A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk.

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

Identification of loci associated with pathological outcomes in Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis using whole-genome sequence data.

Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case-control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P ≤ 5 × 10-7) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses.

Predicting sensitivity of repeated environmental sampling for Mycobacterium avium subsp. paratuberculosis in dairy herds using a Bayesian latent class model.

Between-herd transmission of Mycobacterium avium subsp. paratuberculosis (MAP) by subclinically infected cattle is an important risk which can hamper effective control of paratuberculosis. Knowledge of herd status would substantially reduce this risk; MAP positive farms can be detected with environmental sampling. The objective of this study was to compare cumulative sensitivities of annual environmental sampling with two or four samples per sampling event without knowledge of true herd status and to calculate the number of sampling events to achieve a cumulative sensitivity of at least 0.9. Data from three repeated sampling events in two study populations, one with 55 herds (two samples/event) and another with 30 herds (four samples/event) including test results, herd and sample characteristics and prior prevalence estimates, were derived from the Alberta Johne's Disease Initiative (Alberta, Canada). A recursive Bayesian latent class model was used to predict the cumulative sensitivity of repeated environmental sampling events. A sampling scheme with four samples per sampling event had a higher cumulative sensitivity than an alternative scheme with two samples. To achieve a cumulative sensitivity of at least 0.9 with 95% probability, eight sampling events with two environmental samples per set, or four sampling events with four samples per set were required. Further model assessment demonstrated that these results can only be generalized to cattle populations with a similar within-herd prevalence to those studied here (approximately 0.08). Nonetheless, these results could help predict herd-level prevalence in cattle populations after environmental testing and provide information regarding the uncertainty behind status estimates for herds repeatedly tested using environmental samples.

Individual faecal and boot swab sampling to determine John's disease status in small cattle herds.

Individual faecal samples were collected from adult animals in 275 cattle farms previously positive for Mycobacterium avium subsp. paratuberculosis (MAP). In addition, boot swab samples were collected in 30 randomly chosen farms. Faecal samples were tested for MAP by a combination of bacterial culture and PCR. A logistic regression and the Pearson Correlation were used to calculate the relation between the number of MAP­positive cows and boot swab results. In 66.9% of all previously tested herds, no positive individual faecal sample was detected, indicating possible fadeout of the infection. In 9 (30.0%) of the 30 selected farms, at least one MAP­shedding animal was detected in faecal samples individually collected, while only 5 (16.7%) of these farms were found positive when the boot sampling method was used. The sensitivity of the boot swab sampling increased up to 92% (95% CI: 41%­99%), if at least 12 animals were faecal MAP­shedders in a herd. The current study shows possible fadeout of JD in a substantial percentage of previously infected herds. Furthermore, in small herds, a relatively high within­herd prevalence of MAP­shedding animals is needed to assure reliable positive boot swab results.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

A Mycobacterium species for Crohn's disease?

In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Prediction of Johne's disease state based on quantification of T cell markers and their interaction with macrophages in the bovine intestine.

Cell-mediated immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) are regulated by various types of T lymphocytes. The aim of this study was to quantitate T cell subsets in the mid-ileum of cows naturally infected with MAP to identify differences during different stages of infection, and to determine whether these subsets could be used as predictors of disease state. Immunofluorescent labeling of T cell subsets and macrophages was performed on frozen mid-ileal tissue sections archived from naturally infected dairy cows in either subclinical or clinical disease status, and noninfected control cows. Comprehensive IF staining for CD4, CD8α, TcR1-N24 (gamma delta), FoxP3, CXCR3 and CCR9 served to define T cell subsets and was correlated with macrophages present. Clinically affected cows demonstrated significantly higher numbers of CXCR3+ (Th1-type) and CCR9+ (total small intestinal lymphocytes) cells at the site of infection compared to the subclinical cows and noninfected controls. Further, predictive modeling indicated a significant interaction between CXCR3+ and AM3K+ (macrophages) cells, suggesting that progression to clinical disease state aligns with increased numbers of these cell types at the site of infection. The ability to predict disease state with this model was improved from previous modeling using immunofluorescent macrophage data. Predictive modelling indicated an interaction between CXCR3+ and AM3K+ cells, which could more sensitively detect subclinical cows compared to clinical cows. It may be possible to use this knowledge to improve and develop an assay to detect subclinically infected animals with more confidence during the early stages of the disease.

Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in dairy herds in Minas Gerais state, Brazil / [Soroprevalência do Mycobacterium avium subespécie paratuberculosis e vírus da leucemia bovina e fatores de risco associados em rebanhos leiteiros de Minas Gerais, Brasil]

Mycobacterium avium subesp. paratuberculosis (MAP) e o vírus da leucemia bovina (BLV) são agentes que causam grandes perdas econômicas nos rebanhos. O objetivo deste estudo foi avaliar a situação epidemiológica da paratuberculose bovina (PTB) e leucose enzoótica bovina (EBL) em rebanhos leiteiros de Lagoa Formosa, Minas Gerais, Brasil. Foram coletadas 236 amostras de sangue de vacas, as quais foram submetidas aos testes ELISA e imunodifusão em gel de ágar para detecção de anticorpos contra MAP e BLV. A soroprevalência de anticorpos contra MAP e BVL foi de 20% para os rebanhos e 6% para os animais e de 85% para os rebanhos e 50,42% para os animais, respectivamente. A presença dessas enfermidades deve servir como um alerta para os produtores e veterinários, para que concentrem maior atenção na implementação de medidas higiênico-sanitárias, incorporando elementos de vigilância com base nos riscos identificados no estudo.(AU)

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Putting Crohn's on the MAP: Five Common Questions on the Contribution of Mycobacterium avium subspecies paratuberculosis to the Pathophysiology of Crohn's Disease.

For decades, Mycobacterium avium subspecies paratuberculosis (MAP) has been linked to the pathogenesis of Crohn's disease. Despite many investigations and research efforts, there remains no clear unifying explanation of its pathogenicity to humans. Proponents argue Crohn's disease shares many identical features with a granulomatous infection in ruminants termed Johne's disease and similarities with ileo-cecal tuberculosis. Both are caused by species within the Mycobacterium genus. Sceptics assert that since MAP is found in individuals diagnosed with Crohn's disease as well as in healthy population controls, any association with CD is coincidental. This view is supported by the uncertain response of patients to antimicrobial therapy. This report aims to address the controversial aspects of this proposition with information and knowledge gathered from several disciplines, including microbiology and veterinary medicine. The authors hope that this discussion will stimulate further research aimed at confirming or refuting the contribution of MAP to the pathogenesis of Crohn's disease and ultimately lead to advanced targeted clinical therapies.

Bayesian estimation of the true prevalence of paratuberculosis in Hungarian dairy cattle herds.

Paratuberculosis is a chronic incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), which leads to extensive economic losses on dairy farms, and may also pose serious public health risk to the consumers. The aim of our study was to estimate the true prevalence of paratuberculosis in commercial dairy cattle herds participating in a voluntary MAP testing programme that started in February 2018 in Hungary. Milk samples collected during official milk recording were used for MAP ELISA testing. A Bayesian two-stage hierarchical (herd and animal level) model was fitted to the data. Altogether, 26,437 cows from 51 herds were sampled, which represents 14.4 % of the Hungarian dairy cow population. The median herd size was 477 cows (interquartile range: 331-709). Each studied farm had at least one ELISA positive cow, resulting in a herd-level apparent prevalence of 100 %. The overall within herd apparent prevalence was 5.5 %. Herd-level true prevalence was estimated at 89.1 % [95 % credible interval (CrI): 80.3-95.6%]. Within the infected herds, the median animal-level true prevalence was 4.4 % (3.2-5.8%) for primiparous and 10.3 % (7.9-12.9%) for multiparous cows, respectively. The probability of having an animal-level true prevalence of at least 5% among primiparous cows, within infected herds, was 17.8 %. Similarly, the probability of having an animal-level true prevalence of at least 5% or 10 % among multiparous cows was 100 % and 56 %, respectively. Simulations assuming herd-level true prevalence varying from 50 to 100 % revealed high accuracy of our Bayesian model. Our study showed that a large percentage of the studied Hungarian dairy cattle herds was infected with MAP.

Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods.

Gold nanoparticle based immunochromatographic biosensor for rapid diagnosis of Mycobacterium avium subspecies paratuberculosis infection using recombinant protein.

