Mycobacterium avium ssp. paratuberculosis – Combined Serological Testing and Classification of Individual Animals and Herds

Several serological tests for detection of antibodies to Mycobacterium avium ssp. paratuberculosis, which is the causative agent of Johne's disease are validated, some of which are available commercially. These tests differ in sensitivity and specificity. Test results reported to farmers or veterinarians are therefore dependent upon the test in use. In the present study, three commercially available tests are used to test 2748 bovine sera from 119 Bavarian herds serologically. Serological results are compared with individual animals and on herd level. A scheme for serological testing and classification of herds as well as of individual animals based on a combination of serological test results is proposed.     

Bayesian estimation of true between-herd and within-herd prevalence of Salmonella in Danish veal calves

Specialised veal producers that purchase and raise calves from several dairy herds are potentially at high risk of delivering Salmonella-infected animals to slaughter. However, the true prevalence of Salmonella infected veal producing herds and the prevalence of infected calves delivered to slaughter from infected herds are unknown in Denmark. Due to uncertainties about test sensitivity and specificity, these prevalences are not straightforward to assess. The objective of this study was to estimate the within-herd- and between-herd prevalence of Salmonella in veal calves delivered for slaughter to abattoirs in Denmark. Furthermore, it was investigated to which extent the estimates differed between a setup using both serological tests and faecal culture, compared to just serological tests, and whether the applied sampling scheme in the national surveillance programme in Denmark was sufficient to establish high posterior estimates of freedom from infection in individual herds. We used Bayesian analysis to avoid bias as a result of fixed test validity estimates. Serological test results from 753 animals and faecal culture from 1233 animals from 68 randomly selected Danish veal producing herds that delivered more than 100 calves to slaughter per year were used to estimate the prevalences and estimates of freedom from Salmonella. Serological test results of 7726 animals from 185 herds were used to compare the difference in prevalence estimates between serology alone vs. faecal culture combined with serology. We estimated that 34-57% of specialised veal producing herds were infected with Salmonella. Within the infected herds, 21-49% of the animals were infected. Few herds obtained high posterior estimates for the probability of freedom from infection given the collected data, with only six of 68 herds obtaining posterior probability of being infected less than 10%. Furthermore, this study indicated that serology is sufficiently sensitive and specific to be used for estimating the prevalence of Salmonella-infected specialised veal producing herds.     

Serology as an aid to diagnosis: uses and abuses

The importance of correct interpretation of serological test results, common sources of error and problems associated with tests are discussed. In bovine brucellosis, a disease which is ideally suited to serological diagnosis, foetal contact with infection may cause the calf to be a serologically negative carrier. The immune tolerant animal resulting from foetal contact with virus is a major problem in the serological detection of border disease. In Johnes' disease and to a lesser extent in Brucella ovis and leptospiral infections, problems associated with sensitivity and specificity of the tests are stressed. Serovar specificity, cumbersome test procedures and negative tests in the incubation period cause difficulty in the interpretation of serological test results for leptospirosis. The importance of clinical examination, herd histories and alternative diagnostic procedures is important in all diseases. Wherever possible, flocks or herds should be maintained in specific disease-free state. Selection of stock from accredited herds or flocks is the most certain method of buying non-infected animals.     

Serology as an aid to diagnosis: uses and abuses

The importance of correct interpretation of serological test results, common sources of error and problems associated with tests are discussed. In bovine brucellosis, a disease which is ideally suited to serological diagnosis, foetal contact with infection may cause the calf to be a serologically negative carrier. The immune tolerant animal resulting from foetal contact with virus is a major problem in the serological detection of border disease. In Johnes' disease and to a lesser extent in Brucella ovis and leptospiral infections, problems associated with sensitivity and specificity of the tests are stressed. Serovar specificity, cumbersome test procedures and negative tests in the incubation period cause difficulty in the interpretation of serological test results for leptospirosis. The importance of clinical examination, herd histories and alternative diagnostic procedures is important in all diseases. Wherever possible, flocks or herds should be maintained in specific disease-free state. Selection of stock from accredited herds or flocks is the most certain method of Guying non-infected animals.     

Test strategies in bovine viral diarrhea virus control and eradication campaigns in Europe.

