The effect of a hiding space on the behaviour and heart rate variability of dairy calves during temporary separation from the dam.

In natural settings, newborn calves hide for several days before joining the herd. It is unclear whether dairy calves housed indoors would show similar hiding behaviour. This study aimed to describe the use of an artificial hide provided to calves during temporary separation from the dam and assess the effect it has on lying and sleep-like behaviour, as well as heart rate variability (HRV). Twenty-eight cow-calf pairs were randomly assigned to having a hide (n = 14), or no hide (n = 14). Hide use (n = 14), as well as lying and sleep-like behaviour (n = 28), were recorded continuously via video camera during the first hour after the dam was removed for morning milking on day three to seven. Heart rate and R-R intervals were recorded using Polar equine monitors for a subsample of 12 calves (n = 6 per treatment) on day six. Descriptive statistics were calculated for hide use. Wilcoxon Signed Rank tests were used to evaluate whether having a hide affected lying and sleep-like behaviours as well as HRV. Hide use decreased over days and was highly variable between calves. Lying behaviour did not differ between treatments. Duration of sleep-like behaviour was higher for calves without a hide compared to those with a hide. Calves with a hide tended to show signs of higher HRV and parasympathetic activity compared to calves without a hide. Results suggest that providing a hiding space to young calves may be beneficial during periods when the cow is removed from the pen for milking.

Horse Housing on Prince Edward Island, Canada: Attitudes and Experiences Related to Keeping Horses Outdoors and in Groups.

Limited research has assessed the "human dimension" of horse care. The aims of this study were to (1) understand horse owner attitudes toward horse welfare when kept outdoors versus indoors and in groups versus individually, (2) compare horse owner attitudes toward horse welfare with the ways in which they house their horses, and (3) explore horse owner reasons for and challenges with their horses' housing. Seventy-six horse owners in Prince Edward Island, Canada completed a questionnaire. Non-parametric tests and quantitative content analysis were used for data analysis. Consistent with the way horses were kept, most (82-96%) owners agreed that horses' physical health, mental well-being, and natural living were better when kept outdoors and in groups. Fewer (64-68%) participants agreed that the horses' standard of care was better when kept outdoors or in groups. Results show associations between owners whose attitudes suggest indoor and/or individual housing is better for horse welfare and keeping their horses indoors part-time and/or individually. Two overarching themes were developed from owners' responses regarding their reasons and challenges related to the ways in which horses were housed: horse-centered and owner-centered care. The results indicate that horse owners' choices about their horses' housing correspond to beliefs about improved horse welfare.

Evaluation of antibody and antigen cross-reaction in Kenyan dairy cattle naturally infected with two pestiviruses: Bovine viral diarrhea virus and classical swine fever virus.

Background and Aim: Bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) are important pathogens of cattle and pigs, respectively, and belong to the genus Pestivirus. As CSFV has been shown to infect cattle, it can create diagnostic challenges of BVDV results through possible cross-reactivity where cattle could be exposed to pigs and CSFV. This study aimed to determine the possible cross-reactivity of BVDV and CSFV enzyme-linked immunosorbent assay (ELISA) results for antigen (Ag) and antibody (Ab) among smallholder dairy cattle in Kenya. Materials and Methods: This was a cross-sectional study based on a single visit to farms to collect serum samples and other descriptive farm-level and animal-level information. Testing for BVDV Ag and Ab was conducted on serum samples from 320 dairy cows and heifers, with CSFV Ag and Ab testing conducted on a subset of 133 and 74 serum samples, respectively. CSFV testing was based on BVDV test results and the availability of enough sample volume from farms that kept pigs. The Ag and Ab tests utilized IDEXX ELISA for both BVDV and CSFV. Results: For the 74 samples with Ab tests for both viruses, 40 (54.0%) were BVDV Ab positive, while 63 (85.1%) were CSFV Ab positive. Of the 40 BVDV Ab positive samples, 36 cattle (90.0%) tested positive for CSFV Ab. However, of the 34 BVDV Ab negative samples, 27 (79.4%) were CSFV Ab test-positive. For the 133 samples with Ag tests for both viruses, 125 (94.0%) were BVDV Ag positive, while 2 (1.5%) samples were CSFV Ag positive. None of the eight BVDV Ag negative samples was positive for CSFV Ag and only two (1.6%) of the 125 BVDV Ag positive samples were positive for CSFV Ag. Conclusion: The results indicate either substantial cross-reactivity of the two Ab ELISA tests, or reactivity with some other protein in the samples that led to the positive Ab test results. There was only limited evidence for cross-reactivity of the two Ag ELISA tests. We recommend that Pestivirus genus cross-reactivity be considered when interpreting BVDV ELISA results in cattle, more for Ab than Ag tests. Further research is needed to clarify the levels of cross-reactivity between BVDV and other Pestivirus Ag and Ab tests from animals on mixed-species farms.

