Shedding Of Mycobacterium Avium Subsp. Paratuberculosis Research Articles

Lining the small intestine with mycobacteriophages protects from Mycobacterium avium subsp. paratuberculosis and eliminates fecal shedding

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Faecal microbial diversity in a cattle herd infected by Mycobacterium avium subsp. paratuberculosis: a possible effect of production status

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, or paratuberculosis (PTB) in ruminants, besides having zoonotic potential. It possibly changes the gut microbiome, but no conclusive data are available yet. This study aimed at investigating the influence of MAP on the faecal microbiome of cattle naturally infected with PTB. In a follow up period of 10 months, PTB status was investigated in a herd of dairy cattle with history of clinical cases. Each animal was tested for MAP infection using serum and milk ELISA for MAP anti-bodies and IS900 real-time PCR and recombinase polymerase amplification assays for MAP DNA in the faeces and milk monthly for 4 successive months, then a last one after 6 months. The faecal samples were subjected to 16S rDNA metagenomic analysis using Oxford Nanopore Sequencing Technology. The microbial content was compared between animal groups based on MAP positivity rate and production status. All animals were MAP positive by one or more tests, but two animals were consistently negative for MAP DNA in the faeces. In all animals, the phyla firmicutes and bacteroidetes were highly enriched with a small contribution of proteobacteria, and increased abundance of the families Oscillospiraceae, Planococcaceae, and Streptococcacaceae was noted. Animals with high MAP positivity rate showed comparable faecal microbial content, although MAP faecal positivity had no significant effect (p > 0.05) on the microbiome. Generally, richness and evenness indices decreased with increasing positivity rate. A significantly different microbial content was found between dry cows and heifers (p < 0.05). Particularly, Oscillospiraceae and Rikenellaceae were enriched in heifers, while Planococcaceae and Streptococcaceae were overrepresented in dry cows. Furthermore, abundance of 72 genera was significantly different between these two groups (p < 0.05). Changes in faecal microbiome composition were notably associated with increasing MAP shedding in the faeces. The present findings suggest a combined influence of the production status and MAP on the cattle faecal microbiome. This possibly correlates with the fate of the infection, the concern in disease control, again remains for further investigations.

Mycobacterium avium subsp. paratuberculosis in Sheep and Goats in Austria: Seroprevalence, Risk Factors and Detection from Boot Swab Samples.

This study aimed to investigate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in small ruminants in Austria by testing 22,019 serum samples with ELISA for the presence of specific antibodies. Furthermore, detailed investigations in five MAP-infected goat herds were carried out by ELISA, qPCR and bacterial culture. The found animal-level apparent MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Significant risk factors for seropositivity in goat herds were: herd size, animal trading, farmed as a dairy herd, Animal Health Service membership and cohabitation with farmed game. For sheep flocks, seroprevalence was significantly higher in flocks with animal trading and where cattle or goats were kept in the flock, respectively. The overall apparent within-herd MAP seroprevalence in the five goat farms investigated was 21.8% (11.7%-28.0%, calculated true seroprevalence 38.6%) and an overall rate of MAP shedding of 12.3% was detected (5.0%-24.7%). It was possible to identify MAP by culture using boot swab samples in each herd. The results indicated a moderate MAP infection rate in small ruminants in Austria.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis

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.

Classification and prediction of Mycobacterium Avium subsp. Paratuberculosis (MAP) shedding severity in cattle based on young stock heifer faecal microbiota composition using random forest algorithms

