The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall

Infection with Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is a widespread problem in the United States and worldwide, and it constitutes a significant health problem for dairy animals with a potential effect on human health. Mycobacterium paratuberculosis is easily transmitted through consumption of contaminated milk; therefore, finding safe methods to reduce the mycobacterial load in milk and other dairy products is important to the dairy industry. The main objective of the current study was to investigate the effect of natural products, such as bacteriocins designated as “generally regarded as safe” (GRAS), on the survival of M. paratuberculosis in milk. Commercially synthesized bacteriocin (nisin) was used to examine its effect on the survival of laboratory and field isolates of M. paratuberculosis and in contaminated milk. Surprisingly, nisin had a higher minimum inhibitory concentration (MIC) against the laboratory strain (M. paratuberculosis K10), at 500 U/mL, than against field isolates (e.g., M. paratuberculosis 4B and JTC 1281), at 15 U/mL. In milk, growth of M. paratuberculosis was inhibited after treatment with levels of nisin that are permissible in human food at 4°C and 37°C. Using both fluorescent and scanning electron microscopy, we were able to identify defects in the bacterial cell walls of treated cultures. Our analysis indicated that nisin reduced membrane integrity by forming pores in the mycobacterial cell wall, thereby decreasing survival of M. paratuberculosis. Thus, nisin treatment of milk could be implemented as a control measure to reduce M. paratuberculosis secreted in milk from infected herds. Nisin could also be used to reduce M. paratuberculosis in colostrum given to calves from infected animals, improving biosecurity control in dairy herds affected by Johne's disease.     

Short communication: Correlation between within-herd antibody-prevalence and bulk tank milk antibody levels to Mycobacterium avium ssp. paratuberculosis using 2 commercial immunoassays

The objective of this study was to determine the correlation between the results obtained with the ELISA technique for antibodies to Mycobacterium avium ssp. paratuberculosis in serum and bulk tank milk at the herd level. For this purpose, 203 samples of bulk tank milk were analyzed with 2 commercial ELISA from dairy herds with a prevalence of seropositive animals that was also determined. In regard to the reference test (results in blood serum), the sensitivity of the bulk tank milk test to detect high-positive herds (≥10% seroprevalence) ranged from 85.7 to 71.4%. The specificity to detect herds with no seropositive animals ranged from 70.5 to 53%. In a quantitative approach, Pearson correlation coefficients, reported as a measure of the linear association between herd seroprevalences and transformed optical density values recorded in bulk tank milk, were 0.39 and 0.54 for the studied ELISA. Although the test results were relatively fairly correlated with the within-herd prevalence, the practical utility of bulk tank milk testing for Mycobacterium avium ssp. paratuberculosis seems limited, especially regarding specificity.     

Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell–mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (～0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.     

Prevalence of Mycobacterium avium ssp. paratuberculosis infections in Canadian dairy herds

Johne's disease is a progressive, chronic disease with inflammation of the small intestine of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Accurately estimating prevalence of MAP infections is important when controlling spread of infection or monitoring effectiveness of control programs. In the absence of a consistent test method used in prevalence studies across Canada, prevalence estimates among regions and programs cannot be compared. The aim of the current study was to estimate and compare prevalence of MAP infection in Western Canada, Ontario, Québec, and the Atlantic provinces, as well as among varying herd sizes and housing types. On 362 dairy farms located in all 10 provinces of Canada, environmental samples were collected and cultured for detection of MAP. For each herd, 1 sample was collected from the lactating cow area and manure storage. An additional environmental sample was collected from the area where breeding-age heifers were housed. Using prior distributions from previous research, diagnostic sensitivity and specificity were calculated to assess the ability of only 2 environmental samples (manure storage and lactating cow area) to identify MAP-positive farms, resulting in a sensitivity and specificity of 38 and 100%, respectively. We found no difference in sensitivity and specificity when including breeding-age heifers environmental samples. Test characteristics were applied to environmental culture results from the 362 participating farms in all 4 regions, resulting in true prevalence estimates of 66% for farms in Western Canada, 54% in Ontario, 24% in Québec, and 47% in Atlantic Canada. Herds housed in tiestalls had lower prevalence than freestall-housed herds, and herds with 101–150 and >151 cows had higher prevalence than herds with ≤100 cows. This was the first time MAP prevalence was determined using 1 detection method, performed in 1 laboratory, and within a single year across Canada, enabling direct comparisons of prevalence among regions, housing types, and herd sizes.     