Communication skills training and assessment of food animal production medicine veterinarians: A component of a voluntary Johne's disease control program.

In food animal production medicine (FAPM), the success of control programs for infectious diseases that have serious animal health and economic consequences frequently rely on the veterinarian's effective communication and producer adherence to veterinary recommendations. However, little research has been conducted on communication skills of practicing FAPM veterinarians. During this study, we developed a communication training workshop intervention to support the Atlantic Johne's Disease Initiative. Seventeen FAPM veterinarians across 10 clinics practicing within Maritime Canada participated in a pre-post intervention study design. Communication skills were evaluated utilizing 3 assessment tools; an objective structured clinical exam (OSCE), standardized client feedback, and an instrument designed for veterinary participants to assess their self-efficacy. Study results showed that before training, communication skills of participating veterinarians had limitations, including skill deficits in communication tasks strongly associated with increased adherence to veterinary recommendations. Based on the 3 assessment tools, communication skills of participating veterinarians improved with the training provided. Significant increases were detected in pre- to postintervention self-efficacy percentage scores, OSCE percentage and global scores from expert raters, and OSCE percentage and global scores from standardized client feedback. These improvements emphasize the importance of communication skills training specific to FAPM.

Dairy producer satisfaction and knowledge transfer with the veterinary-administered risk assessment and management plan in a voluntary Johne's disease control program.

The Atlantic Johne's Disease Initiative (AJDI) aims to control Mycobacterium avium ssp. paratuberculosis infection by using veterinary-administered risk assessments to identify high-risk management practices and prompt changes in management behavior. Objectives for this study were to measure producer satisfaction with the veterinary-administered risk assessment and management plan (RAMP) process in a voluntary Johne's disease (JD) control program, compare RAMP-specific satisfaction results based on herd JD status, and measure knowledge transfer from certified veterinarians to producers during the RAMP. A satisfaction questionnaire was adapted to the RAMP process in the AJDI to measure producer satisfaction. The questionnaire included 9 RAMP-specific producer satisfaction items, 1 global RAMP satisfaction item, and 16 questions to assess producer knowledge and knowledge translation about JD, bovine viral diarrhea (BVD), and bovine leukosis virus (BLV) during the RAMP (BVD and BLV used for comparison purposes). A total of 133 dairy producers in the AJDI (79.6% response rate) completed the questionnaire by telephone. The RAMP-specific satisfaction was high among the AJDI producers surveyed, and these results were not found to differ based on herd JD status. The lowest satisfaction scores and the highest number of "unable to assess" responses were for the item relating to cost. Factors that contributed to RAMP-specific producer satisfaction were not identified from the demographic and herd information available in this study. The knowledge scores indicated moderate knowledge about JD and fair knowledge about BVD and BLV. Evidence of knowledge translation from the RAMP was mixed in this study. Bovine viral diarrhea knowledge scores were not found to differ based on whether or not the certified veterinarian discussed BVD during the preceding RAMP, but BLV knowledge scores were higher among dairy producers that discussed BLV during the preceding RAMP. Strengths and gaps in producer knowledge about these 3 infectious diseases were identified. By using this producer questionnaire, interventions aimed at improving the content, delivery, and satisfaction of RAMP in JD control programs, such as the AJDI, can be developed.

Effect of housing improvement and other factors on the growth of heifer calves on Kenyan smallholder dairy farms.

