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

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

Milk as diagnostic fluid for udder health management.

BACKGROUND: Mastitis is the major disease affecting milk production of dairy cattle, and milk is an obvious substrate for the detection of both the inflammation and its causative infectious agents at quarter, cow, or herd levels. In this review, we examine the use of milk to detect inflammation based on somatic cell count (SCC) and other biomarkers, and for the detection of mastitis pathogens through culture-based and culture-free methods. FINDINGS: The use of SCC at a cow or bulk milk level to guide udder health management in lactation is well-established, and SCC is increasingly used to guide selective dry cow treatment. Other markers of inflammation include electrical conductivity, which is used commercially, and markers of disease severity such as acute phase proteins but are not pathogen-specific. Some pathogen-specific markers based on humoral immune responses are available, but their value in udder health management is largely untested. Commercial pathogen detection is based on culture or polymerase chain reaction, with other tests, for example, loop-mediated isothermal amplification or 16S microbiome analysis still at the research or development stage. Matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) is increasingly used for the identification of cultured organisms whilst application directly to milk needs further development. Details of test sensitivity, specificity, and use of the various technologies may differ between quarter, cow, and bulk milk applications. CONCLUSIONS: There is a growing array of diagnostic assays that can be used to detect markers of inflammation or infection in milk. The value of some of these methods in on-farm udder health improvement programs is yet to be demonstrated whilst methods with proven value may be underutilised.

Dataset on risk factors for seroconversion against Mycobacterium avium subspecies paratuberculosis in dairy cows.

Johne's disease (JD) is a chronic wasting disease caused by Mycobacterium avium subspecies paratuberculosis (MAP). MAP is responsible for large economic losses for the dairy sector and has been linked to human disease. Susceptibly to MAP is mainly limited to young animals and diagnostic tests are poor at detecting MAP in early stages of infection. Therefore, ascertaining the contribution of the dam to the risk of calf infection and the relative role of the different infection routes is important to inform disease control measures. This data article presents MAP exposures at time of calving on a cohort of 439 calves born between 2012 and 2013 from 6 UK dairy herds. Each calf participated in routine quarterly MAP milk ELISA testing using the IDEXX Porquire ELISA. Each animal was followed until testing MAP positive, being culled or end of follow up (January 2023). The dataset includes risk factors associated with transmission via colostrum route (MAP status of cow giving colostrum); transmission via the dam (MAP status of the dam) and transmission via fecal oral route (whether at birth the calf spent a long time in a dirty yard). Ascertainment of exposure to risk factors involved video recording and self-capture data forms from time of calving in the maternity area of the farms until calf left the area. The dataset provides a unique opportunity to examine MAP infection and its relationship with different exposures at time of birth, where cows were followed up during their entire lives.

Unravelling transmission of Mycobacterium avium subspecies paratuberculosis to dairy calves: results of a lifelong longitudinal study.

Johne's disease (JD) is a chronic disease of ruminants endemic in the UK and other countries and responsible for large economic losses for the dairy sector. JD is caused by Mycobacterium avium subspecies paratuberculosis (MAP), which typically infects calves that remain latently infected during a long period, making early detection of infection challenging. Cow to calf transmission can occur in-utero, via milk/colostrum or faecal-orally. Understanding of the different transmission routes to calves is important in informing control recommendations. Our aim in this longitudinal study was to measure the association between the transmission routes via the dam and the environment on a calf subsequently testing serologically positive for MAP. The study population comprised of 439 UK dairy calves from 6 herds enrolled between 2012 and 2013. These calves were followed up from birth until 2023. At birth individual calf data was captured. During follow-up, individuals entering the milking herd were quarterly tested for the presence of MAP antibodies using milk ELISA. Cox regression models were used to measure the association between exposure from the dam (in-utero and/or colostrum) or from the environment (long time in dirty yard) and time to first detection of MAP infection. An association between calves born to positive dams and probability of having a MAP positive test result remained after excluding potential MAP transmission via colostrum (Hazard ratio: 2.24; 95% CI: 1.14 - 4.41). Calves unlikely to be infected with MAP via the in-utero or colostrum route, had 3.68 (95% CI: 3.68 1.45-9.33) higher hazard of a positive test result when they stayed longer in a dirty calving area. The effect of the dam infection status on transmission to calves precedes the dam's seroconversion and persists after excluding the potential role of transmission via colostrum. The association between time spent in a dirty calving area and probability of a MAP positive test result highlights the role of environmental contamination as a source of infection in addition to the dam.