Several European countries have initiated national and regional control-and-eradication campaigns for bovine viral diarrhea virus (BVDV). Most of these campaigns do not involve the use of vaccines; in Germany, vaccination is used only in states in which it is considered necessary because of high BVDV prevalence. In European countries without organized BVDV control programs, vaccination is commonly used to control BVDV. Diagnostic test strategies are fundamental to all control-and-eradication campaigns; therefore, the purpose of this review is to describe how the available diagnostic tests are combined into test strategies in the various phases of control-and-eradication campaigns in Europe. Laboratory techniques are available for BVDV diagnosis at the individual animal level and at the herd level. These are strategically used to achieve 3 main objectives: 1) initial tests to classify herd status, 2) follow-up tests to identify individual BVDV-infected animals in infected herds, and 3) continued monitoring to confirm BVDV-free status. For each objective or phase, the validity of the diagnostic tests depends on the mode of BVDV introduction and duration of infection in test-positive herds, and on how long noninfected herds have been clear of BVDV. Therefore, the various herd-level diagnostic tools--such as antibody detection in bulk milk or in blood samples from young stock animals, or BVDV detection in bulk milk--need to be combined appropriately to obtain effective strategies at low cost. If the individual diagnostic tests are used with due consideration of the objectives of a specific phase of a BVDV control program, they are effective tools for controlling and eradicating BVDV in regions not using vaccination and where vaccination is a part of the control or eradication program.     

Discrepancies between the isolation of Salmonella from mesenteric lymph nodes and the results of serological screening in slaughter pigs

Most Salmonella control programmes are based on serological testing in the slaughterhouse. However, from a point of view of carcass contamination, it is rather the presence of Salmonella spp. in the animal at the time of slaughter that is important. The aim of this cross-sectional study was to investigate the possible discrepancies between the isolation of Salmonella spp. in the mesenteric lymph nodes and the results of serological screening. In total, 1821 fattening pigs originating from 60 Belgian farrow-to-finish herds were sampled in the slaughterhouse. The serum samples were analysed using an indirect mix-ELISA for the presence of Salmonella antibodies and evaluated at 3 cut-off values namely 10, 20, and 40% Optical Density (OD). All mesenteric lymph node samples were submitted to qualitative Salmonella isolation and a representative number of isolates was serotyped. From each herd, 30 animals were screened both serologically and bacteriologically and the herd was considered as positive when at least one sample was positive. At the herd level, 83.6% (cut-off OD 40%) to 100.0% (cut-off OD 10%) of the herds from which Salmonella had been isolated were evaluated as seropositive. At the individual level, only 34.5% (cut-off OD 40%) to 82.8% (cut-off OD 10%) of the animals from which Salmonella had been isolated were seropositive. Overall, a weak agreement was found between bacteriology and serology for Salmonella diagnosis. If pig herds are categorised using serological tests in the slaughterhouse, one should be aware of the fact that slaughter pigs can still harbour Salmonella spp. in the mesenteric lymph nodes, without being detected in serological tests. The cut-off value used to evaluate a sample as serologically positive and the number of samples per herd are of major importance to classify herds correctly in order to protect human health.     

Attempts at Preventing Further Spread of Bovine Virus Diarrhoea Virus (BVDV) Infection in 5 Danish Dairy Herds in which BVDV had been Isolated

In 5 herds in which bovine virus diarrhoea virus (BVDV) had been isolated, all animals were bled for virological and serological examination. After the herd blood test, follow up blood tests were made on calves born up to 6 months later in 1 herd, 9 months later in 1 herd and up to 12 months later in 3 herds. Persistently infected animals (PI animals) were removed and after a time period a small herd sample of 10 animals that were born after removal of the PI animals were examined for BVDV antibodies.At the herd blood test a total of 21 PI animals were detected. During the follow up period another 25 PI animals were born.Among animals in the small herd samples collected after removal of the PI animals, antibody positive animals were found in the 2 herds with the shortest follow up period. In the 3 herds with a 1 year follow up period there were no antibody carriers in the herd sample.It seems possible to prevent further spread of infection with BVDV if all animals in the herds as well as animals born during the following year are examined and PI animals removed.     

Field trials of an inactivated, oil-emulsion porcine parvovirus vaccine in British pig herds

Inactivated porcine parvovirus vaccines have been available commercially in Britain since 1984 and are now widely used in breeding herds. To investigate their value in cost benefit terms an oil-emulsion vaccine developed at Weybridge was used in trials on 1243 gilts in 12 herds during the period 1984 to 1986. In each herd approximately half the gilts were given the vaccine before breeding and the remainder were left unvaccinated. Blood samples were taken at vaccination and two to four weeks later to measure the serological responses, and the reproductive performances of the two groups were compared. When the data from all the gilts in the 12 herds were combined and analysed together there was surprisingly little difference between the reproductive performance of the vaccinated and unvaccinated groups. Only when the results from individual herds were analysed and interpreted against a background knowledge of wild parvovirus activity (as derived from a study of the serological results) did an understanding and evaluation of the benefits of vaccination become possible. As herds vary with respect to the absence or presence of porcine parvovirus and the epidemiology of the infection it is recommended that vaccination be used with discrimination; it should then prove highly cost effective.     