Highly infectious and obvious withstand ability of Mycobacterium avium subspecies paratuberculosis (MAP) to environment as well as lack of on-site field diagnostic methods notably hampers the paratuberculosis (PTB) control. The existing intricacy, time-consuming and complicated diagnostic methods of PTB accentuate the development of novel and easy-to-perform on-site test. A gold nanoparticle (GNP) based lateral-flow assay (LFA) using MAP recombinant protein (44 kDa) has been developed for sensitive and specific detection of PTB in field conditions. The diagnostic sensitivity and specificity of the LFA for MAP specific antibodies was found approximately 84.2% and 83.3% in comparison to indirect enzyme-linked immunosorbent assay. Consequently, the newly developed GNP based LFA offers on-site and cost-effective method for the prompt diagnosis of PTB and precludes the time-consuming laboratory screening.

Bayesian latent class estimation of sensitivity and specificity parameters of the PMS-PCR test for the diagnosis of cattle sub-clinically infected with Mycobacterium avium subsp. paratuberculosis.

The objective of this study was to estimate the performance of the peptide magnetic separation PCR test (PMS-PCR) for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) in sub-clinically infected dairy cattle. Twenty-one herds were randomly selected from a source population of 131 commercial dairy herds with a known history of MAP infection, located in the De Los Rios and De Los Lagos regions, in southern Chile. In the selected herds, all milking cows with ≥2 parities and without any clinical signs were sampled, collecting feces and blood-serum samples. The PMS-PCR test was used to analyze the fecal samples, while serum samples were analyzed using a commercial ELISA kit. A Bayesian latent class model was used to estimate the sensitivity (Se) and specificity (Sp) of the diagnostic tests. A total of 1381 animals were sampled in the 21 selected dairy herds, with an average sample size of 65 animals per herd (range 10-721). The PMS-PCR test had a greater Se than the ELISA test, with a median of 85.5 % (posterior probability interval (PPI) 95 %: 79.3-91.0%), while the ELISA test presented a median of 21.7 % (95 % PPI: 18.3-25.4%). On the other hand, the ELISA test had a better Sp than the PMS-PCR test, with a median of 97.7 % (95 % PPI: 96.6-98.5%), whereas PMS-PCR presented a median of 90.8 % (95 % PPI: 88.3-93.9%). Model results showed that PMS-PCR has a better Se than all available tests for MAP diagnosis in subclinical animals. However, this test should be used with care in herds with high infection rates, where a high MAP environmental load is expected, potentially increasing the frequency of false positive cases due to the pass-through phenomenon.

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.

Comparative evaluation of Mycobacterium avium subspecies paratuberculosis (MAP) recombinant secretory proteins as DTH marker for paratuberculosis.

Delayed type hypersensitivity (DTH) based skin test is an important onsite animal herd screening procedure for detecting the early stages of the chronic mycobacterial infections. DTH testing plays a vital role in the diagnosis of paratuberculosis infection. However, there are questions over the specificity of this test due to cross-reactive epitopes present on the purified protein derivative (PPD) prepared from the whole cell secretory proteins. PPD may contain proteins shared with other mycobacteria especially environmental species. Therefore, it is needed to test alternate paratuberculosis specific secretory antigens. Present study explored the potential of recombinant secretory proteins (MAP2168c, MAP1693c, MAP3547c, MAP4308c and MAP2677c) as DTH markers. The published literature shows that these proteins as strong cell mediated markers with specificity to paratuberculosis bacilli. To determine the positive skin thickness cutoff, herds of farm animals with history of endemic paratuberculosis were selected and thickness of >2.0 mm was reported as the positive cutoff. Preliminary findings on pilot scale animals report the usefulness of recombinant secretory proteins as DTH markers over traditional Johnin assay. Traditional Johnin reported more false positives and negatives compared to gold standard fecal PCR and field reference plate ELISA test. Present findings encourage and demand further research.

Assessing efficacy of N-Acetyl-l-Cysteine-Sodium Hydroxide on bacterial viability and enhanced recovery of Mycobacterium avium subsp. paratuberculosis from bovine colostrum.