Prevalence of Brucella spp. in milk from aborted and non-aborted animals in Dhamar governorate, Yemen.

Brucella infection in animals is considered a great problem in most countries of the world. Our study designed to determine the prevalence of brucella in field animal's milk in Dhamar governorate, Yemen. Total of 808 raw milk samples from non-aborted field animals, 120 milk samples from aborted animals, and 30 pasteurized milk samples were teste by Milk-Ring Test (MRT), milk-ELISA test, isolation and identification of brucella species, and antibiotic susceptibility. The prevalence of brucella in milk samples from field animals was 0.8%, 2.6%, and 2% in cows, sheep, and goat milk samples respectively with MRT, and 0.8%, 1.3% and 1.6% in cows, sheep and goat milk samples respectively with the milk- ELISA test. The prevalence rate in milk samples from aborted animals was 33%, 64% and 41.2% with the MRT and 39%, 49%, and 41.2% in cows, sheep and goats respectively with the milk-ELISA test. All pasteurized milk samples were negative for the milk-ELISA test. The result of isolation showed 0.1% of Brucella in milk samples from field animals while 9.2% from aborted animals. All isolates of Brucella species were sensitivities to rifampicin, doxycycline, kanamycin, gentamicin, streptomycin, tetracycline, and ciprofloxacin, while resistant to ampicillin, erythromycin, and novobiocin. In conclusion, the high prevalence of milk brucella especially in aborted animals needs focusing and build controlling strategies plans to decrease the losses to the economy and avoid transferred to humans with unpasteurized milk consumption.

The Occurrence of Mycobacterium avium Subspecies paratuberculosis Positive Milk Antibody ELISA Results in Dairy Cattle under Varying Time Periods after Skin Testing for Bovine Tuberculosis.

Enzyme-linked immunosorbent assays (ELISA) are used to screen cows for Mycobacterium avium subspecies paratuberculosis (MAP) infections, informing Johne's disease (JD) management practices in dairy herds. The causative agent of bovine tuberculosis (bTB), Mycobacterium bovis, and MAP share multiple antigens. Moreover, Mycobacterium avium subspecies avium is used in the single intradermal cervical comparative tests (SICCT) that are routinely used in early detection of cows infected with bTB. Although these are different types of immune responses, potentially the SICCT may interfere with the levels of MAP antibodies. This study aimed to clarify the relationship between the SICCT-MAP milk ELISA testing interval and apparent prevalence of JD risk statuses. Data from 51 herds were used, totalling 46,738 cow observations. The Poisson models showed that MAP milk ELISA testing at 14 day intervals post-SICCT statistically significantly increased the odds of detecting JD-positive cows compared to JD testing 85+ days post-SICCT. The odds ratio (OR) started at 2.5 in the first 14 day interval post-SICCT, increasing each two-week period to an OR of 4.0 at 57-70 days, to subsequently drop. Additionally, a herd history of bTB increased the odds of detecting JD-positive cows (OR = 1.2); this was relatively limited compared to the magnitude of the post-SICCT effect.

Association between milk yield and milk anti-Fasciola hepatica antibody levels, and the utility of bulk tank milk samples for assessing within-herd prevalence on organic dairy farms.