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.     

Comparison of three serological tests in the diagnosis of Brucella infection in unvaccinated cattle in Eritrea

Three serological methods, the Rose-Bengal test (RBT), the complement-fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (I-ELISA) were compared for the detection of Brucella-infected animals in unvaccinated cattle herds in Eritrea. In this study, 71 herds first were classified as positive or negative for Brucella infection on the basis of at least one animal being seropositive by RBT and CFT. All the 159 RBT-positive samples from the 26 seropositive herds and 214 RBT-negative samples randomly selected from the seropositive herds and from the 45 negative herds were tested further by CFT and I-ELISA. Using the ELISA titer as main predictor, and incorporating the RBT results, a logistic model was built to predict the CFT-negative or -positive status of individual sera and to estimate sensitivity and specificity. Whilst the ELISA titers (< or =20) accurately predicted all the negative sera in herds that were also negative by the CFT, the number of seropositive animals was higher by ELISA in herds that had positive animals. Serum samples which give higher degrees of agglutination with the RBT need not be re-tested with CFT; consideration of the seropositive status of a herd should be taken into consideration on defining the cut-off optical density readings for ELISA.     

Evaluation of a single serological screening of dairy herds for Neospora caninum antibodies

Twenty-one dairy herds with a history of Neospora caninum-associated abortions were used for a longitudinal serological study. A total of 1,676 animals were blood sampled 3 times and used to evaluate a single serological screening for N. caninum antibodies. The results of the first serological screening were compared with the results based on three consecutive samples, whereby two or more positive or negative test results per animal were considered to determine its serological status as positive or negative, respectively. In both test regimes 95.3% of the animals had the same interpretation, of which 33.9% were seropositive, and 61.3% seronegative. Relative sensitivity of one-time sampling compared to three consecutive samplings was 94.7%, while relative specificity was 95.6%. Relative specificity differed between herds. Predictive values positive and negative of one-time sampling were 92.4 and 97%, respectively. The agreement between one-time sampling and three consecutive samplings, kappa, was 0.90. For evaluation of discrepant results age distribution and pedigree data were used to provide clues regarding likelihood of transmission. Age clustering of seropositive animals was interpreted to indicate a point source infection. Daughter-mother relationships were used for the interpretation of congenital infections. The proportion of congenital infection decreased with increasing parity of the mother. Seropositive heifers had 80% congenitally infected offspring, while in older cows 66% of the offspring was congenitally infected, possibly due an increased immunity to transplacental infection with age. It is concluded that a single serological screening of a whole herd in connection with an analysis of age distribution and pedigree data is a rapid and valid method to interpret the serologic status of individual animals and to study the mode of transmission of N. caninum.     

BHV-1 eradication program in Bavaria: a marker-independent strategy

In Bavaria a BHV-1 eradication program was initiated in 1986 and was changed to a compulsory program in 1998. The eradication success increased progressively from < 50% in 1986 to 87% of the farms in 2002. BHV 1-free farms are controlled by bulk milk serology twice a year along with blood serology in animals that are negative but from herds where positive field virus infected animals are present. All serological tests are performed with an indirect ELISA test, all positive results are confirmed by a gB ELISA. Currently about 100.000 virus infected cattle are in Bavarian herds, approximately 80% of these animals are in heavily infected herds (> 10 infected animals). These herds comprise about 5% of all Bavarian herds. The eradication of the virus in these heavily infected herds is the most diifficult, whereas the prevention of new infections appears controllable. In this review current problems in BHV1 eradication are named and possible improvements are discussed.     

Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle

There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds.     

Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.     