This trial evaluated average daily gain (ADG) effects of heifer calves (< 1 year old) from affordable housing improvements to the roof and flooring on 150 randomly allocated smallholder dairy farms. During the 16-month data collection period, bimonthly farm visits were used to measure weight and other animal- and farm-level factors on the 187 study calves. Multivariable linear regression was used to model ln ADG and ADG during pre-weaning and post-weaning periods, respectively. Median pre-weaning and post-weaning ADGs were 0.307 (interquartile range (IQR): 0.227-0.398) and 0.487 (IQR: 0.354-0.675) kg/d, respectively. In the final pre-weaning model (p<0.050), factors positively associated with ln ADG were calf age at first acaricide application, and total number of calf pens, while factors negatively associated with ln ADG included calf mortality risk over the last 5 years and calf age at first ad lib access to water. In an interaction term, for calves from parity 3+ dams, ADG was lower when milk was fed twice/day than thrice/day, with no difference in calves of lower parity dams. In the final post-weaning model, housing improvements increased ADG by 5.6%. Other factors positively associated with post-weaning ADG were feeding of calf pellets, wheat bran, maize bran, and hay. Calf age at first introduction of concentrate and calf mortality risk over the last 5 years were negatively associated with ADG. In an interaction term, ADG was high when there were faecal coccidia oocysts and when calves had visual or physical contact with their dams, but low when faecal coccidia cysts were present, and these dam-calf connections were absent. In a second interaction term, ADG increased with more calf pens for female principal farmers, while remaining low for male principal farmers. In conclusion, while controlling for other factors of ADG, making affordable calf housing improvements enhanced ADG, particularly during the post-weaning period.

Management factors associated with perinatal lamb mortality in Prince Edward Island flocks.

The objective of this observational cohort study was to identify management factors associated with lamb mortality risk for sheep flocks in Prince Edward Island, Canada. Data were collected from 50 lambing groups from 36 sheep flocks during 3 farm visits before, during and after the lambing seasons in 2014-15. Variables of interest included flock management practices, ewe health indicators, ewe nutrition, litter size and lamb birth weight. Principal component analysis was performed and resulting component scores were used for further analysis using a mixed Poisson regression model with lamb mortality risk as the outcome. The median group-level lamb mortality in the first 8 weeks of life was 10.0 % (0 %-30.3 %), with 25 groups having lamb mortality greater than 10 %, which is considered higher than the standard productivity goal. Four principal component scores were retained in the final model identifying generalized factors associated with lamb mortality: 1) flock factors, 2) forage factors, 3) lamb health factors, and 4) general health factors. Specifically, the following management factors were indirectly through the 4 principal components associated with lower lamb mortality: using goal setting; having a strong working relationship with a veterinarian; seeking veterinary advice for animal treatment; using benzimidazole-class anthelmintics; feeding forage with high crude protein, digestible energy, and net energy for maintenance and low acid detergent fiber to late-gestation ewes; applying visual lamb identification methods; using anti-coccidial prophylactic medication to lambs; administering clostridial vaccines to lambs; avoiding separation of hypothermic lambs from their dams; and treatment/prevention of neurological and/or wasting disease. Although this study is exploratory, and confirmation is required, the results should help sheep farmers and researchers direct attention to management variables that could reduce lamb mortality in sheep flocks.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

Evaluation of the Precision Xtra meter for monitoring blood ß-hydroxybutyrate concentrations in late-gestation ewes.

Blood samples were collected from late-gestation ewes to determine the agreement of a point-of-care (POC) Precision Xtra meter and a standard laboratory test for ß-hydroxybutyrate (BHBA). Fresh whole blood samples were immediately tested with the POC instrument, and serum samples were analyzed with a standard commercial biochemical analyzer. Ewes were classified as having ketonemia if their BHBA concentrations were ≥800 µmol/L. Scatter plots, paired t-tests, Bland-Altman limits of agreement, and Gwet AC1 tests were used to compare results. The 2 tests had very good agreement. The values between instruments were not statistically different based on paired t-tests ( p = 0.312). The intercept and slope of a linear mixed model, containing the standard test results as an outcome and the POC meter results as a predictor, were 0.02 (95% CI: 0.00, 0.04) and 0.98 (95% CI: 0.96, 1.01), respectively. When the samples were classified into ketonemic classes (non-ketonemic and ketonemic) based on BHBA concentrations obtained from each test, the Gwet AC1 statistic was 0.94 (95% CI: 0.91, 0.97; p < 0.001). The ketosis classification agreed in 95% of samples. Based on the Bland-Altman plot and limits of agreement, the optimal cutoff to diagnose ketonemia with the POC meter was 1,000 µmol/L, which is 200 µmol/L higher than the laboratory BHBA medical decision limit. The Precision Xtra meter provided excellent correlation and substantial agreement with the standard laboratory technique for measuring blood BHBA in late-gestation ewes.

