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We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne's disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity., (Copyright © 2024 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The lack of sensitive diagnostic methods to detect Mycobacterium avium subsp. paratuberculosis (Map) subclinical infections has hindered the control of paratuberculosis (PTB). The serum proteomic profiles of naturally infected cows presenting focal and diffuse pathological forms of PTB and negative controls ( n = 4 per group) were analyzed using TMT-6plex quantitative proteomics. Focal and diffuse are the most frequent pathological forms in subclinical and clinical stages of PTB, respectively. One (focal versus (vs.) control), eight (diffuse vs. control), and four (focal vs. diffuse) differentially abundant (DA) proteins ( q -value < 0.05) were identified. Ingenuity pathway analysis of the DA proteins revealed changes in the acute-phase response and lipid metabolism. Six candidate biomarkers were selected for further validation by specific ELISA using serum from animals with focal, multifocal, and diffuse PTB-associated lesions ( n = 108) and controls ( n = 56). Overall, the trends of the serum expression levels of the selected proteins were consistent with the proteomic results. Alpha-1-acid glycoprotein (ORM1)-based ELISA, insulin-like growth factor-binding protein 2 (IGFBP2)-based ELISA, and the anti-Map ELISA had the best diagnostic performance for detection of animals with focal, multifocal, and diffuse lesions, respectively. Our findings identify potential biomarkers that improve diagnostic sensitivity of PTB and help to elucidate the mechanisms involved in PTB pathogenesis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Mycobacterium avium ssp. paratuberculosis (MAP) has been implicated in the development of Crohn's disease (CD) for over a century. Similarities have been noted between the (histo)pathological presentation of MAP in ruminants, termed Johne's disease (JD), and appearances in humans with CD. Analyses of disease presentation and pathology suggest a multi-step process occurs that consists of MAP infection, dysbiosis of the gut microbiome, and dietary influences. Each step has a role in the disease development and requires a better understanding to implementing combination therapies, such as antibiotics, vaccination, faecal microbiota transplants (FMT) and dietary plans. To optimise responses, each must be tailored directly to the activity of MAP, otherwise therapies are open to interpretation without microbiological evidence that the organism is present and has been influenced. Microscopy and histopathology enables studies of the mycobacterium in situ and how the associated disease processes manifest in the patient e.g., granulomas, fissuring, etc. The challenge for researchers has been to prove the relationship between MAP and CD with available laboratory tests and methodologies, such as polymerase chain reaction (PCR), MAP-associated DNA sequences and bacteriological culture investigations. These have, so far, been inconclusive in revealing the relationship of MAP in patients with CD. Improved and accurate methods of detection will add to evidence for an infectious aetiology of CD. Specifically, if the bacterial pathogen can be isolated, identified and cultivated, then causal relationships to disease can be confirmed, especially if it is present in human gut tissue. This review discusses how MAP may cause the inflammation seen in CD by relating its known pathogenesis in cattle, and from examples of other mycobacterial infections in humans, and how this would impact upon the difficulties with diagnostic tests for the organism., (© 2024. Crown.)

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., (© 2024. The Author(s).)

Introduction: As a contagious and chronic disease in the livestock industry, Paratuberculosis is a significant threat to dairy herds' genetic and economic resources. Due to intensive breeding and high production of dairy cattle, the incidence and prevalence are higher. Developing non-destructive diagnostic methods for the early detection and identification of healthy animals is paramount for breeding programs. Conventional methods are almost entirely destructive, have low accuracy, lack precision, and are time-consuming. Near-infrared spectroscopy (NIRS) and aquaphotomics can detect changes in biofluids and thus have the potential to diagnose disease. This study aimed to investigate the diagnostic ability of NIRS and aquaphotomics for Paratuberculosis in dairy cattle., Methods: Blood plasma from dairy cattle was collected in the NIR range (1,300 nm to 1,600 nm) 60 days before and 100 days to 200 days after calving in two groups, positive and negative, using the same consecutive enzyme-linked immunosorbent assay test results three times as a reference test., Results: NIRS and aquaphotomics methods invite 100% accuracy, sensitivity, and specificity to detect Paratuberculosis using data mining by unsupervised method, Principal Component Analysis, and supervised methods: Soft Independent Modeling of Class Analogiest, Linear Discriminant Analysis, Quadratic Discriminant Analysis, Partial Least Square-Discriminant Analysis, and Support Vector Machine models., Discussion: The current study found that monitoring blood plasma with NIR spectra provides an opportunity to analyze antibody levels indirectly via changes in water spectral patterns caused by complex physiological changes, such as the amount of antibodies related to Paratuberculosis by aquagram., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Behdad, Pakdel and Massudi.)

Aims: This study aimed to identify specific genomic targets for the detection and strain typing of Map and analyse their sensitivity and specificity, and detect Map directly from faeces., Methods and Results: A comparative genomics approach was used to identify specific genomic targets for the detection and strain typing of Map. A Map specific qPCR using the primer pair 7132 that targets a DNA segregation ATPase protein was able to detect all strains of Map and is more sensitive than the current Johne's disease PCR assays with a sensitivity of 0.0002 fg µl-1. A strain specific qPCR using the Atsa primer pair that targets the arylsulfase gene was able to differentiate between Type S and Type C strains of Map and was more sensitive than the IS1311 PCR and REA with a sensitivity of 40 fg µl-1 and was specific for Type S Map. Both assays successfully detected Map directly from faeces., Conclusion: This study developed and validated two genomics informed qPCR assays, 7132B Map and Atsa Type S and found both assays to be highly specific and sensitive for the detection of Map from culture and directly from faeces. This is the first time that a probe-based qPCR has been designed and developed for Map strain typing, which will greatly improve the response time during outbreak investigations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

Johne's disease is an infectious enteric disease caused by Mycobacterium avium subspecies paratuberculosi s (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., Competing Interests: The authors declare no conflict of interest.

