Søren Saxmose Nielsen

Abstract: A voluntary risk-based control program on paratuberculosis in dairy cattle was initiated in Denmark in 2006. Cows were categorized as high-risk (antibody-positive at least once within the last 3 tests) or low-risk animals based on the results of 3 to 4 annual milk ELISA detecting Mycobacterium avium ssp. paratuberculosis (MAP)-specific antibodies. High-risk animals require management practices aimed at decreasing calf exposure to MAP-contaminated colostrum and milk, and feces originating from these cows. Moreover, repeated test-positive cows are recommended for slaughter before next calving. The objective was to assess the effect of different management practices on the prevalence of MAP-specific antibodies. A questionnaire on management practices was distributed to 1,261 participating herds in December 2008. A total of 1,092 (87%) herd managers returned the questionnaire. Repeated prevalence data from 1,081 herds were available for a period up to 4.25 yr after the first test round. The changes in the prevalence of MAP-specific antibodies from the start of interventions were assessed using a hierarchical logistic model, where different management practices were assessed: a) culling of repeated test-positive cows, b) separation of high-risk from low-risk cows in calving areas, c) cleaning of calving areas after high-risk cows calved, d) removal of calves born to high-risk dams within 2h after calving, e) use of colostrum for feeding of heifer calves from low-risk cows only, f) use of waste milk for feeding of heifer calves from low-risk cows only, g) herd size, and h) proportion of purchased animals. Multivariable analyses suggested that only the proportion of purchased animals (>15% purchased animals as well as 0 to 15% purchased animals compared with no purchased animals in the herd), culling of repeated test-positive animals, and use of waste milk from specific cow groups influenced the decrease in prevalence of MAP-specific antibodies. The control program has been running for just 4.25 yr, and it is assumed that the full effect of the risk-based management practices will only be observed after 4 to 8 yr. Therefore, lack of association between some practices and decrease in prevalence may be a reflection of a short study period. Furthermore, decreases in the prevalence of MAP-specific antibodies may not reflect discontinued transmission of MAP in all age groups.

Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is a slow growing bacterium that can infect ruminants and remain latent for years without development of any clinical signs or disease. Diagnosis is often based on detection of MAP antibodies in milk or serum samples or culture of bacteria from faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen candidates described to date with special emphasis on antigen candidates tested for CMI responses. Relevant information on 115 different MAP antigens was systematically extracted from literature and summarized in 6 tables of CMI antigens, secreted antigens, cell wall and membrane antigens, lipoprotein antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified for this application. Most of the novel diagnostic candidates were evaluated in few animals and it is recommended that an appropriate sample size is included for evaluation of antigen candidates in future studies.

Abstract: Diagnostic test evaluations (DTEs) for chronic infections are challenging because a protracted incubation period has to be considered in the design of the DTE, and the adverse effects of infection may be widespread and progressive over an animal's entire life. Frequently, the specific purpose of the test is not formally considered when a test is evaluated. Therefore, the result is often a DTE where test sensitivity and specificity estimates are biased, either because of problems with establishing the true infection status or because the test detects another aspect of the infection (and analyte) than originally intended. The objective of this paper is to outline a structured approach to the design and conduct of a DTE for diagnostic tests used for chronic infections in animals, and intended for different purposes. We describe the process from reflections about test purpose and the underlying target condition through considerations of the pathogenesis, and specification of a practical case definition, which can subsequently be used in the DTE for the specific purpose. The process is illustrated by two examples of Mycobacterium avium subsp. paratuberculosis (MAP) infections in cattle. MAP infections are chronic and can result in different adverse effects at different time points during the incubation period. The description provides input on the process and deductive reasoning which are integral parts to develop a high-quality design of a DTE for chronic infectious diseases.

Abstract: The Danish dairy industry initiated a voluntary, producer-paid control programme on Mycobacterium avium subsp. paratuberculosis (MAP) in 2006. Approximately 28% of the dairy herds, including 40% of the cows, are part of the programme. Farmers' reasons for participation are of great importance for the success of voluntary disease control programmes. This study was carried out to determine these reasons. Questionnaires with seven close-ended questions were mailed to the 1177 voluntary participants in 2008. A total of 1013 (86%) responded, and multiple reasons could be given by each farmer. The distribution of reasons for participation in the programme was: (1) control to increase animal health (91%); (2) certification "free of MAP infection" within 4-10 years (87%); (3) control to avoid production losses associated with MAP infections (86%); (4) control of MAP infections to increase consumer safety (64%); (5) certification for sale of livestock (58%); (6) control following production losses (48%); and (7) certification "free of MAP infection" within 1-3 years (31%). It was not possible to rank the reasons for participation since weighting of responses was not included. Therefore, the relative importance of the reasons given could not be determined. Based on the farmers' responses, control of MAP infections to increase animal health and avoid production losses were frequent reasons for participating in the Danish paratuberculosis control programme. The results also indicate that implementation of a certification programme would be desired by farmers. Animal health advisors should consider the diversity of participation reasons and address the specific reasons of individual farmers when establishing a strategy for control of MAP infections in a herd.

Abstract: Paratuberculosis is a chronic granulomatous infection caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects multiple ruminant species causing important economic losses. Therefore, control programmes at herd and regional levels have been established worldwide and prevalence estimates are needed for their implementation. Although different herd-level prevalence estimations for paratuberculosis have been reported in Europe, very few studies provided comparable and interpretable values, due to poor study designs and lack of knowledge about the accuracy of the diagnostic tests used. To overcome these problems we applied a latent class analysis to the results of two prevalence studies carried out in two neighbouring Northern Italian regions (Lombardy and Veneto) that account for over 50% of the Italian dairy cattle population. Serum samples from a randomly selected number of farms in the two regions were analyzed by different ELISA tests. The herd-level Apparent Prevalences (AP) were 48% (190/391) for Lombardy and 65% (272/419) for Veneto. Median within-herd APs were 2.6% and 4.0% for Lombardy and Veneto, respectively. Posterior estimates for the herd-level True Prevalences (TP) based on a Bayesian model were very similar between the two regions (70% for Lombardy and 71% for Veneto) and close to previous estimates of infected herds in Europe. The two 95% credibility intervals overlap each other, virtually showing only one distribution of the herd-level true prevalence for both regions. On the contrary, estimates of the within-herd TP distributions differed between the two regions (mean values: 6.7% for Lombardy and 14.3% for Veneto), possibly due to the different age distribution within the herds from the two regions.

Abstract: A Bayesian methodology was developed based on a latent change-point model to evaluate the performance of milk ELISA and fecal culture tests for longitudinal Johne's disease diagnostic data. The situation of no perfect reference test was considered; that is, no "gold standard." A change-point process with a Weibull survival hazard function was used to model the progression of the hidden disease status. The model adjusted for the fixed effects of covariate variables and random effects of subject on the diagnostic testing procedure. Markov chain Monte Carlo methods were used to compute the posterior estimates of the model parameters that provide the basis for inference concerning the accuracy of the diagnostic procedure. Based on the Bayesian approach, the posterior probability distribution of the change-point onset time can be obtained and used as a criterion for infection diagnosis. An application is presented to an analysis of ELISA and fecal culture test outcomes in the diagnostic testing of paratuberculosis (Johne's disease) for a Danish longitudinal study from January 2000 to March 2003. The posterior probability criterion based on the Bayesian model with 4 repeated observations has an area under the receiver operating characteristic curve (AUC) of 0.984, and is superior to the raw ELISA (AUC=0.911) and fecal culture (sensitivity=0.358, specificity=0.980) tests for Johne's disease diagnosis.