Fasciola hepatica is an important disease of livestock that is responsible for substantial economic losses worldwide. Estimates of the impact of infection on milk yield vary, likely reflecting different geographical locations, farm-level management, and diagnostic methods. Measuring anti-Fasciola antibodies on bulk tank milk (BTM) by ELISA provides a convenient herd-level diagnosis, but the utility of this test remains unclear. Therefore, we evaluated the utility of BTM ELISA test results in Danish organic dairy farms, including estimating the association between 305 day energy corrected milk yield (305d ECM) and F. hepatica infection both at individual and herd level. BTM samples from 218 organic farms were analysed using IDEXX ELISA and subsequently the farmers were interviewed during spring 2016 with the aim of characterising their management practices. The corresponding farm-level production data covering the period 2014-2017 were collected from the Danish national cattle registry. In the following year, 284 individual milk samples (4-7 per herd) along with BTM samples were collected from a subset of the same herds (n = 55). Linear mixed models were used to estimate the association between milk production and ELISA value at both individual and farm levels, and a generalised additive model was used to assess the relationship between within-herd prevalence and BTM ELISA. A dichotomised BTM result with positive outcome was associated with a reduction of 580.5 kg in average 305d ECM, and a positive outcome on individual-level ELISA was associated with a 919.5 kg reduction in milk yield for cows in their third or later lactations. A strong relationship between quantitative BTM ELISA sample to positive percentage (S/P%) and apparent within-herd prevalence based on dichotomised individual-level milk ELISA was also observed, although this relationship was non-linear in nature. We conclude that a useful indication of the within-herd prevalence of infection can be obtained from BTM ELISA following categorisation as negative, low, medium or high according to S/P% cut-offs of approximately 30, 80, and 150. This approach represents a cheap and useful diagnostic tool for monitoring the long-term success of control strategies for F. hepatica infections on a dairy farm.

Antibody detection and molecular analysis for Mycobacterium avium subspecies paratuberculosis (MAP) in goat milk: Systematic review and meta-analysis.

Paratuberculosis is an incurable infectious disease that affects several species, including goat (Capra hircus). The etiologic agent is Mycobacterium avium subspecies paratuberculosis (MAP) that has tropism for the intestine, causing anorexia, progressive weight loss and death. In goats, the main transmission route is the ingestion of water and food contaminated by infected feces. Affected animals also eliminate the agent through milk, with a potential biological risk to public health. Thus, the aim of this study was to conduct a research of the literature available in electronic media for a systematic review, followed by a meta-analysis of the results found on prevalence and diagnostic tests adopted in the detection of MAP antibodies and DNA in goat milk. The following search parameters were used: "Mycobacterium avium subsp. paratuberculosis" AND (goat OR small ruminant) AND (milk OR pasteurized milk). Strictly obeying pre-established criteria, 437 articles were selected from the respective electronic databases of scientific content: ScienceDirect (285), PubMed (68), Web of Science (60) and Scopus (24), of which nine papers were elected to the construction of the systematic review and meta-analysis. The prevalence of MAP antibodies in milk detected by milk-ELISA ranged from 1.1 to 67.7% and the prevalence of MAP DNA in goat milk detected by MAP-specific polymerase chain reaction (PCR) ranged from 1.94 to 37.74%. A meta-analysis indicated a combined MAP infection prevalence of 8.24%, but with high heterogeneity among study findings (I2 = 98.7%). The identification of the MAP in goat milk implies the need for surveillance of the agent in order to prevent economic losses and impact on public health.

The interpretation of serial Johne's disease milk antibody results is affected by test characteristics, pattern of test results and parallel bovine tuberculosis testing.

