Cattle transfers between herds under paratuberculosis surveillance in The Netherlands are not random.

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.

Certification of herds as free of Mycobacterium paratuberculosis infection: actual pooled faecal results versus certification model predictions.

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.

Infection of C57BL/10ScCr and C57BL/10ScNCr mice with Leishmania major reveals a role for Toll-like receptor 4 in the control of parasite replication.

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR4(0/0)) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rbeta2(0/0)] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

Specificity of two tests for the early diagnosis of bovine paratuberculosis based on cell-mediated immunity: the Johnin skin test and the gamma interferon assay.

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 (IFNgamma) assay, were estimated in 35 uninfected dairy herds. In each herd a random sample of 20 young (6-24 months old) and 20 adult (> or =24 months old) female dairy cattle were tested by both tests simultaneously. Skin test specificity was 93.5% using a skin thickness increase of > or =4mm as the cut-off value. IFNgamma assay specificity when interpreted using a newly developed algorithm was 93.6%. When interpreted according to two alternative algorithms provided by the IFNgamma kit suppliers, the assay had specificities of 66.1 and 67.0%. If the skin test and IFNgamma 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 IFNgamma assay on 1631 total animals was fair (kappa=0.41). Antigen batch influenced the specificity of both the skin test, ranging from 92 to 95%, and the IFNgamma 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 IFNgamma 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.

Paratuberculosis: eradication, control and diagnostic methods.

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.

Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies 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%.

The role of the liver in the response to LPS: experimental and clinical findings.

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-alpha induces hepatocyte apoptosis and lethal effects are due to secreted TNF-alpha and not to cell-associated TNF-alpha. An exaggerated production of TNF-alpha has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-gamma and IFN-alpha/beta are involved in the macrophage-induced release of TNF-alpha. The prominent role of LPS and TNF-alpha in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-alpha/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-alpha and interleukin (IL)-1beta levels than responders. Moreover, in responders, an equilibrium between IFN-gamma and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.

Use of long-term vaccination with a killed vaccine to prevent fecal shedding of Mycobacterium avium subsp paratuberculosis in dairy herds.

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.

Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Culture of strategically pooled bovine fecal samples as a method to screen herds for paratuberculosis.

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.

Genetic variation of susceptibility to Mycobacterium avium subsp. paratuberculosis infection in dairy cattle.

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.

Factors influencing the isolation of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples.

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.

Cost-benefit analysis of vaccination against paratuberculosis in dairy cattle.

Paratuberculosis is an infectious and incurable disease which causes considerable economic losses in dairy cattle, due mainly to premature disposal and losses of milk production. In 1984 the Animal Health Service North-Netherlands started a vaccination trial in which young calves were vaccinated once, to test whether vaccination reduced the production losses and whether the overall costs of vaccination were outweighed by the benefits. Vaccination against paratuberculosis reduced the number of clinically infected animals by almost 90 per cent. It also reduced the numbers of subclinically infected animals and animals with a positive histological and/or bacteriological test result. Although vaccination did not prevent losses in milk production, it reduced the infection pressure and the clinical signs of the disease. Partial budgeting showed that vaccination against paratuberculosis was highly profitable. The costs of vaccination were US$15 per cow and the benefits (total returns minus costs) were US$142 per cow.

[Improvement of fertility status in dairy farms by using the milk progesterone test]. / Verbetering van de fertiliteitsstatus op melkveebedrijven door toepassing van de melk-progesterontest.

On nine farms comprising a total number of 370 cows, studies were done for two years to determine whether the reproductive status (RS) could be improved by systematic performance of the milk progesterone test. Milk samples were taken on the day of insemination (day 0), day 8 and day 21 to verify oestrus detection, the observation of ovarian dysfunction and the early diagnosis of (non-)pregnancy. The RS on these nine farms increased from thirty-six to fifty-four, which was mainly due to the reduction of the number of inseminations required for each conception (from 2.1 to 1.7). On ten farms selected by a similar method, on which reproduction was supervised by the veterinary practitioner, the RS increased from forty-seven to fifty-five, which increase was particularly due to inseminations carried out sooner after calving. The RS of ten herds which served as controls, increased from forty-one to forty-seven. An analysis of costs showed that the costs using the progesterone test were estimated to be equivalent to the expense of supervising a reproductive dairy herd health programme. A diagnosis negative for pregnancy based on three milk samples tested was correct in 98 per cent of the cases. A diagnosis positive for pregnancy was correct in 90 per cent of the cases. When a death rate of 8 per cent of the embryos within three weeks after conception is taken into account, the proportion will also be 98 per cent. Summarizing, it is concluded that systematic performance of the milk progesterone test provides an attractive method of improving reproduction on those farms which do not take part in veterinary supervision of reproductive dairy herd health programmes.

[Survey of the incidence of Dictyocaulus arnfieldi infections in donkeys in the Netherlands (author's transl)]. / Een onderzoek naar het voorkomen van Dictyocaulus arnfieldi infecties bij ezels in Nederland.

The results of an investigation into the incidence of Dictyocaulus arnfieldi in donkeys in the Netherlands show that 51 per cent excrete larvae in the faeces. This infection was not detected in animals under five months. The proportion of infected animals did not increase with age. However, the level of excretion was found to increase with age. There was no correlation between the excretion of lungworm larvae and pulmonary symptoms.

[Use of chlormadinone acetate (CAP) in bovine reproduction (author's transl)]. / Het gebruik van chlormadinonacetaat (CAP) bij de voortplanting van het rund.

The results obtained with chlormadinone acetate (CAP) in the treatment of dairy cattle with inactive ovaries (anoestrus) and during the puerperium are reported. CAP was administered to eighteen animals with inactive ovaries for fourteen days. Oestrus occurred in fourteen animals within from one to four days after treatment had been completed. Insemination during this oestrus resulted in pregnancy in five animals. CAP was also administered to sixty animals on three farms for fourteen days during the puerperium, treatment starting on the twelfth day post partum. This was done for the purpose of improving subsequent fertility. However, as regards the interval between parturition and first insemination, the conception rate following the first insemination and the interval between parturition and conception, there were no detectable differences between the treated groups and the controls in the same herds (Table 1).

[A comparative study of some forms of treatment in cystic ovarian follicles of cattle (author's transl)]. / Een vergelijkend onderzoek van enkele therapieën ter behandeling van ovariële follikelcysten bij het rund.

Various drugs were used in the treatment of 140 cows with cystic ovarian follicles (2.9 per cent of the population) on twenty-five dairy farms during a four-year period. This was done as part of a herd fertility programme. Aanoestrus (approximately sixty days after parturition) was found to be due to cystic follicles in fifty-three cows (7 per cent of the cows examined for anoestrus). An irregular cycle or nymphomania was found to be present in eighty-seven cows (62 per cent). Treatment consisted in intravenous injection of 3,000 I.U. of human chorionic gonadotrophin plus 125 mg. of progesterone (Nymfalon" or intramuscular injection of 0.2 mg. of gonadotrophin-releasing hormone (Lutal". There were no significant differences between the results of the two forms of treatment (Tables 1 and 2), neither as regards becoming pregnant nor where the interval between injection and the next oestrus was concerned. Nine cows which failed to respond to several courses of treatment with Nymfalon or Lutal, were treated with chlormadinone acetate (oral administration of 20 mg. daily for sixteen days). Only two animals became pregnant. Abortion occurred in one of the animals within three months (Table 3).