The rate and structure of cattle transfers between 206 Dutch cattle herds with a 'Mycobacterium avium subsp. paratuberculosis (Map)-free' status by November 2002, were analyzed over a 3-year period (November 1999-November 2002). Of the 206 'Map-free' herds, 184 were closed herds during the period studied. In total, 280 cattle had been introduced into 22 herds at an average rate of 0.33 animals per year per 100 cattle present in the 206 herds. Assuming a random herd-contact structure, the observed rate of cattle transfers between certified 'Map-free' herds was sufficiently low to relax the surveillance scheme to biennial herd examinations by pooled fecal culture of all cattle > or =2 years of age. The cattle transfers were not randomly distributed over the herds. Forty-four of the 280 cattle originated from 12 other 'Map-free' herds. The other 236 cattle did not originate from a 'Map-free' herd and were introduced into a herd before it obtained the 'Map-free' status. No cattle were introduced into any of the 'Map-free' herds from which cattle were transferred to other 'Map-free' herds. Thus, continued propagation of the infection by cattle transfers was impossible in the group of herds studied during the study period. Therefore the surveillance scheme may be further relaxed, and may be differentiated regarding the risk herds pose to other herds.

Dutch dairy herds closed for at least 3 years with no history of paratuberculosis were recruited for a study on herd-certification. One hundred dairy herds were tested for Mycobacterium paratuberculosis at 6-month intervals by pooled faecal culture (five individual animal samples per pool) with solid media. Ninety of the herds completed 9 herd tests and 10 herds dropped out of the study for reasons other than a paratuberculosis diagnosis. Of the 90 herds completing the full study, 61% eventually were found to be M. paratuberculosis-infected. The number of infected herds detected decreased with each round of testing. Assuming that all infected herds had been detected by the ninth herd test, the observed percentage of herds that were truly noninfected (P-free) after each round of testing was calculated. The observed P-free was compared to the predicted P-free based on a previously reported herd-certification model. The P-free predicted by the model was significantly different from the observed P-free. When a single assumption in the model was changed and a diagnostic sensitivity of 40–50% was selected, the predicted P-free closely approximated the observed P-free for the 90 Dutch dairy herds studied. The critical assumption that was changed for Version 2.0 of the model was within-herd infection prevalence for infected but test-negative herds after each round of serial testing. Model Version 1.0 had assumed a 50% decrease in within-herd prevalence but Version 2.0 assumed a stable within-herd prevalence. Culture of pooled faecal samples provides a high-sensitive, high-specific, low-cost test for herd-certification programs.

Culture of Mycobacterium avium subsp. paratuberculosis (Map) from fecal samples is a key element in many paratuberculosis control programs. Contamination of fecal cultures by other bacteria and fungi at different stages of incubation is a common problem and leads to extra costs for collection and culturing of new samples from the animals concerned. However, re-sampling animals following contamination of fecal samples detected at a late stage of incubation may be inefficient. Therefore, the results of re-sampling and culturing of feces from cattle after contamination of a previous individual culture were analyzed in a retrospective study.Re-testing of 2640 animals following contamination at four, eight, 12 or 16 weeks of incubation resulted in detection of 3.2%, 6.2%, 1.3% and 2.1% culture positive animals respectively.The obtained results were applied to our rates of positive and contaminated individual fecal cultures. Not re-testing animals following contamination after more than eight weeks of incubation would have resulted in an estimated reduction of the rate of re-testing of 25%, while the estimated relative sensitivity of the individual fecal culture was still 98.5% in comparison to re-testing all animals following contamination at: 16 weeks of incubation.It is concluded that re-testing cattle following contamination of a fecal culture is not cost efficient if contamination occurs after more than eight weeks of incubation.

At the beginning of the twentieth century, the dairy industry and cattle breeding organisations initiated a programme to control infectious diseases by means of a complex system of rewards and penalties. The history of programmes to control paratuberculosis in sheep, goats, and cattle in various countries is described. The vaccination of young animals (especially goats and sheep) seems to be an effective measure in the prevention of clinical paratuberculosis, although changes in management and hygiene practices are also important. A control programme for infectious cattle diseases has a number of phases (a life cycle) and different components. Two components are essential for success, namely, open and regular communication with farmers, veterinary practitioners, and other people involved, and a good registration and identification system for cattle, herds, and veterinary practitioners. The principles and different levels of control of paratuberculosis are discussed. The Dutch paratuberculosis programme is divided in two parts: an intensive programme based on test and cull and an extensive programme based on education. The intensive programme has ten herd status levels: 5 to 10 for non?suspect herds and 1 to 4 for infected herds or herds of unknown status. The higher the status, the greater the chance that a herd is free of paratuberculosis. An outline is given of the Dutch paratuberculosis programme. Diagnostic methods are summarised. Future developments regarding vaccination and control or acceptance of paratuberculosis are discussed.