BACKGROUND: In the UK, quarterly Johne's disease milk antibody ELISAs (JD-mELISAs) are commonly used to classify animals which are likely to be infectious, termed "red cows". "Red cows" are classified following two positive results from the previous four tests (e.g. + - - +). All cattle are also regularly screened for bovine tuberculosis using intradermal avian and bovine tuberculin, and it is advised to maintain a 60 day interval between a tuberculosis test and JD-mELISA. AIMS: To evaluate the impact of bovine tuberculosis testing on JD-mELISAs, and to quantify the impact of test specificity and "red cow" classification test pattern on the probability of infection. METHODS: Four years of individual cow milk records with JD-mELISA results were collated from 735 dairy farms and matched to tuberculosis testing records. A two-level multivariable logistic regression model quantified the effect of tuberculosis testing on JD-mELISA result. The specificity and age-dependent sensitivity of a single JD-mELISA were estimated and used to calculate likelihood ratios following each test. Using Bayes' theorem, the posterior probability of infection with Johne's disease was calculated for different specificities, ages of cow, and patterns of test results. RESULTS: There were increased odds of a positive JD-mELISA if it was ≤30 days (OR: 2.1) or 31-60 days (OR: 1.2) after a tuberculosis test, compared to >90 days. A larger avian skin reaction at the tuberculosis test was also associated with increased odds of a positive JD-mELISA. The proportion of cows which tested exclusively negative after their first positive JD-mELISA was higher if that JD-mELISA was ≤30 days after a tuberculosis test compared to >90 days. The posterior probability of infection reduced substantially when the test specificity was slightly reduced. In "red cows" classified following two consecutive positive tests, if the test specificity was reduced to 0.95, then the posterior probability of infection was only >95 % if the prior probability was >13 %. If the "red cow" classification was due to two non-consecutive positive tests (+ - - +), the posterior probability of infection was only >95 % if the prior probability was >43 %. CONCLUSIONS: Testing for Johne's disease within 60 days of a tuberculosis test is associated with a higher chance of a positive JD-mELISA and this may reflect a reduction in the ELISA specificity. Relatively small reductions in JD-mELISA specificity can markedly reduce the posterior probability of infection which also depends on the pattern of test results which classifies "red cows".

Selective flukicide treatment of non-lactating cows and the corresponding production impact of Fasciola hepatica in dairy herds in Sweden.

A control strategy against Fasciola hepatica infection based on selective treatment of non-lactating animals was evaluated in four Swedish dairy herds. The study was conducted over the course of two consecutive seasons in moderately to highly F. hepatica infected herds with robotic milking, where heifers and dry cows received an oral drench with albendazole (10 mg/kg) during three visits in January, February and March in both 2017 and 2018. This resulted in an anthelmintic coverage between 38 % and 58 % of the animals. Furthermore, on each visit, the infection status of all dewormed animals along with 15 randomly selected milking cows were monitored by detection of F. hepatica coproantigens. Individual milk samples were also collected quarterly from the whole herds for measurements of individual antibody levels against the parasite using milk ELISA. In addition, individual data on milk yield and quality were collected on a monthly basis between 2016 and 2018. To further study the impact of the infection on milk production, truly F. hepatica positive and negative cows in the first lactation were identified based on the results from coproantigen and milk ELISA assays. Total F. hepatica coproantigen prevalence in the herds varied between 28 % and 85 % in the first year, and between 27 % and 68 % in the second year of the study. We found that two years of treatments resulted in a significant decrease of coproantigen-positivity especially on the two most heavily infected farms. These results were confirmed by a similar drop in within-herd prevalences obtained by milk ELISA results. The infection had a significant negative impact on milk yields in untreated F. hepatica positive cows. No consistent long-term effect was observed at the herd level probably due to the influx of animals infected before puberty and/or adult animals that were re-infected at dry-off. This is the first study of the effects of F. hepatica infection on milk yield and quality in dairy herds in Sweden.

From six to zero per cent Mycobacterium avium subsp. paratuberculosis milk ELISA positivity in three years - probably induced by Mycobacterium vaccae.