A cross-sectional study of the welfare of calves raised in smallholder dairy farms in Meru, Kenya, 2017.

AIM: This study was aimed at describing calf comfort and determining the individual and pen level factors that affect comfort status (in particular, calf leg hygiene scores) of smallholder dairy farms in Meru County, Kenya. MATERIALS AND METHODS: A cross-sectional study was carried out on 52 calves that were up to 1 year old in 38 dairy farms (mean±standard deviation: Herd size=1.71±0.7 milking cows and milk production=6.7±3.1 L/day) in Meru, Kenya, in 2017, with the intention to describe their comfort and determine the factors associated with leg hygiene as a critical parameter for calf comfort assessment. Calves' biodata, health status, and leg hygiene were assessed, along with pen characteristics such as area, hygiene, and knee impact and knee wetness scores, while a questionnaire was administered to the farmers to gather information regarding calf housing management practices in the farm. RESULTS: The calves had a mean body weight of 85.2±32.8 kg and average daily weight gain of 0.50±0.45 kg per day. 71% of calves had a good body condition score (≥2.5), and the mean space allowance per calf was 2.52±1.56 m2. Approximately 75% of the calves (39/52) were kept in pens, and the rest were reared outdoors. For 39 calves kept indoors, 26% (10/39) of them had wooden or concrete floors while 74% (29/39) had dirt floors. Nearly two-thirds (62%) of indoor calves (26/39) were reared in pens with bedding, and 23% (9/39) and 33% (13/39) of the calves reared indoors were kept in pens displaying a failed knee impact test and failed knee wetness test. Indoor housed calves had an increased probability of having dirty calf legs (cleanliness score of >2.5) by 8.6 times (p=0.031), compared to outdoor-housed calves. In the final multivariable logistic regression model of 39 calves in pens, concrete or wood floors (odds ratio [OR]=7.9, p=0.047), poor body condition (OR=17.1, p=0.020) and use of bedding (OR=12.5, p=0.046) appeared to be positively correlated with dirtiness of calf legs, compared to dirt floors, good body condition, and no bedding, respectively. CONCLUSION: Overall, some calf comfort aspects were covered for the majority of calves examined, but 69% of the pens were categorized as dirty, especially those with wooden or concrete floors and poor bedding management. Smallholder dairy farmers in Kenya should be trained on calf housing management to improve calf comfort and productivity.

Bovine pancreatolithiasis: case report and review of the literature.

An 8-y-old lactating Holstein cow was presented to the herd veterinarian with a history of acute ataxia, and because of poor prognosis, the animal was euthanized and submitted for postmortem examination. A lumbar vertebra was incompletely fractured and had an adjacent subdural hematoma. The pancreas had a hard texture with exaggerated nodular appearance, and on cut surface, the pancreatic ducts appeared fibrotic and contained scores of round-to-faceted pancreatoliths of 0.5-2.0 mm diameter. These calculi extended from the major ducts to the pancreatic duct, sphincter, and into the duodenal lumen. Microscopically, the pancreas had severe acinar atrophy and dissecting glandular fibrosis. The major pancreatic ducts were ulcerated with fibrosis and inflammation. In humans, chronic pancreatitis has been linked to osteoporosis and increased risk of bone fractures. Manual evaluation of ribs at the time of autopsy did not reveal abnormalities in bone pliability or fragility in our cow.

The association of serum ß-hydroxybutyrate concentration with fetal number and health indicators in late-gestation ewes in commercial meat flocks in Prince Edward Island.