Phagomagnetic separation-quantitative PCR: A rapid,sensitive and specific surveillance tool for viable Mycobacterium avium ssp. paratuberculosis in bulk tank and individual cow milk samples

Bulk tank milk samples from 392 Northern Ireland dairy farms and individual milk from animals (n = 293) on 4 of these farms were tested by a novel phagomagnetic separation (PhMS)-quantitative (q)PCR assay able to detect and quantify viable Mycobacterium avium ssp. paratuberculosis (MAP), to demonstrate its potential utility as a milk surveillance tool. Viable MAP were detected in 26.5% of the bulk tank milks, with MAP contamination levels ranging from 1 to 8,432 MAP/50 mL of milk; less than 2% of farms had MAP contamination levels >100 MAP/50 mL in their bulk tank milk. Follow-up PhMS-qPCR testing of milk from individual animals on 4 farms that had the highest numbers of MAP in their bulk tank milks indicated that 17 to 24% of animals in each herd were shedding viable MAP in their milk. Mean MAP numbers detected ranged between 6.7 and 42.1 MAP/50 mL of milk. No significant correlation was observed between the detection of viable MAP in bulk or individual milks by PhMS-qPCR and parallel milk ELISA results, or between PhMS-qPCR results and any other milk recording results (somatic cell count, total bacterial count, % butterfat, or % protein). Viable MAP was detected by IS900 qPCR in 52 (85.2%) Pozzato broth cultures of 61 PhMS-qPCR-positive individual milks after 12 wk of incubation, suggesting few PhMS-qPCR results were false positives. The mean sensitivities of the PhMS-qPCR assay and milk ELISA applied to individual milks were estimated by Bayesian latent class analysis to be 0.7096 and 0.2665, respectively, and mean specificities were similar (0.9626 and 0.9509). Our findings clearly demonstrate that the novel PhMS-qPCR assay could be a useful milk surveillance tool for dairy processors, or a milk monitoring tool for Johne's disease control or milk quality assurance programs.     

Short communication: Heritability of susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in Holstein cattle

Johne's disease in cattle is the result of infection of the small intestine by Mycobacterium avium ssp. paratuberculosis (MAP), leading to an incurable inflammatory bowel disease (Johne's disease or paratuberculosis). The disease is a concern both for its direct cost to dairy producers and for its zoonotic potential. The objective of this study was to estimate the heritability for susceptibility to infection of cattle by MAP using Johne's testing records (ELISA test for presence of antibodies to MAP in milk or blood) from US Holstein cattle from 2009 to 2016. Data sets were edited to include records from herds with 100 or more total records and sires with 50 or more daughters. Data sets were further edited to include (1) only herds with at least 1 positive test, (2) herds with at least 2.5% positive test results, and (3) herds with at least 5% positive test results to examine the effect of data from herds with higher proportions of positive tests, and presumably higher pathogen exposure, on heritability estimates. Two models were used in this study, a linear sire model and a binary threshold-probit sire model. Both were mixed models considering fixed effects of herd and age at test, the latter as a covariate accounting for linear and quadratic effects; random effects included sire and residual. Analyses were conducted using a restricted maximum likelihood method. Heritability estimates (±standard error) from the linear model were 0.041 ± 0.004, 0.050 ± 0.004, and 0.062 ± 0.007 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the threshold model were 0.157 ± 0.014, 0.174 ± 0.016, and 0.186 ± 0.021 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the linear model were affected by population incidence for positive tests, in contrast to estimates from the threshold model, likely accounting for the difference in magnitude of heritability estimates between models and suggesting that the threshold model analysis is the better choice. Heritability estimates increased as data were restricted to herds with presumed higher MAP exposure for both linear model and threshold model analyses. These estimates are similar to previous estimates in other dairy cattle populations and suggest the potential for selection to lessen susceptibility to MAP infection.     