This research communication reports the results of a study aimed at investigating the effects of introducing Mycobacterium vaccae on paratuberculosis carriage in a dairy herd. M. vaccae is a non-pathogenic member of the Mycobacteriaceae, with immunomodulatory and immunotherapeutic capabilities, acting by stimulating the cellular immune system, important in protection against paratuberculosis. Starting in 2014 we administered, by gavage, 1010 live M. vaccae bacteria to all new-born heifers on a dairy farm, first within 24 h of birth and again 2 weeks later. Paratuberculosis carriage was monitored yearly by milk ELISA. Faecal samples of 50% of cows, aged 3 years, born 1, 2 or 3 years before the experiment's onset, were tested by qPCR for MAP shedding and compared to 100% treated cows of the same age. Within 3 years, milk ELISA positivity was reduced from 6 to 0% and remained unchanged for the subsequent 2 years. One qPCR positive control cow was found each year for a total of 3 animals (2.46%). One positive cow (1%) was found among the treated cows. Two of the 3 positive control animals, still present on the farm at the end of 2019, tested negative whereas the positive test cow continued shedding MAP. M. vaccae shedding heifers mixing with adult cows were the probable means of the microorganism's propagation. The results of this investigation indicate that the introduction of live M. vaccae may be an inexpensive and fast alternative to current paratuberculosis control practices, justifying further exploration of the topic.

Use of a voluntary testing program to study the spatial epidemiology of Johne's disease affecting dairy herds in Minnesota: a cross sectional study.

BACKGROUND: One of the key steps in the management of chronic diseases in animals including Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is the ability to track disease incidence over space and time. JD surveillance in the U.S. dairy cattle is challenging due to lack of regulatory requirements, imperfect diagnostic tests, and associated expenses, including time and labor. An alternative approach is to use voluntary testing programs. Here, data from a voluntary JD testing program, conducted by the Minnesota Dairy Herd Improvement Association, were used to: a) explore whether such a program provides representative information on JD-prevalence in Minnesota dairy herds, b) estimate JD distribution, and, c) identify herd and environmental factors associated with finding JD-positive cows. Milk samples (n = 70,809) collected from 54,652 unique cows from 600 Minnesota dairy herds between November 2014 and April 2017 were tested using a MAP antibody ELISA. Participant representativeness was assessed by comparing the number of JD-tested herds with the number of herds required to estimate the true disease prevalence per county based on official statistics from the National Agricultural Statistical Services. Multivariable logistic regression models, with and without spatial dependence between observations, were then used to investigate the association between herd status to JD (positive/negative), as indicated by milk ELISA results, and available covariates at the herd level. RESULTS: Within the study population, at least one test-positive cow was found in 414 of 600 (69%) herds. Results indicated that large herds that test frequently and herds located in loamy or silt soils are more likely to have at least one MAP test-positive cow. After adjusting for herd size, testing frequency, and soil type, there was no spatial dependence in JD risk between neighboring dairies within 5 to 20 km. Furthermore, the importance of collecting data on herd management, feed, and biosecurity for insightful interpretations was recognized. The study suggested that, although limited, the voluntary testing database may support monitoring JD status. CONCLUSIONS: Results presented here help elucidate the spatial characteristics of JD in Minnesota and the study may ultimately contribute to the design and implementation of surveillance programs for the disease.

Evaluation of a serum ELISA for detection of bovine leukemia viral antibodies in milk samples.

Bovine leukemia virus (BLV) infection has worldwide distribution in both dairy and beef herds. Our study was initiated in order to encourage control of BLV infection by using milk samples, in lieu of serum samples, to readily test lactating animals prior to dry-off and calving. Two Holstein dairy herds (A and B), with known status of BLV infection as determined by serology, were sampled by the collection of serum and fresh milk samples. Selected samples were tested using a USDA-licensed BLV antibody ELISA kit (Bovine leukemia virus antibody test kit; VMRD, Pullman, WA) for serum. Forty-one lactating cows from each herd were sampled. Herd A was confirmed to have endemic BLV infection; herd B was confirmed to be free of BLV infection. The milk ELISA results demonstrated 100% identification of positive and negative animals compared with the serum results. The correlation of the ELISA values between serum and milk samples was 97%, which supports the use of this BLV ELISA on milk samples.

The genetic architecture of milk ELISA scores as an indicator of Johne's disease (paratuberculosis) in dairy cattle.

