced a Fleiss' kappa coefficient of 0.15, indicating very poor overall agreement between the four laboratories. Laboratories A-D diagnosed 4, 3, 2, and 1 flocks at the farm level, respectively, as MAP positive. In Project 2, 38 pooled ovine samples from 10 flocks were analyzed to compare the performance of laboratories A and B. The numbers of positive results for laboratories A and B were 24 (63.1%) and 17 (44.7%), respectively (Cohen's kappa 0.54), indicating that laboratory A was more sensitive than B in line with results from Project 1. Variation between laboratories offering MAP qPCR assays is a significant concern, and further work is warranted to validate and standardize the performance of assays between laboratories for both ovine and bovine samples.IMPORTANCEOur study reports the findings of an inter-laboratory ring trial comparing the performance of four different quantitative polymerase chain reaction (qPCR) assay services for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle and sheep. MAP is the causative agent of Johne's disease (also known as paratuberculosis), a significant production-limiting disease in livestock populations with a worldwide distribution. The content of this paper is significant and novel as it is the first to highlight the marked variation between the diagnostic sensitivity and reproducibility of the three principal commercial laboratories offering MAP qPCR diagnostic and screening services in Great Britain. The low sensitivity and high variability between the laboratories are of great concern and relevance to veterinary practitioners and livestock producers.

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

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

Advances in understanding the genetic architecture of antibody response to paratuberculosis in sheep by heritability estimate and LDLA mapping analyses and investigation of candidate regions using sequence-based data.

BACKGROUND: Paratuberculosis is a contagious and incurable disease that is caused by Mycobacterium avium subsp. paratuberculosis (MAP) with significant negative effects on animal welfare and farm profitability. Based on a large naturally infected flock over 12 years, we analyzed repeated enzyme-linked immunosorbent assay tests (ELISA), OvineSNP50 BeadChip genotypes and whole-genome sequences imputed from 56 influential animals. The main goals were to estimate the genetic parameters of proxy traits for resistance to MAP, identify genomic regions associated with the host's immune response against MAP and search for candidate genes and causative mutations through association and functional annotation analyses of polymorphisms identified by sequencing. RESULTS: Two variables were derived from ELISA tests. The first, a binary variable, assessed the infection status of each animal over the entire productive life, while the second considered the level of antibody recorded over time. Very similar results were obtained for both variables. Heritability estimates of about 0.20 were found and a significant region capturing 18% and 13% of the genetic variance was detected on ovine chromosome 20 by linkage disequilibrium and linkage analysis on OvineSNP50 positions. Functional annotation and association analyses on the imputed sequence polymorphisms that were identified in this region were carried out. No significant variants showed a functional effect on the genes that mapped to this region, most of which belong to the major histocompatibility complex class II (MHC II). However, the conditional analysis led to the identification of two significant polymorphisms that can explain the genetic variance associated with the investigated genomic region. CONCLUSIONS: Our results confirm the involvement of the host's genetics in susceptibility to MAP in sheep and suggest that selective breeding may be an option to limit the infection. The estimated heritability is moderate with a relevant portion being due to a highly significant region on ovine chromosome 20. The results of the combined use of sequence-based data and functional analyses suggest several genes belonging to the MHC II as the most likely candidates, although no mutations in their coding regions showed a significant association. Nevertheless, information from genotypes of two highly significant polymorphisms in the region can enhance the efficiency of selective breeding programs.

MicroRNAs modulate immunological and inflammatory responses in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

MicroRNAs (miRNAs) regulate the post-transcriptional expression of genes by binding to their target mRNAs. In this study, whole miRNA sequencing was used to compare the expression of miRNAs in ileocecal valve (ICV) and peripheral blood (PB) samples of cows with focal or diffuse paratuberculosis (PTB)-associated lesions in gut tissues versus (vs) control cows without lesions. Among the eight miRNAs differentially expressed in the PB samples from cows with diffuse lesions vs controls, three (miR-19a, miR-144, miR32) were also down-regulated in cows with diffuse vs focal lesions. In the ICV samples, we identified a total of 4, 5, and 18 miRNAs differentially expressed in cows with focal lesions vs controls, diffuse lesions vs controls, and diffuse vs focal lesions, respectively. The differential expression of five microRNAs (miR-19a, miR-144, miR-2425-3p, miR-139, miR-101) was confirmed by RT-qPCR. Next, mRNA target prediction was performed for each differentially expressed miRNA. A functional analysis using the predicted gene targets revealed a significant enrichment of the RNA polymerase and MAPK signaling pathways in the comparison of cows with focal vs no lesions and with diffuse vs focal lesions, respectively. The identified miRNAs could be used for the development of novel diagnostic and therapeutical tools for PTB control.

Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS900 PCR assay with a commercial ISMap02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS900 PCR assay. Among culture-positive samples before incubation, the IS900 PCR assay yielded significantly more positive results than the ISMap02 PCR assay; however, among culture-negative samples, the IS900 PCR assay yielded positive results both before and after incubation. The ISMap02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation.

Evidence of Mycobacterium avium subsp. paratuberculosis binding to albumin: technical and biological implications.

Albumin binding ability is a well-characterized feature of many bacteria. To the best of our knowledge, there are no previous reports about this ability among mycobacteria, even when bovine serum albumin (BSA) is a common component of supplements used for the enrichment of synthetic media for mycobacterial growth in vitro and also of buffers used in laboratory techniques. In this work we explored the albumin binding ability of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogenic bacterium causing a known and relevant ruminant disease worldwide, by immunizing rabbits with MAP (grown in media containing or not BSA) or BSA and conducting ELISA and immunoblot experiments with the obtained sera. As a result, we found that MAP can bind BSA when cultured in a conventional BSA-containing medium and when incubated for a short time in the presence of the protein. We also evaluated the host specificity of MAP interaction with albumin and found a preference for the protein of bovine origin when compared with its horse and rabbit homologs. Considerations about its technical and biological implications are discussed.