Comparison of fecal pooling strategies for detection of Mycobacterium avium ssp. paratuberculosis in cattle

In herds with typical moderate to low within-herd prevalence, testing for Mycobacterium avium ssp. paratuberculosis (MAP), the infectious agent of Johne's disease, will be more cost-effective if individual fecal samples are cultured in composite pools. However, sensitivity to classify a pool containing 1 or more positive individual samples as positive may depend on pool size and number of individual positive samples within a pool. Fecal samples collected from 994 dairy cows sampled at slaughter were cultured to detect MAP. Culturing was done both individually and as composite pooled samples using the TREK ESP Culture System II broth medium (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH). Composite samples consisted of pools containing feces from 3, 5, 8, 10, or 15 cows. The number of individual fecal culture-positive cows within each pool ranged from 0 to 4. Culture of individual fecal samples detected MAP in 36 (3.6%) of the 994 cows. Individual samples that were detected within the first 50 d by TREK ESP Culture System II were more likely to lead to a positive pool result. In total, 840 pooled fecal samples were examined for presence of MAP, and of those, 272 pools actually contained feces from fecal culture-positive cows. The crude sensitivity (proportion of pools that contained at least 1 fecal-positive cow that tested positive) for pools of 3, 5, 8, 10, and 15 was 47, 67, 44, 59, and 39%, respectively. Across pools, an increase of the number of fecal culture-positive samples from 1 to 2 enhanced overall crude sensitivity from 44 to 71%. However, sensitivity did not further increase for pools with 3 or 4 fecal culture-positive samples (63 and 60%, respectively). Additionally, a simulation analysis assessing probability of pooled fecal samples being positive in herds of 50 and 100 cows was conducted. The simulation assumed that 1, 2, or 5 cows per herd were MAP fecal culture-positive and that pools of 5 and 10 were used. This low-prevalence herd simulation indicated that weighted mean herd probabilities of detecting a positive herd ranged between 52 and 99.3%, with the lowest probability for pools of 10 with 1 positive cow in the herd and the highest probability for pools of 5 with 5 positive cows in the herd. However, overall, pools of 5 and 10 had similar diagnostic capabilities, enabling cost savings by utilizing pools of 10.     

The effect of Mycobacterium avium ssp. paratuberculosis infection on clinical mastitis occurrence in dairy cows

Endemic diseases can be counted among the most serious sources of losses for livestock production. In dairy farms in particular, one of the most common diseases is Johne's disease, caused by Mycobacterium avium ssp. paratuberculosis (MAP). Infection with MAP causes direct costs because it affects milk production, but it has also been suspected to increase the risk of clinical mastitis (CM) among infected animals. This might contribute to further costs for farmers. We asked whether MAP infection represents a risk factor for CM and, in particular, whether CM occurrences were more common in MAP-infected animals. Our results, obtained by survival analysis, suggest that MAP-infected cows had an increased probability of experiencing CM during lactation. These results highlight the need to account for the interplay of infectious diseases and other health conditions in economic and epidemiological modeling. In this case, accounting for MAP-infected cows having an increased CM occurrence might have nonnegligible effects on the estimated benefit of MAP control.     

Evaluation of national surveillance methods for detection of Irish dairy herds infected with Mycobacterium avium ssp. paratuberculosis

The aim of this study was to evaluate the utility and cost-effectiveness of a range of national surveillance methods for paratuberculosis in Irish dairy herds. We simulated alternative surveillance strategies applied to dairy cattle herds for the detection of Mycobacterium avium ssp. paratuberculosis (MAP)-infected herds (case-detection) or for estimation of confidence of herd freedom from infection (assurance testing). Strategies simulated included whole-herd milk or serum serology, serology on cull cows at slaughter, bulk milk tank serology, environmental testing, and pooled fecal testing. None of the strategies evaluated were ideal for widespread national case-detection surveillance. Herd testing with milk or serum ELISA or pooled fecal testing were the most effective methods currently available for detection of MAP-infected herds, with median herd sensitivity >60% and 100% herd specificity, although they are relatively expensive for widespread use. Environmental sampling shows promise as an alternative, with median herd sensitivity of 69%, but is also expensive unless samples can be pooled and requires further validation under Irish conditions. Bulk tank milk testing is the lowest cost option and may be useful for detecting high-prevalence herds but had median herd sensitivity <10% and positive predictive value of 85%. Cull cow sampling strategies were also lower cost but had median herd sensitivity <40% and herd positive predictive values of <50%, resulting in an increased number of test-positive herds, each of which requires follow-up herd testing to clarify status. Possible false-positive herd testing results associated with prior tuberculosis testing also presented logistical issues for both cull cow and bulk milk testing. Whole-herd milk or serum ELISA testing are currently the preferred testing strategies to estimate confidence of herd freedom from MAP in dairy herds due to the good technical performance and moderate cost of these strategies for individual herd testing. Cull cow serology and bulk tank milk sampling provide only minimal assurance value, with confidence of herd freedom increasing only minimally above the prior estimate. Different testing strategies should be considered when deciding on cost-effective approaches for case-detection compared with those used for building confidence of herd freedom (assurance testing) as part of a national program.     