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.

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Die Autoren erklären, dass keine geschützten, finanziellen, beruflichen oder anderweitigen Interessen an einem Produkt oder einer Firma bestehen, welche die in dieser Veröffentlichung genannten Inhalte oder Meinungen beeinflussen können., (Thieme. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS 900 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 IS 900 PCR assay with a commercial ISMap 02 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 IS 900 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 IS 900 PCR assay. Among culture-positive samples before incubation, the IS 900 PCR assay yielded significantly more positive results than the ISMap 02 PCR assay; however, among culture-negative samples, the IS 900 PCR assay yielded positive results both before and after incubation. The ISMap 02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS 900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap 02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets., Methods: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated., Results: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more., Limitations: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied., Conclusions: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control., (© 2023 British Veterinary Association.)

Background: Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic progressive granulomatous enteritis mainly affecting domestic and wild ruminants worldwide. Although paratuberculosis could be prevail in Ethiopia, there is a scarcity of epidemiological data on paratuberculosis in the country. Thus, this study was conducted to estimate the prevalence of paratuberculosis based on gross and microscopic lesions in cattle slaughtered at ELFORA Abattoir, central Ethiopia. Small intestines and associated lymph nodes of 400 apparently healthy cattle which were slaughtered at ELFORA export abattoir were examined for gross and microscopic lesions of paratuberculosis. The microscopic lesions were classified into four grades (I-IV) based on the type and number of cells infiltrated into the lesion. The prevalence of paratuberculosis was estimated on the basis of gross as well as microscopic lesion of paratuberculosis., Results: The prevalence of paratuberculosis was 11.25% (95% Confidence interval, CI = 0.083-0.148) on the basis of gross lesion. However, relatively lower prevalence (2.0%, 95% CI = 0.01, 0.039) was recorded based on microscopic lesion. The gross lesions were characterized by intestinal thickening, mucosal corrugations and enlargement of associated mesenteric lymph nodes. On the other hand, the microscopic lesions were characterized by granuloma of different grades ranging from grade I to grade III lesions., Conclusions: The present study indicated the occurrence of paratuberculosis in cattle of Ethiopia based on the detection of gross and microscopic lesions consistent with the lesion of paratuberculosis. The result of this study could be used as baseline information for future studies on the epidemiology and economic significance of paratuberculosis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in triggering rheumatoid arthritis (RA) could be a population-specific phenomenon. This study explored the relationship between MAP and RA using serological and molecular techniques; In this case-control study, 239 Iranian participants, including 120 RA patients and 119 controls, were enrolled. The indirect ELISA was designed to diagnose antibodies against MAP3865c125-133 and Zinc transporter 8 (ZnT8) 178-186 . The Nested-Polymerase Chain Reaction (PCR) detected MAP in blood; The frequency of MAP in RA patients and controls was 31.9% and 12.5%, respectively (P = 0.002). The antibodies against MAP3865c125-133 and ZnT8 178-186 were 42.9% and 37% in RA patients and 14.2% and 11.7% in the controls, respectively (P < 0.0001). Interestingly, positive ELISA results in previously diagnosed (PD) RA were more common than newly diagnosed (ND) RA patients (P < 0.05).; The findings showed a higher frequency of MAP and its antibodies in the RA patients than in the controls. This data indicated MAP as one of RA's predisposing factors. Also, this first report implies the high positivity of MAP in Iranian RA patients., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Paratuberculosis is an enteric disease caused by Mycobacterium avium subs. Paratuberculosis (MAP). Quantifying the load of MAP in faeces samples offers the advantage of determining the stage of infection and planning control measures. Currently, detection of MAP in faecal specimens relies on cultural assays and quantitative PCR (qPCR), but both methods have limitations such as prolonged isolation times for cultural assay and the absence of nucleic acid standards for qPCR. Digital PCR (dPCR) represents an advancement over qPCR as it allows direct quantification of nucleic acid in a sample without the need for a standard curve. The present paper reports about the validation process, following ISO 20395:2019 guidelines, of a F57 digital PCR assay for quantifying MAP cells in faecal samples. Based on our validation, the Limit Of Detection (LOD) corresponds to 7.85 10 4 MAP cells/g, and the Limit Of Quantification (LOQ) to 7.85 10 5 MAP cells, with an efficiency of recovery at LOQ estimated about 4.5%. To assess precision, we evaluated the same faecal sample extracted by two different operators at different times. The standard deviation under repeatability conditions (S Repeatability) and intersession variability conditions (S Intermediate) were calculated, resulting in values of 0.43 and 0.26, respectively. Trueness was determined at LOQ and a value ten times higher, yielding percentages of 3.35% and 5.16%, respectively. Linearity showed a R 2 value of 0.998, indicating strong linear correlation. Measurement uncertainty was 26% in absolute value and 3% on a logarithmic base 10 scale. Overall, the assay exhibits good specificity and robustness. Our validation underlines the good performance of the quantification method and allow the laboratory to provide quantitative results of MAP/cells on faecal samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic and progressive granulomatous enteritis and economic losses in dairy cattle in subclinical stages. Subclinical infection in cattle can be detected using serum MAP antibody enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR) tests., Objectives: To investigate the differences in blood parameters, according to the detection of MAP using serum antibody ELISA and fecal PCR tests., Methods: We divided 33 subclinically infected adult cattle into three groups: seronegative and fecal-positive (SNFP, n = 5), seropositive and fecal-negative (SPFN, n = 10), and seropositive and fecal-positive (SPFP, n = 18). Hematological and serum biochemical analyses were performed., Results: Although the cows were clinically healthy without any manifestations, the SNFP and SPFP groups had higher platelet counts, mean platelet volumes, plateletcrit, lactate dehydrogenase levels, lactate levels, and calcium levels but lower mean corpuscular volume concentration than the SPFN group ( p < 0.017). The red blood cell count, hematocrit, monocyte count, glucose level, and calprotectin level were different according to the detection method ( p < 0.05). The SNFP and SPFP groups had higher red blood cell counts, hematocrit and calprotectin levels, but lower monocyte counts and glucose levels than the SPFN group, although there were no significant differences ( p > 0.017)., Conclusions: The cows with fecal-positive MAP status had different blood parameters from those with fecal-negative MAP status, although they were subclinically infected. These findings provide new insights into understanding the mechanism of MAP infection in subclinically infected cattle., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test. Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0-20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown., Competing Interests: Declaration of Competing Interest The authors have no financial or personal conflicts of interest that could have inappropriately influenced the development of this research., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Estimation of the accuracy of diagnostic tests in the absence of a gold standard is an important research subject in epidemiology (Dohoo et al., 2009). One of the most used methods the last few decades is the Bayesian Hui-Walter (HW) latent class model (Hui and Walter, 1980). However, the classic HW models aggregate the observed individual test results to the population level, and as a result, potentially valuable information from the lower level(s) is not fully incorporated. An alternative approach is the Bayesian logistic regression (LR) latent class model that allows inclusion of individual level covariates (McInturff et al., 2004). In this study, we explored both classic HW and individual level LR latent class models using Bayesian methodology within a simulation context where true disease status and true test properties were predefined. Population prevalences and test characteristics that were realistic for paratuberculosis in cattle (Toft et al., 2005) were used for the simulation. Individual animals were generated to be clustered within herds in two regions. Two tests with binary outcomes were simulated with constant test characteristics across the two regions. On top of the prevalence properties and test characteristics, one animal level binary risk factor was added to the data. The main objective was to compare the performance of Bayesian HW and LR approaches in estimating test sensitivity and specificity in simulated datasets with different population characteristics. Results from various settings showed that LR models provided posterior estimates that were closer to the true values. The LR models that incorporated herd level clustering effects provided the most accurate estimates, in terms of being closest to the true values and having smaller estimation intervals. This work illustrates that individual level LR models are in many situations preferable over classic HW models for estimation of test characteristics in the absence of a gold standard., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In the United Kingdom (UK) a voluntary programme to control paratuberculosis in cattle based on herd management and serological screening has been operating since 1998. The programme assigns a risk level to each participating herd according to the within herd seroprevalence and the confirmation of the presence of infection with Mycobacterium avium subspecies paratuberculosis (MAP) by faecal culture or polymerase chain reaction (PCR). From the outset a general concern over the specificity of the paratuberculosis antibody enzyme-linked immunosorbent assay (ELISA) resulted in the use of a faecal screen for the causal organism to negate or confirm infection in individual seropositive animals. Progress in improving the diagnostic tests has been gradual throughout the life of the programme and the under-pinning approach to using tests to determine the risk of paratuberculosis for a herd required to be re-examined. This study used a large data set of more than 143,000 test results over five years from the lowest paratuberculosis risk level category of herds to estimate the specificity of a commercially available paratuberculosis antibody ELISA for cattle. In each year of the study the estimated specificity reached or exceeded 0.998. We also examined the apparent impact that annual or more frequent application of the single intradermal comparative cervical tuberculin (SICCT) test for tuberculosis (TB), using purified protein derivatives of Mycobacterium bovis and Mycobacterium avium subspecies avium, had on specificity of the antibody ELISA for paratuberculosis. We found a statistically significant difference in three of the five years with herds that were officially tuberculosis free and not subject to frequent SICCT testing. This difference was small and considered to be of little practical importance for the paratuberculosis assurance programme. We concluded that, in the UK the mandatory TB surveillance programme of cattle herds is not a limiting factor in the use of serological testing to support herd-level assurance schemes for paratuberculosis. Furthermore, in paratuberculosis, where shedding of MAP is intermittent and the sensitivity of the commercially available PCR tests for detection MAP is highly variable, faecal screening of seropositive animals is an unreliable method for negating infection in seropositive cattle., Competing Interests: Declaration of Competing Interest The author was previously the manager of SRUC Veterinary Services and the SRUC Premium Cattle Health Scheme is a commercial enterprise., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, contagious, and typically life-threatening enteric disease of ruminants caused by a bacterium of the genus Mycobacterium, but it can also affect non-ruminant animals. MAP transmission occurs through the fecal-oral pathway in neonates and young animals. After infection, animals generate IL-4, IL-5, and IL-10, resulting in a Th2 response. Early detection of the disease is necessary to avoid its spread. Many detection methods, viz., staining, culture, and molecular methods, are available, and numerous vaccines and anti-tuberculosis drugs are used to control the disease. However, the prolonged use of anti-tuberculosis drugs leads to the development of resistance. Whereas vaccines hamper the differentiation between infected and vaccinated animals in an endemic herd. This leads to the identification of plant-based bioactive compounds to treat the disease. Bioactive compounds of Ocimum sanctum and Solanum xanthocarpum have been evaluated for their anti-MAP activity. Based on the MIC50 values, Ursolic acid (12 µg/mL) and Solasodine (60 µg/mL) were found to be suitable for anti-MAP activity.