The standard procedure for the improved cultural recovery of viable Mycobacterium spp. from diverse samples mainly depends on reducing the viability of background microbiota using different chemical compounds. This study was designed to i) evaluate the efficacy and comparison between N-Acetyl-l-Cysteine-Sodium hydroxide (NALC-2% NaOH) and hexadecylpyridinium chloride (0.75% HPC) treatment and exposure time on reducing the viability of undesirable microorganisms with minimal impact on colostrum consistency; and ii) assess the impact of NALC-2% NaOH on improved and enhanced recovery of Mycobacterium avium subsp. paratuberculosis (MAP) in spiked postpartum colostrum samples and consistency of colostrum. A total of 40 samples, each treated with NALC-2% NaOH for 15 min or 0.75% HPC for 5 h, were investigated for total mesophilic aerobic bacteria (MAB) and enterobacteria (EB) (CFU mL-1). The results showed that treatment of colostrum samples with NALC-2% NaOH completely eliminated EB and significantly reduced MAB (3.6 log10 CFU mL-1). Conversely, samples treated with 0.75% HPC produced a complex mixture following interaction with the colostrum protein and showed non-significant and variable results. In addition, the spiked colostrum treated with NALC-2% NaOH for 15 min revealed recovery of viable MAP cells with a minimum limit of detection of 1.36 log10 CFU 10 mL-1 where no change in the consistency of colostrum was observed. In conclusion, 15-min NALC-2% NaOH treatment of colostrum may significantly reduce the viability of undesirable microorganisms and help to enhance the efficient recovery of MAP without impacting the consistency of high quality postpartum colostrum. This rapid procedure is suitable for efficient recovery and early detection of MAP as well as preventing its transmission to neonates and young calves in MAP infected herds.

Prevalence of paratuberculosis in dairy cattle in ecuador.

Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic infectious contagious disease of the intestinal tract of ruminants that are also associated with Crohn's disease in humans. The existence of paratuberculosis in Ecuador is virtually unknown; hence, the present study was performed to gain insight into the prevalence of this disease. Methods: Three dairy cattle farms in different geographic regions in Ecuador were investigated for the infection with MAP, and 600 blood samples, 200 of each cattle herd, were processed with an indirect enzyme-linked immunosorbent assay. Fecal samples of the seropositive cows were processed for culture on modified Löwenstein-Jensen medium. Results: One hundred and fifty bovines (25%) resulted seropositive and we confirmed with culture the presence of MAP in 4.7% (7/150) of the seropositive cows. Approximately 20% of the fecal samples of seropositive cows yielded nontuberculous mycobacteria (NTM) species including M. avium subsp. avium, a NTM species closely related to MAP. Conclusions: The seroprevalence of paratuberculosis in this first study for Ecuador is high (25%). We discuss a possible interference of NTM species, isolated from fecal samples, with the diagnosis of paratuberculosis. With this report, a baseline study, we confirm for the first time the presence of paratuberculosis in Ecuador, and we provide the necessary information for future studies and control of this disease.

Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany.

AIMS: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. METHODS AND RESULTS: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. CONCLUSIONS: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

From six to zero per cent Mycobacterium avium subsp. paratuberculosis milk ELISA positivity in three years - probably induced by Mycobacterium vaccae.

This research communication reports the results of a study aimed at investigating the effects of introducing Mycobacterium vaccae on paratuberculosis carriage in a dairy herd. M. vaccae is a non-pathogenic member of the Mycobacteriaceae, with immunomodulatory and immunotherapeutic capabilities, acting by stimulating the cellular immune system, important in protection against paratuberculosis. Starting in 2014 we administered, by gavage, 1010 live M. vaccae bacteria to all new-born heifers on a dairy farm, first within 24 h of birth and again 2 weeks later. Paratuberculosis carriage was monitored yearly by milk ELISA. Faecal samples of 50% of cows, aged 3 years, born 1, 2 or 3 years before the experiment's onset, were tested by qPCR for MAP shedding and compared to 100% treated cows of the same age. Within 3 years, milk ELISA positivity was reduced from 6 to 0% and remained unchanged for the subsequent 2 years. One qPCR positive control cow was found each year for a total of 3 animals (2.46%). One positive cow (1%) was found among the treated cows. Two of the 3 positive control animals, still present on the farm at the end of 2019, tested negative whereas the positive test cow continued shedding MAP. M. vaccae shedding heifers mixing with adult cows were the probable means of the microorganism's propagation. The results of this investigation indicate that the introduction of live M. vaccae may be an inexpensive and fast alternative to current paratuberculosis control practices, justifying further exploration of the topic.