Short communication: Heritability estimates for susceptibility to Mycobacterium avium ssp. paratuberculosis infection in Chinese Holstein cattle

Paratuberculosis in ruminants, which is caused by Mycobacterium avium ssp. paratuberculosis (MAP), is a contagious, chronic enteric disease associated with economic losses, animal welfare, and health implications in dairy cattle production. In this study, we estimated the variance components and heritability of susceptibility to MAP infection in Chinese Holstein cattle. We collected 4,937 serum samples from cows in 7 dairy herds in the Beijing region of China and used the ELISA test to detect antibodies to MAP. Three statistical models were implemented to estimate heritabilities: (1) a linear model (ELISA sample-to-positive ratios as a continuous trait); (2) a binary threshold model (positive/negative from ELISA results); and (3) an ordered threshold model (ELISA results as an ordered categorical model with categories 1 to 5 corresponding to negative, uncertain, mildly positive, intermediate positive, and strongly positive). The heritability estimates ranged from 0.0389 to 0.1069, indicating that genetic factors affect MAP infection susceptibility in Chinese Holstein cattle.     

Longitudinal relationship between fecal culture,fecal quantitative PCR,and milk ELISA in Mycobacterium avium ssp. paratuberculosis-infected cows from low-prevalence dairy herds

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of ruminant Johne's disease, presents a particular challenge with regard to infection mitigation on dairy farms. Diagnostic testing strategies to identify and quantify MAP and associated antibodies are imperfect, and certain facets of the relationship between diagnostic tests remain to be explored. Additional repeated-measures data from known infected animals are needed to complement the body of cross-sectional research on Johne's disease-testing methods. Statistical models that accurately account for multiple diagnostic results while adjusting for the effects of individual animals and herds over time can provide a more detailed understanding of the interplay between diagnostic outcomes. Further, test results may be considered as continuous wherever possible so as to avoid the information loss associated with dichotomization. To achieve a broader understanding of the relationship between diagnostic tests, we collected a large number of repeated fecal and milk samples from 14 infected cows, in addition to bulk milk samples, from 2 low-prevalence dairy herds in the northeast United States. Predominately through the use of mixed linear modeling, we identified strong associations between milk ELISA optical density, fecal quantitative PCR, and fecal culture in individual animals while concurrently adjusting for variables that could alter these relationships. Notably, we uncovered subtleties in the predictive abilities of fecal shedding level on milk ELISA results, with animals categorized as disease progressors reaching higher ELISA optical density levels. Moreover, we observed that spikes in fecal shedding could predict subsequent high ELISA values up to 2 mo later. We also investigated the presence of MAP in individual milk samples via PCR and noted an association between poor udder hygiene and MAP positivity in milk, suggesting some level of environmental contamination. The paucity of positive milk samples and the complete absence of detectable MAP in the bulk tank throughout the study period indicate that contamination of milk with MAP may not be of chief concern in low-prevalence herds. An enhanced understanding of the interrelationships between diagnostic tests can only benefit the development of testing strategies and objectives, which in turn may lessen MAP infection prevalence in dairy herds.     

