Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by culture and serology

Faecal samples from 186 dairy cows representing ten commercial dairy herds with sporadic clinical paratuberculosis (group A), and from 100 dairy cows from herds without a history of paratuberculosis (group B) were cultured for Mycobacterium avium subspecies paratuberculosis (MAP). Two different decontamination methods, a NaOH/oxalic acid method and treatment with 0.75% hexadecylpyridinium chloride (HPC) were performed prior to inoculation of Loewenstein-Jensen agar slants with and without mycobactin. Cultures were incubated for 16 weeks. Acid-fast staining bacteria were identified as MAP on the basis of mycobactin dependency and by PCR-RFLP analysis of the IS 1311-insertion element of M. avium. MAP was grown from 15 out of 186 group A animals (8.1%) whereas faecal culture for MAP was consistently negative in group B. The growth rate of MAP was significantly higher (8.1% vs. 1.6%) and the contamination rate of cultures was significantly lower (17.6% vs. 21.5%) in faecal samples decontaminated with NaOH/oxalic acid than with HPC-treated faecal samples (p<0.01, McNemar's test). Atypical mycobacteria which were grown from 46.8% of NaOH/oxalic acid treated specimens were not obtained from any of the HPC-treated samples. A commercial ELISA with MAP-lipoarabinomannan as the antigen was used to detect MAP-antibodies in unabsorbed sera from all animals. The percentage of ELISA-positive cows was 16.8%. Overall agreement between antibody detection and MAP-positive faecal culture was 15.4%.     

Comparison of milk and serum enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.

Milk and serum samples from 35 dairy herds in 17 states were evaluated for cow- and herd-level Mycobacterium avium subspecies paratuberculosis (MAP) antibody test agreement. Evaluation of 6,349 samples suggested moderate agreement between milk and serum enzyme-linked immunosorbent assay (ELISA) results, with a kappa value of 0.50. Cow-level sensitivity (Se) for 18 dairy operations with 1,921 animals was evaluated relative to fecal culture results. At the cow level, the milk ELISA relative Se was not significantly different from that of the serum ELISA (21.2 and 23.5%, respectively). Logistic regression models revealed a positive association between lactation number and milk ELISA status. Non-Holstein cows were more likely to test milk ELISA positive than Holstein cows. Cows in the first 2 weeks of lactation and after week 45 of lactation were more likely to test milk ELISA positive than cows between 3 and 12 weeks of lactation. Milk production > 80% of herd average was negatively associated with testing milk ELISA positive. Animals in the West and Midwest regions were less likely than animals in the Southeast region to test ELISA positive by either test. Estimates for herd-level sensitivity for the milk and serum ELISA, relative to fecal culture results, ranged from 56 to 83%. At the cow and herd levels, milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use Dairy Herd Improvement Association testing.     

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India

Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.     

Molecular detection and typing of Mycobacterium avium subspecies paratuberculosis from milk samples of dairy animals

Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne’s disease were screened by Ziehl–Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region.     

The importance of allergic skin test with Johnin,antibody ELISA,cultural fecal test as well as vaccination for the sanitation of three chronically paratuberculosis-infected dairy herds in Rhineland-Palatinate

Three chronically paratuberculosis infected herds were tested for six years twice a year (intradermal Johnin test, antibody ELISA (IDEXX Corp.), microbial culture) according to a sanitary program. Culling of shedding animals and vaccination of calves with NEOPARASEC (Merial Corp.) were part of the program. In course of experiment, 1015 samples of 228 non vaccinated cows and 1502 samples of 293 vaccinated cattle have been tested. 3.8% of the vaccinated animals proved positive in microbial culture. Nearly all vaccinated calves developed granulomas sized from hazelnut to loaf at the injection site. Positive reactions in intradermal test as well as in antibody ELISA were found in very young calves. 24.3%, 33.7%, 25.9%, respectively of the non vaccinated animals were identified as shedders of M. avium subsp. paratuberculosis (MAP) by microbial culture. In the first and in the second herd most shedders of MAP were found in the first herd examination (66.7%, 42.9%, respectively), whereas in the third herd they were detected in the fifth examination (31.0%). At the beginning, 17.9% of non vaccinated animals proved positive in intradermal test, 14.4% in antibody ELISA. Afterwards, the number of positive test results decreased but increased again towards the end of the experiment. 48.5% of the 66 shedders showed positive reactions in intradermal test, 57.6% in antibody ELISA, 77.3% in at least one of these both tests. Antibodies in ELISA were found in rising frequency from two years before the time of shedding. 50.0% of the shedders reacted positive in ELISA at the time of shedding. In selected shedders first positive results were found at the age of about two years. Unfortunately, only incomplete hygienic measures were realized by the farmers. Under field conditions the realisation of attending sanitary programs is difficult. MAP is spread mainly by buying of animals, therefore a certification program for paratuberculosis free herds is urgently necessary as well as an improvement of diagnostic methods.     