BackgroundBovine paratuberculosis is a devastating infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). The development of the paratuberculosis in cattle can take up to a few years and vastly differs between individuals in severity of the clinical symptoms and shedding of the pathogen. Timely identification of high shedding animals is essential for paratuberculosis control and minimization of economic losses. Widely used methods for detection and quantification of MAP, such as culturing and PCR based techniques rely on direct presence of the pathogen in a sample and have little to no predictive value concerning the disease development. In the current study, we investigated the possibility of predicting MAP shedding severity in cattle based on the faecal microbiota composition. Twenty calves were experimentally infected with MAP and faecal samples were collected biweekly up to four years of age. All collected samples were subjected to culturing on selective media to obtain data about shedding severity. Faecal microbiota was profiled in a subset of samples (n = 264). Using faecal microbiota composition and shedding intensity data a random forest classifier was built for prediction of the shedding status of the individual animals.ResultsThe results indicate that machine learning approaches applied to microbial composition can be used to classify cows into groups by severity of MAP shedding. The classification accuracy correlates with the age of the animals and use of samples from older individuals resulted in a higher classification precision. The classification model based on samples from the first 12 months of life showed an AUC between 0.78 and 0.79 (95% CI), while the model based on samples from animals older than 24 months showed an AUC between 0.91 and 0.92 (95% CI). Prediction for samples from animals between 12 and 24 month of age showed intermediate accuracy [AUC between 0.86 and 0.87 (95% CI)]. In addition, the results indicate that a limited number of microbial taxa were important for classification and could be considered as biomarkers.ConclusionsThe study provides evidence for the link between microbiota composition and severity of MAP infection and shedding, as well as lays ground for the development of predictive diagnostic tools based on the faecal microbiota composition.

Attempted Control of Paratuberculosis in Dairy Calves by Only Changing the Quality of Milk Fed to Calves.

Simple SummaryMilk is an important transmission route of Mycobacterium avium subsp. paratuberculosis (MAP) for dairy calves. The aim of the present study was to assess the efficacy of milk treatments to prevent MAP infection transmission to calves. A one-year field longitudinal study was carried out. Newborn calves were assigned to one of four experimental groups. MAP numbers were estimated for treated and untreated milk. Infection progression was monitored monthly. After one year, calves were euthanized, and tissue samples examined grossly and by histopathology. Milk treatments were highly effective though MAP shedding was also observed in all groups. It was not enough to try to block MAP infection transmission controlling only the quality of milk consumed by calves.One of the important routes of Mycobacterium avium subsp. paratuberculosis (MAP) transmission in dairy calves is milk. The aim of the present study was to assess the efficacy of milk treatments to prevent MAP infection transmission to calves. A one-year longitudinal study was carried out. Newborn calves were assigned to one of four experimental groups: 5 calves received naturally MAP-contaminated milk, 5 calves received copper treated milk, 4 calves were fed calf milk replacer, and 3 were fed UHT pasteurized milk. MAP load in milk was estimated. Infection progression was monitored monthly. After one year, calves were euthanized, and tissue samples were cultured and visually examined. MAP was undetectable in milk replacer and UHT milk. Copper ion treatment significantly reduced the number of viable MAP in naturally contaminated milk. Fecal shedding of MAP was observed in all study groups but began earlier in calves fed naturally contaminated milk. Paratuberculosis control programs must place multiple hurdles between the infection source, MAP-infected adult cows, and the most susceptible animals on the farm, young calves. As our study shows, strict dependence on a single intervention to block infection transmission, no matter how important, fails to control this insidious infection on dairy farms.

Is the effectivity of copper ions treatment of milk enough to block Mycobacterium avium subsp. paratuberculosis infection in calves?

Milk is an important transmission route of Mycobacterium avium subsp. paratuberculosis (MAP) for dairy calves. Given its resistance to pasteurization, alternative milk treatments are needed to control MAP transmission via milk. The present study reports the evaluation of a novel milk decontamination treatment based on copper ions as a means of preventing infection in dairy calves. Ten newborn calves were assigned to one of two experimental groups (n=5) which were studied for 1 year. The first group was fed milk naturally contaminated with MAP and the second one received the same milk but after being treated with copper ions. In both groups, milk MAP load was estimated. The progression of the infection was monitored monthly and at the end of the study, calves were euthanised, and tissue samples were examined both grossly and by histopathology. The treatment of milk with copper ions significantly reduced the number of viable MAP. Faecal shedding of MAP was observed in both study groups, but the calves fed naturally contaminated milk began to shed MAP earlier. Only calves fed copper-treated milk showed histopathological evidence consistent with MAP infection. The latter offers more questions than answers, and maybe the presence of a more tolerant and virulent MAP strain could be the final answer to this situation.