Effects of additional milk replacer feeding on calf health, growth, and selected blood metabolites in calves

The objective of the experiment was to evaluate effects of increased milk replacer feeding on growth, intake, feed efficiency, and health parameters in stressed calves. Holstein bull calves (n = 120; approximately 3 to 8 d of age) were purchased from sale barns and dairy farms and housed in fiberglass hutches. In addition, wood shavings contaminated with coronavirus were mixed with clean shavings and added to each hutch before the start of the experiment. Calves were fed either a fixed amount (454 g/d) of a 20% crude protein (CP), 20% fat milk replacer to weaning at 28 d or a variable amount (454, 681, 908, and 454 g/d on d 0 to 7, 8 to 14, 15 to 31, and 32 to 41, respectively) of a milk replacer containing 28% CP and 17% fat without or with added dietary supplement containing bovine serum. Calves were also fed commercial calf starter and water ad libitum. Plasma IgG concentration in most calves on arrival at the facility was < 10 g/L. Intake, change in body weight, feed efficiency, morbidity and mortality, and selected plasma metabolites were determined. Body weight at 28 d, 56 d, daily body weight gain, intake of milk replacer, fecal scores, days with diarrhea, and days treated with antibiotics were increased with feeding variable amount of milk replacer over the 56-d study. Starter intake from d 1 to 56 was reduced from 919 to 717 g/d in calves fed fixed and variable amounts of milk replacer, respectively. Morbidity, measured as the number of days that calves had diarrhea, was increased by 53% when a variable amount of milk replacer was fed. Calves fed variable milk replacer were treated with antibiotics for 3.1 d compared with 1.9 d for calves fed 454 g of milk replacer/d. Concentrations of plasma glucose, urea N, and insulin-like growth factor-I were increased when calves were fed variable amount of milk replacer. Dietary supplement containing bovine serum had no effect on any parameter measured. There was no effect of milk replacer feeding on concentrations of nonesterified fatty acids, total protein, or growth hormone concentrations. Plasma tumor necrosis factor-α was highest in calves with the highest plasma IgG concentrations on the day of arrival and might be related to the calf's ability to identify pathogens in the environment. Under conditions of this study, calves fed variable amount of milk replacer and exposed to immunological challenge before weaning had greater BW gain, but also increased incidence of diarrhea that required added veterinary treatments.     

Longitudinal study of Mycobacterium avium ssp. paratuberculosis fecal shedding patterns and concurrent serological patterns in naturally infected dairy cattle

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease that affects ruminants worldwide. Despite global interest in the control of this disease, gaps exist in our knowledge of fecal shedding patterns and concurrent serological patterns. This longitudinal study in dairy cattle herds with high MAP seroprevalence in France aimed at accurately describing fecal shedding patterns over 1 year; relating those shedding patterns to individual animal characteristics (age, breed, parity); and exploring the association between fecal shedding patterns and serological patterns. To describe temporal fecal shedding patterns and continuity of shedding, along with the standard quantitative PCR (qPCR) threshold cycle we used a cutoff value that related to low or nonculturable fecal shedding. We also defined a threshold cycle indicative of shedding in high quantities to describe infection progression patterns. Twenty-one herds completed the study, and 782 cows were tested 4 times each. We obtained 4 sets of paired fecal qPCR and serum ELISA results from 757 cows. Although we targeted highly likely infectious animals, we found a large diversity of shedding patterns, as well as high variability between herds in the proportion of animals showing a given pattern. The fecal qPCR results of almost 20% of the final study sample were positioned at least once in the range that indicated low or nonculturable fecal shedding (between the adjusted and the standard cutoff value). Although these animals would typically be classified as non-shedders, they could be important to infection dynamics on the farm. Animals that shed at least twice consecutively and animals that shed in high quantities rarely reverted to negativity. Repeated fecal qPCR can be used to detect temporal fecal shedding traits, and the decision to cull an animal could practically be based on temporal, semiquantitative results. Overall, we found a mismatch between fecal shedding and ELISA seropositivity (637 animals were ELISA-negative 4 times, but only 13% of those animals were qPCR-negative 4 times). We found that having more than 2 ELISA-positive samples was strongly related to persistent and continuous shedding. We suggest that although serological testing is much less sensitive than qPCR, it can also be used, particularly over the course of multiple testing events, to identify animals that are most likely to contribute to the contamination of the farm environment.     