Johne's disease (or paratuberculosis), caused by Mycobacterium avium ssp. paratuberculosis (MAP) infection, is a globally prevalent disease with severe economic and welfare implications. With no effective treatment available, understanding the role of genetics influencing host infection status is essential to develop selection strategies to breed for increased resistance to MAP infection. The main objectives of this study were to estimate genetic parameters for the MAP-specific antibody response using milk ELISA scores in Canadian Holstein cattle as an indicator of resistance to Johne's disease, and to unravel genomic regions and candidate genes significantly associated with MAP infection. After data editing, 168,987 milk ELISA records from 2,306 herds, obtained from CanWest Dairy Herd Improvement, were used for further analyses. Variance and heritability estimates for MAP infection status were determined using univariate linear animal models under 3 scenarios: (a) SCEN1: the complete data set (all herds); (b) SCEN2: herds with at least one suspect or test-positive animal (ELISA optical density ≥0.07); and (c) SCEN3: herds with at least one test-positive animal (ELISA optical density ≥0.11). Heritability estimates were calculated as 0.066, 0.064, and 0.063 for SCEN1, SCEN2, and SCEN3, respectively. The correlations between estimated breeding values for resistance to MAP infection and other economically important traits, when significant, were favorable and of low magnitude. Genome-wide association analyses identified important genomic regions on Bos taurus autosome (BTA)1, BTA7, BTA9, BTA14, BTA15, BTA17, BTA19, and BTA25 showing significant association with MAP infection status. These regions included 2 single nucleotide polymorphisms located 2 kb upstream of positional candidate genes CD86 and WNT9B, which play key roles in host immune response and tissue homeostasis. This study revealed the genetic architecture of MAP infection in Canadian Holstein cattle as measured by milk ELISA scores by estimating genetic parameters along with the identification of genomic regions potentially influencing MAP infection status. These findings will be of significant value toward implementing genetic and genomic evaluations for resistance to MAP infection in Holstein cattle.

Short- and long-term association between individual levels of milk antibody against Ostertagia ostertagi and first-lactation heifer's production performances.

It is agreed that exposure of adult dairy cattle to helminths on pasture can negatively affect production performances as milking herd. Young animals, especially replacement heifers, represent the future of a dairy farm and are among the most vulnerable to helminth infections in a dairy herd. For this reason, dairy farmers tend to frequently treat heifers against helminths, although the impact of helminths on heifers' production performances is still poorly understood. Using different epidemiological and serological tools, this study examines the relationship between heifer exposure to helminths on pasture and production performances over time. During a one-year period, 1,454 individual milk samples were collected from first-lactation heifers in England and tested for Ostertagia ostertagi (O. ostertagi) antibodies. After controlling for other confounders, increasing milk antibody levels against O. ostertagi were significantly associated with decreased milk yield at sampling but not at day 305 of heifer lactation. We did not observe any relationship between milk antibody levels against O. ostertagi in heifers and yields in fat and protein. However, heifers with a high level of milk antibodies against O. ostertagi were more likely to produce dead calf at first calving and present a delay in second calving. Moreover, these heifers had significantly higher levels of milk antibodies against Mycobacterium paratuberculosis (M. paratuberculosis) during their first lactation and were more likely to die before the end of the study. We argue that epidemiological approaches can be useful but must be complemented by other methodologies to better understand the impact of helminth infections in dairy heifers. In order to address the complex dynamics of helminth infections in dairy cattle production we require more comprehensive approaches that include triangulation between data sources and interdisciplinary studies.

A probabilistic approach to the interpretation of milk antibody results for diagnosis of Johne's disease in dairy cattle.