The objective of this study was to report the occurrence of Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats, via description of their clinical presentation, histopathological findings, and molecular identification of the infectious agent. Screening was performed using IS900 real-time PCR (qPCR) in milk samples from 179 properties in the semiarid of Northeast region of Brazil. Pooled milk samples from all lactating goats from processing plants were submitted to molecular diagnosis. One property had a positive result at qPCR. The production unit which had the positive sample for MAP was located, and an on-site visit to this property was performed to collect individual milk samples, seven of which tested MAP positive by IS900 qPCR. With permission from the owner, two goats (Animal 1 was positive and Animal 2 was negative on first qPCR for MAP) were acquired and euthanized. Animals 1 and 2 had milk and portions of the duodenum, ileum, colon, and mesenteric lymph nodes positive at qPCR for MAP. Animal 1 also had MAP DNA detected in part of the jejunum and cecum. In animal 2, the ileocecal valve tested positive. MAP was not detected in the blood or feces of either animal; however; it was confirmed for the association of clinical findings, histopathology, and qPCR. The gene IS900 from the positive samples were sequenced and showed a 99% similarity with MAP. The MAP was identified for the first time in the goat milk and tissues in the semiarid region of Northeast Brazil., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Amplification of the IS 900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis , which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS 900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS 900 due to 5' deletions at some IS 900 loci. These IS 900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS 900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS 900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS 900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Although Mycobacterium avium subsp. paratuberculosis (MAP) has threatened public health and the livestock industry, the current diagnostic tools (e.g., fecal PCR and enzyme-linked immunosorbent assay [ELISA]) for MAP infection have some limitations, such as inconsistent results due to intermittent bacterial shedding or low sensitivity during the early stage of infection. Therefore, this study aimed to develop a novel biomarker focusing on elucidating the gut microbial signature of MAP-positive ruminants, since the clinical signs of MAP infection are closely related to dysbiosis. 16S rRNA-based gut microbial community analysis revealed both a decrease in microbial diversity and the emergence of several distinct taxa following MAP infection. To determine the discriminant taxa diagnostic of MAP infection, machine learning-based feature selection and predictive model construction were applied to taxon abundance data or their transformed derivatives. The selected taxa, such as Clostridioides (formerly Clostridium) difficile, were used to build models using a support vector machine, linear support vector classification, k -nearest neighbor, and random forest with 10-fold cross-validation. The receiver operating characteristic-area under the curve (ROC-AUC) analysis of the models revealed their high accuracy, up to approximately 96%. Collectively, taxonomic signatures of cattle gut microbiotas according to MAP infection status could be identified by feature selection tools and applied to establish a predictive model for the infection state. IMPORTANCE Due to the limitations, such as intermittent bacterial shedding or poor sensitivity, of the current diagnostic tools for Johne's disease, novel biomarkers are urgently needed to aid control of the disease. Here, we explored the fecal microbiota of Johne's disease-affected cattle and tried to discover distinct microbial characteristics which have the potential to be novel noninvasive biomarkers. Through 16S rRNA sequencing and machine learning approaches, a dozen taxa were selected as taxonomic signatures to discriminate the disease state. In addition, when constructing predictive models using relative abundance data of the corresponding taxa, the models showed high accuracy for classification, even including animals with subclinical infection. Thus, our study suggested novel noninvasive microbiological biomarkers that are robustly expressed regardless of subclinical infection and the applicability of machine learning for diagnosis of Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn's disease. Therefore, control is becoming priority for governments. Control is largely dependent on "Test and Cull" or "Test and Segregate" policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces., Methods and Results: Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples., Conclusion: Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: paratuberculosis (PTB) is clinically characterized by diarrhea and progressive weight loss in ruminants, and causes economic losses due to decreased milk production, premature animal disposal, and increased veterinary treatment costs. The presence of PTB has been increasing in milk producing bovine farms in Mexico, yet epidemiology data from specific farms remains scarce. Objective: to determine the proportion of cows positive for Mycobacterium avium subsp. paratuberculosis (Map) among culled cows. Methods: in the present study, 40 culled cows from 25 farms located at the complejo agropecuario industrial de Tizayuca (CAITSA) in Hidalgo (Mexico) were evaluated for PTB presence by histopathological and microbiological studies. Tissue and rectal scraping samples were taken from all cows, and studied for presence of histopathology PTB-compatible lesions. Samples were processed for Map isolation on selective agar culture media; typical Map colonies were confirmed by polymerase chain reaction (PCR). Results: potential PTB infection was found in 25% (10/40) of the animals evaluated by histopathology and acid-fast bacilli (AFB) staining, but only 7.5% (3/40) were positive for type 2 Map (C cattle strain) by PCR. Conclusions: the prevalence of PTB in cattle farms at CAITSA was confirmed, which may be valuable epidemiological information in Mexico. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate Mycobacterium avium subsp. paratuberculosis (MAP) in dairy buffalo herds from six different geographical areas in Nineveh, Iraq. A total of 87 individual faecal samples from river buffaloes, representing 12 dairy herds, were investigated for detection of MAP using cultural, Ziehl‑Neelsen and MAP‑specific PCR‑based methods. Overall, MAP was detected at a higher frequency at herd‑level (4/12; 33%) compared to the total individual faecal samples (14/87; 16%) with a cell density ranging from 101 to 103 CFU g‑1. A significantly (p < 0.05) higher frequency (9/17; 53%) of MAP was observed in faecal samples collected from clinically diseased as compared to healthy (5/70; 7%) buffaloes selected for the study. However, no statistically significant difference (p ≥ 0.05) was observed in the frequency of MAP occurrence between clinical (9; 64%) and apparently healthy (5; 36%) cases. This report, which is the first MAP study based on data from Iraqi dairy buffalo herds suggests that MAP transmission is a significant health risk for grazing livestock. In conclusion, this study would help farm owners and regulatory authorities to realise the importance of developing and applying best farm management practices in order to prevent transmission of MAP to healthy animals and the environment. In addition, effective diagnostic tests should be taken into account when carrying out the screening tests.