Effects of spray-dried animal plasma in calf milk replacer on health and growth of dairy calves

Male Holstein calves (n = 120) purchased from local dairy farms were fed one of three calf milk replacers for 42 d. Experimental milk replacers were formulated to contain whey protein concentrate (WPC) as the primary protein source or WPC plus 5% spray-dried bovine plasma (SDBP) or spray-dried porcine plasma (SDPP). The SDPP was heated to remove heat-insoluble materials and provide products with similar IgG content. Calves were also fed commercial calf starter and water for ad libitum consumption. Intake, change in body weight (BW), feed efficiency, morbidity and mortality were determined. Mortality was 10, 3, and 2 in calves fed WPC, SDBP, and SDPP treatments, respectively. Morbidity, measured as the number of days that calves had diarrhea was reduced by 30% when SDBP or SDPP were fed. Calves had diarrhea for 6.9, 3.9, and 4.7 d during the 42-d study when fed commercial calf milk replacer containing WPC, SDBP, and SDPP, respectively. Fecal scores tended to be reduced and feed efficiency tended to be improved when SDBP or SDPP were fed. Mean intakes of total dry matter during the 42-d study were greater when calves were fed SDBP or SDPP and were 661, 710, and 684 g/d for calves fed WPC, SDBP, and SDPP, respectively. Mean BW gains from d0 to 42 were 231,261, and 218 g/d, respectively. Calves fed SDPP tended to have lower BW gain during the first 28 d of the study. However, difference in daily BW gain from d 1 to 28 was only 39 g/d. Inclusion of SDBP or SDPP in milk replacer reduced morbidity and mortality of milk-fed dairy calves.     

Environmental sample characteristics and herd size associated with decreased herd-level prevalence of Mycobacterium avium ssp. paratuberculosis

Environmental sampling is an effective method for estimating regional dairy herd-level prevalence of infection with Mycobacterium avium ssp. paratuberculosis (MAP). However, factors affecting prevalence estimates based on environmental samples are not known. The objective was to determine whether odds of environmental samples collected on farm changed culture status over 2 sampling times and if changes were specific for location and type of housing (freestall, tiestall, or loose housing), the sample collected (i.e., manure of lactating, dry, or sick cows; namely, cow group), and effects of herd size. In 2012–2013 [sampling 1 (S1)] and 2015–2017 [sampling 2 (S2)], 6 environmental samples were collected and cultured for MAP from all 167 (99%) and 160 (95%) farms, respectively, in the province of Saskatchewan, Canada. Only the 148 dairy farms sampled at both sampling periods were included in the analysis. A mixed effects logistic regression was used to determine whether differences between sampling periods were associated with herd size and sample characteristics (cow group contributing to environmental sample, type of housing, and location). In S1 and S2, 55 and 34%, respectively, of farms had at least 1 MAP-positive environmental sample. Correcting for sensitivity of environmental sampling, estimated true prevalence in S1 and S2 was 79 and 48%, respectively. Herds with >200 cows were more often MAP-positive than herds with <51 cows in both S1 and S2. The percentage of positive samples was lower in S2 compared with S1 for all sampled areas, cow groups contributing to samples, types of housing where samples were collected, and herd size categories. However, samples collected from dry cow areas had the largest decrease in MAP-positive samples in S2 compared with all other cow group samples. Herds that were MAP-negative in S1 with a herd size 51 to 100 or 101 to 150 were more likely to stay MAP-negative, whereas MAP-positive herds with >200 cows more frequently stayed MAP-positive. No difference was observed in the odds of a sample being MAP-positive among housing types or location of sample collection in both sample periods. Of all farms sampled, 104 (70%) did not change status from S1 to S2. In conclusion, when herd-level MAP prevalence decreased over the 3-yr interval, the change in prevalence differed among herd size categories and was larger in samples from dry cow areas. It was, however, not specific to other characteristics of environmental samples collected.     

Reduction of viable Mycobacterium avium ssp. paratuberculosis in slurry subjected to anaerobic digestion in biogas plants