More insights about the interfering effect of Mycobacterium avium subsp. paratuberculosis (MAP) infection on Mycobacterium bovis (M. bovis) detection in dairy cattle.

The estimated herd and within herd Mycobacterium bovis (M. bovis) infection prevalence in the southern Chile regions are 0.3 and 0.67%, respectively. However, higher rates of infection still remain in some herds. In parallel, it is well established that a big proportion of cattle herds are infected with Mycobacterium avium subsp. paratuberculosis (MAP), which has been also associated with a clear interference effect on M. bovis diagnosis. The present study aims to provide more insights about the diagnostic interference for Mycobacterium bovis detection due to co-infection with MAP. To better understand the dynamics of this identified interference, the effect of MAP genotype present, as well as MAP faecal shedding values (as proxy of the infection progression), for each of the CFT results was compared. No relationship was observed between MAP genotype with any type of differential response to the diagnostic tests of M. bovis infection. However, MAP shedding values in animals with positive CFT diagnostic results for M. bovis infection was significantly lower than animals with a negative CFT result, observing that as the MAP shedding load raises, the response to the bovine tuberculin test tends to be negative. The findings reported in this study allows to interpret that one of the causes of the prolonged elimination of M. bovis infection from some cattle herds may be due in part to the advanced MAP infection status in co-infected individuals affecting the outcome of screening in-vivo diagnostic techniques such as CFT. These false negative animals that show negative results to M. bovis detection tests, may maintain the infection at herd level and spread the pathogen to healthy individuals.

Occurrence of faecal shedding of Mycobacterium avium subs. paratuberculosis by calves

Paratuberculosis or Johne´s disease is a chronic granulomatous enteric disease afflicting cattle, sheep, goats and other ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Calves are infected mainly via an oral route, especially from manure and environment contamination by infected adult cattle. It is generally assumed that calves rarely shed MAP bacteria and that calf-to-calf transmission is of minor importance. The aim of the study was to evaluate occurrence of faecal excretion of MAP by calves born to MAP-positive and MAP-negative dams in a low MAP prevalence herd. The study involved 66 dam-calf pairs. Samples of faeces were collected: for each cow once about 2–3 weeks to parturition, and for each calf 3 times at 2–5, 180–185 and 360–365 days of age. All samples were studied for the presence of MAP by culture or by PCR of IS900 insertion sequence MAP DNA. Faecal samples of the dams demonstrated 15/66 dams with at least one positive result. Analysis of MAP shedding prevalence in the faeces of calves originating from MAP-positive and MAP-negative dams shows that in 15 MAP-positive dams- 10 out of 15 calves (66.66%) at the age of 180–185 days and at the aged of 360–365 days 11 out of 15 calves (73.33%) were MAP-positive in faecal samples. In the case of MAP-negative dams: 7 out of 51 calves (13.72%) at the age of 180–185 days, and 8 out of 51 calves (15.68%) at the age of 360–365 days were MAP-positive. MAP-infection cases in calves coming from infected and healthy dams prove the occurrence of calf-to-calf transmission and that contact with infected calves increases the risk of MAP spreading among healthy calves.

Influence of Stress Connected with Moving to a New Farm on Potentially MAP-Infected Mouflons.

There is no European legislation concerning paratuberculosis that requires that imported animals be kept in quarantine and commonly they are directly released into areas with other animals. In this study, detection of latent infection of paratuberculosis in healthy mouflons previously diagnosed as paratuberculosis-free, but originating from a real time quantitative PCR- (qPCR-) positive herd, occurred after their transport to a new farm. During a twelve-day quarantine period, all mouflons irregularly shed Mycobacterium avium subsp. paratuberculosis (MAP) in faeces, and in a small number of cases also in milk. After the animals were released from quarantine, MAP was detected for a further two days, after which, testing was negative, except in one case. Therefore, the stress connected with transport, novel environment, dietary change, or limited area with high density of animals might have contributed to the induction of paratuberculosis and the shedding of MAP from the animals, previously diagnosed as MAP-negative. According to these results, the keeping of imported animals in quarantine and their examination for MAP presence not only before the transport but also afterwards should be recommended. The designation of a particular area of a farm as a quarantine enclosure could help to mitigate the impact of stress caused by a confined space with a high density of animals.