Johne's disease (bovine paratuberculosis) is an endemic disease caused by Mycobacterium avium subspecies paratuberculosis (Map). Map is transmitted between herds primarily through movement of infected but undetected animals. Within infected herds, possible control strategies include improving herd hygiene by reducing calf exposure to faeces from cows, reducing stress in cows resulting in a longer latently infected period where shedding is minimal, or culling highly test-positive cows soon after detection. Risk-based trading can be a strategy to reduce the risk that Map spreads between herds. Our objective was to assess whether within-herd measures combined with risk-based trading could effectively control Map spread within and between dairy cattle herds in Ireland. We used a stochastic individual-based and between-herd mechanistic epidemiological model to simulate Map transmission. Movement and herd demographic data were available from 1st January 2009-31st December 2018. In total, 13,353 herds, with 4,494,768 dairy female animals, and 72,991 bulls were included in our dataset. The movement dataset consisted of 2,304,149 animal movements. For each herd, a weekly indicator was calculated that reflected the probability that the herd was free from infection. The indicator value increased when a herd tested negative, decreased when animals were introduced into a herd, and became 0 when a herd tested positive. Based on this indicator value, four Johne's assurance statuses were distinguished: A) ≥ 0.7 - 1.0, B) ≥ 0.3 - < 0.7, C) > 0.0 - < 0.3, and D) 0.0. A is the highest and D the lowest Johne's assurance status. With risk-based trading some of the observed movements between herds were redirected based on Johne's assurance status with the aim of reducing the risk that a non-infected herd acquired an infected animal. Risk-based trading effectively reduced the increase in herd prevalence over a 10-year-period in Ireland: from 50% without risk-based trading to 42% with risk-based trading in the metapopulation only, and 26% when external purchases were risk-based as well. However, for risk-based trading to be effective, a high percentage of dairy herds had to participate. The most important within-herd measures were improved herd hygiene and early culling of highly infectious cows. These measures reduced both herd and within-herd prevalence compared to the reference scenario. Combining risk-based trading with within-herd measures reduced within-herd prevalence even more effectively., Competing Interests: Declarations of interest None., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Paratuberculosis is a chronic enteric progressive disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). Despite cultural methods being considered the gold standard for the diagnosis of paratuberculosis, quantitative PCR (qPCR) assays have been developed for this purpose. These assays showed sensitivity and specificity comparable to cultural method but provide more rapid analysis results. Aim of our work was the validation of an IS900-qPCR assay for detection of MAP in faeces according to the OIE guidelines relative to the validation of assays for infectious diseases. The analytical and diagnostic characteristics and the reproducibility of the qPCR method were assessed. The robustness of the assay was evaluated using two extraction methods (silica column and magnetic beads DNA capture) and two qPCR systems (STEPONE™ and CFX96™). According to our validation, the analytical specificity, inclusivity and exclusivity were found to be appropriate for the use of this qPCR assay as a diagnostic test. Specifically, the limit of detection was approximately 100 CFU/g or even less if binomial approaches were used for the determination of the 95 % probability of detection (logit and clog-log models) with sufficient repeatability and reproducibility. Estimation of test accuracy was performed using a Bayesian two latent class model, in various scenarios combining different priors for prevalence and accuracy of the two tests used. All models were run considering three different cut-offs for qPCR. Our validation study underlines the good performance of this IS900-qPCR assay for diagnosis of MAP representing a valid and robust alternative to culture. Moreover, coupled with the semiautomatic magnetic beads DNA extraction method, this assay allows the rapid processing of numerous samples., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

The objective of the present study was to evaluate the diagnostic accuracy of the individual fecal culture (IFC), fecal PCR (FPCR), and serum ELISA for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep from four governorates in Egypt, using a latent class model (LCM) fitted within a Bayesian framework. Furthermore, the within-governorate prevalence of MAP infection in sheep was estimated as a secondary objective. Fecal and blood samples were collected from 370 sheep in four Egyptian governorates. Fecal samples were analyzed by IFC and RT-PCR based on ISMav2 gene, while ELISA was performed on serum samples. The sensitivity (Se) and specificity (Sp) of the three diagnostic tests were estimated using a three-tests-four-populations Bayesian LCM to obtain posterior estimates [medians and 95% Bayesian credible intervals (95% BCI)] for each parameter. The median Se estimates (95% BCI) for IFC, FPCR, and serum ELISA were 31.8% (22.8-41.4), 49.7% (31.8-79.9), and 61.2% (39.8-81.4), respectively. The median Sp estimates (95% BCI) for IFC, FPCR, and serum ELISA were 97.7% (96.1-98.9), 97.7% (95.6-99.5), and 98.4% (96.9-99.3), respectively. The median within-governorate paratuberculosis prevalence (95% BCI) was 5.2% (1.1-13.6), 8.4% (2.9-17.7), 9.4% (3.0-20.7), and 18.2% (10.5-29.5) for the Gharbia, Menoufia, Qalyubia, and Kafr El-Sheikh governorates, respectively. In conclusion, at a ratio of the optical density (OD) sample/OD positive control threshold of > 45%, ELISA showed the highest Se among the three tests and comparable Sp to IFC and FPCR. The test ELISA evaluated in this study is an interesting alternative for detecting MAP in sheep due to its higher Se, lower cost, and shorter turnaround laboratory time compared to IFC and FPCR., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Paratuberculosis is a widespread chronic infection of Mycobacterium avium subspecies paratuberculosis (MAP) that causes significant economic losses to the sheep industry. The current study investigated this disease, which causes diarrhea in sheep, particularly, in Bayannaoer, Inner Mongolia, China. Diagnosis was based on clinical symptoms, pathological autopsy, histopathological inspection, and serological and molecular methods., Results: MAP was confirmed using polymerase chain reaction using DNA extracted from tissue and fecal samples. Serum samples from 472 individual sheep were obtained to detect antibodies against MAP using an enzyme-linked immunosorbent assay. MAP antibodies were separately detected in 17.86% (35/196) and 18.48% (51/276) of sheep herds at approximately 6 months and ≥ 1 year of age, respectively. The tissue lesion and pathological section results were consistent with paratuberculosis infection., Conclusions: To our knowledge, this is the first report of Mycobacterium avium subspecies paratuberculosis seroprevalence in Bayannaoer sheep in Inner Mongolia. Our findings show that MAP is not only prevalent, but also a potential threat to this region. Further investigations, including long-term epidemiological surveillance and isolation are needed for the awareness and effective treatment of paratuberculosis in sheep of Inner Mongolia., (© 2022. The Author(s).)

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 10 7  CFU/mL, 1.68 × 10 6  CFU/mL and 1.68 × 10 5  CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 10 4  CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 10 9  CFU/mL from feces and no less than 10 4  CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 10 5 and 2 × 10 3  CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log 10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 10 11 to 4.14 × 10 4 for IS900 (FAM), 1.27 × 10 11 to 1.27 × 10 4 for IS900 mRNA (Cy5), and 3.68 × 10 10 to 3.68 × 10 4 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 10 4 , 1.27 × 10 4 , and 3.68 × 10 4 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 10 4 and 3.68 × 10 4 copies for IS900 and ModD, respectively., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Paratuberculosis, commonly known as Johne's disease, is a chronic granulomatous infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Clinical signs, including reduced milk yields, weight loss and diarrhoea, are typically absent until 2 to 6 years post exposure., Objectives: To identify metabolomic changes profiles of MAP challenged Holstein-Friesian (HF) cattle and correlate identified metabolites to haematological and immunological parameters., Methods: At approximately 6 weeks of age, calves (n = 9) were challenged with 3.8 × 10 9 cells of MAP (clinical isolate CIT003) on 2 consecutive days. Additional unchallenged calves (n = 9) formed the control group. The study used biobanked serum from cattle sampled periodically from 3- to 33-months post challenge. The assessment of sera using flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) for high throughput, sensitive, non-targeted metabolite fingerprinting highlighted differences in metabolite levels between the two groups., Results: In total, 25 metabolites which were differentially accumulated in MAP challenged cattle were identified, including 20 which displayed correlation to haematology parameters, particularly monocyte levels., Conclusion: The targeted metabolites suggest shifts in amino acid metabolism that could reflect immune system activation linked to MAP and as well as differences in phosphocholine levels which could reflect activation of the Th1 (tending towards pro-inflammatory) immune response. If verified by future work, selected metabolites could be used as biomarkers to diagnose and manage MAP infected cattle., (© 2022. The Author(s).)

Mycobacterium avium subsp. paratuberculosis (MAP) has been identified as one of the environmental agents that causes multiple sclerosis (MS). The global prevalence of MS has been upsurging over the years; however, efforts to divulge the role of MAP in MS have been limited. As a result, the present study aimed at assessing the odd ratios (ORs) associated MAP with the risk of MS. MAP-related MS data were obtained from 6 databases using the terms 'multiple sclerosis' or 'MS' and 'paratuberculosis' without regard for time or language restrictions following PRISMA standards. A total of 2,538 participants' data from 12 studies presenting anti-MAP antibodies and MAP DNA from 4 studies were fitted in random-effects (RE) and fixed-effects (FE) meta-analytic models. Furthermore, the between-study heterogeneity was measured using I 2 -values with a significant limit set at an I² > 75%. Analytical rigor and publication bias was determined using leave-one-out-analytics, Egger's tests, and p-curve analysis. In the FE and RE models, anti-MAP antibodies data significantly associated MS risk with MAP as 10.71 OR (95%-CI [7.78; 14.74], p-value < 0.0001) and 12.76 OR (95%-CI [8.13; 20.02], p-value < 0.0001) respectively, with an I 2 value of 34.9% (95%-CI [0.0%; 67.2%]; p-value = 0.11). Similarly, the MAP DNA dataset in FE significantly present MS risk due to MAP as 5.53 OR (95%-CI [3.54; 8.66], p-value< 0.0001) while, RE showed 5.27 OR (95%-CI [3.22; 8.60], p = 0.0017), with an I 2 -value = 0.0% (95%-CI [0.0%; 84.7%]; p-value = 0.71). Eggers' test, on the other hand, found publication bias in anti-MAP antibodies data (intercept = 1.61, 95% CI: 0.45 - 2.77, t = 2.72, p = 0.021), but not in MAP DNA dataset (intercept = -5.57, 95% CI: -20.44 - 9.29, t = -0.74, p = 0.54). The robustness of the meta-analyses was demonstrated by all sensitivity analyses. In addition, there is no evidence of p-hacking observed (right-skewness test (P Full < 0.001, P Half <0.001; statistical power ≥ 94% (95%-CI: 72.5%-99%)). In conclusion, the synthesis revealed a strong association between MAP and MS, indicating that MAP is a significant environmental agent that may trigger MS. Thus, early screening of MAP in MS cases may assist in the therapeutic approach to its management/treatment. Therefore, future studies should be tailored towards the role of MAP in the severity of MS phenotypes, as well as address global data gaps and low disease surveillance., (Copyright © 2022. Published by Elsevier B.V.)

The objective of this cross-sectional study was to evaluate the presence of infectious disease in newly arrived cattle on dairy farms in Ontario. Cattle that were more than 2 years old and arrived at dairy farms within the previous year were tested. A total 321 cattle from 56 dairy farms were sampled and had blood submitted to a diagnostic laboratory. Of all sampled cattle, 0.0%, 39.6%, 2.2%, and 1.3% tested positive for Anaplasma , bovine leukemia virus, Mycobacterium avium subsp. paratuberculosis , and Salmonella Dublin, respectively. Based on these results, it is imperative that dairy producers are vigilant to ensure they do not purchase animals with these important and untreatable infectious diseases., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

The National Bovine Tuberculosis (bTB) Eradication Program for dairy cattle has been operating in Taiwan for many years and has allowed the prevalence of bTB to decrease gradually. However, 29% of intradermal tuberculin test (ITT)-positive dairy cows were later found to be TB negative based on necropsy, histopathological examination, and mycobacterial isolation results. Studies in Taiwan have indicated that Mycobacterium avium subsp. paratuberculosis (MAP) may lead to false-positive ITT. Due to the high prevalence (over 90%) of paratuberculosis (PTB) serum antibody among Taiwan's farms, comparative ITT (CITT) has been recommended to differentiate between bTB and PTB infections. In this study, we used ITT, CITT, and enzyme-linked immunosorbent assay (ELISA) to evaluate the prevalence of bTB from 2012 to 2018. We also used pathological and bacterial examination from ITT-positive dairy cows to evaluate CITT's diagnostic ability and adjust its cutoff point accordingly. After careful selection, 36 cows (including 31 cows from 11 ITT-positive farms and 5 from 2 ITT-negative farms) were examined by CITT. The cutoff point was adjusted using a receiver operating characteristic (ROC) analysis. Overall, our results identified the ITT-positive prevalence in Taiwan as 0.03-0.22%, and PTB-positive prevalence as 54.55-73.53%. The results of sensitivity, specificity, kappa, and ROC analyses have identified the optimal cutoff point for the CITT in Taiwan as ≥ 3 mm. At this cutoff point value, the sensitivity and specificity were 62.5% and 96.43%, respectively. Our findings can be used to reduce the false-positive response rate caused by PTB cross-reaction and accelerate the eradication of bTB in Taiwan., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: This study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC)., Materials and Methods: Assuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP., Results: Mt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43-0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61-0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19-0.58), goats 0.19 (0.08-0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15-0.51), goats 0.27 (0.13-0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species., Conclusion: Overall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants' flocks., (© 2022. The Author(s).)

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by 'Test and Cull' or 'Test and Segregate' Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8-1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed 'good strength of agreement' with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Herd-level diagnosis of paratuberculosis using a pool-milk ELISA (pool size: n ≤ 50) is a novel, economical, and convenient method to identify blood serological Mycobacterium avium ssp. paratuberculosis (MAP) antibody-positive herds. To date, the diagnostic performance of the pool-milk ELISA has been described only under laboratory conditions where herd prevalence was simulated by the preparation of milk pools consisting of milk samples of cows with a known MAP status determined by fecal culture. In our observational study, test performance under field conditions was studied using pooled milk and individual blood samples. A total of 486 herds within the MAP prevalence reduction program of Lower Saxony, from which pooled milk and individual blood ELISA results were available, were assigned to this study. Data were analyzed for the period between January 1 and December 31, 2018, the first year after herd testing became obligatory in this federal state of Germany. To evaluate whether pooled milk samples reliably distinguish between herds with a MAP-apparent blood serological within-herd prevalence (MAP-Ab-WHP app ) ≥5% and herds with a MAP-Ab-WHP app <5%, the distribution of the MAP-Ab-WHP app was compared between pool-positive and pool-negative herds. The MAP-Ab-WHP app was 3.4% (median; 95% confidence interval = 0-11.4%) in pool-positive herds and 1.2% (median; 95% confidence interval = 0-6.4%) in pool-negative herds. Only 10.8% (n = 12) of the pool sample-negative herds had a MAP-Ab-WHP app ≥5% and were therefore false negatives, given the aims of the MAP prevalence reduction program. Hence, the pool-milk sampling strategy seems well suited to distinguish between herds with a MAP-Ab-WHP app ≥ 5% and herds with a MAP-Ab-WHP app <5% since only 10% of serum MAP-ELISA positive herds were missed. Employing a logistic regression model, we estimated that the minimum blood serological MAP-Ab-WHP app to detect a pool-positive herd with a probability of 95% was 8%, which fits well with the aim of the MAP prevalence reduction program to focus on herds with a MAP-Ab-WHP app of ≥5%. Despite the limitations of the control approach, which include milk pool sample collection and a low sensitivity of the ELISA used in milk pools and serum samples, the aims of the MAP prevalence reduction program can be achieved. The results of these field data support that pool-milk sample ELISA is a useful, economical, and low labor-intensive tool to identify herds seropositive for MAP in a MAP prevalence reduction program., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Diagnostic tests for Johne's disease in cattle are characterised by poor sensitivity and often imperfect specificity at the animal level. Because farmers and veterinarians have limited assurance or confidence from results of testing individual animals for Mycobacterium avium subsp. paratuberculosis (MAP), control programmes based on whole herd-level testing provide the best framework for classifying herds. At the herd level, there is a diverse range of testing options for MAP based on both direct and indirect testing of individual and pooled samples. The most common measures of herd test performance, herd sensitivity (HSe) and herd specificity (HSp), are important for decision-making in herd test selection, for estimating prevalence and as inputs for simulation studies. This systematic review investigated the results of herd test evaluations for MAP in cattle, through a comprehensive search of the literature and a systematic four-stage screening process to identify relevant publications. Forty-six publications with relevant results were eligible for inclusion in the final review, containing evaluations of whole-herd ELISA serological testing, bulk milk tank ELISA, culture, PCR and phage testing, pooled faecal testing and environmental sample testing. Data extracted from each publication included sample populations, methods of analysis, reference tests, cut-off points, HSe and HSp. Direct comparisons between the reported HSe and HSp estimates of different studies is challenging due to the variations in herd prevalence and test protocols used. The data in this systematic review will benefit decision-makers and researchers and highlights knowledge gaps requiring further research., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The objectives of this retrospective analysis were to: 1) estimate the diagnostic sensitivity (Se) and specificity (Sp) of bacterial culture of environmental samples for determining Mycobacterium avium subsp. paratuberculosis (MAP) infection status in Québec dairies, using a Bayesian Latent Class Model (BLCM); and 2) determine the association between the number of positive environmental samples and the individual fecal culture (IFC) apparent and true MAP within-herd prevalence. Environmental and individual fecal samples were collected from 87 commercial dairy herds participating in previous research projects. Environmental samples included two composite samples of 20 g collected from different locations within each of the following sites: an area where manure from the majority of adult cattle accumulates, a manure storage area and another site of manure accumulation chosen by the veterinarian. Samples were cultured using the MGIT Para TB culture liquid media and the BACTEC MGIT 960 system. The Se and Sp of environmental sampling were estimated using a one-test-one-population BLCM. Herds were considered positive for environmental sampling if at least one out of the six samples collected was positive. The apparent and true IFC within-herd MAP prevalence estimates for each herd were obtained using a two-stage cluster BLCM, then merged in a single dataset with the environmental sample results. The association between the within-herd MAP prevalence results (apparent and true), and the number of positive environmental samples was assessed using a zero-inflated negative binomial (ZINB) model. In all BLCMs, median posterior estimates and 95 % Bayesian credible intervals (BCI) were obtained with OpenBUGS statistical freeware. Se and Sp of environmental sampling were 43.7 % (95 % BCI: 32.5-55.5) and 96.2 % (95 % BCI: 84.2-99.8), respectively. Overall, the number of positive environmental samples increased with the apparent and true MAP within-herd prevalence. The true prevalence was higher than the apparent prevalence for a given number of positive environmental samples. The probability of not observing a positive environmental sample decreased with the prevalence. Despite its imperfect accuracy, environmental sampling is an inexpensive and non-invasive sampling method to determine MAP infection status in tie-stall herds that can be used as a proxy to estimate the true within-herd prevalence. The absence of positive environmental samples in a single sampling visit is likely an indicator of a very low within-herd prevalence rather than being MAP exempt., (Copyright © 2021 Elsevier B.V. All rights reserved.)