Cattle infected with Mycobacterium avium ssp. paratuberculosis (MAP) shed the bacterium in their feces. This may lead to considerable concentrations of MAP in slurry, which has been postulated to contribute to MAP transmission when this slurry is used as fertilizer. For other bacterial species, anaerobic digestion has been shown to reduce bacterial load and to increase the safety of organic waste. Therefore, the objective of this study was to investigate the effects of anaerobic digestion in biogas plants on MAP survival in slurry from 16 dairy farms with a history of MAP infection. Presence of MAP was determined using MAP culture and a commercial MAP IS900 quantitative PCR (qPCR) applied on untreated slurry samples, slurry samples after primary fermentation, and digestate. Unfermented slurry samples from most enrolled farms tested positive for MAP, via both culture and qPCR. After the fermentation process, MAP could no longer be cultured in most samples, with the exception of 2 samples from farms where high numbers of MAP-shedding cows were kept at the time of sampling. A Bayesian binomial model predicted a probability of 93% for a MAP-negative culture result after fermentation. In most samples, MAP DNA was still detectable when using the IS900 qPCR. The probability of a negative result in qPCR was estimated to be 27%. Results of this study indicate that subjecting MAP-positive slurry to anaerobic digestion in biogas plants leads to a reduction of viable MAP below the detection limit; however, MAP DNA remained detectable. It remains undetermined whether MAP DNA detected in fermentation products is a residue of MAP degradation or belongs to viable MAP below the detection limit or in a dormant state. In conclusion, subjecting MAP-positive slurry to anaerobic mesophilic digestion reduces viable MAP concentration below the detection limit. The use of digestion products as fertilizer on pasture and agricultural soils instead of untreated slurry may therefore reduce the risk of MAP transmission.     

Modeling of alternative testing strategies to demonstrate freedom from Mycobacterium avium ssp. paratuberculosis infection in test-negative dairy herds in the Republic of Ireland

In light of the various adverse effects of Johne's disease on animal productivity and the debate on the role of its causative organism, Mycobacterium avium ssp. paratuberculosis, in the etiology of Crohn's disease, major dairy-producing countries around the world have implemented national control programs aimed at reducing the prevalence of this infection in cattle. A pilot control program was initiated in Ireland in 2013, with a key objective to provide farmers with test-negative dairy herds with tools and knowledge to increase their confidence of freedom over time. The aim of this study was to estimate the confidence of freedom obtained in test-negative Irish dairy herds over time with various sampling scenarios and to evaluate the cost-effectiveness of alternative scenarios for achieving an acceptable level of confidence of freedom in herds with no evidence of infection. A stochastic model was developed to simulate repeated annual testing of individual animals using ELISA and confirmatory assays over a period of 20 yr. Two scenarios modeled the current herd-screening options, whereas 14 alternative scenarios explored the effect of varying parameters from the current testing strategies, such as the frequency of testing, the eligibility criteria for selecting animals, the type of assay, the probability of introduction, and the assay sensitivity. Results showed that the current testing strategy with milk twice a year or serum once a year in all animals over 2 yr old provided the highest annual herd sensitivity, with a median value of 55%. Although the median confidence of freedom increased over time for all scenarios, the time required to reach 90 and 95% confidence of freedom was highly variable between scenarios. Under the testing scenario where serum tests were used once a year, the confidence of freedom reached 90% after 4 yr and 95% after 7 yr of testing. Some of the alternative scenarios achieved an acceptable level of confidence of freedom in a reasonable timeframe and at lesser cost than the current testing strategies. The results of this work are used to provide recommendations for the next phases of the program.     

A preliminary study investigating effects of oral monensin sodium in an enteric Mycobacterium avium ssp. paratuberculosis infection model of calves

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, an enteric infection of ruminants that causes significant economic burden for dairy and beef producers. Efforts to control MAP in endemic herds typically focus on herd management practices such as limiting exposure or early culling of infected animals and, occasionally, vaccination. The ionophore monensin sodium may have protective effects against MAP both in vivo and in vitro; however, this has not been thoroughly evaluated experimentally. Using a direct intestinal MAP challenge model, we have observed similarities regarding persistence of MAP in tissues and apparent resilience to infection compared with experimental oral infection or natural disease. Here we sought to investigate the effects of oral monensin supplementation in experimentally MAP-infected calves. We examined the persistence of MAP in the intestinal tissues, MAP-induced intestinal inflammation, fecal MAP shedding, and seroconversion using a commercial serologic assay. Monensin-supplemented MAP-infected calves demonstrated evidence for resilience to MAP infection earlier in this study compared with monensin-free MAP-infected calves. However, statistical modeling did not identify a significant effect of monensin on outcomes of infection, and more work is required to understand how monensin affects early tissue colonization of MAP in calves.     

Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds.