Sasha R. Lanyon

Sero-prevalence of Neospora caninum and Besnoitia besnoiti in South Australian beef and dairy cattle

A serological survey for Neospora caninum and Besnoitia besnoiti was carried out in beef and dairy cattle in South Australia. Serum samples of dairy cattle (n=133) from 9 properties and tank milk samples from a further 122 dairy herds were tested. An additional 810 sera from beef cattle from 51 properties were also tested. Testing at the individual animal level by IDEXX NEOSPORA X2 Ab test ELISA revealed a low prevalence of N. caninum antibodies of only 2.7% (95% CI; 1.6-3.7%) sera positive, as did the milk testing that showed 2.5% (95% CI; 1.4-3.6%) of tank milks being positive. At the herd level, 29.4% (95% CI; 16.9-41.9%) of beef, and 44.4% (95% CI; 12.0-76.9%) of dairy cattle herds showed serum antibodies. The highest within-herd prevalence in beef was 20% and 25%in dairy, which explains the low herd prevalence in dairy detected by bulk milk testing. Testing for B. besnoiti antibodies by PrioCHECK(®) Besnoitia Ab 2.0 ELISA initially identified 18.4% (95% CI: 15.8-21.0%) of 869 individual cattle sera as positive by ELISA at the manufacturers suggested cut-off threshold (15 PP). Additional tests by immunoblot and IFAT, however, could not confirm any of the ELISA results. The use of a higher (40 PP) threshold in the ELISA is suggested to improve specificity. There is thus no evidence of B. besnoiti infection in South Australian cattle.

Validation and evaluation of a commercially available ELISA for the detection of antibodies specific to bovine viral diarrhoea virus (bovine pestivirus)

OBJECTIVE To compare the performance of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies specific to bovine viral diarrhoea virus (BVDV) with a virus neutralisation test (VNT) and agarose gel immunodiffusion (AGID) test. DESIGN A total of 125 cattle serum samples were tested by a commercially available ELISA for antibodies specific to BVDV and by a VNT as the reference standard. A comparison between AGID and ELISA for detection of BVDV antibodies was also carried out, using 1182 serum samples from unvaccinated South Australian cattle. METHODS Two-graph receiver operating characteristics (TG-ROC) analysis was used to confirm that the manufacturers recommended cut-off value for the ELISA was appropriate. Two-by-two tables were constructed to analyse the concordance of serological results among the three assays. McNemar tests were used to assess the agreement among serological tests. RESULTS AND CONCLUSIONS Using the manufacturers cut-off threshold, supported by TG-ROC analysis, the ELISAs sensitivity and specificity were calculated to be 96.7% and 97.1%, respectively, compared with the VNT. Compared with AGID, ELISA with specific BVDV antibodies may be more sensitive and detect 5.8% more samples than AGID. McNemar test also showed a significant difference (P < 0.001) between AGID and ELISA.

Comparison of serum, ear notches, and nasal and saliva swabs for Bovine viral diarrhea virus antigen detection in colostrum-fed persistently infected (PI) calves and non-PI calves

The diagnosis of neonatal and young calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) by antigen-capture enzyme-linked immunosorbent assay (ACE) may be complicated by interference from colostrum-derived specific antibodies. Ten calves, with 3 calves identified as PI and 7 as non-PI were used in the current study. All non-PI calves were shown to be seropositive for BVDV-specific antibodies by antibody enzyme-linked immunosorbent assay (Ab-ELISA) on serum. Serum samples, ear notch samples, and nasal and saliva swabs were collected from each calf from birth until 12 weeks of age and tested by ELISA for BVDV-specific antigen and antibodies. Following colostrum ingestion, Ab-ELISA sample-to-positive (S/P) ratios rose by a mean of 0.95 (95% confidence interval [CI] = 0.64–1.25) and 1.72 (95% CI = 1.55–1.89) in seropositive, non-PI calves and in PI calves, respectively. The mean S/P ratios then declined to approximately 1.1 in non-PI calves and 0.5 in PI calves at between 60 and 80 days of age. In PI calves, testing for antigen in serum and nasal and saliva swabs was subject to interference by colostrum-derived antibodies in calves up to 3 weeks of age. Nasal swabs were less affected than serum and saliva swabs. Ear notches maintained positive ACE corrected optical densities at all sample times, despite a drop in the signal following the ingestion of colostrum.

Milk as a diagnostic sample for a commercially available ELISA to identify bovine viral diarrhoea (BVD) antibodies in dairy herds

OBJECTIVE The aims of this study were to evaluate a commercially available ELISA for the detection of bovine viral diarrhoea virus (BVDV)-specific antibodies in individual milk compared with individual serum samples, and in bulk milk samples compared with within-herd antibody prevalence and bulk milk quantitative reverse transcription polymerase chain reaction (qRT-PCR) results. METHODS Paired individual serum and individual milk samples were collected from 125 lactating cows and tested by ELISA; 96 bulk milk samples were also tested. Within-herd antibody prevalence was calculated based on milk ELISA results for 25 individual cows in each herd. Additionally, 167 bulk milk samples were tested for BVDV-specific antibodies by ELISA and for the presence of BVDV by qRT-PCR to establish the correlation between antibody result and virus presence. RESULTS Good agreement was observed between individual milk and serum results (Kappa = 0.865). The ELISA was observed to detect BVDV-specific antibodies in individual milk samples with a relative sensitivity of 96.6% and specificity of 89.2%. The bulk milk samples revealed a strong (r(2) = 0.95) relationship between the ELISA result and the within-herd antibody prevalence. The proportion of herds that tested positive by bulk milk qRT-PCR increased as the bulk milk antibody S/P ratio increased. CONCLUSION Commercially available ELISA testing of individual and bulk milk samples is an appropriate alternative to serum testing with good test performance in these samples. Determining a threshold for the detection of herds containing active BVD infection by testing bulk milk is a novel use for an antibody ELISA kit and provides more practical, relevant test results.

Bovine viral diarrhoea virus (‘pestivirus’) in Australia: to control or not to control?

BACKGROUND Acute infection with bovine viral diarrhoea virus (BVDV) usually causes only mild clinical disease in cattle, but infection of animals of breeding age can result in immune suppression (resulting in an increased incidence and severity of secondary disease) and decreased reproductive performance. If infection occurs during pregnancy, the virus may cross the placenta and either cause abortion, establish immunotolerance and persistent infection (PI) in the fetus or cause congenital deformities. These outcomes depend on the stage of pregnancy at the time of infection. INTERNATIONAL PERSPECTIVE BVDV is recognised as a disease of significant financial impact in a number of countries. As a result, national and regional BVDV control programs are now in place in several regions around the world. In Europe, these programs largely rely on the identification and removal of the PI animals, whereas vaccination has tended to be the chosen method of control in the United States. BVD IN AUSTRALIA BVDV is endemic in Australian cattle populations, with more than 80% of herds surveyed showing some level of exposure to the pathogen. The cost to the national industry is estimated to be AUD57.9 million annually. This review identifies and discusses the challenges to BVDV control in Australia, including farmer attitudes, herd size, sheep as a potential reservoir host and diagnostic capabilities. We conclude that systematic BVDV control in Australia is, or soon will be, an option; however, detailed cost-benefit analyses will need to be undertaken.

Investigation of AGID and two commercial ELISAs for the detection of Bovine viral diarrhea virus–specific antibodies in sheep serum

Effective control and the eventual eradication of Bovine viral diarrhea virus (BVDV) from cattle populations depend on the accurate identification of infected animals. Although typically a disease agent of cattle, BVDV is known to infect a wide variety of nonbovine species, including sheep. However, validation of serologic tests in these nonbovine species, particularly sheep, is lacking. We analyzed 99 sheep sera (57 samples from Pestivirus-naive sheep, and 42 samples from BVDV-inoculated sheep) in order to investigate 3 serologic tests: the agarose gel immunodiffusion (AGID) and 2 commercial enzyme-linked immunosorbent assays (ELISAs) for detection of BVDV antibodies. At the manufacturer’s cutoff thresholds, the AGID performed with 95.2% diagnostic sensitivity; ELISA-A performed with sensitivity of 90.5% and ELISA-B with 69.1%. All 3 tests performed with 100% diagnostic specificity. Two-graph receiver operating characteristic analysis showed that performance characteristics were optimized, such that both diagnostic sensitivity and diagnostic specificity were >95% for both ELISAs, if the thresholds were altered to 34.9% inhibition for ELISA-A and 63.5 signal-to-noise ratio for ELISA-B.