Abstract: Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in beef has been reported as a public health concern because asymptomatically infected cattle may contain MAP in tissues that are used for human consumption. Associations between MAP carcasses contamination and animal characteristics such as age, breed, production type, and carcass classification were assessed. Cheek muscles from 501 carcasses were sampled cross-sectionally at a Danish abattoir and tested for presence of viable MAP and MAP DNA by bacterial culture and IS900 realtime PCR, respectively. Cheek muscle tissues from carcasses of two dairy cows were positive by culture whereas 4% of the animals were estimated with ≥10 CFU/gram muscle based on realtime PCR. Age was found to be associated with carcass contamination with MAP. The observed viable MAP prevalence in beef carcasses was low. However, detection of MAP and MAP DNA in muscle tissues suggested that bacteremia occurred in slaughtered cattle.

Abstract: Effect of time for culling cows infected with Mycobacterium avium ssp. paratuberculosis on prevalence and profitability was identified through simulations. Seven test-and-cull strategies with different culling criteria and no attempts to close infection routes were compared with strategies with (1) no control and (2) closure of infection routes and no culling. The effects on true prevalence and gross margin were evaluated in a herd with typical reproduction management (heat detection rate of 38%). This was repeated in a herd with poor reproduction management (heat detection rate of 28%), because poor reproduction leads to lack of replacement animals, which was hypothesized to affect the economic effects of culling. Effects of varying prices of milk, replacement heifers, and hourly wages were also evaluated. The simulated results predicted that immediate culling after the first positive antibody ELISA test would be the most effective culling strategy to reduce prevalence. However, closing transmission routes was even more effective in reducing the prevalence. In the first 3 to 6 yr, all test-and-cull strategies reduced gross margin by US$5 to 55/stall per year. These losses were fully compensated by increased gross margin in yr 6 to 19. In the short run (7 yr with typical reproduction and 10 yr with poor reproduction), it was most profitable to cull test-positive cows when their milk yield decreased below 85% of that expected according to their parity and lactation stage, especially in herds with poor reproduction management. However, this strategy only stabilized the prevalence and did not reduce it. In the long term (>7 yr from implementation of a strategy), it was most profitable to cull cows immediately or as soon as possible after testing positive the first time. Varying milk prices did not affect the ranking between the different culling strategies. Increased market price (20%) of replacement heifers made all culling strategies less profitable and made culling based on a milk yield criterion the most profitable culling strategy for a longer period (11 to 13 yr). A 20% reduction in heifer price made immediate culling after a positive test the most profitable strategy overall in herds with typical reproduction, and after 9 yr in herd with poor reproduction. To conclude, the ideal culling strategy depends on the aim of intervention, the time horizon, and the reproductive capabilities combined with prices of replacement animals.

Abstract: Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assays use purified protein derivative of MAP (PPDj) which are complex antigen mixtures with low specificity. The objectives of this study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The study included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was an animal with ≥2 positive tests for ≥4 antigens, resulting in 13 cases and 13 non-cases. IFN-γ levels of cases were higher compared to IFN-γ levels of non-cases (P<0.05). The results of the IFN-γ assay using PPDj did not correlate well with the results using the novel antigens. PPDj produced elevated IFN-γ responses of samples from both the non-infected and the MAP infected herd, indicating unspecific IFN-γ responses and showed low consistency. Three latency proteins, LATP-1, LATP-2 and LATP-3 gave positive IFN-γ tests that correlated very well with the case definition suggesting high immunogenicity. Three tested antigens, LATP-2, MAP-1 and MAP-2 have no homologue in the M. avium subsp. avium or M. bovis genome and could be promising diagnostic antigens, especially LATP-2 correlated highly with the case definition.

Abstract: An in vivo experiment was conducted to evaluate the effects of tetracycline and zinc on pig colonization and transmission of methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 398. Eight piglets naturally colonized with MRSA ST398 and 8 MRSA-negative piglets of the same age and breed were assigned to three groups treated with tetracycline and zinc (Group 1), zinc (Group 2) or tetracycline alone (Group 3) and one non-treated group (Group 4), each containing two MRSA-positive and two MRSA-negative animals. Two additional non-treated control groups composed of only MRSA-positive (Group 5) and MRSA-negative (Group 6) animals were used to check for stability of MRSA carriage status. Nasal swabs and environmental wipes were collected on Days 0, 7, 14, and 21, and the occurrence of MRSA in each sample was quantified by bacteriological counts on Brillianceâ„¢ MRSA agar. Significantly higher nasal MRSA counts were observed in the zinc-treated (p=0.015) and tetracycline-treated (p=0.008) animals compared to the non-treated animals. Environmental MRSA counts appeared to increase over time in Groups 1 and 2 but such an increase was not statistically significant. MRSA-negative animals housed with MRSA-positive animals became positive in all groups, whereas the carriage status of the animals in Groups 5 and 6 did not change. This study demonstrates that feed supplemented with tetracycline or zinc increases the numbers of MRSA ST398 in the nasal cavity of pigs. Transmission of MRSA from positive to negative animals housed within the same pen was not influenced by exposure to these agents.

Abstract: The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as "poor" in a review published in 2008 in Veterinary Microbiology.

Abstract: Survival analysis methods can be used to identify factors associated with the time to induction of infection. In the absence of a perfect test, detection of infection is generally delayed and depends on the duration of the latent infection period. We assess, via simulations, the impact of ignoring the delayed detection of infection on estimated survival times and propose a Bayesian Weibull regression model, which adjusts for the delayed detection of infection. The presence of non-differential detection delay seriously biased the baseline hazard and the shape of the hazard function. For differential detection delay, the associated regression coefficients were also biased. The extent of bias largely depended on the longevity of the delay. In all considered simulation scenarios our model led to corrected estimates. We utilized the proposed model in order to assess the age at natural infection with Mycobacterium avium subsp. paratuberculosis (MAP) in Danish dairy cattle from the analysis of available time to milk-seropositivity data that detected infection with delay. The proposed model captured the inverse relationship between the incidence rate of infection and that of seroconversion with time: susceptibility to infection decreases with time (shape parameter under the proposed model was rho=0.56<1), while older animals had a higher probability of sero-converting (rho=2.67>1, under standard Weibull regression). Cows infected earlier in their lives were more likely to subsequently shed detectable levels of MAP and, hence, be a liability to herd-mates. Our approach can be particularly useful in the case of chronic infections with a long latent infection period, which, if ignored, severely affects survival estimates.

Abstract: A voluntary, risk-based control program for paratuberculosis in dairy herds was started in 2006 in Denmark. The program does not include non-dairy herds, and the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in these herds is unknown. The objective of this study was to establish the prevalence of MAP infected adult non-dairy cattle in Denmark. Serum samples were collected between October, 2008 and January, 2009 from every 6th animal over 24 months of age, sent to slaughter to Danish slaughterhouses from non-dairy herds. The final sample included 2345 cattle of 13 different breeds, with the largest breed group being crossbreds (of unknown breeds) (30%) and three dairy breeds (Danish Holstein, Danish Jersey and Danish Red Cattle) comprising 27% of the samples. The serum samples were tested using a MAP specific antibody ELISA (IDScreen) and positives were defined as the sample-to-positive ratio greater than 0.60. Estimation of the breed-specific apparent prevalences, true prevalences (TP), and true prevalences with a random effect of breed was done in a Bayesian analysis. Information about test sensitivity and specificity were based on literature data and expert information. Regardless of the method of analysis, the estimated prevalences showed similar differences between breeds. The dairy breeds Danish Jersey, Danish Holstein and Danish Red Cattle were ranked highest (i.e. with highest prevalence) (TP medians: 13, 10, and 6, respectively). Combined, the dairy breeds had a significantly higher prevalence than the other breeds, median TP (dairy)=15.7% vs. median TP (non-dairy)=0.8%. For the individual non-dairy breeds, the median estimates were generally higher, illustrating the problems of ranking groups based on relatively small sample sizes.