Johne's disease is a serious wasting disease of ruminants that is of high economic importance for the dairy sector in particular. The chronic nature of the disease, the fluctuations in antibody levels and the limited ability of diagnostic tests to identify cows at early stages of infection are huge challenges for the control of the disease. In the United Kingdom, the latter is commonly based on repeated milk ELISA testing of lactating cows, followed by selected culling and improved management practices around calving. In this paper, the dataset built through a large quarterly screening programme conducted in the United Kingdom since 2010 is used to investigate the use of milk ELISA testing for Johne's disease management. Over the study period, 13,509 out of 281,558 cows were identified as high-risk of being infected and shedding mycobacteria in the faeces, based on a case definition of at least two consecutive positive milk ELISA results. Around a third of them were kept in the dairy herd a year or more after being classified as high-risk. However, 16% of these cows did not have any further positive test, suggesting that they might be uninfected animals. The mean specificity and sensitivity of the milk ELISA test were estimated at 99.5% and 61.8%, respectively. The cows in the dataset are categorised in different result groups according to the number of positive test results and whether they are classified as high-risk according to the programme's case definition. The posterior probability of infection is calculated after each test in order to investigate the impact of repeated testing on the belief in a cow's infection status. The interpretation of the results show that most cows classified as high-risk are very likely to be infected, while some other groups that do not match the case definition could reasonably be considered as infected too. Our results show that there is considerable potential for more targeted use of serological testing, including adjusting the testing frequency and implementing the posterior probability approach.

A longitudinal study of gastrointestinal parasites in English dairy farms. Practices and factors associated with first lactation heifer exposure to Ostertagia ostertagi on pasture.

The gastrointestinal nematode Ostertagia ostertagi is an important cause of lost production, health, and welfare in cattle. Detailed records were obtained over a 5-yr period (2010-2015) by questionnaires and qualitative interviews to investigate the practices adopted by dairy farmers to control cattle helminth infections and the factors associated with heifer exposure to O. ostertagi on pasture. In total, 1,454 heifers' individual milk samples were collected over a 1-yr period (2014-2015) in 43 dairy farms in England and tested for O. ostertagi antibody by ELISA. Multilevel linear regression models were used to investigate the association between individual milk optical density ratio (ODR) against O. ostertagi and heifer management from birth to time of sampling. Farm and heifer median ODR against O. ostertagi were 0.98 (interquartile range = 0.76-1.02) and 0.64 (interquartile range = 0.42-0.84), respectively. The majority of heifers (88%) received an anthelmintic treatment before sampling in this study. After controlling for the effect of anthelmintic treatments, heifer individual milk ODR against O. ostertagi significantly increased with high stocking rate at first grazing and co-grazing with adult cows before calving. Conversely, heifer individual milk ODR against O. ostertagi significantly decreased when heifers had co-grazed with sheep and pasture grass had frequently been mowed. Overall, these results provide evidence to support targeting grazing management toward limiting the use of anthelmintics in dairy young stock to enable sustainable control of cattle helminth infections in England. However, to be accepted and adopted by farmers, these best practices would need to take into account farmers' perspectives and contextual challenges.

Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy.

BACKGROUND: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs. RESULTS: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain. CONCLUSIONS: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity.

Infections with the bovine lungworm Dictyocaulus viviparus, the causative agent of parasitic bronchitis, are accompanied by substantial economic losses due to impacts on production, clinical respiratory disease or even death of diseased cattle. To detect lungworm antibodies in cattle, an enzyme-linked immunosorbent assay (ELISA) based on recombinant major sperm protein (MSP) has been developed. However, it remained unknown whether the infection dose influences antibody levels, and how acquired immunity influences antibody level patterns during reinfections. The latter may lead to low within-herd seroprevalence and thus to negative MSP-ELISA results in examination of bulk tank milk (BTM). Thus, infection experiments with 12 different doses ranging from 10 to 3000 D. viviparus larvae were performed to assess whether the antibody response is dose-dependent. Second, the impact of reinfections on the antibody response was evaluated in infection experiments, and third, antibody patterns in dairy cows during naturally occurring reinfections were assessed in a longitudinal field study based on individual milk samples. Results of this study demonstrate that the rise in MSP antibodies during first infection is dose-independent at infection doses of 25 lungworm larvae and above. However, following reinfections the magnitude and duration of the MSP antibody response are reduced or lacking, depending on the interval to reinfection. The field study revealed short periods of seropositivity as a common pattern in dairy cows subjected to natural D. viviparus reinfections. Low within-herd seroprevalence in dairy herds can thus be a result of continuous reinfections. Low infection doses should not be a barrier to serodiagnosis of lungworm infection in first-time infected 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.