Caprine Paratuberculosis Seroprevalence and Immune Response to Anti-Mycobacterium avium Subspecies paratuberculosis Vaccination on the Canary Islands, Spain.

Paratuberculosis (PTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic disease with economic impact on ruminant farming worldwide. The Canary Islands count with the fourth largest goat population in Spain and are "officially free" of bovine tuberculosis. Twelve farms were included with 2774 serum samples tested by an enzyme-linked immunosorbent assay (ELISA) for detection of anti-MAP antibodies in two sessions. In the first session, an overall apparent prevalence of 18.4% (2.5% up to 61.1%) was obtained. Farms with prevalences (0-10%], (10-20%] and >20% were identified, with differences in seroconversion in the same prevalence group between farms and age ranges. Non-vaccinated (nV) and vaccinated (V) animals were included in the second sampling session. Higher levels of antibodies were detected in V animals older than 12 months, with considerable variations between age ranges and farms. Our results describe the current PTB status of the Canary Islands' goat farming. Furthermore, new insights on the effect of the farm prevalence on seroconversion in V animals are provided, although further studies are needed to evaluate the multiple factors affecting the immune response to anti-MAP vaccination.

Challenges for the management of Johne's disease in the UK: Expectation management, space, 'free riding', and vet-farmer communication.

Johne's disease in cattle is a significant global animal health challenge. Johne's disease is chronic, affecting the gastrointestinal tract of cattle and other ruminants and is caused by the bacteria Mycobacterium avium ssp. Paratuberculosis. Many countries have introduced schemes and programmes to try and control the spread of Johne's disease, including the UK. Despite efforts to control it, however, Johne's disease remains consistently ranked by UK producers as the top ranked disease negatively affecting productivity, indicating that schemes are not perceived to have solved the problem fully. Building on a global systematic review of the literature on barriers and solutions for Johne's disease control on-farm, we conducted an empirical study with over 400 farmers and 150 veterinary professionals across the UK. The study used workshops and semi-structured interviews to understand better the challenges dairy farmers and veterinarians face in implementing on-farm Johne's disease management schemes with the aim of identifying solutions. The study found that four main challenges are faced in the on-farm control of Johne's - (1) Management of farmer expectations around Johne's disease, with eradication near impossible, (2) Issues regarding space for segregation and the related economics of control (3) A 'free-riding' problem which can be influenced by the voluntary nature of control plans and (4) Challenges in vet-farmer communication, including levels of knowledge. Our findings have relevance for the control of Johne's disease in the UK and other countries, including for regions with voluntary and compulsory control programmes.

Changes in the Johne's disease situation in GB dairy herds over 10 years, as revealed by regular milk ELISA data.

Effective management of cattle infected with Johne's Disease (JD) is crucial to minimizing transmission and within-herd prevalence. Within Great Britain (GB), the voluntary National Johne's Management Plan (NJMP) requires farmers and a certified vet to conduct a risk assessment to determine the herd risk, examine the herd JD status and formulate a management plan. Individual milk ELISA tests for JD antibodies are widely used to monitor infection. The JD Tracker application, available within the dairy data management software InterHerd+ and other web-based environments, is being used by farmers and veterinarians to facilitate the practical use of milk ELISA data to aid JD-related management decisions. The JD Tracker application uses a herd's milk ELISA data to calculate a collection of 'JD parameters' that are indicative of the current JD status of the herd alongside contemporary and retrospective drivers linked to transmission and maintenance of infection. Herein, we use milk ELISA data from 154 regularly testing herds to review the temporal trends in JD parameters from 2013 to 2022. Since 2015, JD Tracker parameters have improved in these herds, most notably average test value (ATV) and within-herd prevalence (%Pos30). Trends in driver parameters suggest that farmers are progressively less likely to serve repeat test-positive (J5) cows and are more readily removing them. The data also reveal that the burden of JD is disproportionately greater in herds with higher ATV. In 2022, the 25 % of herds with the highest ATVs accounted for 42 % of positive tests and 42 % of repeat ELISA positive (J5) cows. Retrospectively, it is not possible to identify with certainty factors that directly contributed to the trends in JD parameters, but it is notable that the introduction of the NJMP was coincided with the improving JD situation. In 2019, participation in the NJMP or an equivalent scheme became mandatory for dairy farms to be compliant with the food and farms standards assurance scheme Red Tractor, with the result that JD management plans are now completed by 95 % of UK dairy farms. As far as we know, the UK is unique in its development of a tool (the JD Tracker) which adds utility to milk ELISA data using specifically designed JD parameters. Anticipated further work includes the development of a national database of JD testing herds and application of the JD Tracker at national scale to enable more comprehensive industry-level monitoring of JD within GB dairy farms.

Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

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.

Herd-level true seroprevalence of caseous lymphadenitis and paratuberculosis in the goat population of Poland.

A large-scale study was carried out in the Polish goat population in 2014-2021 to determine the herd-level true seroprevalence (HTP) of caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis (Cp) and paratuberculosis (PTB) caused by Mycobacterium avium ssp. paratuberculosis (Map). Two-stage cluster sampling was applied to herds counting at least 20 adult goats (aged >1 year) and in each herd all males and 10-13 females were tested. At least one seropositive goat regardless of its sex was necessary to consider the herd as infected. HTP was estimated using the Bayesian approach with the Gibbs sampler in the EpiTools and reported as the median and 95 % credibility interval (95 % CrI). A total of 1282 adult goats from 86 herds were serologically tested using two commercial ELISAs (Cp-ELISA and Map-ELISA). At least 1 seropositive result of Cp-ELISA and Map-ELISA was obtained in 73/86 herds (84.9 %) and 40/86 herds (46.5 %), respectively. HTP of CLA was estimated at 73.3 % (95 % CrI: 65.0 %, 80.4 %) and HTP of PTB was estimated at 42.9 % (95 % CrI: 25.8 %, 58.0 %). There was a significant positive association between the occurrence of CLA and PTB in the herds (odds ratio 6.0, 95 % confidence interval: 1.2, 28.8; p = 0.010). Probability of the seropositive result for PTB was also significantly higher in Cp-seropositive goats than in Cp-seronegative goats (odds ratio 3.9, 95 % confidence interval: 2.4, 6.3; p < 0.001) which could indicate either a higher risk of co-infection or a higher rate of false positive results for PTB in Cp-positive goats. To investigate this issue, optical densities obtained in Map-ELISA were compared between Cp-positive and Cp-negative goats and results of Map-ELISA were adjusted accordingly. Map-negative sera from Cp-positive goats turned out to have significantly higher optical densities than Map-negative sera from Cp-negative goats (p < 0.001). After the adjustment, the herd-level apparent seroprevalence of PTB was 41.9 % (36/86 herds) so it still fell within the 95 % CrI of HTP of PTB calculated before the adjustment. Concluding, CLA appears to be widespread in the Polish goat population. In many of them it may be subclinical at the moment, however will likely emerge in the future as the disease follows cyclic pattern in Poland. On the other hand, given the total lack of clinical PTB in Polish goats, an explanation for a high HTP of PTB remains unclear and warrants further studies using tests of higher analytical specificity than ELISA.

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

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

A novel diagnostic approach to Paratuberculosis in dairy cattle using near-infrared spectroscopy and aquaphotomics.

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.

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

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

A comprehensive review on Phyto-MAP: A novel approach of drug discovery against Mycobacterium avium subspecies paratuberculosis using AYUSH heritage.