Evaluation of the effects of a killed whole-cell vaccine against Mycobacterium avium subsp paratuberculosis in 3 herds of dairy cattle with natural exposure to the organism

To evaluate effects of vaccination with a killed whole-cell vaccine against Mycobacterium avium subsp paratuberculosis (MAP) on fecal shedding of the organism, development of clinical paratuberculosis (Johne's disease [JD]), milk production, measures of reproduction, and within-herd longevity of dairy cattle naturally exposed to MAP. Controlled clinical trial. 200 vaccinated and 195 unvaccinated (control) dairy cows from 3 herds in Wisconsin. Every other heifer calf born in each herd received the MAP vaccine; 162 vaccinates and 145 controls that had ≥ 1 lactation were included in analyses. Bacteriologic culture of fecal samples for MAP was performed annually for 7 years; results were confirmed via histologic methods and PCR assay. Production records and culture results were evaluated to determine effects of vaccination on variables of interest in study cows. Annual whole-herd prevalence of MAP shedding in feces was also determined. Vaccinates had a significantly lower hazard of testing positive for MAP via culture of fecal samples than did controls over time (hazard ratio, 0.57; 95% confidence interval, 0.34 to 0.97). Fewer vaccinates developed clinical JD than did controls (n = 6 and 12, respectively), but these differences were nonsignificant. Overall within-herd longevity, total milk production, and calving-to-conception intervals were similar between vaccinates and controls. In all herds, prevalence of MAP shedding in feces decreased over time. Vaccination with a killed whole-cell MAP vaccine appeared to be an effective tool as part of a program to control the spread of JD in dairy cattle.

Composite testing for ante-mortem diagnosis of Johne’s disease in farmed New Zealand deer: correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR

BackgroundIn the absence of overt clinical signs of Johne’s Disease (JD), laboratory based tests have largely been limited to organism detection via faecal culture or PCR and serological tests for antibody reactivity. In this study we describe the application of quantitative faecal PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in New Zealand farmed deer to quantify the bacterial load in cervine faecal samples as an adjunct to an existing serodiagnostic test (Paralisa™) tailored for JD diagnosis in deer. As ELISA has potential as a cheap, high throughput screening test for JD, an attempt was made to assess the sensitivity, specificity and positive/negative predictive (PPV/NPV) values of Paralisa™ for estimating levels of faecal shedding of MAP as a basis for JD management in deer.ResultsCorrelations were made between diagnostic tests (ELISA, qPCR, culture and histopathology) to establish the precision and predictive values of individual tests. The findings from this study suggest there is strong correlation between bacterial shedding, as determined by faecal qPCR, with both culture (r = 0.9325) and histopathological lesion severity scoring (r = 0.7345). Correlation between faecal shedding and ELISA reactivity in deer was weaker with values of r = 0.4325 and r = 0.4006 for Johnin and Protoplasmic antigens, respectively. At an ELISA Unit (EU) cutoff of >50 (Johnin antigen) the PPV of Paralisa™ for significant faecal shedding in deer (>104 organisms/g) was moderate (0.55) while the NPV was higher (0.89). At an EU cutoff of ≥150, the PPV for shedding >105 organisms/g rose to 0.88, with a corresponding NPV of 0.85.ConclusionsThe evidence available from this study suggests that Paralisa™ used at a cutoff of 50EU could be used to screen deer herds for MAP infection with sequential qPCR testing used to cull all Paralisa™ positive animals that exhibit significant MAP faecal shedding.