Understanding the Impact and Control of Bovine Viral Diarrhoea in Cattle Populations

Bovine viral diarrhoea (BVD) affects cattle populations around the world, with significant financial consequences. The losses associated with BVD stem from reproductive loss (in acutely infected cattle), poor productivity of immunotolerant, persistently infected cattle, and indirect losses due to increased occurrence of other diseases because of BVD-related immunosuppression. Control or eradication of BVD is a valuable strategy to reduce losses and ease animal suffering and appears economically justified in the many programs active around the world. This review briefly outlines the epidemiology of BVD and explores the financial implications of infection and the measures that can be taken to reduce them, with an emphasis on the role of diagnostic tests in control programs.

Pretreatment of serum samples to reduce interference of colostrum-derived specific antibodies with detection of Bovine viral diarrhea virus antigen by ELISA in young calves

Antigen enzyme-linked immunosorbent assay (ELISA) is used for the detection of Bovine viral diarrhea virus persistently infected (BVDV PI) cattle; however, colostrum-derived antibodies may interfere with antigen detection in serum from young PI calves. Our study aimed to assess serum pretreatment methods for reducing such interference. Dilution of PI serum with serum containing specific antibody showed that antibody levels equivalent to those observed in colostrum-fed calves were able to eliminate all antigen signals in a serum sample. Serum was treated with ethylenediamine tetra-acetic acid at pH 4.5, 5.5, 6.5, and 7.5, then boiled, centrifuged, and the supernatant-recovered. BVDV antibody was undetectable by ELISA in supernatants from treated samples, and the antigen ELISA signal was improved. Maximum antigen signal recovery of >90% was achieved at pH 5 ± 0.5. When this optimal treatment method was applied to field samples from 3 PI calves (which were negative in the antigen-capture ELISA without treatment), the antigen signal improved and gave a positive result in each case. Pretreatment may provide an improvement in the detection of young PI calves.

Pooling serum to identify cohorts of nonmilking cattle likely to be infected with Bovine viral diarrhea virus by testing for specific antibodies

Testing for specific antibodies against Bovine viral diarrhea virus (BVDV) in pooled serum may present an opportunity to decrease the cost of screening for herds of high seroprevalence and increased likelihood of active infection. Experimental serum pools (n = 280) were created by combining equal aliquots of serum from between 5 and 25 individuals. A further 188 serum pools were generated from field serum samples. All pools and individual sera were tested for BVDV-specific antibodies by enzyme-linked immunosorbent assay (ELISA), according to manufacturer’s instructions. Pools returned repeatable results, with coefficients of variation generally below 10%. The presence of serum from a persistently infected (PI) individual in the pool had no significant effect on the ELISA sample-to-positive (S/P) ratio. The results revealed that a single strong antibody-positive individual could maintain a positive result (at the manufacturer’s threshold) in pools of up to 128, while even a single weak-positive animal would generate a positive result in pools of up to 8. The S/P ratio of the pool was positively related to the within-pool prevalence of antibody-positive individuals. However, as the strength of the individual positive animals contributing to the pool had a large effect on the pool S/P ratio, the S/P ratio could not be used to accurately predict the within-pool prevalence of field serum pools. An alternative method of S/P ratio interpretation was pursued, and a two-graph receiver operating characteristic analysis allowed segregation of pools into low, medium, and high risk with good results when applied to field serum pools.

Perspectives on Current Challenges and Opportunities for Bovine Viral Diarrhoea Virus Eradication in Australia and New Zealand

This review outlines the history of bovine viral diarrhoea virus (BVDV) and the current situation in Australia and New Zealand. BVDV has been reported as present in cattle from both countries for close to 60 years. It rates as the second most economically significant disease afflicting cattle, and is highly prevalent and spread throughout the beef and dairy industries. While other cattle diseases have been the subject of government control and eradication, infection with BVDV is presently not. Eradication has been undertaken in many other countries and been judged to be a good investment, resulting in positive economic returns. Presently, Australia and New Zealand have adopted a non-compulsory approach to control schemes, initiated and managed by farmers and veterinarians without the ultimate goal of eradication. Moving towards eradication is possible with the infrastructure both countries possess, but will require additional resources, coordination, and funding from stakeholders to move to full eradication.