ETHNOPHARMACOLOGICAL RELEVANCE: Indian system of Traditional medicine, AYUSH (Ayurveda, Yoga, Unani, Siddha, and Homeopathy) has great potential with a History of Safe Use (HOSU) of thousands of medicinal plants included in pharmacopoeias. The multi-targeted approach of phytoconstituents present in different traditionally used medicinal plants makes them suitable candidates for research against various infective pathogens. MAP which is a dairy-borne pathogen is associated with the development of Johne's disease in ruminants and Crohn's disease like autoimmune disorders in human beings. There are no reliable treatment alternatives available against MAP, leaving surgical removal of intestines as the sole option. Hence, there exists an urgent need to search for leads against such infection. AIM OF THE STUDY: The present review has been conducted to find out the ethnopharmacological evidence about the potential of phytoconstituents against Mycobacterium avium subspecies paratuberculosis (MAP), along with the proposal of a potential phyto-MAP mechanism for the very first time taking anti-inflammatory, immunomodulatory, and anti-microbial traditional claims into consideration. MATERIALS AND METHODS: We have analyzed and reviewed different volumes of the two main traditional scriptures of India i.e. Ayurvedic Pharmacopoeia of India (API) and Unani Pharmacopoeia of India (UPI), respectively-for identification of potential anti-MAP plants based on their claims for related disorders. These plants were further investigated systematically for their scientific publications of the last 20 years (2002-2022) available through electronic databases including Google Scholar, Pubmed, and Scopus. The studies conducted in vitro, cell lines, and in vivo levels were taken into consideration along with the associated mechanisms of phytoconstituents. RESULTS: A total of 70 potential medicinal plants have been identified. Based on the ethnopharmacology, a potential phyto-paratuberculosis (Phyto-paraTB) mechanism has been proposed and out of 70, seven potential anti-MAP plants have been identified to have a great future as anti-MAP. CONCLUSION: A novel and scientifically viable plan has been proposed for addressing anti-MAP plants for stimulating research against MAP and related disorders using mass-trusted AYUSH medicine, which can be used as an alternative remedy in resistance cases otherwise can be advocated as an adjuvant with modern treatments for better management of the disease.

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

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

The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line.

BACKGROUND: Johne's disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne's disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne's disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved. RESULTS: Immune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways. CONCLUSIONS: Identifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.

A scoping review on associations between paratuberculosis and productivity in cattle.

Paratuberculosis (PTB), or Johne's disease, is a disease with worldwide distribution caused by Mycobacterium avium subsp. paratuberculosis (MAP) that leads to chronic enteritis, primarily in ruminants. Even subclinical infection significantly reduces the animals' performance, and consequences of the disease lead to high economic losses for the cattle industry. To estimate the economic burden of bovine PTB and to evaluate the benefits of a potential control program, accurate estimates of the production effects associated with the disease are required. Therefore, the aim of this scoping review was to provide a comprehensive overview of associations between MAP infection and production parameters in cattle. The studies were collected from three electronic databases. Of the total 1,605 identified studies, 1,432 did not meet the set criteria in the title and abstract screening and a further 106 were excluded during full-text review. Finally, data on 34 different production parameters were extracted from 67 publications. Results show that the magnitude of reported performance losses varies depending on several factors, such as the type of diagnostic test applied, disease status or number of lactations. Studies reported a reduction in milk yield, changes in milk quality (e.g., higher somatic cell count, lower amount of produced milk fat and protein), reduced fertility (e.g., prolonged calving interval and service period, higher abortion rate and calving difficulties), reduced weaning weight, slaughter weight and slaughter value, or a higher risk for mastitis. Results from the studies included in our review show a median decrease of milk yield per infected cow of -452 kg/lactation for raw and -405 kg/lactation for modeled data. Similarly, the amount of produced milk protein fell by a median of -14.41 kg/lactation for modeled data and the amount of produced milk fat by a median of -13.13 kg/lactation. The reviewed studies revealed a prolonged calving interval by around 30 days and a 1.5 to 3 times higher likeliness of culling per lactation in PTB positive animals. Results from this scoping review provide evidence-based inputs for the development of economic models aiming at the estimation of the costs and benefits associated with different disease control scenarios for PTB.

Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples.

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.

Comparison of Machine Learning Tree-Based Algorithms to Predict Future Paratuberculosis ELISA Results Using Repeat Milk Tests.

Machine learning algorithms have been applied to various animal husbandry and veterinary-related problems; however, its use in Johne's disease diagnosis and control is still in its infancy. The following proof-of-concept study explores the application of tree-based (decision trees and random forest) algorithms to analyze repeat milk testing data from 1197 Canadian dairy cows and the algorithms' ability to predict future Johne's test results. The random forest models using milk component testing results alongside past Johne's results demonstrated a good predictive performance for a future Johne's ELISA result with a dichotomous outcome (positive vs. negative). The final random forest model yielded a kappa of 0.626, a roc AUC of 0.915, a sensitivity of 72%, and a specificity of 98%. The positive predictive and negative predictive values were 0.81 and 0.97, respectively. The decision tree models provided an interpretable alternative to the random forest algorithms with a slight decrease in model sensitivity. The results of this research suggest a promising avenue for future targeted Johne's testing schemes. Further research is needed to validate these techniques in real-world settings and explore their incorporation in prevention and control programs.

Progress in Paratuberculosis Control Programmes for Dairy Herds.

While paratuberculosis control has been studied for over a century, knowledge gaps still exist regarding the uptake and efficacy of control programmes. This narrative review aims to summarise studies on control programmes presented at the IDF ParaTB Fora in 2021 and 2022 and the International Colloquium on Paratuberculosis in 2022. Studies were grouped by topic as follows: successful control, field studies, education and extension, voluntary and compulsory control programmes, and surveillance. Various Map control programmes resulted in a decreasing animal and herd level Map prevalence. Long-term stakeholder commitment, stable funding, involvement of herd veterinarians and incentives for farmers to participate were shown to be pivotal for long-term success. Control measures focused on vertical and calf-to-calf transmission may improve Map control in infected herds. Easy-to-capture visualisation of surveillance test results to inform participants on the progress of Map control in their herds was developed. The probability of freedom from disease and estimated within-herd prevalence were identified as good candidates for categorisation of herds to support low-risk trade of cattle. Results of the surveillance schemes may inform genetic selection for resistance to Map infection. In conclusion, successful paratuberculosis control is feasible at both the herd and country level provided that crucial prerequisites are met.

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

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

Development and evaluation of genomics informed real-time PCR assays for the detection and strain typing of Mycobacterium avium subsp. paratuberculosis.

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.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

An ELISA Using Synthetic Mycolic Acid-Based Antigens with DIVA Potential for Diagnosing Johne's Disease in Cattle.

THE PROBLEM: Ante-mortem diagnosis of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne's disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne's disease in cattle. METHOD: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne's disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals. RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX. CONCLUSION: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.

Successful Control of Mycobacterium avium Subspecies paratuberculosis Infection in a Dairy Herd within a Decade-A Case Study.

This longitudinal case study provides an in-detail report of the process towards the elimination of Mycobacterium avium subsp. paratuberculosis (MAP) from a closed 450-head commercial dairy herd. In parallel, two diagnostic approaches were applied to all cows in annual intervals during 2012-2022: detection of MAP in individual faecal samples by bacteriological cultivation on solid medium and detection of MAP-specific antibodies by ELISA. For each annual sampling, the kappa coefficients for test agreement and the survival rates of MAP-positive and MAP-negative cows were calculated. Applying a multivariable linear regression model revealed a significantly lower fat-corrected 305-day milk yield for MAP-positive cows. The true prevalence of MAP shedders reduced from 24.2% in 2012 to 0.4% in 2019 and during 2020-2022, no MAP shedder was identified. Test agreement was generally low and bacteriological cultivation showed positive results earlier than the ELISA. In the first years of control, the survival of MAP shedders was longer than in the final stage. In conclusion, the elimination of MAP from a dairy herd might be feasible within a decade. Changes in the test agreement must be considered. Timely removal of MAP shedders, hygienic calf rearing, and colostrum supply are key for successful control.