Effect of season and lactation stage on the diagnostic sensitivity of direct real-time PCR assay for detection of fecal shedding of Mycobacterium avium subsp paratuberculosis

Mycobacterium avium subsp paratuberculosis (MAP) is the causative organism of Johne's disease. Dairy Farmers of Canada lists this production-limiting disease as one of the top two animal health priorities for the Canadian dairy industry. Infection of newborn calves from ingestion of MAP-infected feces, colostrum, or milk is considered the main route of transmission and a major concern in a herd. Current diagnostics rely on identification of the bacterium in feces via culture and molecular tests or identification of MAP antibodies in milk or serum via enzyme-linked immunosorbent assays (ELISA). However, these tests are inadequate to meet industry needs for herd biosecurity and environmental-transmission control of MAP, especially when MAP-infected cows are in the subclinical stages of the disease. Milk ELISAs have poor predictive values due to imperfect sensitivity and specificity, especially when used in herds with a low prevalence of MAP-infected cows. Although culture of fecal samples for MAP is currently the gold standard diagnostic test for identification of MAP-infected cows, the long incubation times, costs, and intermittent shedding of MAP in feces hinder its use as an efficient screening tool. The goal of this study was to assess how shedding patterns of MAP in feces vary with lactation stage and season, as determined via direct real-time polymerase chain reaction (PCR) assay.

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination.Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections.The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.

Outcome of Three Commercial Serum ELISAs and Faecal Detection of Mycobacterium avium subsp. paratuberculosis in Consecutive Samples from a Cattle Herd with Low Prevalence of Paratuberculosis (Johne’s Disease)

Paratuberculosis (Johne's disease) in ruminants is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Owing to the lack of accurate laboratory tests, diagnosis is challenging in subclinically infected cattle. To evaluate the long-term performance of serum ELISAs for the detection of paratuberculosis in a dairy herd with low MAP-prevalence, three investigations of all the cows and the consecutive testing of 33 cows suspected to be infected with MAP and 30 cows classified as MAP free were performed over a period of 22 months. Blood samples were tested by three commercial serum ELISAs, MAP shedding was detected by bacteriological culture and polymerase chain reaction (PCR). The ELISA results varied in a wide range in the herd investigations with 1.2% to 18.8% positive samples, the faecal samples were positive for MAP between 1.8% and 4.9% in the three herd investigations. Over the study period, ELISA-positive serum samples varied between 0.0% and 69.7% in MAP-suspicious and 0.0% and 17.6% in MAP-unsuspicious cows with a poor correlation between ELISAs and faecal shedding. The correlation coefficient of the optical density values of the three ELISAs varied between 0.348 and 0.61. Evidence of cow specific variations of residuals was found in all linear models. The linear mixed models showed relevant contribution of cow specific variation in explanation of the residual variances. They also showed significant effects of the explanatory ELISA, the group (MAP-suspicious or MAP-unsuspicious) and the time of sampling. It can be concluded that the choice of the laboratory test significantly influences the outcome of the testing for MAP and that none of the three ELISAs can be thoroughly recommended as single test for the early diagnosis of paratuberculosis in cattle. Test results should always be interpreted with caution to avoid erroneous decisions and the disappointment of those engaged in the abatement of paratuberculosis.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26–0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution.

Effects of infectious young stock on results of certification, surveillance and control programmes for paratuberculosis in dairy herds

In many epidemiological models for paratuberculosis, it is assumed that infected young stock (<2 years of age) do not shed Mycobacterium avium subsp. paratuberculosis (MAP) before adulthood. If this assumption were true, the effective separation of young stock from adult cattle (≥2 years) would largely prevent postnatal infections, provided that uninfected adult cattle are highly resistant to infection. However, this assumption is in contrast with observed faecal shedding of MAP in young stock. Consequently, this assumption may have resulted in an underestimation of the effects of MAP transmission in herds participating in certification-, surveillance-, and control programmes for paratuberculosis. Therefore, the aim of the present study was to evaluate the long-term effects of transmission of MAP amongst young stock on key output parameters of certification-, surveillance-, and control programmes for paratuberculosis in simulated closed dairy herds. Closed Dutch dairy herds participating in a paratuberculosis programme were simulated with a stochastic model, JohneSSim. Various test schemes, preventive management measures, distributions of age at onset of faecal shedding and rates of effective contacts between young stock were simulated. The results indicate that transmission of MAP amongst young stock has no relevant effects on the animal-level prevalence and milk quality of herds that are certified in a paratuberculosis programme. However, transmission of MAP amongst young stock increased the economic losses due to paratuberculosis and costs of participation in a programme. Moreover, it substantially decreased the beneficial effect of the separation of young stock from adult cattle on the probability of being certified. However, even in the presence of transmission of MAP amongst young stock, preventive management measures to separate young stock from adult cattle remain important.

Detection of spatial and temporal spread of Mycobacterium avium subsp. paratuberculosis in the environment of a cattle farm through bio-aerosols

Environmental samples were collected to investigate the spatial and temporal spread of Mycobacterium avium subsp. paratuberculosis (MAP) in a dairy cattle barn before and after the introduction of two groups of MAP-shedding animals. Samples collected off the floor of the barn reflected the moment of sampling whereas samples collected by microfiber wipes at a minimal of 3 m height contained the accumulated settled dust over a 3-week period. Samples were analysed by IS 900 qPCR for the presence of MAP DNA and by culture for viable MAP bacteria. MAP DNA was detected in a large number of sites both before and after introduction cattle. MAP DNA was detected inside the barn in floor and dust samples from cubicles and slatted floors and in settled dust samples located above the slatted floors and in the ventilation ridge opening. Outside the barn MAP DNA was detected by PCR in samples reflecting the walking path of the farmer despite hygiene measures. No viable MAP was detected before the introduction of shedder cattle. Three weeks later viable MAP was found inside the barn at 7/49 locations but not outside. Fifteen weeks later viable MAP was also detected in environmental samples outside the barn. In conclusion, introduction of MAP shedding cattle lead to widespread contamination of the internal and external environment of a dairy barn, including the presence of viable MAP in settled dust particles suggesting potential transmission of MAP infection through bio-aerosols.

Isolation ofMycobacterium aviumsubsp.paratuberculosisfrom Muscle Tissue of Naturally Infected Cattle

Johne's disease or paratuberculosis is a chronic granulomatous inflammation of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recent studies suggest an association between MAP and Crohn's disease in humans. MAP can become widely distributed within the tissues of infected animals, and meat may be a possible route of exposure of MAP to humans. In this study, 47 dairy and beef cattle were examined for the occurrence of viable MAP in diaphragm muscle. At the slaughterhouse, gut tissues, diaphragm muscle, blood, and feces of the 47 animals were collected for bacteriological culture, as well as gut samples for histopathological analysis. MAP was detected by bacteriological culture and conventional and real-time IS900 polymerase chain reaction in the diaphragm muscle of six infected cattle at slaughter (13%). The six animals showing evidence of MAP in diaphragm muscle had diffuse lesions and severe granulomatous inflammation in ileocecal lymph nodes, jejunal lymph nodes, ileocecal valve, and ileum. All six had heavy bacterial load in mesenteric lymph nodes, ileocecal valve, ileum, and jejunum, and four showed clinical signs of paratuberculosis. Two animals did not show clinical signs but had viable MAP in intestinal tissues and in diaphragm muscle as well. MAP was found in blood of only one of the six animals showing evidence of MAP in diaphragm muscle and in feces of three of them. In general, there was a positive association between enteric lesion severity, clinical signs of paratuberculosis, heavy bacterial load in intestinal tissues, fecal shedding of MAP, and the presence of disseminated MAP infection in diaphragm muscle. The results of this study demonstrated that MAP can be detected and cultured from muscle of MAP-infected cattle destined for human consumption and suggest a possible risk of exposure of humans to MAP via contaminated meat.

Feeding Pasteurized Non-saleable Milk did not Increase the Risk for Mycobacterium Avium subsp. paratuberculosis Infection in Adult Dairy Cows

While the most important route of transmission of Mycobacterium avium subsp. paratuberculosis (MAP) is generally considered to be through the ingestion of infective feces from the calf's environment, other potential sources of transmission could include shedding (or post-harvest contamination) of MAP in colostrum or milk. The importance of consuming raw milk in MAP transmission to calves has not been quantified. The objective of this study was to describe if feeding pasteurized non-saleable milk from a Johne's infected herd controlled the transmission of MAP in calves, as compared to calves fed commercial milk replacer, under conditions of natural exposure.