Coxiella burnetii: Serological reactions and bacterial shedding in primiparous dairy cows in an endemically infected herd—impact on milk yield and fertility

Coxiella burnetii (C. burnetii) is the causative agent of Q fever both in humans and animals. The objectives of this study were to investigate seropositivity and bacterial shedding in heifers and primiparous cows in an endemically infected herd and to assess the effects on post‐partum diseases, fertility and milk production. At the age of 9 months, 96 Holstein heifers were included. Sampling was performed reproduction‐orientated: at the beginning of the study, at detection of first pregnancy, 3 weeks before expected calving date (blood serum), at parturition and after 21, 42, 100 and 150 days in milk (DIM) (blood serum, vaginal swabs and milk). Serum samples were investigated by a commercial ELISA for the presence of specific antibodies and vaginal swabs and milk samples by PCR to detect C. burnetii DNA. Individual animal data (calving ease, stillbirth, retained foetal membranes, puerperal metritis, endometritis after 42 DIM, presence of corpus luteum after 42 DIM, interval calving‐first service, interval calving‐conception, number of inseminations until 150 DIM, proportion of pregnant cows until 100 and 150 DIM, proportion of pregnant cows after first service and data of the dairy herd improvement test) were documented. All heifers were seronegative at the age of 9 months and 3 weeks before the expected calving date. Subsequently, the proportion of seropositive animals and the antibody score increased significantly towards 42 and 100 DIM, respectively. Vaginal C. burnetii shedding was highest at parturition (30.9%), while the most positive milk samples were detected after 100 DIM (15.3%). Coxiella burnetii seropositivity and shedding had no impact on parameters of reproduction. However, milk fat yield was declined in puerperal vaginal shedders and cows which seroconverted during their first 42 DIM, respectively.     

Longterm investigation in an Austrian dairy herd with low prevalence of paratuberculosis detection of antibodies in blood and milk

Paratuberculosis (Johne's disease), which is widely distributed throughout the world, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Diagnosis of subclinically infected cattle is challenging and is especially problematic in herds with low prevalence of MAP. The aim of this long-term study was the comparison of different diagnostic tests for MAP and specific antibodies in a herd with low prevalence of MAP. Three different commercially available serum-ELISA (Svanovir-ELISA, Svanova, Uppsala, Sweden; IDEXX-ELISA, IDEXX Laboratories, Maine, USA; Pourquier-ELISA, Institut Pourquier, Montpellier, France) and two milk ELISA (Svanovirm-ELISA Svanova, Uppsala, Sweden; Pourquier-ELISA, Institut Pourquier, Montpellier, France) were compared. Apart from these indirect diagnostic tests, two methods for the detection of the etiologic agent (bacteriologic culture and real-time PCR of faecal samples) were performed. In January 2005 the first and in April 2005 the second herd investigation of all animals older than 2 years (n=335) were carried out. Blood, milk and faecal samples were taken. From November 2005 until April 2006 follow up investigations were performed. For this purpose, blood-, milk- and faecal samples were monthly taken from 63 selected animals. The highest number of blood- and milk samples with a detectable antibody-level was found by the Svanovir-ELISA. There was a significant correlation between serum- and milk- Svanovir-ELISA results, whereas the agreement between ELISA and faecal culture/PCR was low. Significant correlations between Svanovir-serum-ELISA results and milk somatic cell counts could be registered. Moreover, there was significant agreement between IDEXX-serum-ELISA results with the age and number of lactations of the cows, as well as the mother's MAP-status.     

Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.     

Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle

Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4 cfu per tube less than placebo treated cows (P = 0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P = 0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR = 0.27; P = 0.03). Monensin sodium administered to infected animals at 335 mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown.     

Serological and molecular detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in cattle of dairy herds in Colombia

The objective of this study is the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by serum enzyme-linked immunosorbent assay (ELISA), fecal polymerase chain reaction (PCR), and fecal culture in Colombian dairy herds. Serum and fecal samples from asymptomatic cows (n = 307) of 14 dairy herds were tested for MAP by an unabsorbed ELISA test (ELISA-A). Serum and fecal samples from positive ELISA-A animals (n = 31) were further tested by an absorbed ELISA test (ELISA-B) and PCR. Fecal samples from animals of herds positive by ELISA-A and PCR (n = 105) were inoculated onto three different culture media. ELISA-A produced positive results in 10% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in two and six serum and fecal samples from positive ELISA-A animals, respectively. Fecal samples were negative for MAP on all culture media. The results of this study confirmed the presence of MAP in local dairy herds and the difficulties of MAP detection in asymptomatic animals by ELISA, PCR, and fecal culture.     

Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution.     

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.     

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).     

Association of Mycobacterium avium subspecies paratuberculosis infection with milk production and calving interval in Iranian Holsteins

There are inconsistent results for the association of Mycobacterium avium subspecies paratuberculosis (MAP) infection with production and reproduction in dairy cows. Determination of these associations in each region is essential to encourage participation of dairy cattle producers in disease control programs. This study was conducted in Shiraz, southern Iran, to quantify the association of subclinical MAP infection with 305-day milk production and calving interval in Iranian Holsteins. A total of 21 dairy herds were selected for the study and in each herd, quarter milk samples were collected from ten to 12 dairy cows for PCR analysis. Data about parity, calving interval, length of lactation period, total milk production and 305-day milk production were also provided for each animal. Overall, 252 individual milk samples were collected. Herd- and individual-level prevalence of MAP infection were 23.8% (95% CI, 6.2–41.4%) and 3.2% (95% CI, 1.3–5.1%), respectively based on IS900 nested PCR. The results for 305-day milk production revealed a 248 kg reduction in positive cows compared with negative ones (P = 0.009). When cows from positive herds were compared with cows from negative herds, a 335-kg reduction in 305-day milk production (P = 0.005) and a 30-day increase in calving interval (P = 0.057) were observed in the former group. These findings support the previous results that paratuberculosis infection is negatively associated with the performance of the animals.     

The value of a bulk-tank milk ELISA and individual serological and faecal examination for diagnosing (sub)clinical Dictyocaulus viviparus infection in dairy cows

To test the value of a recently developed bulk-tank milk (BTM) ELISA for diagnosing (sub)clinical Dictyocaulus viviparus infection in lactating dairy herds under field conditions, bulk milk samples were collected from farms with or without clinical symptoms suspected to be caused by lungworm infection. Results of the BTM ELISA were compared against individual examinations for lungworm larvae in faeces and lungworm antibodies in serum from up to 20 heifers (parity 1) and up to 20 cows (parity ≥ 2) on the same farms. This also allowed, for the first time, to examine the value of individual faecal and serological examinations in the diagnosis of (sub)clinical lungworm infections. In total, 33 farms participated. Of these, 16 reported clinical symptoms possibly related to lungworm infection (defined as a suspected positive clinical status or CS(+)) and 17 reported having no such symptoms (CS(-)). In total, 503 heifers and 649 cows were sampled. Of all faeces samples positive for lungworm larvae, 94 were from heifers (18.9% of all heifers) and 75 from cows (11.7% of all cows) (P<0.001). Of all sera positive for lungworm antibodies, 130 were from heifers (26.1% of all heifers) and 113 from cows (17.5% of all cows) (P<0.001). Of the CS(-) farms 41% had at least one heifer or cow shedding larvae and 71% had at least one seropositive heifer or cow. Of the CS(+) farms this was 81% and 94%, respectively. There were only 4 farms, all CS(-), where none of the animals were found shedding larvae and all animals tested seronegative. This implies that on 76% of the CS(-) farms lungworm infection circulated unnoticed. On all CS(+) farms the suspicion that lungworm caused the respiratory symptoms was confirmed by the individual faecal and serological examinations, whereas the BTM ELISA confirmed presence of lungworm on half of the CS(+) farms. The latter in particular occurred on farms with the more severe outbreaks. Overall, of 32 available BTM samples 10 tested positive (8 of 15 CS(+) and 2 of 17 CS(-) farms). For diagnosing suspected lungworm disease it was concluded that testing a BTM sample might suffice in case of moderate to severe outbreaks. However, in case of a mild outbreak with just a few animals coughing, examining individual animals has to be preferred over testing a BTM sample. The likelihood to detect lungworm infection is higher if heifers are sampled compared to cows. Sensitivity of the BTM ELISA was 35.7% if the presence of at least one seropositive and/or one larvae shedding animal in the herd was used to define lungworm positive farms. On average, at least 30% of the herd had to be seropositive before the BTM ELISA was found positive for lungworm antibodies. Results indicate that the BTM ELISA in its current form does not appear to be suitable for surveys on the prevalence of lungworm presence on farms. However, this BTM ELISA might be used in large-scale surveys to detect, for instance, annual changes in percentage positive farms, as long as it is recognized that positivity is more closely related to incidence of lungworm disease than to prevalence of lungworm infection.     