Appropriate management measures to prevent new infections in infected herds are emphasized in the Dutch paratuberculosis control program with the aim to raise a new uninfected cow generation to replace the infected live stock. Alternative methods are pursued to answer to the demand of farmers wishing to eradicate paratuberculosis faster. One of the proposed alternatives is a program aiming to identify all infected cattle during a one year test period, using three different tests simultaneously. The testing schedule comprised: serological test by ELISA in all animals > 24 months, fecal Culture of all animals > 6 months, and a gamma interferon assay of all young stock > 6 months. At the same time farmers were advised to remove all test-positive animals promptly and to carry out all recommended preventive management measures. This approach has been tested in 15 dairy herds with a low Mycobacterium avium subsp. paratuberculosis (Map) -prevalence (< 10 % culture-positives). The herds ware tested twice during the first year and were followed after 2 and 4 years with repeated tests to evaluate the results of the program. After the first test, 3 herds were removed from the program because they did not sell culture-positive animals. After 4 years, 4 of the 12 herds were test-negative, although three of these herds had a test-positive cow in between. In 6 herds test-positive animals were detected in each herd test. One of the farmers with test-positive herds ceased farming, before the herd test at 4(th) year. It can be concluded that, with the strategy as applied in this trial, paratuberculosis may be eradicated in some herds in a one-year period. Although successful in a minority of herds, this program has the disadvantage of superfluous culling. Removal of fecal shedders, in combination with management measures, remains the most cost-effective way to eradicate paratuberculosis from dairy herds in a short time.

Paratuberculosis in cattle is a chronic debilitating infectious disease caused by Mycobacterium paratuberculosis. Control of paratuberculosis is based on tests that principally detect advanced stages of infections: faecal culture and serology. Tests measuring cell-mediated immunity (CMI) could improve control of paratuberculosis if able to diagnose mycobacterial infections earlier, before animals become infectious. A drawback of CMI tests for paratuberculosis has been a reported low specificity. This study re-examined CMI specificity and factors that may affect it. The specificities of the Johnin skin test and its in vitro equivalent, the gamma interferon (IFNγ) assay, were estimated in 35 uninfected dairy herds. In each herd a random sample of 20 young (6–24 months old) and 20 adult (≥24 months old) female dairy cattle were tested by both tests simultaneously. Skin test specificity was 93.5% using a skin thickness increase of ≥4 mm as the cut-off value. IFNγ assay specificity when interpreted using a newly developed algorithm was 93.6%. When interpreted according to two alternative algorithms provided by the IFNγ kit suppliers, the assay had specificities of 66.1 and 67.0%. If the skin test and IFNγ assay were used in parallel, and only animals positive on both tests were considered as M. paratuberculosis-infected, the specificity was 97.6%. Agreement between skin test and IFNγ assay on 1631 total animals was fair (κ=0.41). Antigen batch influenced the specificity of both the skin test, ranging from 92 to 95%, and the IFNγ assay, ranging from 71 to 99% among batches. Test specificity also varied among herds ranging from 58 to 100% for the skin test and 57 to 100% for the IFNγ assay. While factors affecting CMI test specificity and agreement need further evaluation, the high specificity and general agreement among these CMI tests, coupled with the excellent results obtained in the control of bovine tuberculosis using CMI tests, support their potential value in the early diagnosis and control of paratuberculosis.

Control of paratuberculosis in dairy herds is based on preventing the transmission of Mycobacterium avium subsp. paratuberculosis ( Mptb) from cows to calves by management measures, supported by removal of cows excreting these bacteria by the fecal route ( Mptb shedders). Fecal culture is the most accurate test for identifying Mptb shedders, but this technique is expensive and takes up to 16 weeks for results to be available. Serologic tests are inexpensive, rapid, and easy to perform. Of serologic tests, the complement fixation test (CFT) and absorbed enzyme-linked immunosorbent assay (ELISA) are the serologic tests used most frequently; the CFT is considered less accurate than the ELISA with respect to sensitivity and specificity. The commonly accepted absorbed ELISA is from the Australian Central Serum Laboratory. However, a European supplier has marketed a second ELISA that is supposed to be more sensitive in detecting Mptb shedders. These 2 absorbed ELISAs, designated ELISA-A and ELISA-B, and an in-house CFT were compared with data from 2 serum panels. The Mptb shedding panel consisted of sera from 198 culture-positive cows from 53 infected herds. The method used for culture of fecal samples was a modified Jørgensen method on individual samples. The Mptb shedder detection rate by the 3 serologic tests ranged from 29.8% to 39.4%. Detection rate for ELISA-A was lower than that for ELISA-B and CFT. For all 3 tests, detection rate was dependent on the level of Mptb shedding and the age of the animals. Detection rates increased as cattle age increased to 4 years. The specificity panel was initially composed of sera from 811 cows randomly selected from 41 herds without clinical paratuberculosis that were negative for Mptb based on whole-herd fecal culture. The modified Jørgensen method for culture was used on pooled fecal samples. Serologic test specificity ranged from 93.4% to 99.8%. The specificity of ELISA-A was higher than that of ELISA-B and CFT. Specificity of ELISA-B between herds was 75–100%. Specificity of CFT between herds was 62–100%. The low specificity of ELISA-B and CFT could not be explained by a higher sensitivity for Mptb-infected cows before onset of shedding, because in the 19 herds with 8 more subsequent negative whole-herd fecal cultures in the 4 years after sampling, specificity was not improved. The insufficient specificity of ELISA-B was not corrected sufficiently by heightening the cutoff value because Mptb shedder detection rate was lowered to 28.9%, equal to that of ELISA-A, and specificity only rose to 97%, much lower than that of ELISA-A. Taking into account the different test characteristics, serologic tests are a cost-effective alternative to fecal culture in high-prevalence herds. For certification programs, only ELISA-A is recommended because in a large number of nonsuspect herds specificity remained almost 100%.

OBJECTIVES:To determine whether vaccination with a killed vaccine prevents fecal shedding of Mycobacterium avium subsp paratuberculosis, to compare effectiveness of a culture and cull program in vaccinated and nonvaccinated herds, and to compare paratuberculosis-related preventive management in vaccinated and nonvaccinated herds.SAMPLE POPULATION:58 commercial Dutch dairy herds.DESIGN:Cross-sectional study (study A) in vaccinated (n = 25) and nonvaccinated (29) herds of dairy cows. Longitudinal study (study B) in vaccinated (n = 2) and nonvaccinated (2) herds of dairy cows.PROCEDURE:In study A, fecal samples were obtained from adult cows in herds with and without a history of vaccination with a killed vaccine. Management measures were evaluated. In study B, fecal samples were obtained 4 times at 6-month intervals from cows older than 6 months. Cows that had positive test results were removed from the herd directly after the outcome of the culture.RESULTS:In study A, differences were not detected among the 25 herds that were vaccinated; culture results were positive for M avium subsp paratuberculosis in 4.4% of herds. In 29 herds that had not been vaccinated, culture results were positive in 6.7%. In study B, the percentage of positive results on culture decreased from 10.9% and 5.7% to 3.5% and 0%, respectively in the 2 vaccinated herds. In the 2 nonvaccinated herds, percentages decreased from 6.1% and 16.5% to 0% and 2.3%, respectively. Management practices were different between herds that were vaccinated and herds that were not; owners of herds that were not vaccinated followed more preventive management procedures and practiced less feeding of raw milk to calves.CONCLUSIONS AND CLINICAL RELEVANCE:Vaccination of calves with a killed vaccine does not prevent transmission of M avium subsp paratuberculosis; therefore, hygienic practices remain essential in herd management.

Fecal samples from 733 cows in 11 dairy herds with a low prevalence of paratuberculosis were cultured for the presence of Mycobacterium avium subsp. paratuberculosis both individually and after combining (pooling) in groups of 5. The culture procedure was the modified Jorgensen method, which uses NaOH and oxalic acid for decontamination and modified Lowenstein-Jensen agar slants for cultivation. Pooling was performed by mixing fecal samples from 5 animals ordered by age, herein referred to as strategic pooling. Culture of individual fecal samples detected M. a. paratuberculosis infections in 43 of the 733 cows and 7 of 11 infected herds (herd sensitivity = 64%). Culture of pooled fecal samples detected M. a. paratuberculosis in 28 of 151 pooled samples representing 8 of the infected 11 herds (herd sensitivity = 73%). Feces of the 43 culture-positive cows was included in 32 pools: of these 32 pools, 26 were culture positive and 6 were culture negative. In addition to the 26 positive pools containing feces from cows that were found culture positive on individual fecal samples, another 2 pools were culture positive, although comprised of feces from cows with negative results after culture of individual fecal samples. From the total of 45 infected cows that were found (43 by individual fecal culture and an additional 2 by pooled fecal culture), individual fecal culture detected 43 of these 45 (96%), while pooled fecal culture detected 39 (87%). Culture of strategically pooled fecal samples using the modified Jorgensen method was equivalent in herd sensitivity to the culture of individual fecal samples and is significantly less expensive.

Paratuberculosis is an infectious disease that is not easily amenable to classical control methods such as treatment and vaccination. Experimental animal models suggest that there could be genetic factors responsible for susceptibility or resistance to infection with the causative agent, Mycobacterium avium subsp. paratuberculosis. The aim of this study was to estimate genetic variation in susceptibility to paratuberculosis in Dutch dairy cattle. Data collected during a vaccination trial, conducted from 1984 to 1994, was used. A total of 3020 cows, with complete pedigree records and infection status at slaughter, were available for analysis. A standard polygenic statistical probit model was used to estimate heritabilities. The estimated heritability of susceptibility to M. avium. subsp. paratuberculosis infection was 0.06 for the overall population. In the subpopulation of vaccinated animals the estimated heritability was 0.09. Other calculations based on the model used in this study argue against a prominent role for vertical transmission.Because the establishment of genetic variation is one of the first steps towards the exploration of the possible use of selection for genetic improvement, the present study provides evidence for the presence of genetic variation in the susceptibility of cattle to paratuberculosis. Because the economic impact of the disease is substantial, the development and application of genetic tools, along with other control methods, could be instrumental in the eradication of paratuberculosis.

Paratuberculosis is an infectious disease that is not easily amenable to classical control methods such as treatment and vaccination. Animal models suggest that genetic factors influence the susceptibility or resistance to the causative agent, Mycobacterium avium subsp. paratuberculosis. The aim of this study was to estimate the heritability of susceptibility to paratuberculosis in Dutch dairy cattle. Data collected during a vaccination trial, conducted from 1984 to 1994, were used. A total of 3,020 cows with complete pedigree records were available for analysis. A mixed model probit analysis was used to estimate heritabilities. The estimated heritability of susceptibility to M, a. paratuberculosis infection was 0.06 for the overall population. In the subpopulation of vaccinated animals the estimated heritability was 0.09. Estimation of model effects when genetic effects were included showed that higher farm prevalence was associated with a higher risk for an individual animal to become infected (p=0.001), but argued against a prominent quantitative role for vertical transmission of the infection in a vaccinated population. In conclusion the results of this study indicate that genetic effects influence the susceptibility of animals to infection with M. a. paratuberculosis. The genetic effects were most prominent in the vaccinated population and this may have implications for future vaccine development studies. Furthermore, the results could be a first step in the search for susceptibility related genetic markers that may aid in the eradication of bovine paratuberculosis.

A modified procedure was used for culture of Mycobacterium paratuberculosis (Mptb) from bovine feces. Bovine fecal samples were decontaminated with NaOH, exposed to a mixture of oxalic acid and malachite green, incubated in a mixture of neomycin and amphotericin B. Decontaminated specimens were inoculated onto modified Löwenstein-Jensen medium. Specimens processed by high-speed centrifugation showed growth earlier than specimens prepared by low-speed centrifugation. However, the overall number of positive cultures at 16 weeks was not different for the 2 methods. When infected dairy herds were sampled 4 times at 6-month intervals and culture-positive cows were culled, the prevalence of infected cattle declined over time. After selective culling, the cattle left in the herds shed low numbers of Mptb, which explains why it took longer for cultures to become positive. No heifers younger than 11 months were culture positive, but heifers 13–14 months of age were more frequently culture positive than were heifers of any other age. The 16-week culture period is needed with this method to detect cattle shedding low numbers of Mptb. High-speed centrifugation of samples does not increase the efficiency of identification of animals shedding Mptb.

A modified procedure(4) for culture of M. a. paratuberculosis (Mptb) from bovine feces based on the method of Jorgensen(3) for fecal samples from individual cows was used for the culture of pooled fecal samples. Each pooled sample contained fecal material from five cows. In eleven dairy herds fecal samples from 733 cows were cultured both individually and as part of a pool. Samples from cattle of the same age were pooled (strategic sampling). Individual cultures demonstrated the presence of Mptb in six of these eleven herds and 43 of the 733 cows. The pool ed fecal cultures detected Mptb in seven out of eleven herds and in 28 out of 151 pools. Six culture-positive animals were not detected in pooled fecal cultures. On the other hand, two pools in which no positive animals were detected by individual culture were culture-positive. If compared with individual culture as the "gold standard", sensitivity of the pooled fecal culture method was 86% and specificity was 96%. The average number of colonies in positive cultures of pooled fecal samples was 22 and the total number of colonies in the five corresponding individual cultures was 31. The high sensitivity of this pooled fecal culture method was unexpected because experiments with pooled fecal culture based on another culture method reported a sensitivity of only 38%, compared with individual fecal cultures(8). The decrease in total number of colonies after pooling the samples was lower than expected mathematically this decrease was expected to be 80% instead of 29% in pools containing only one positive sample. We conclude: (I) the effect of pooling of fecal samples on diagnostic sensitivity cannot be estimated without taking into account the method of culture and the method of pooling; (2) culture of strategically pooled fecal samples with the modified Jorgensen method proved to be a good alternative for culture of individual fecal samples to detect the presence of Mptb in dairy herds and leads to a significant reduction of costs; and (3) the difference between the sensitivity of the described culture method and the method reported earlier for pooled fecal samples should be investigated further before recommendations about sample pooling as a method for assessing herd prevalence can be given.

In The Netherlands attention is not only focused on the reduction of the economic damage due to clinical paratuberculosis but also on the eradication of the disease from the herds. It has been suggested that 4 to 6 years of vaccination can decrease or even eliminate M. a. paratuberculosis (Mptb) infection in a herd. The purposes of the present study were: (1) to determine if vaccination against paratuberculosis for at least 10 years prevents fecal shedding of Mptb; (2) to compare the effect of a culture-and-cull eradication program on fecal shedding of Mptb between vaccinated and not vaccinated herds. The total number of fecal samples collected from twelve herds with a history of more than ten years of vaccination was 1,129. Of these samples, 40 (3.5%) were Mptb-positive. In three herds no Mptb-positive fecal samples were found. In the nine culture-positive herds the prevalence varied from 1% to 29%. After a vaccination program of 12 years for 5 herds the prevalence was 4.9%. In 34 herds with clinical paratuberculosis that had never vaccinated against paratuberculosis a total number of 2,846 fecal samples were cultured in the same period. Of these samples 169 were Mptb-positive (5.9%). In eight herds no positive fecal sample was cultured. There was no significant difference between the vaccinated and the non-vaccinated herds in the rate of decline of culture-positives in the two year culture-and-cull eradication program. In both groups the rate of fecal shedders declined over time with a sharp decline after the onset of the culture-and-cull program followed by a minor decline in the following period. The two year culture-and-cull program had the same effect on vaccinated and unvaccinated herds. This study demonstrates that a 10 year vaccination program is no guarantee that shedding of Mptb is prevented. Therefore a vaccination program has to be combined with or replaced by a culture-and-cull program.

In 1995 the objective was formulated to stop the spread of paratuberculosis in dairy herds in the three northern provinces of The Netherlands. In support of this objective was the formation of a group of dairy herds believed to be free of M. a. paratuberculosis infection. These herds were to serve as a source of uninfected replacement animals for others. Selection of the herds was based on two criteria: (1) absence of clinical paratuberculosis in the last five years, declared by both herdsman and veterinarian and (2) closed herd management for the last three years. Strategically pooled fecal culture of all adult cows (>24 months) was performed at six months intervals. One hundred and thirteen herds were selected. Herd size varied from 20 to 280 adult dairy cows. The first assessment was a review of the laboratory records of the Dutch Animal Health Service. Thirteen herds were suspected of M. a. paratuberculosis infection because of recorded positive test results from individual blood or fecal samples in the preceding 5 years. This was in contradiction with the farmer and veterinarian declaration. The hundred remaining herds entered the culture program. Four subsequent cultures six months apart detected M. a. paratuberculosis-positive pooled fecal samples in 13, 8, 10 and 9 herds (40 in total) from the one hundred herds in the program. In addition to the 13 herds that were excluded from the program based on registered laboratory diagnostic test records, and the 40 herds that were positive at pooled fecal herd culture, another three herds had to be removed from the program because one herd ceased operating and two introduced animals from herds with a unknown paratuberculosis status. Therefore, the number of participating herds dropped from an initial 1 13 participants to 57 within two years. Four additional herds were eliminated, primarily due to contamination of fecal cultures which reduced the reliability of the negative culture results in three of these herds. Our conclusions were: (1) The absence of clinical signs, as declared by the herdsman and confirmed by the practicing veterinarian, is no guarantee about the absence of Mptb infections in the herd. (2) Culture of pooled fecal samples was able to detect Mptb infections in a large proportion of herds believed to be free of M. a. paratuberculosis infection. (3) Repeated cultures of strategically pooled fecal samples in combination with closed herd management were needed to exclude Mptb infections in presumably uninfected dairy herds. The optimal number of herd cultures required to provide a sufficient level of confidence that the herd is indeed free of the infection is still an open question.