Late-gestation ewes are susceptible to ketonemia resulting from high energy requirement for fetal growth during the last few weeks of pregnancy. High lamb mortality is a possible consequence of effects of ketonemia on both ewes and lambs. Determining risk factors to ketonemia is a fundamental step to identify ewes at risk, in order to avoid losses caused by ketonemia. Serum ß-hydroxybutyrate (BHBA) concentrations of 384 late-gestation ewe samples were determined. Physical examinations, including body condition, FAMACHA© and hygiene scoring, were performed. Udders and teeth were also examined. Fecal floatation was performed to detect gastrointestinal helminth eggs of the ewe fecal samples. General feeding management practices and season at sampling were recorded. Litter sizes were retrieved from lambing records. Factors associated with log serum BHBA concentration were determined using a linear mixed model, with flock and lambing groups as random effects. The mean serum BHBA concentration was 545.8 (±453.3) µmol/l. Ewes with a body condition score (BCS) of 2.5-3.5 had significantly lower log BHBA concentrations than ewes with a BCS of ≤2.0, by 19.7% (p = 0.035). Ewes with a BCS of >3.5 had a trend toward higher log BHBA concentrations compared to ewes with a BCS of 2.5-3.5. Ewes with a FAMACHA© score of 3 had significantly higher log BHBA concentrations than ewes with a FAMACHA© score of 1 or 2, by 12.1% (p = 0.049). Ewes in which gastrointestinal helminth eggs were detected had significantly higher log BHBA concentrations than ewes in which helminth eggs were not detected, by 12.3% (p = 0.040). An increased litter size was associated with higher log BHBA concentration (p ≤ 0.003), with the log BHBA concentrations of ewes having twins, triplets, and quadruplets or quintuplets were higher than those of ewes having singleton by 19.2%, 30.4%, and 85.2%, respectively. Season at sampling confounded the association between log BHBA concentration and FAMACHA© score, and therefore was retained in the final model even though it was not statistically significant. Intra-class correlation coefficients at the flock and lambing group levels were 0.14 and 0.32, respectively.

Non-indigenous predators threaten ecosystem engineers: Interactive effects of green crab and oyster size on American oyster mortality.

Non-indigenous green crabs (Carcinus maenas) are emerging as important predators of autogenic engineers like American oysters (Crassostrea virginica) throughout the eastern seaboard of Canada and the United States. To document the spreading distribution of green crabs, we carried out surveys in seven sites of Prince Edward Island during three fall seasons. To assess the potential impact of green crabs on oyster mortality in relation to predator and prey size, we conducted multiple predator-prey manipulations in the field and laboratory. The surveys confirmed an ongoing green crab spread into new productive oyster habitats while rapidly increasing in numbers in areas where crabs had established already. The experiments measured mortality rates on four sizes of oysters exposed to three sizes of crab, and lasted 3-5 days. The outcomes of experiments conducted in Vexar® bags, laboratory tanks and field cages were consistent and were heavily dependent on both crab size and oyster size: while little predation occurred on large oysters, large and medium green crabs preyed heavily on small sizes. Oysters reached a refuge within the 35-55 mm shell length range; below that range, oysters suffered high mortality due to green crab predation and thus require management measures to enhance their survival. These results are most directly applicable to aquaculture operations and restoration initiatives but have implications for oyster sustainability.

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.

Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne's disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six "Bison type" isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale.

Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12) as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

Sensitivity of solid culture, broth culture, and real-time PCR assays for milk and colostrum samples from Mycobacterium avium ssp. paratuberculosis-infectious dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) can be shed in feces, milk, and colostrum. The goal of this study was to assess assays that detect MAP in these sample types, including effects of lactation stage or season. Understanding the performance of these assays could improve how they are used, limiting the risk of infection to calves. Forty-six previously confirmed MAP-positive cows from 7 Atlantic Canadian dairy farms were identified for colostrum sampling and monthly sampling of milk and feces over a 12-mo period. Samples were assayed for MAP using solid culture, broth culture, and direct real-time PCR (qPCR). Across assay types, test sensitivity when applied to milk samples averaged 25% of that when applied to fecal samples. For colostrum samples, sensitivity depended on assay type, with sensitivity of qPCR being approximately 46% of that in feces. Across sample types, sensitivity of qPCR was higher than that of the other assays. Sensitivity of qPCR, when applied to milk samples, was significantly higher in summer than in other seasons. Summer was also the season with highest agreement between milk and fecal samples collected within the same month. Our results suggest that qPCR would detect more cows shedding MAP in their milk and colostrum than solid or broth culture assays, particularly during the summer, thus providing better management information to limit exposure of calves to this infectious organism.