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Comparative performance of tests using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis

Although some ELISA tests using cytoplasmic or outer membrane antigens of Brucella have been developed to improve the diagnosis of canine brucellosis, the performance of these assays has not been compared. In the present study three ELISA tests using lipopolysaccharide (LPS)-free cytoplasmic proteins (CPs) of Brucella abortus, the lumazine synthase (LS) of Brucella spp. or a hot-saline (HS) extract of Brucella canis containing outer membrane antigens were used to test sera from dogs with suspected or confirmed brucellosis (n=36) and from dogs with pathological conditions other than brucellosis (n=212). In the first group the proportion of positive results was 92, 92 and 81% for the ELISAs with HS, CP and LS, respectively, and 94% of the samples were positive by at least one ELISA test. Three dogs that were negative by agglutination (2ME-RSAT) had a positive result by at least one ELISA, and this discrepancy was attributed to the lower analytical sensitivity of agglutination tests. This hypothesis was confirmed by a serological follow-up of seven dogs recently infected with B. canis in three of which the illness was diagnosed earlier by one or more ELISA tests than by 2ME-RSAT. Among dogs having pathological conditions other than brucellosis, specificities were 94.3, 96.7 and 96.7% for the ELISAs with HS, CP and LS, respectively. This study shows that HS-ELISA and CP-ELISA are highly specific and sensitive for the diagnosis of canine brucellosis and can detect the infection by B. canis shortly after the exposure to the pathogen.     

Assessment of test results when using a commercial enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in repeated samples collected from adult dairy cattle

OBJECTIVE: To determine the proportion of adult cattle that change test status when an ELISA for antibodies against Mycobacterium avium subsp paratuberculosis (MAP) is used to assay samples collected twice at variable intervals and to determine whether cows with an initial strong positive result were more likely to maintain positive status, compared with all cows with an initial positive result. DESIGN: Cross-sectional observational study. ANIMALS: 3,757 adult dairy cattle. PROCEDURE: Serum samples were obtained twice from cattle at intervals ranging from 77 to 600 days between collections. Samples were tested with an ELISA for detection of antibodies to MAP. RESULTS: Of 157 cattle with initial positive results (value for the sample divided by the value for positive-control serum [S/P] > or = 0.25), 62 (39.5%) had negative results for the second sample. Of 71 cattle with an initial S/P value > or = 0.40, 13 (18.3%) had a negative result (S/P < 0.25) for the second sample. Of 33 cattle with an initial S/P > or = 0.70, 3 (9.1%) had a negative result (S/P value < 0.25) for the second sample. Interval between collection of samples did not affect results. CONCLUSIONS AND CLINICAL RELEVANCE: Many cows changed ELISA status between samples collected at variable intervals. Cows with an initial high S/P value (> or = 0.70) were more likely to maintain positive status than cows classified as positive on the basis of cutoff values of > or = 0.25 or > or = 0.40. Veterinarians should expect variability in ELISA results when repeated testing of cattle is used as part of an MAP control program.