Abstract: From May 15, 2005 to April 15, 2008, 1878 cases of neoplasms in dogs were reported to the web-based Danish Veterinary Cancer Registry. The proportions of malignant (38 per cent) and benign (45 per cent) tumours were similar. The most common malignant neoplasms were adenocarcinomas (21 per cent), mast cell tumours (19 per cent) and lymphomas (17 per cent). The benign neoplasms most commonly encountered were lipomas (24 per cent), adenomas (22 per cent) and histiocytomas (14 per cent). Skin (43 per cent) and the female reproductive system including mammary tissue (28 per cent) were the most common locations of neoplasia. There was a distinct breed predisposition for tumour development, with a high standard morbidity ratio (indicating a higher risk of cancer) for boxers and Bernese mountain dogs. A standard morbidity ratio below 1 was observed in German shepherd dogs and Danish/Swedish farm dogs, suggesting a lower risk of cancer in these breeds.

Abstract: Paratuberculosis is a chronic infection of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is possible to detect infection with paratuberculosis at different stages of disease by means of various diagnostic test strategies. The objective of the present study was to evaluate if early cell-mediated immunity could predict the antibody results of milk samples in cattle with different faecal culture (FC) status. A group of 975 cows from 18 Danish MAP infected dairy herds was studied during a 3-year period. Cell-mediated immunity was measured in blood samples from heifers by use of an IL-12 potentiated IFN-gamma protocol. Following calving, milk samples were collected and analysed for MAP specific antibodies by ELISA and faecal samples were cultured. The relationship between the variables IFN-gamma and FC and the outcome of ELISA was assessed using generalised additive models. The results of the study showed that a significant association exists between early IFN-gamma and later FC status with occurrence of antibodies. In addition, the early IFN-gamma and FC status affect the antibody ELISA result at different stages post calving. We observed that only some IFN-gamma positive animals developed a positive antibody response against MAP, which indicate that cell-mediated immune responses can control or eradicate MAP in many animals.

Abstract: Prevalence estimates are used by decision makers such as policy makers and risk assessors to make choices related to certain diseases and infections. Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic infection particularly resulting in economic losses among farmed ruminants. Therefore, this infection is of concern for many farming industry decision makers. As a result, multiple studies have been carried out to determine the within-herd and between-herd prevalence of MAP infections. The objective of the present study was to conduct a review and, if possible, compare animal and herd level prevalences of MAP among farmed animals in Europe. European data on prevalences of MAP in all farmed animal species were included from a review of literature. Information on target population and study design, tests used and apparent prevalences were recorded, and subsequently true prevalences were calculated when possible. A critical review of the included studies indicated that although a wide range of studies have been conducted, likely and comparable true prevalence estimates could rarely be calculated. Based on a few studies where the prevalences appeared to be plausible, it was concluded that prevalences of MAP would have to be guesstimates based on available data. The true prevalence among cattle appeared to be approximately 20% and was at least 3-5% in several countries. Between-herd prevalence guesstimates appeared to be >50%. No countries had published sufficient information to claim freedom from MAP or just a near-zero prevalence of MAP infections. No within-flock prevalence estimates were available for goats and sheep. The between-flock prevalence guesstimates were >20%, based only on estimates from Switzerland and Spain. Major critical issues were identified in the majority of the studies, primarily due to lack of knowledge of the test accuracy of the diagnostic test used, or due to studies where the study population did not reflect the target population. Because most of the reported studies did not yield prevalence estimates which were comparable to other studies, there is a continuing need for well-designed studies of the prevalence of MAP infections.

Abstract: Infection with Mycobacterium avium ssp. paratuberculosis (MAP) in dairy cattle often results in reduced milk production and premature culling. Some test-positive animals can live for years without being affected by infection, whereas others are test negative when they die from the infection. Our objective was to describe the deviation in milk production of cows with various MAP antibody profiles compared with their repeatedly test-negative herdmates in the same parity. Data were obtained from herds participating in the Danish control program on paratuberculosis, for which 4 annual MAP antibody ELISA of individual cows were performed per herd per year. A total of 136,489 ELISA results from 38,998 dairy cows in 64 herds were used along with 484,285 test-day records on energy-corrected milk (ECM) yield. Cows were divided into 6 antibody groups based on their repeated milk ELISA results: A0) repeated ELISA negative; A1) ELISA negative, but only once; A2) ELISA negative on the last 3 tests, but with 1 previous positive result; A3) ELISA negative on the last test, but with 1 or more previous positive results; A4) last sample was ELISA positive, but all previous were negative; and A5) at least the last 2 samples were ELISA positive. The expected test-day kilograms of ECM by herd and parity were estimated for cows in antibody group A0. Deviations from expected milk production were then assessed for cows in the other antibody groups relative to the time of the first test-positive ELISA result (D 0). Cows in groups A2, A3, and A5 produced approximately 0.5 kg of ECM/d more than cows in group A0 at 300 d before D 0. Cows in group A4 had a decline in milk production from d 300 before D 0, with daily milk production reduced by 5 kg of ECM at 200 d after D 0. Milk production of cows in group A5 was reduced by 2.5 kg of ECM at 300 d after D 0 compared with 300 d before D 0, whereas cows in groups A2 and A3 produced 0.5 kg of ECM more than cows in group A0. The conclusions of the study were that 1) increasing the number ELISA tests increases the predictive value of ELISA for inference on milk production losses, 2) a combination of ELISA with assessment of observed milk production may be a valuable tool for decisions on culling, and 3) the declines in milk production attributable to MAP occurred over a long time period, and may not be realized by the herd manager without more advanced management tools such as the model proposed here.

Abstract: Bovine renal lipofuscinosis (BRL) is an incidental finding in cattle at slaughter. Condemnation of the kidneys as unfit for human consumption was until recently considered the only implication of BRL. Recent studies have indicated a negative influence on the health of affected animals. The present study investigated the prevalence, genetics and effect of BRL on milk yield and weight at slaughter.

Abstract: The effect of infection with Mycobacterium avium ssp. paratuberculosis (MAP) on slaughter weight and slaughter value of dairy cows was evaluated. Two data sets were analyzed: 1) recordings from 1,031 cows from herds in a pilot study to control MAP infections, and 2) recordings from 36,455 cows from herds participating in the Danish MAP control program. The effect of stage of MAP infection on carcass weight and slaughter value was assessed by ANOVA. Infection stage was diagnosed by repeated milk antibody ELISA in both data sets. Furthermore, repeated fecal culture was recorded in data set 1 and occurrence of enteritis or enteric edema found at slaughter was recorded in data sets 1 and 2. Compared with presumably unaffected cows with at least 2 ELISA negative tests, slaughter weight and value were reduced by 10 and 17%, respectively, in cows with positive ELISA at slaughter. If the cow was also positive using fecal culture, slaughter weight and value were reduced up to 15 and 31%, respectively. The slaughter weight and value were reduced an additional 20 and 31%, respectively, for cases with recorded enteritis or edema. Thereby, summarized weight losses of up to 31% and slaughter value losses up to 48% occurred. Cows with negative fecal cultures had reduced slaughter results only if they were ELISA-positive in the last 2 tests. Losses of both slaughter weight and slaughter value caused by MAP were more severe than previously estimated. These losses could be predicted by repeated milk ELISA tests with or without confirmation with fecal culture.

Abstract: Critical, systematic reviews of available diagnostic test evaluations are a meticulous approach to synthesize evidence about a diagnostic test. However, often the review finds that data quality is poor due to deficiencies in design and reporting of the test evaluations and formal statistical comparisons are discouraged. Even when only simple summary measures are appropriate, the strong correlation between sensitivity and specificity and their dependence on differences in diagnostic threshold across studies, creates the need for tools to summarise properties of the diagnostic test under investigation. This study presents summary receiver operating characteristics (SROC) analysis as a means to synthesize information from diagnostic test evaluation studies. Using data from a review of diagnostic tests for ante mortem diagnosis of paratuberculosis as an illustration, SROC and hierarchical SROC (HSROC) analysis were used to estimate overall diagnostic accuracies of antibody ELISAs for bovine paratuberculosis while accounting for covariates: the target condition (infectious or infected) used in the test evaluation (one for the evaluation of Se and one for Sp); and the type of test (serum vs. milk). The methods gave comparable results (regarding the estimated diagnostic log odds ratio), considering the small sample size and the quality of data. The SROC analysis found a difference in the performance of tests when the target condition for evaluation of Se was infected rather than infectious, suggesting that ELISAs are not suitable for detecting infected cattle. However, the SROC model does not take differences in sample size between study units into account, whereas the HSROC allows for both between and within study variation. Considering the small sample size, more credibility should be given to the results of the HSROC. For both methods the area under the (H)SROC curve was calculated and results were comparable. The conclusion is that while the SROC is simpler and easier to implement, analyse and interpret, the HSROC does have properties encourage the extra effort involved in the analysis.

Abstract: Within-household transmission of Escherichia coli may contribute to the pathogenesis of urinary tract infection but understanding of transmission is limited by the lack of longitudinal data on individual shedding patterns. In this study, faecal E. coli was isolated over 6 months from 18 humans and 13 dogs in eight households. Typing 322 E. coli isolates by amplified fragment length polymorphism showed high overall diversity as indicated by the average diversity index (0.66). However, individual shedding patterns varied considerably: two persons carried a single resident E. coli clone throughout the study whereas distinct clones were isolated from other individuals on each sampling time. Nineteen clones were shared within six of the eight households and seven of these clones were shared between humans and dogs. The frequent sharing of clones belonging to phylotypes B2 (n=7) or D (n=4) supports the hypothesis that urovirulent E. coli are transmitted between household members, including dogs, or may be acquired by a common source such as food.

Abstract: The Danish Veterinary Cancer Registry (DVCR) was established in 2005 and prospectively collects information about neoplasms in Danish dogs and cats. The present study evaluated the agreement between veterinary practice records and electronic submissions made to the DVCR from May 2005 through June 2008. The variables compared were gender, breed, diagnosis, date of birth, death and diagnosis, localization and biological behaviour of the neoplasms. Agreement of 95% between DVCR and the original data were considered acceptable with regard to use of data in studies. Recorded proportions of agreement were: (1) breed: 97.4%; (2) diagnosis: 95.6%; (3) location: 95.6%; (4) biological behaviour: 93.0%; (5) gender: 92.5%; (6) date of death: 91.5%; (7) date of diagnosis: 80.1%; (8) date of birth: 76.7%. All variables except date of death, birth, and diagnosis met the required 95% agreement limit. Data on dates of birth, death and diagnosis were considered less suitable for further studies.

Abstract: Infections with Mycobacterium avium subsp. paratuberculosis (MAP) can be latent for years without affecting the animal, but the animal may become infectious or clinical at some point. Diagnosis of paratuberculosis can be a challenge primarily in latent stages of the infection, and different diagnosis interpretations are usually required by the variety of decision makers. The objective of this paper was to provide a critical review of reported accuracies of ELISA tests, interferon-gamma assays (IFN-gamma) and faecal culture (FC) techniques used for diagnosis of three defined target conditions: MAP infected, MAP infectious and MAP affected animals. For each animal species, target condition and diagnostic test-type, sensitivities (Se) and specificities (Sp) were summarised based on a systematic, critical review of information in literature databases. The diagnostic test information often varied substantially for tests of the same type and make, particularly ELISA, which was the most frequently reported test-type. Comparison of the various tests accuracies was generally not possible, but stratification of test-evaluations by target condition improved the interpretation of the test accuracies. Infectious and affected animals can often be detected, but Se for infected animals is generally low. A main conclusion of the review was that the quality of design, implementation and reporting of evaluations of tests for paratuberculosis is generally poor. Particularly, there is a need for better correspondence between the study population and target population, i.e. the subjects chosen for test evaluation should reflect the distribution of animals in the population where the test is intended to be used.

Abstract: Somatic cell counts (SCC) in bulk tank milk delivered for human consumption are one of the indicators of milk quality and are used for milk pricing. Consequently, milk from cows with high SCC is frequently used by farmers for feeding of calves to lower the SCC in bulk tank milk. Young calves are more susceptible to Mycobacterium avium ssp. paratuberculosis (MAP) and may acquire the infection early in life through ingestion of MAP-contaminated milk. The occurrence of MAP antibodies can be an indicator of MAP shedding. Because MAP can be shed in milk from infected cows, and antibodies to MAP can be an indicator of the infectious status, an association between antibodies to MAP and high SCC can result in high-SCC milk being at risk of containing MAP. Feeding milk containing high SCC to susceptible calves may result in MAP infections. Somatic cell counts and MAP antibodies in milk were measured repeatedly in 7,251 cows from 26 Danish dairy herds to investigate the association between the occurrence of MAP antibodies and high SCC. The results of robust regression showed a log-linear relationship between the age at first positive ELISA and the age at first high SCC sample (R2 = 0.51). Of the 1,733 cows positive for MAP antibodies and with high SCC, high SCC was detected prior to MAP antibodies in 46% of the cows. Still, in 40% of the cows, MAP antibodies were detected before a high SCC. Therefore, the findings do not point to a causal relationship between high SCC and antibodies to MAP, but suggest a strong association and highlight a potentially increased risk of MAP transmission when milk with high SCC is fed to calves.

Abstract: Due to their multiple antibiotic resistance properties, methicillin-resistant Staphylococcus aureus (MRSA) are a major public health problem. After the recently described emergence of MRSA in animals, the authors investigated the risk of nasal MRSA carriage among people with professional contact with animals.

Abstract: Paratuberculosis is a chronic infection affecting cattle and other ruminants. In the dairy industry, losses due to paratuberculosis can be substantial in infected herds and several countries have implemented national programmes based on herd-classification to manage the disease. The aim of this study was to develop a method to estimate the probability of low within-herd prevalence of paratuberculosis for Danish dairy herds. A stochastic simulation model was developed using the R programming environment. Features of this model included: use of age-specific estimates of test-sensitivity and specificity; use of a distribution of observed values (rather than a fixed, low value) for design prevalence; and estimates of the probability of low prevalence (PrLow) based on a specific number of test-positive animals, rather than for a result less than or equal to a specified cut-point number of reactors. Using this model, five herd-testing strategies were evaluated: (1) milk-ELISA on all lactating cows; (2) milk-ELISA on lactating cows<or=4 years old; (3) milk-ELISA on lactating cows>4 years old; (4) faecal culture on all lactating cows; and (5) milk-ELISA plus faecal culture in series on all lactating cows. The five testing strategies were evaluated using observed milk-ELISA results from 19 Danish dairy herds as well as for simulated results from the same herds assuming that they were uninfected. Whole-herd milk-ELISA was the preferred strategy, and considered the most cost-effective strategy of the five alternatives. The five strategies were all efficient in detecting infection, i.e. estimating a low PrLow in infected herds, however, PrLow estimates for milk-ELISA on age-cohorts were too low in simulated uninfected herds and the strategies involving faecal culture were too expensive to be of practical interest. For simulated uninfected herds, whole-herd milk-ELISA resulted in median PrLow values>0.9 for most herds, depending on herd size and age-structure. None of the strategies provided enough power to establish a high PrLow in smaller herds, or herds with a younger age-structure. Despite this, it appears as if the method is a useful approach for herd-classification for most herds in the Danish dairy industry.

Abstract: Diagnosis of infections with Mycobacterium avium subsp. paratuberculosis (MAP) is difficult due to a long incubation period and lack of tests which can accurately predict the future status of animals. Early detection of infectious animals is necessary to reduce transmission of MAP. The objective of this study was to determine the time from first detection of MAP-antibodies in milk ELISA to start of MAP shedding, for animals with various shedding patterns. An observational longitudinal study was carried out over 3 years. A total of 24,076 milk and 10,074 faecal samples were obtained from 1906 cows and tested using ELISA and FC, respectively. Cows were classified into 5 shedding groups based on their repeated FC: non-shedders (NS; n=1512 cows, 79.3% of total), transient (TS; n=36, 1.9%), intermittent (IS; n=137, 7.2%), low (LS; n=143, 7.5%), and high shedders (HS; n=78, 4.1%). Results showed that 5% of TS, 30% of IS, 60% of LS and 70% of HS were ELISA-positive at the date of first positive FC, and many HS (28%) and LS (14%) were positive >or=1 year prior to first detection of shedding. FC confirmed shedding within the first year after the positive ELISA in 10% of 328 cows with fluctuating ELISA compared with 35% of 445 cows with the last 2 or more ELISAs positive. To conclude, MAP-antibodies were generally detected prior to start of bacterial shedding, with difference between the various patterns of shedding, and a positive ELISA was useful for predicting that an animal would subsequently become infectious.

Abstract: Mycobacterium avium ssp. paratuberculosis (MAP) infections cause major losses to the dairy industry. Transmission of MAP occurs primarily via feces and in utero, but MAP can also be excreted in colostrum and milk. The objective of this study was to determine whether colostrum and milk fed to calves are important risk factors for infection with MAP. A questionnaire was sent to 1,050 farms participating in the Danish control program on paratuberculosis in early 2007. Details on practices regarding colostrum and milk feeding between 1999 and 2006 were obtained from 808 (77%) herds. Nine vaccinated herds were excluded. Information on MAP antibody-ELISA results, date of birth, and herd of birth of 93,994 animals was obtained from the Danish Cattle Database. A 2-level logistic regression model was fitted with a dichotomized ELISA response, with outcome, age, source of colostrum, and milk as fixed effects, and herd as a random effect. Animals fed colostrum from multiple cows had an odds ratio of 1.24 of being ELISA positive compared with animals fed colostrum from their own dam only. Calves suckling with foster cows had an odds ratio of 2.01 of being ELISA positive compared with calves fed milk replacer. Feeding bulk tank milk and pooled milk from cows with high somatic cell counts did not increase the risk of being ELISA positive. Overall, the results of the study suggested that source of milk was not of great importance for the transmission of MAP, but colostrum should be fed only from the dam of that calf.

Abstract: Long-term effects of paratuberculosis on within-herd prevalence and on-farm economy of implementing risk-based control strategies were compared with alternative strategies by using a herd-simulation model. Closing transmission routes is essential for effective control of paratuberculosis. However, many farmers lack the resources to carry out these procedures for all cows in the herd. When using risk-based control strategies 1) all cows are tested quarterly with a milk ELISA, 2) specific cows with a high risk of being infectious are identified, and 3) the farmer can focus only on these infectious animals to close infection routes. In this way the workload can be reduced, making these control strategies more feasible. This study evaluates potential long-term effects of the risk-based approach compared with non-risk-based strategies by simulations conducted with the herd-simulation model PTB-Simherd. Seven control strategies were simulated in herds with initial true herd prevalences of 5, 25, and 50%, respectively. The results predicted the risk-based control strategies to be very efficient and comparable to the best whole-herd strategies in reducing the within-herd prevalence of paratuberculosis with considerably less labor. If infection routes are closed efficiently, prevalence can be reduced to 10% of initial prevalence within 5 to 7 yr. Test-and-cull strategies without closing infection routes were found, by simulation, to be ineffective in reducing prevalence and were not cost-effective methods. The profitability of the various control strategies depends on hourly wages and time spent per cow/calving. Furthermore, simulations show that immediate culling of highly infectious cows is only necessary and cost-effective if infection routes from these cows are not efficiently closed. The risk-based control strategies are recommended in the Danish voluntary control program "Operation Paratuberculosis," which was initiated in February 2006 and now includes 1,220 dairy farmers in Denmark.

Abstract: A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.

Abstract: The aim of the investigation was to determine the genetic relationship of a phenotypically diverse strain collection of epidemiologically unrelated strains from the taxon 2 and 3 complex of Bisgaard isolated from different hosts. A total of 325 isolates belonging to the taxon 2 and 3 complex of Bisgaard was characterized phenotypically in 82 characters. The genetic relationship among a subset of 60 isolates was investigated by amplified fragment length polymorphism (AFLP). The isolates were selected aiming at including the broadest diversity with regards to phenotype and host spectrum. The results suggested a statistically clear association between AFLP clusters and the host species families Columbidae (pigeon, dove), Anatidae (duck, goose) and Psittacidae (parrot, parakeet, budgerigar), respectively. This association was further supported by results from previous whole cell protein profiling and DNA:DNA hybridization studies. In conclusion, it appears that distinct genetic lineages within the taxon 2 and 3 complex of Bisgaard have evolved specificity for host bird species of different families. The observed host specificity of taxon 2 and 3 organisms may be used in future diagnostics and studies elucidating aspects of pathogenicity and epidemiology associated with the different lineages and their respective hosts.

Abstract: Low sensitivity (Se) of diagnostic tools is often mentioned as a major problem in the control of paratuberculosis (PTB) and much effort is put into the improvement of these tests. The hypothetical perspectives of improving the Se of a milk-antibody ELISA (hereafter: milk-ELISA) used in test-&-cull strategies against PTB in dairy cattle were investigated by simulations. The current Se varies between 10 and 80%, increasing with increasing lactation stage, parity and infection stage. We simulated the effects on a dairy herd's production of improving this Se to 80% (independent of these factors) and assumed no concomitant decrease in specificity. By using a PTB model called PTB-Simherd, 12 scenarios were simulated to study three test-&-cull strategies in each of four herds with 200 dairy cows. To show the maximal effect of using test-&-cull with such an improved test we simulated three strategies: (1) no testing, (2) testing with milk-ELISA test with the current Se and culling of positive cows immediately and (3) testing with milk-ELISA test with a Se improved to 80% and culling positive cows immediately. The four herds were defined by a moderate (25%) or high (80%) initial true within-herd prevalence (including young stock), and a poor or good heat-detection success of 40 or 60%. We assumed that these factors influenced the effects of improving the Se of the milk-ELISA. Management both concerning calf management and in general was specified to represent a typical Danish herd. Using an improved milk-ELISA was predicted to reduce the prevalence of PTB more effectively than the current ELISA, and over 10 years bring the production of a herd with moderate initial prevalence up to a production level comparable to a non-infected herd (unlike if the current ELISA had been used). In a herd with high initial prevalence (80%) milk production was increased more by using the improved milk-ELISA, but after 10 years the replacement rate was still very high causing problems with having enough recruitment animals-especially in high-prevalence herds with poor reproductive performance. Economically important measurements in all four herds benefited from the improvement of the test over a 10-year period. However, in the first 3-5 years the improved test would be more expensive to use than the current test, due to increased replacement (reduced net annual revenue per cow euro15 on average) but after that, net annual revenue increased continuously; after 10 years it was euro70-90 higher, than if the current milk-ELISA was used. Also, the milk-ELISA test with its current Se seemed to be profitable already after 2 years in high-prevalence herds using a test-&-cull strategy based on the milk-ELISA alone.

Abstract: Paratuberculosis (PTB) causes severe economic losses to farmers and the infection has very complex effects (many indirect) on the production of a dairy herd. These indirect effects have not or only briefly been described by earlier PTB-simulation models, and therefore they were included in a new model called PTB-Simherd. Our aim was to develop the basis for a decision-support tool which can predict herd-specific production-related effects from introduction of different control strategies against PTB. The PTB-Simherd is a dynamic, stochastic, and mechanistic Monte-Carlo model simulating a dairy herd including young stock. Paratuberculosis and relevant control strategies against this infection were built into an existing herd simulation model. The model simulates epidemiological and production related consequences of PTB and control strategies against it in the herd. It also reflects indirect effects of PTB and control strategies through effects on replacements and herd demographics. Every animal in the herd is specified with biological parameters (including PTB state and test results) and it is updated in weekly time-steps. Management is specified at herd level with 353 parameters of which 78 are related to PTB. To demonstrate the basic characteristics of the model, scenarios with varying infection risks (sensitivity analyses) plus scenarios with seven different control strategies in two herds with good and poor reproduction were simulated for 10 years. Breaking of infection routes turned out to be the only strategy predicted to reduce the true prevalence of PTB in a herd. Supplementing this strategy with test-&-cull strategies had limited effect on prevalence and using test-&-cull alone just delayed the increase in prevalence. The effects of different PTB-control strategies on the production (especially sale/purchase of heifers, feed consumption and prevalences of other diseases) were predicted to be affected by other conditions like heat-detection success, replacement% and herd demographics--which were again affected by PTB infection of the herd. These links and indirect effects of control strategies thus seem important to include when modeling and predicting effects of PTB control in dairy herds.

Abstract: Transmission of Mycobacterium avium subsp. paratuberculosis (Map) to susceptible animals is primarily considered to occur via faeces and milk originating from infectious cows. However, studies of factors resulting in increased transmission of Map are difficult to perform due to a long and unpredictable incubation period and inaccurate diagnostic tests. A multi-level Bayesian mixture model has been shown to predict the infection status of an individual cow more precisely than traditional cut-off based methods used for interpretation of diagnostic test-information, thereby increasing the precision of the diagnostic information. The objective of our study was to assess management-related risk factors for within-herd transmission of Map. Management-related risk factors were recorded in 97 Danish dairy herds. Twenty-six months following that recording, the antibody status of all lactating cows (n=7,410) in the same herds was measured by the use of an ELISA. A multi-level Bayesian mixture model was used to assess the association between the probability of infection of individual cows and 41 herd-level management-related risk factors using univariable analyses. In this model, the continuous OD value was used to estimate the probability of infection, corrected for known animal covariates and laboratory factors. The statistical significance of the potential risk factors was assessed by calculating odds ratios and their 95% credibility posterior intervals. Four significant risk factors were identified: housing of cows in bed stalls compared to housing in tie stalls; low level of hygiene in the feeding area of calving areas; low amounts of straw in the bedding of the calving area; high animal density among young stock >12 months of age. Surprisingly, the hygiene level in the calving area was not found to affect the odds of infection.

Abstract: Diagnostic inference by use of assays such as ELISA is usually done by dichotomizing the optical density (OD)-values based on a predetermined cut-off. For paratuberculosis, a slowly developing infection in cattle and other ruminants, it is known that laboratory factors as well as animal specific covariates influence the OD-value, but while laboratory factors are adjusted for, the animal specific covariates are seldom utilized when establishing cut-offs. Furthermore, when dichotomizing an OD-value, information is lost. Considering the poor diagnostic performance of ELISAs for diagnosis of paratuberculosis, a framework for utilizing the continuous OD-values as well as known coavariates could be useful in addition to the traditional approaches, e.g. for estimating within-herd prevalences. The objective of this study was to develop a Bayesian mixture model with two components describing the continuous OD response of infected and non-infected cows, while adjusting for known covariates. Based on this model, four different within-herd prevalence indicators were considered: the mean prevalence in the herd; the age adjusted prevalence of the herd for better between-herd comparisons; the rank of the age adjusted prevalence to better compare across time; and a threshold-based prevalence to describe differences between herds. For comparison, the within-herd prevalence and associated rank using a traditional dichotomization approach based on a single cut-off for an OD corrected for laboratory variation was estimated in a Bayesian model with priors for sensitivity and specificity. The models were applied to the OD-values of a milk ELISA using samples from all lactating cows in 100 Danish dairy herds in three sampling rounds 13 months apart. The results of the comparison showed that including covariates in the mixture model reduced the uncertainty of the prevalence estimates compared to the cut-off based estimates. This allowed a more informative ranking of the herds where low ranking and high ranking herds were easier to identify.

Abstract: Paratuberculosis is a chronic infection, and animals are not equally affected by it. Therefore, diagnostic tests that are able to detect different stages of the infection are needed for objective decision making. A longitudinal study was carried out to describe the ability of 2 tests to predict 2 conditions in dairy cattle: "infection" and "infectious," exemplifying 2 different purposes of testing. "Infection" is the term of choice for certification and eradication purposes, and "infectious" is more relevant for control purposes. In the study period of 3 yr, repeated sampling of milk (n = 23,219) and feces (n = 8,832) was performed. A total of 1,985 Danish dairy cows provided material for the study. Milk samples were analyzed for antibodies using an ELISA, and fecal samples were analyzed for mycobacteria by culture. A reference test to correctly classify cattle antemortem does not exist; thus, "infection" and "infectious" were defined by repeated testing using one test as the condition to be detected by the other test. Fecal culture responses were evaluated against antibody status, and ELISA responses were evaluated against detected bacterial shedding. The results of this study indicate that the ability of both tests to detect "infection" increases almost linearly from 2 to 5 yr of age, whereas the ability of both tests to detect "infectious" is not affected by age. Purpose-specific tests are required to appropriately interpret and use test results for management of paratuberculosis, and relevant covariates, such as age, should be included when possible.

Abstract: Paratuberculosis (Johne's disease) is a significant animal health problem. Evaluation of diagnostic tests for Johne's disease has been difficult due to lack of a gold standard test. In recent years, there has been interest in receiver operating characteristic (ROC) curve estimation without any gold standard test. Typically, either Bayesian or maximum likelihood methods are proposed. Although these methods overcome the lack of a gold standard test in ROC curve estimation, little work has been done to incorporate covariates in the analysis. In this paper, we propose a method for estimation of ROC curves based on statistical models to adjust for covariate effects when the true disease states of test animals are unknown. The covariates may be correlated with the disease process or with the diagnostic testing procedure, or both. We propose a 2-part Bayesian model: first, a logistic regression model for disease prevalence is used to fit the covariates; second, a linear model is used to fit the covariates to the distribution of test scores. We used Markov chain Monte Carlo methods to compute the posterior estimates of the sensitivities and specificities that provide the groundwork for inference concerning the diagnostic procedure's accuracy. We applied the methodology to milk ELISA scores from several dairy-cow herds for the diagnostic testing of paratuberculosis. We found that both milk yield and its interaction with age had significant effects on the disease process whereas only milk yield was significant on the testing procedure.

Abstract: Paratuberculosis is a chronic infection of ruminants and other species caused by Mycobacterium avium ssp. paratuberculosis (Map). Establishing test strategies for paratuberculosis will require insight into the temporal aspects of certainty with a given test. In this study, the age at which cows tested positive by ELISA and fecal culture (FC) was investigated by use of time-to-event analyses. The effects of herd, parity, and shedding group were evaluated at the age of test-positive ELISA and FC, respectively. Finally, the test frequency was investigated for the probability of cows being tested ELISA-positive. Milk and fecal samples were collected repeatedly over a 3-yr period from 1,776 Danish dairy cows from 8 herds. The milk samples were tested for the presence of antibodies by using an ELISA, and an FC test was used for detection of Map. Repeated ELISA testing detected 98 and 95% of cows classified as high and low shedders, respectively, suggesting that most infected cows develop antibodies. Among the high shedders, 50% were positive before 4.3 yr of age (quartiles 1 to 3: 3.4 to 5.7 yr of age). Repeated FC detected only 72% of the cows that were ELISA-positive, and 50% of the ELISA-positive cows were detected by FC at 7.6 yr of age. The age with the highest probability of testing positive was determined as the interval with the steepest slope in the survival probability plots. The highest probability of testing positive by ELISA was from 2.5 to 4.5 yr of age. The highest probability of testing positive by FC was from 2.5 to 5.5 yr of age. For both ELISA and FC, testing positive was highest in the first 300 d in milk. For cows younger than 4 yr of age, monthly testing with ELISA, compared with testing every 2 yr, could increase the probability of detecting cows with antibodies by 19%. In older cows, there were no apparent differences in the probability of testing positive by monthly sampling compared with sampling every second year. Therefore, for older animals the effect of more frequent sampling would be for early detection rather than to obtain additional information. Cows shedding high numbers of Map will produce antibodies, although not necessarily concomitantly with the shedding. These antibodies can be detected by ELISA with a test strategy that is different for younger and older cows. We suggest testing younger cows more frequently than older cows and that testing should be done prior to 350 d in milk.

Abstract: This study describes experimental infections in 4-week-old chickens inoculated intravenously with approximately 10(8) colony-forming units Streptococcus gallinaceus strain CCUG 42692T (C13156) or Enterococcus hirae strain DSM 20160 (C17410). Birds were necropsied following death and obvious clinical signs of disease or were euthanized weekly after infection for up to 4 weeks. At necropsy, lesions included splenomegaly, hepatomegaly, valvular and/or mural endocarditis. Cardiac lesions included focal necrotizing myocarditis and/or yellow-white vegetative valvular endocarditis or greyish proliferations associated with the mitral valves in 35% (6/20) and 79% (19/24) of birds infected with S. gallinaceus and in 20% (4/20) and 55% (12/22) of birds infected with E. hirae via the brachial and jugular veins, respectively. S. gallinaceus was reisolated from heart valves in 45% (9/20) and 75% (18/24) and E. hirae in 35% (7/20) and 73% (16/22) after inoculation via brachial and jugular veins, respectively. Both challenge strains were also isolated from liver, spleen, bone marrow and hock joints. A significant difference between the infections with the two strains was seen only with reisolation of E. hirae from hock joints (P < 0.007). Significant differences were apparent between the two inoculation routes only with E. hirae, where infection via the jugular vein was associated with higher culture positive isolations from the heart (P = 0.029), bone marrow (P = 0.002) and hock joints (P < 0.001) compared with the brachial vein. Birds injected with sterile phosphate-buffered saline were negative for culture of the challenge strains and no lesions were observed in these controls. The results confirm that both S. gallinaceus and E. hirae can cause endocarditis in experimentally infected chickens.

Abstract: Histopathology of the dermo-epidermal junction in the lamellar region of front claws was examined in 6 dairy heifers given an alimentary oligofructose overload and compared with sections from a control group of 6 heifers. Four of the 6 heifers administered oligofructose developed clinical signs of acute laminitis before they were euthanized. Postmortem samples from front claws were processed for histology. Eleven histopathologic characteristics were selected from the existing literature and used in a blinded evaluation of sections. In total, 104 front claw samples, including 8 samples from 2 cows having spontaneously occurring acute laminitis, were evaluated histologically using hematoxylin and eosin as well as periodic acid-Schiff staining. The major morphological features associated with oligofructose-induced acute clinical laminitis were stretching of lamellae, dermal edema, hemorrhage, changes in basal cell morphology, presence of white blood cells in dermis, and signs of basement membrane detachment. Changes at the lamellar junction of claw tissue affected by oligofructose-induced clinical laminitis resembled tissue from the 2 cows suffering from spontaneous acute clinical laminitis, and generally were consistent with existing descriptions of laminitis histopathology. Important exceptions to existing descriptions in the literature were stretching of lamellae and basement membrane changes. Not previously described, we considered these early signs of acute laminitis. In conclusion, this study documents that oligofructose-induced clinical laminitis is associated with histopathological changes at the lamellar interface. A weakened dermo-epidermal junction is a possible intermediate stage in the pathophysiology of bovine sole ulceration at the typical site.

Abstract: We devised a general method for interpretation of multistage diseases using continuous-data diagnostic tests. As an example, we used paratuberculosis as a multistage infection with 2 stages of infection as well as a noninfected state. Using data from a Danish research project, a fecal culture testing scheme was linked to an indirect ELISA and adjusted for covariates (parity, age at first calving, and days in milk). We used the log-transformed optical densities in a Bayesian network to obtain the probabilities for each of the 3 infection stages for a given optical density (adjusted for covariates). The strength of this approach was that the uncertainty associated with a test was imposed directly on the individual test result rather than aggregated into the population-based measures of test properties (i.e., sensitivity and specificity).

Abstract: The purpose of the study was to compare the growth of Mycobacterium avium subsp. paratuberculosis (Map) and the degree of contamination on Herrold's egg yolk medium (HEYM) and modified Löwenstein-Jensen medium (LJ).

Abstract: To analyze how infection with Mycobacterium avium subsp. paratuberculosis (MAP) affects the shape of lactation curves, a three-level hierarchical test-day model was set up with fat-corrected test-day milk yield (FCTM) as response. Milk samples from 6955 cows in 108 Danish dairy herds were tested with ELISA to detect antibodies against MAP. Optical densities (ODs) recorded on a continuous scale were standardized according to parity and stage of lactation. In addition to standardized ODs (stOD), seven fixed covariates, quadratic terms and first-order interactions were included in the model. Cow and cow nested in herd were included as random effects. Cows of first, second and higher parities were analyzed separately. The lactation curves after peak yield were significantly less persistent in young infected cows, where an increase of one stOD unit was associated with a depression of the milk yield per day through day 305 of 3.7 kg FCTM in first parity and 2.7 kg FCTM in second parity. In second-parity cows, the lactation curve also was both depressed through the entire lactation and more steep after 60 days in milk (DIM). In third and older parities, a significant effect of the quadratic term of stOD indicated exponentially increased losses with increased ODs.

Abstract: The purpose of this study was to estimate the genetic variation and the heritability of the ability to establish an immune response by producing antibodies to Mycobacterium avium subsp. paratuberculosis. Antibody levels were determined using an ELISA and measuring optical density (OD) values from milk samples of 11,535 cows from 99 herds. The pedigree of the 11,535 cows and information about days in milk, parity, milk yield, and others were obtained from the Danish Cattle database. The statistical analyses were made using a bivariate mixed animal model. The bivariate model with daily milk yield and OD as dependent variables showed a significant heritability of the ability to produce Mycobacterium avium subsp. paratuberculosis antibodies of 0.102 (genetic variance=0.054) and a nonsignificant genetic correlation of -0.037 between daily milk yield and OD. When a sire model was used, the estimated heritability was 0.091. To evaluate whether intrauterine infection could be reflected in the OD, variation among 2715 dam-daughter or maternal sister groups was also estimated. Variation in this data was nonsignificant, possibly because only very few clinical cases or end-stage cows, i.e., heavy bacterial shedders may have been represented among the cows sampled. It does not appear that intrauterine transmission is of major importance in the transmission of paratuberculosis.

Abstract: During the hunting season of 2001-02, blood and spleen samples from 59 red deer (Cervus elaphus), 77 roe deer (Capreolus capreolus), four fallow deer (Dama dama), and five chamois (Rupicapra rupicapra) were collected from nine hunting districts (n = 133) and one deer farm (n = 12) in southern Austria. Sera were tested for antibodies against bovine viral diarrhea virus (BVDV) with an enzyme-linked immunosorbent assay (ELISA) and virus neutralization tests against three BVDVs and one border disease virus strain. Reverse transcriptase polymerase chain reaction was used for detection of pestivirus-specific RNA in spleen samples. Antibodies were detected in one serum sample when using ELISA and virus neutralization tests. Results of the virus neutralization tests of this sample provided strong evidence for the exposure to the BVDV-1 genotype. The spleen samples were negative for pestivirus-specific RNA.

Abstract: A stratified cross-sectional study consisting of four strata of biosecurity based on production system type, including organic/free-range layer, battery-cage layer, layer parent, broiler parent and broiler grandparent flocks, was performed to estimate the prevalence of haemolytic Gallibacterium spp. Thirty birds were sampled by tracheal and cloacal swabs in each flock. A flock was considered infected when just one bird tested positive. A total of 27 flocks was included in the study. All chickens from the broiler grandparent flocks sampled negative, whereas 28% of the broiler parents, 40% of the layer parents, 67% of the battery-cage layers and 96% of the organic/free-range chickens sampled positive. A total of 95.9% (standard deviation +/- 7.6%) of birds from infected flocks was colonized by haemolytic Gallibacterium species. A significantly higher number of tracheal swabs was positive compared with cloacal swabs. The probability of vertical transfer was investigated by sampling offspring from an infected as well as a non-infected parent flock. None of the samples were found positive. In conclusion, we showed that haemolytic Gallibacterium spp. were widely distributed within the Danish commercial chicken production systems. However, prevalence proportions were highly influenced by the production system and found to be significantly associated with the biosecurity level observed in the flocks. In general, flock infections resembled an 'all or none' type of colonization as practically all of the chickens in infected flocks sampled positive. There was no evidence of vertical transmission of Gallibacterium.

Abstract:

Abstract: The accuracy of three diagnostic tests for paratuberculosis was evaluated using maximum-likelihood estimation of sensitivity and specificity. We also explored the variety of estimates that can be obtained if the tests are to be used in populations of different composition with regard to infection and disease states. Two enzyme-linked immunosorbent assays (ELISAs) were evaluated separately with the faecal culture (FC). The study was carried out as a cross-sectional field study to cover all likely states of infection with Mycobacterium avium subsp. paratuberculosis.The three basic assumptions for the maximum-likelihood technique were evaluated to validate the results. Our accuracy estimates for the ELISAs were not very different from those previously published, but those for faecal culture differed if a different cut-off value was chosen for the ELISA. If faecal culture was used for screening in a Danish dairy region where the median ELISA reading was a measure of the general disease situation, the sensitivity of the faecal culture was 20-25%. If faecal culture was used as a confirmatory test on cows with a high ELISA reading (and thus high level of antibodies), the sensitivity of the faecal culture would be in the range 60-70%. These results emphasise the importance of the composition of a target population before selecting a specific diagnostic test for a given purpose. We concluded that faecal culture is useful for confirmation but not for screening purposes.

Abstract: The objective of this study was to examine transmission of paratuberculosis in dairy cattle attributable to the dam. Milk samples were collected from 8131 cows in 110 Danish dairy herds. The level of antibodies to Mycobacterium avium subspecies paratuberculosis was determined by use of an ELISA. Information on dam and sire was obtained from the Danish Cattle database. The following two data sets were analyzed: Data set A contained all cows < or = 400 days in milk (n = 7410); data set B contained 1056 dam-daughter pairs present simultaneously in herds at the day of sampling. Cows > 400 days in milk were excluded. Linear mixed models were used to obtain variance components for the effect of sire in data set A and the effect of sire and dam-daughter pairs in data set B. Models for both data sets A and B included information previously shown to confound antibody level and information of the relative prevalence of paratuberculosis in the herd. In data set A, the effect explained by sire was 1.9%, whereas it was 6.3% in data set B. The effect from dam-daughter pairs was 7.7%. Those effects were all significant. It was concluded that the parental contribution was significant, and both heritability of susceptibility and vertical transmission should be considered in any control programs on paratuberculosis in dairy cattle.

Abstract: A longitudinal study was performed to determine the course of the milk antibody response in cows presumably infected with Mycobacterium avium subsp. paratuberculosis. Milk samples were collected repeatedly (1 to 10 times) from all lactating cows in seven Danish dairy herds. A total of 4,289 observations from 812 cows was analyzed after exclusion of samples collected after 280 days in milk (DIM). The level of antibodies in the milk samples was assessed using an indirect ELISA. A piece-wise linear random coefficient regression model was specified. The model controlled for the effect of herd, breed, laboratory effects, and age at first calving to estimate parity-specific antibody responses in relation to DIM. Separate antibody profiles were estimated for fecal culture-positive and fecal culture-negative cows. The resulting population average models showed higher antibody levels for fecal culture-positive cows and higher antibody levels with increasing parity. On average, the antibody response was high at the beginning and end of lactation. However, evaluating the cows individually indicated that most cows actually had quite stable ELISA levels throughout lactation, with some cows having higher levels than others. Thus, two criteria seem applicable to assess whether a cow is infected: stability and ELISA level. The random coefficients for each cow were highly significant. Thus, the study suggests that all cows can be classified into one of the four categories by combining the cow-level ELISA characteristics "stability" and "level" as an aid in the diagnosis ofparatuberculosis and thereby substantially increasing the sensitivity of the ELISA.

Abstract: Two cross-sectional studies were carried out to determine the enzyme-linked immunosorbent assay (ELISA) response to Mycobacterium avium subsp. paratuberculosis (Map) by cow characteristics and stage of lactation. One of the studies (referred to as "milk-group") used milk samples from all lactating cows (n=7994) in 108 Danish dairy herds. The other study (referred to as "serum-group") used serum samples collected from all cows (n=5323) in a subset of 72 herds from the 108 herds. These samples were analysed using a similar ELISA for detection of antibodies. The results from the ELISAs were interpreted with two cut-off values as the optimal cut-off value is not known, and as several levels are recommended to be used in practice. The results showed that the probability of being ELISA-positive was two to three times lower for cows in parity 1 relative to cows in other parities using both milk and serum ELISA. At the beginning of the lactation the probability of being positive was highest in the milk ELISA. In the serum ELISA the odds of being positive was highest at the end of lactation. The findings are important in the interpretation of ELISA results at cow level with a subsequent tentative diagnosis and correction for parity and stage of lactation should be considered when providing a diagnosis of paratuberculosis. Some issues related to the pathogenesis are also discussed.

Abstract: Milk samples from 120 cows were tested up to 10 times in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycobacterium avium ssp. paratuberculosis. The purpose of the study was to estimate variance components of the assay attributable to laboratory factors using mixed model theory. Because of significant interaction between the between-run, between-day and between-plate variables, the ELISA-plate variable was nested in run-number and run-number was nested in day-number. The nested variable accounted for 68% of the laboratory variability (P < 0.001), whereas the intraplate variability accounted for only 0.04% of the laboratory variability (P > 0.9). Therefore, it was concluded that the intraplate variability could be ignored whereas the variability from the combined run-day-plate variable should be considered in any analyses based on the ELISA.

Abstract: Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests (z = 0.182).

Abstract: Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk-collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown.

Abstract: Free-living deer are suggested as a possible source of infection of cattle with bovine virus diarrhea (BVD) virus. To examine this hypothesis blood samples from 476 free-living deer were collected during two different periods and tested for BVD virus and antibody in Denmark. In 1995-96, 207 animals were tested. These included 149 roe deer (Capreolus capreolus), 29 fallow deer (Dama dama), 20 red deer (Cervus elaphus) and one sika deer (Cervus sika). For the remaining eight animals no species information was available. In 1998-99, 269 animals were tested including 212 roe deer and 57 red deer. The animals were selected from areas with a relatively high prevalence of cattle herds with a BVD persistent infection status in 1997 and 1998. All 207 samples from 1995-96 were found antibody-negative except two samples from red deer. Only 158 of the 207 samples were tested for virus and were all found negative. Of the 269 samples from 1998-99 all but one were antibody negative. The positive sample was from a red deer. All samples were virus-negative. It appears that BVD infection does not occur in roe deer in Denmark. The presence of antibody in a few red deer from various districts in Jutland probably results from cattle to deer transmission, rather than spread among deer. Hence, the possibility of free-living deer as a source of infection for cattle in Denmark seems to be remote.

Abstract: In a cross-sectional study on milk samples from 1155 cows from 22 Danish dairy herds, selected risk factors for paratuberculosis were identified. The diagnostic procedure used was an indirect enzyme-linked immuno-sorbent assay (ELISA) to detect antibodies against Mycobacterium avium subsp. paratuberculosis. A sample was considered test-positive if it had a corrected optical density >/=0. 025 (test sensitivity 71.4% and test specificity 89.7%). Of the 1155 samples, 8.8% (102/1155) were test-positive, and 19 out of the 22 dairy herds had >/=1 test-positive cows. The significant risk factors in a multiple logistic regression analysis were: Jersey versus large breeds, high parity versus low parity, the first month after calving versus other months of lactation, and a large herd size compared to a small herd size. The highest probability (37-38%) of a positive test was observed among older cows (parity >4) and tested within the first month after calving (irrespective of breed). The lowest probability (2%) of a positive test-result was observed among first parity, large-breed cows tested before calving or later than one month after.