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne's disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates. RESULTS: Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including "bison type" isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1-2 to 239-240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd. CONCLUSIONS: The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne's disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.

Sensitivity and specificity of repeated test results from a commercial milk enzyme-linked immunosorbent assay for detection of Mycobacterium avium subspecies paratuberculosis in dairy cattle.

OBJECTIVE: To investigate the sensitivity and specificity of results of initial and repeated milk ELISAs (at 6- or 12-month intervals) to detect cows that were shedding Mycobacterium avium subsp paratuberculosis (ie, were infectious) and to evaluate factors influencing the probability that the results of a repeated milk ELISA would be positive for an infectious cow if the results of the initial milk ELISA were negative. DESIGN: Prospective cohort study. ANIMALS: 3,145 dairy cows from 32 herds. PROCEDURES: Herds from the 3 Maritime provinces in Canada (Prince Edward Island, New Brunswick, and Nova Scotia), participating in a Dairy Herd Improvement program, and that had undergone a prior Mycobacterium avium subsp paratuberculosis awareness project were selected for the study. Sample collection occurred between April 2009 and March 2011 with milk and fecal samples collected from all lactating cows in study herds every 6 months. Herds completing < 3 herd visits with collection of individual cow fecal or milk samples, within this sampling timeframe, were excluded from analyses. Fecal samples were cultured in liquid medium and a cow was defined as infectious if ≥ 1 sample was culture positive (reference test). A milk ELISA (index test) was completed with a commercial kit, following manufacturer's instructions. RESULTS: For a 6-month test interval, sensitivities of the milk ELISA to detect infectious cows were 22.0% and 32.6% for initial and combined initial and repeated tests (parallel interpretation), respectively. Specificity of the initial ELISA was 99.6% and was 99.2% for combined tests. For a 12-month test interval, sensitivities of the milk ELISA to detect infectious cows were 25.6% and 45.3% for initial and combined initial and repeated tests (parallel interpretation), respectively. Specificity of the initial ELISA was 99.6% and was 98.9% for combined tests. In infectious cows, magnitude of the initial negative ELISA result was a positive predictor for a positive repeated ELISA result. CONCLUSIONS AND CLINICAL RELEVANCE: Results of a repeated milk ELISA improved detection of Mycobacterium avium subsp paratuberculosis infectious cows, with minimal loss of specificity. A 12-month test interval provided a greater increase in sensitivity, relative to an initial test, than did a 6-month interval. Infectious cows with an initial negative milk ELISA result close to the cutoff for a positive test were more likely to have positive results on a repeated ELISA. Repeated testing improved detection of infectious cows and reduced risk of misclassification compared with a single ELISA result.

The association of detection method, season, and lactation stage on identification of fecal shedding in Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative organism of Johne's disease. Although fecal culture is considered the standard diagnostic test, the long incubation times, costs, and intermittent shedding of MAP hinder efficient screening programs based on culture results. The primary objectives of this study were to determine the detection ability of solid culture, broth culture, and real-time PCR (qPCR) for MAP in fecal samples and to assess how shedding patterns of MAP in feces vary with lactation stage and season. This knowledge could improve the use of these diagnostic assays in Johne's management programs. For this study, 51 MAP-infectious cows from 7 Atlantic Canadian dairy farms had fecal samples collected monthly over a 12-mo period. Samples were analyzed for MAP bacterial load via solid culture, broth culture, and qPCR. For all fecal samples, 46% [95% confidence interval (CI): 40 to 51%] were positive by solid culture, 55% (95% CI: 50 to 60%) by broth culture, and 78% (95% CI: 73 to 82%) by qPCR. Sensitivity of qPCR was numerically higher in the dry and postpartum lactation periods, and qPCR detection in summer and fall was 85% of that in winter and spring. Furthermore, culture-determined moderate or light shedding categories generally corresponded to qPCR cycle threshold values <35, but heavy shedding categories corresponded to qPCR values <29. Direct fecal qPCR is a MAP detection method that is quick and less costly than culture techniques, and it avoids the use of decontamination steps that could decrease numbers of bacteria in a sample below the detection limit. This study indicates that, for known MAP-positive cows, fecal qPCR had high sensitivity of MAP detection, thereby supporting the use of direct fecal qPCR as part of a Johne's herd control program.