Mycobacterium Avium Ssp. Paratuberculosis Research Articles

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.

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 incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD 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 polymerase chain reaction (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 disease. MAP 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 TaqMan technology was used in 107 reports. 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.

Prevalence and spatial distribution of infectious diseases of dairy cattle in Ontario, Canada

Here we investigated the prevalence and spatial distribution of selected pathogens associated with infectious diseases of dairy cattle in Ontario, Canada. The cross-sectional study surveyed bulk tank milk for antibodies against bovine leukemia virus (BLV), Mycobacterium avium ssp. paratuberculosis (MAP), and Salmonella Dublin, and for the presence of mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis). Between October 2021 and June 2022, bulk tank milk (BTM) samples were obtained from every commercial dairy farm in Ontario (n = 3,286). Samples underwent ELISA testing for presence of BLV, MAP and S. Dublin antibodies, and quantitative PCR testing for the detection of specific antigens of pathogens associated with mastitis. Bayesian models were used to estimate prevalence, and spatial analysis was carried out to identify regional clusters of high pathogen prevalence. Prevalence varied for different pathogens. BLV was widespread across dairy farms in Ontario, with an estimated prevalence of 88.3%. Prevalence of MAP, Staph. aureus and S. Dublin in Ontario dairy herds were 39.8%, 31.5% and 5.1%, respectively. The vast majority of dairy herds in Ontario were free of intramammary infections caused by Strep. agalactiae and M. bovis. Clusters of increased test positivity rates were detected for S. Dublin, MAP, and Staph. aureus, indicating potential geographic risk factors for pathogen transmission. For S. Dublin, an area of increased test positivity rates was detected in southwestern Ontario, close to the Canada-US border where most of the dairy herds in Ontario are located. Conversely, a localized cluster of positive test outcomes involving 14 subdivisions located in the southeastern region of Ontario was detected for Staph. aureus. Findings from our survey highlight the importance of the testing of aggregated samples and spatial analysis as part of disease surveillance programs and for implementing risk-based trading approaches among dairy producers.

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

1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. Aphage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment.

Association of Mycobacterium avium paratuberculosis serostatus with age at first calving, calving interval, and milk production in dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, also known as Johne's disease. This infection is responsible for negative effects, ranging from reduction of milk production to reproductive compromise and increased susceptibility to other diseases such as mastitis. Contradictory information on the association between this infection and reproductive performance has been reported in dairy cows. The aim of this work was to investigate associations between individual cow MAP seropositivity and lifetime reproduction and production performance. The MAP serum ELISA (IDEXX MAP Ac) results from all the 13,071 adult cows present on 191 farms and corresponding birth- and calving-date records obtained from the National Association for Genetic Improvement of Dairy Cattle were used. Cows and farms were classified as positive or negative, based on ELISA results. Outcomes assessed were age at first calving (AFC), intercalving intervals (ICI) from first to fourth interval, and average milk production per day of productive cycle (Milk-305/ICI, a ratio between 305-d corrected milk production and the number of days of the respective calving interval). Multilevel mixed models were used to investigate the association of cow MAP status with AFC, ICI, and Milk-305/ICI. Three levels were considered in the models: "measurement occasion," the first level, was nested within cows and cows were nested within farms. The "measurement occasion" is the time point to which all the observed measures (between 2 successive parturitions, such as milk production and somatic cell count) were referred. Our results indicate that MAP-positive cows have a significantly lower 14-d mean AFC than MAP-negative cows. The overall average ICI in our study was 432.5 d (standard deviation: 94.6). The average ICI, from first to fourth, was not significantly affected by MAP seropositivity. No significant effect of MAP positivity was found on the overall ICI. In relation to Milk-305/ICI, MAP-positive cows did not produce significantly less milk than negative cows across their productive lifetime. We observed higher but nonsignificant Milk-305/ICI (kg/d) in MAP-positive cows. In our study, the proportion of MAP-positive cows within lactations remained similar across all lactations, suggesting that seropositivity did not increased drop-off rate.

Mycobacterium avium ssp. paratuberculosis and Crohn's Disease-Diagnostic Microbiological Investigations Can Inform New Therapeutic Approaches.

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease (JD), which is a chronic infectious gastrointestinal disease of ruminants and is often fatal. In humans, MAP has been associated with Crohn's disease (CD) for over a century, without conclusive evidence of pathogenicity. Numerous researchers have contributed to the subject, but there is still a need for evidence of the causation of CD by MAP. An infectious aetiology in CD that is attributable to MAP can only be proven by bacteriological investigations. There is an urgency in resolving this question due to the rising global incidence rates of CD. Recent papers have indicated the "therapeutic ceiling" may be close in the development of new biologics. Clinical trial outcomes have demonstrated mild or inconsistent improvements in therapeutic interventions over the last decades when compared with placebo. The necessity to revisit therapeutic options for CD is becoming more urgent and a renewed focus on causation is essential for progress in identifying new treatment options. This manuscript discusses newer interventions, such as vaccination, FMT, dietary remediation and gut microbiome regulation, that will become more relevant as existing therapeutic options expire. Revisiting the MAP theory as a potential infectious cause of CD, rather than the prevailing concept of an "aberrant immune response" will require expanding the current therapeutic programme to include potential new alternatives, and combinations of existing treatments. To advance research on MAP in humans, it is essential for microbiologists and medical scientists to microscopically detect CWDM and to biologically amplify the growth by directed culture.

Prevalence of paratuberculosis in Thuringian sheep and goat flocks

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. 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. 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. 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.

Isolation of Mycobacterium avium ssp. paratuberculosis and other non-tuberculous mycobacteria from head lymph nodes of wild ruminants and badgers in Switzerland.

The family Mycobacteriaceae contains over 188 species, most of which are saprophytic non-tuberculous mycobacteria (NTM). In wildlife, a variety of different NTM can be found, with different reports about their pathogenic potential. A pathogenic member of NTM is Mycobacterium avium ssp. paratuberculosis (MAP), which can infect farmed and wild ruminants. It causes paratuberculosis which is an economically important chronic disease. Infected farm animals are considered to be the source of infection in wild animals. Wildlife, on the other hand, is thought to be a reservoir for certain members of the Mycobacterium tuberculosis complex (MTBC), such as M. caprae, which causes tuberculosis in cattle and red deer. Switzerland implemented a surveillance program for tuberculosis in wild animals in 2014. Here, we describe the results from the mycobacterial culture of lymph node samples collected from red deer, roe deer, chamois, ibex, and badgers collected within this surveillance program from 2020 to 2022. Overall, samples from 548 animals were checked macroscopically for tuberculosis-like lesions. In total, 88 animals (16.1%), which either had lesions in their lymph nodes or were male and aged older than 5 years, were investigated using mycobacterial culture. In total, 25 animals (28.4%) were positive for NTM, while no MTBC was detected. The most often identified NTM was M. vaccae, followed by M. avium. Most animals positive for NTM did not show any macroscopic lesions. Furthermore, MAP was isolated from the head lymph nodes of two male red deer. Neither of the two MAP-positive animals had any macroscopic lesions in their head lymph nodes or any other signs of disease. The shooting sites of the two MAP-positive animals were located in Alpine pastures used for grazing of cattle during summer, which confirms that species transmission can occur when contaminated pastures are used by different species. In agreement with other studies, the occurrence of MAP in red deer was quite low. However, so far, MAP was mostly isolated from feces and intestinal lymph nodes of wild animals. This is the first detection of MAP in the head lymph nodes of red deer in Switzerland.

Investigating the Prevalence of Paratuberculosis in Hungarian Large-Scale Dairy Herds and the Success of Control Measures over Four Years.

Paratuberculosis (PTB) is a severe, slow-developing, untreatable disease of ruminants. Worldwide, the disease affects more than 50% of herds in the dairy industry, and causes substantial economic losses for dairy producers. Diagnostic tests show limited sensitivity, especially in the early stages of the disease. Our study aimed to investigate the seroprevalence of Mycobacterium avium ssp. paratuberculosis (MAP) in large-scale dairy herds in Hungary, in association with the self-reported presence or absence of screening and intervention measures against MAP transmission. We processed data from 42 large-scale Holstein Friesian farms in Hungary between 1 January 2018 and 31 December 2021. An average of 32,009 (min.: 31,702; max.: 32,207) animals were blood sampled yearly (127,372 in total during the four years), corresponding to 15% of the Hungarian dairy cattle population. All female cattle older than 2 years were blood sampled on the farms enroled in the study. The samples were tested using a commercial ELISA (IDEXX paratuberculosis screening Ab test). Farm managers were interviewed about their on-farm diagnostic and intervention approaches using a uniform questionnaire, including questions on the level of awareness, frequency of ELISA and PCR testing, and their strategies for culling adult animals and reducing transmission to newborn calves. By comparing the annual rate of change in seroprevalence and the amount of change observed during the four-year period, we concluded that test-and-cull strategies implemented in parallel with newborn calf management that aimed at preventing MAP transmission were superior to test-and-cull strategies alone; moreover, fortifying culling decision making via additional ELISA and PCR tests is superior to using a single ELISA result. For farms that carried out a complex program with both "test-and-cull" and proper newborn calf management, there was a proportional reduction in apparent seroprevalence at an average of 22.8% per year. Fifteen of the sampled farms had no measures in place to control paratuberculosis. On these farms, the seroprevalence increased by 12.1% per year on average.

Johne’s disease in beef cattle: Correlation of serum ELISA and fecal PCR results from clinical cases and herd surveillance

Johne’s disease is caused by Mycobacterium avium ssp. paratu­berculosis (MAP) and can be a costly and frustrating disease in beef herds. Clinical Johne’s disease in cattle arises years after inoculation as diarrhea and progressive weight loss. Prior to reaching clinical status, subclinically infected cattle can shed bacteria into their environment, leading to continued spread of the disease within herds. The duration of subclinical infection is highly variable, and the humoral immune response and the amount of fecal shedding of MAP can vary greatly between in­dividuals and throughout the year. Currently available diagnos­tic tests (serum ELISA, fecal PCR, fecal culture) each have sig­nificant limitations. Assays may differ in their utility depending on whether they are used to confirm clinical cases of Johne’s disease or to screen healthy cattle for potential infection.

High-Impact Risk Factors for Mycobacterium avium ssp. paratuberculosis in Dairy Herds in Germany

In a cross-sectional study, it was identified that three regions in Germany differed with respect to their herd-level prevalence for paratuberculosis in dairy cattle. In the study presented here, the same farms were analyzed to identify those components of biosecurity and farm management with the highest impact on Mycobacterium avium ssp. paratuberculosis (MAP) introduction and establishment in a farm. Hence, the data analyzes included 183, 170 and 104 herds from the study regions north, east and south, respectively. A herd was considered MAP-positive if at least one fecal environmental sample was positive. Twenty-six different possible risk factors from five different components of biosecurity and farm management were analyzed. We show that the average management of calf feeding increased the odds for a MAP-positive farm by 5.22 times (95% confidence interval (CI) = 1.25–21.83). With every 100-cow increase in farm size, the risk for a farm to test MAP-positive increased by 1.94 times (CI = 1.15–3.27), 1.14 times (CI = 1.02–1.27) and 5.53 times (CI = 0.44–68.97) in the north, east and south study regions, respectively. Furthermore, the purchase of cattle with an unknown MAP status increased the risk for a farm testing MAP-positive by 2.86-fold (CI = 1.45–5.67). Our results demonstrate that herd size, unknown MAP status of the purchased cattle and different aspects of calf feeding play an important role in the MAP status of a farm and should be in focus in regions with different MAP between-herd prevalence. Additionally, farm individual risk patterns should be identified during (veterinary) biosecurity consultancy.

Evaluation of Single Nucleotide Polymorphisms (SNPs) Associated with Genetic Resistance to Bovine Paratuberculosis in Marchigiana Beef Cattle, an Italian Native Breed.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), a widespread chronic enteritis of ruminants. The progression of the infection depends on the containment action of innate and cell-mediated immunity (CMI), and it is related to environmental and genetic factors. In particular, PTB susceptibility seems to be associated with specific genes coding for immune regulators involved in the cell-mediated response during the infection. The aim of this preliminary study was to verify, in Italian beef cattle, an association between MAP infectious status and the presence of single nucleotide polymorphisms (SNPs) in candidate genes. To the best of our knowledge, this is the first investigation conducted on a native beef cattle breed, known as Marchigiana, reared in Central Italy. The present research, based on a longitudinal study, aimed to identify and correlate phenotypic and genetic profiles characteristic of the subjects potentially able to contrast or contain PTB. In a MAP-infected herd, ELISA, IFN-γ tests, qPCR, and cultures were performed at a follow-up, occurring within a period ranging from three to six years, to evaluate the individual state of infection. Animals testing positive for at least one test were considered infected. DNA samples of 112 bovines, with known MAP statuses, were analyzed to verify an association with SNPs in the genes encoding gamma-interferon (BoIFNG), interleukin receptor 10 (IL10RA), interleukin receptor 12 (IL12RB2), and toll-like receptors (TLR1, TLR2, TLR4). Regarding statistical analysis, the differences among target genes and pairs of alleles in the analyzed groups of animals, were evaluated at a significance level of p < 0.05. For IL10RA and for IL12RB2 genes, relevant differences in genotypic frequencies among the considered cattle groups were observed. For all candidate genes studied in this investigation, SNP genotypes already associated with PTB resistance were found more frequently in our population, suggesting potential resistance traits in the Marchigiana breed.

Genome-wide association study for antibody response to Mycobacterium avium ssp. paratubeculosis in goats.

Mycobacterium avium ssp. paratuberculosis (MAP), causes Johne's disease (JD), or paratuberculosis, a chronic enteritis of ruminants, which in goats is characterized by ileal lesions. The work described here is a case-control association study using the Illumina Caprine SNP50 BeadChip to unravel the genes involved in susceptibility of goats to JD. Goats in herds with a high occurrence of Johne's disease were classified as healthy or infected based on the level of serum antibodies against MAP, and 331 animals were selected for the association study. Goats belonged to the Jonica (157) and Siriana breeds (174). Whole-genome association analysis identified one region suggestive of significance associated with an antibody response to MAP on chromosome 7 (p-value=1.23 × 10-5 ). These results provide evidence for genetic loci involved in the antibody response to MAP in goats.

Descriptive analysis of the changes in Johne's disease management practices on Ontario dairy farms through repeat risk assessment.

Johne's disease (JD) control is often based on the culling of positive animals and the adoption of management practices that minimize exposure of calves to the pathogen Mycobacterium avium ssp. paratuberculosis (MAP). From 2010 to 2013, Ontario, Canada, instituted a voluntary Johne's control program consisting of whole-herd testing and a Risk Assessment and Management Plan (RAMP). The RAMP consisted of 38 questions that evaluated 5 different management areas to characterize herd risk for MAP introduction and within-herd spread. The RAMP produced a numerical score for each area, with higher scores associated with higher risk. The RAMP focused on animal purchases, calving management, calf management, and heifer and cow cleanliness and management. In the summer of 2019, the RAMP was repeated on 180 farms that had participated in the JD program of 2010 to 2013 and had bulk tank milk ELISA results from 2013 and 2017. This cross-sectional study demonstrated that many producers changed management practices over the 4- to 7-year period. Producers changed their cattle buying practices, with a reduction in purchasing from multiple sources and more herds refraining from buying in animals. However, overall scores were higher in 2019 than in 2013. The 2019 RAMP indicated that fewer farms were utilizing individual calving pens in 2019 than in 2013 (13% vs. 26%), yet more farms had policies in place to deal with sick or suspect JD cows entering the maternity area (92% vs. 74%). Management changes occurred over time, some of which represent increased risk (crowded maternity pens) and others decreased risk (closed herd, protocols in place for JD-positive cows) for MAP introduction and transmission. These results highlight the importance of frequent risk assessments and the documentation of changes to management practices on-farm as a means to assess herd disease risk more accurately.

Effect of tuberculin skin testing on serological results against Mycobacterium avium ssp. paratuberculosis (MAP): Evidence of distinct effects in MAP-infected and noninfected cows.

Johne's disease and bovine tuberculosis are diseases of economic, public health, and animal welfare importance. The single intradermal cervical comparative tuberculin (SICCT) test, which is used to determine bovine tuberculosis status as part of eradication schemes in the United Kingdom and some other countries, has been reported to interfere with the results of the widely used ELISA to detect antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in milk. Better understanding of the relationship between SICCT and MAP tests can improve management and control of Johne's disease. The aim of this study was to characterize the relationship between SICCT testing and milk ELISA performance and to assess whether the immunological response to the SICCT test is different for MAP-infected cows and noninfected cows. We used repeated MAP milk ELISA test results of a cohort of 805,561 cows in the United Kingdom between 2010 and 2018 that had milk ELISA tests within 90 d of SICCT testing to identify cows likely to be infected. We then assessed, separately, for cows deemed to be MAP-infected and noninfected, the association between MAP test results and proximity to SICCT testing by means of survival analysis and generalized additive mixed models. The results were used to quantify the effect SICCT testing may have on performance of milk ELISA tests conducted soon after SICCT testing. At high prevalence levels (20%) of MAP in the infected herd, overall accuracy of the milk ELISA is not reduced when testing occurs within 14 d from SICCT testing. Milk ELISA values of cows deemed to be infected were highest when MAP testing was closer in time to SICCT testing, suggesting the SICCT test enhances antibody response for MAP in infected cows. This corresponds to higher sensitivity of the MAP milk ELISA when testing within 30 d of the SICCT test. For cows deemed to be noninfected, the effect of previous SICCT testing was delayed compared with infected cows, with MAP milk ELISA values peaking at around 15 d post-SICCT testing. For both, MAP-infected and noninfected cows, interference from SICCT test diminished 30 d after SICCT testing, suggesting post 30 d to be the most appropriate time for evaluating the milk ELISA for MAP after SICCT testing. Our results provide strong evidence that the effect of the SICCT test on serological response against MAP is different for MAP-infected versus noninfected cows and that, as a result of this distinct effect, it is possible to improve interpretation of MAP milk ELISA test results (higher accuracy) by taking into consideration time since SICCT testing.

Mycobacterium avium subsp. paratuberculosis and Hashimoto's thyroiditis: Is MAP the trigger?

Hashimoto’s thyroiditis (HT) is an autoimmune disorder of the thyroid gland that can cause hypothyroidism. As HT is a multifactorial disorder, activation of immune responses in genetically predisposed individuals exposed to some environmental factors can contribute to it. Microorganisms, as environmental factors, including Mycobacterium avium ssp. paratuberculosis (MAP) by molecular mimicry, can be important in this autoimmune disorder. This study aimed to investigate the association between MAP and HT. This case–control study included 110 participants consisting of 60 HT patients and 50 healthy controls (HCs). Blood samples were collected. Nested PCR of the IS900 gene determined the presence of MAP DNA. The enzyme-linked immunosorbent assay (ELISA) was designed to identify antibodies (Abs) against the MAP3865c epitope, which has a homologous sequence with ZnT8 in the sera. The demographic information of all participants was recorded. Anti-TG, anti-TPO, TSH, anemia, and ruminant exposure were higer in HT patients than in the HCs (p < 0.05). MAP IS900 was detected significantly more in the patients (46.6% consisting of 30, 8.3, and 8.3% in clinical, subclinical, and unknown) than in the HCs (14%). The sera showed a remarkable frequency of reactivity against MAP3865c in the patients (38.3%) in comparison to the HCs (10%) (p = 0.0001). Furthermore, a significantly higher rate of livestock contact and traditional dairy consumption was found in individuals with MAP or anti-MAP3865c Abs positive result (p < 0.05). This study suggests a possible link between MAP and HT. These findings indicated that MAP frequency was not statistically different in the severity of HT and its shift into the clinical and subclinical forms; therefore, it could be assumed that MAPs are the initiators of the process. The results imply on a possible zoonosis transmission route of MAP from livestock products to humans. Further research is needed to confirm these results in larger groups of HT patients.

Herd Prevalence Estimation of Mycobacterium avium Subspecies paratuberculosis Burden in the Three Main Dairy Production Regions of Germany (PraeMAP).

Simple SummaryParatuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is widely spread among ruminants worldwide. After a long-lasting incubation period, infected animals suffer from chronic granulomatous enteritis. Economic losses are caused by premature culling, reduced milk yield and slaughter value in the dairy and beef industry, triggering attempts to control the disease. Paratuberculosis is a listed disease (category E), according to the European Animal Health Law, and intended to be monitored within the European Union. Evaluation of several herd-level monitoring approaches including the testing of environmental fecal samples to detect the infectious agent have been evaluated, proving environmental sampling to be a useful monitoring tool on herd level. This study comprises the application of environmental sampling within a herd prevalence estimation study in German dairy herds. Based on regional differences in the structure of livestock farming, Germany was divided into three regions where a representative number of farms were visited for sample collection. The results clearly indicate a different regional MAP herd level prevalence. The highest percentage of affected herds is found in the eastern part with large dairy herds, and the lowest in the south with the smallest average herd size. We conclude that the regional differences in MAP prevalence imply different approaches to control the disease.On-farm environmental sampling is an effective method for herd-level diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) infection and between-herd prevalence estimation. So far, no prevalence study enrolling important livestock-farming regions has been conducted. As the structure of dairy farming differs between main livestock-farming regions in Germany, our objective was to assess the between-herd prevalence of paratuberculosis for these regions in a standardized approach. Methods: In total, 457 randomly selected dairy farms from three regions of Germany (North: 183, East: 170, South: 104) were sampled between 2017 and 2019. Environmental samples (boot-swabs, aggregate feces and/or liquid manure samples) were cultured and analyzed using an IS900-qPCR for MAP determination. Of the 457 selected farms, 94 had at least one MAP-positive environmental sample with significant differences between regions regarding the apparent (North: 12.0%, East: 40.6%, South: 2.9%) or corrected true (North: 14.8%, East: 50.1%, South: 3.6%) between-herd prevalence. In conclusion, regional differences of between-herd prevalence of paratuberculosis are substantial in Germany, indicating the need for control approaches with different aims. Taking into account regional MAP prevalence, MAP-control programs should focus on on-farm prevalence reduction or on mitigating the risk of between-herd transmission, depending on region.

Economic premiums associated with Mycobacterium avium ssp. paratuberculosis-negative replacement purchases in major dairy-producing regions

Johne's disease, or paratuberculosis, is an infectious disorder of the intestines that can affect domestic and wild ruminants that is caused by an infection with Mycobacterium avium ssp. paratuberculosis (MAP). Although the economic losses due to Johne's disease in dairy cattle herds and the benefits and costs of various potential control practices have been estimated before, little is known about the economic value of purchasing MAP-negative dairy replacements in major dairy-producing regions. This study used Markov Chain Monte Carlo simulation techniques to compare 2 sets of MAP-negative and MAP-positive herds across a comprehensive selection of regions: herds purchasing MAP-negative replacement animals and herds purchasing replacement animals with unknown MAP infection status. The economic benefits per MAP-negative replacement purchased were then estimated over a 10-yr horizon, and the additional value of MAP-negative replacements when compared with unknown status replacements were calculated as a percentage premium of the average aggregated dairy replacement price in each region. An average benefit of US$76 per MAP-negative replacement purchase was estimated in major dairy-producing regions, equivalent to a premium of 13%, with higher premiums in regions characterized by below-average replacement prices and on-average farm-gate prices. It was also estimated that the greatest benefits from MAP-negative replacement purchases are associated with MAP-negative herds that successfully remain uninfected.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

Within-herd prevalence threshold for the detection of Mycobacterium avium ssp. paratuberculosis antibody–positive dairy herds using pooled milk samples: A field study

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-WHPapp) ≥5% and herds with a MAP-Ab-WHPapp <5%, the distribution of the MAP-Ab-WHPapp was compared between pool-positive and pool-negative herds. The MAP-Ab-WHPapp 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-WHPapp ≥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-WHPapp ≥ 5% and herds with a MAP-Ab-WHPapp <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-WHPapp 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-WHPapp 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.