Serological Survey of Bluetongue Virus Serotype‐8 Infection in South American Camelids in Switzerland (2007–2008)

Background: Outbreak of bluetongue virus serotype‐8 (BTV‐8) infection in domestic ruminants in Northern Europe. Objective: To investigate the South American camelids' (SAC) susceptibility to BTV‐8 infection, their role in the epidemiology of the disease, and the use of currently available serological screening tests in SAC in an endemic region. Animals: Three hundred and fifty‐four unvaccinated and 27 vaccinated SAC (170 llamas, 201 alpacas), ranging in age from 1 month to 17 years between June and August 2008. The SAC originated from 44 herds throughout the country, representing 10% of the Swiss SAC population. Methods: Prospective, observational study of a convenience sample of SAC. Serum samples were analyzed with 2 serological screening tests. When results diverged, a 3rd ELISA was carried out for confirmation (ID Screen Bluetongue Competition ELISA kit). Results: All sera from the 354 unvaccinated animals were negative in the endemic region. Reliable seroconversion was observed after administration of 2 doses of vaccine. Conclusions and Clinical Importance: This study suggests a low susceptibility of SAC to BTV‐8 despite the presence of the virus in the cattle and small ruminant population, indicating that SAC do not play a major role in the epidemiology of BTV‐8. Furthermore, these results indicate that commercially available serological tests for BTV‐8 can be used in SAC.     

Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle populations

Systematic eradication of BVDV without vaccination started in Scandinavia in 1993. In principle, the schemes include; (1) identification of non-infected and infected herds using different combinations of serological herd tests such as bulk milk tests and spot tests (sample of animals in a certain age), (2) monitoring/certification of non-infected herds by repeated sampling, applying one of the above-mentioned methods and (3) virus clearance in infected herds aimed at removing persistently infected (PI) animals in a cost- and time-efficient manner. In the virus clearance protocol described, an initial test is performed on all animals with subsequent follow-up of calves born as well as of dams seronegative in the initial test. It is generally recommended to perform an initial antibody test on all samples. This should be done not only to screen for seronegative animals on which virus isolation should be attempted (i.e. possible PI animals), but more in order to identify non-immune animals in reproductive age, that is, the key animals in herd-level persistence of infection. In Sweden, a common finding has been self-clearance, where the infection ceases without any other intervention than controlled introduction of new animals. Other epidemiological observations concern the course of events following virus introduction. Important risk factors for spreading BVDV are discussed, where livestock trade is perceived as the most central to control. Live vaccines, imported semen and embryos constitute special hazards, since they may act as vehicles for the introduction of new BVDV strains. The importance of making farmers aware of herd biosecurity and their own responsibility for it is stressed, and in order to maintain a favourable situation after a scheme has been concluded, effort must be put into establishing such a persisting attitude in the farming community.     

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.     

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).     

Anaplasmosis: Control of the First Outbreak in Canada by Serological Identification and Slaughter

On August 13, 1968 Canada experienced its first outbreak of anaplasmosis. The initial diagnosis based on hematological and clinical evidence was made by the Provincial Veterinary Laboratory, Winnipeg, Manitoba, and later confirmed in our laboratory by use of the complement-fixation test, hematology, and animal transmission studies. Sixteen herds (1,717 cattle) were examined but the outbreak was found to be localized mainly in one herd of 830 cattle. A low degree of infection was also found in four other herds. None of the remaining 11 herds in the area were infected.

The infection was controlled by serological testing, and a slaughter policy. In the four herds with low grade infection, no clinical signs were evident, and serological tests made five and six months after the discovery of the outbreak were negative. In the main herd, the tests were negative at six and nine months.

Even though no clinical manifestations of anaplasmosis were detected, surveillance of the animals in the area was continued. Sera from all the cattle were tested 16 months after the initial test. Four reactors were detected in the herd in which the main infection had previously been located. In addition, single borderline reactions were observed in a herd which previously had only one questionable reactor, and in another herd which had heretofore been negative. All of these reactive animals were slaughtered including the two with low grade reactions of doubtful significance. Following the removal of the reactive animals, tests were performed until negative results were obtained twice at six week intervals. The last test was conducted at the end of January 1970, 18 months after the original test.

     

Brucellosis in Ontario: a case control study.

Data from cattle herds infected with brucellosis and from control (noninfected) herds were collected and analyzed using case control techniques. It appeared that herds located close to other infected herds and those herds whose owners made frequent purchases of cattle had an increased risk of acquiring brucellosis, particularly those who made purchases from other herds or from cattle dealers. Infected herds had a lower level of vaccination than noninfected herds. However, the percentage vaccinated was highly variable in each group. Vaccination per se did not appear to adversely influence the interpretation of serological test results nor did it appear to protect the individual animal. Once infected, the time required to become free of brucellosis was increased by large herd size and/or loose housing. Closed herds also took longer to become brucellosis free than more open herds. The percentage of animals removed from the herd was increased by active abortion. Those herds with multiple serological reactors (positives and questionables) at the first herd test after the imposition of quarantine had the highest percentage of cattle removed.