Implementing a probabilistic definition of freedom from infection to facilitate trade of livestock: putting theory into praxis for the example of bovine herpes virus-1.

International trade of livestock and livestock products poses a significant potential threat for spread of diseases, and importing countries therefore often require that imported animals and products are free from certain pathogens. However, absolute freedom from infection cannot be documented, since all test protocols are imperfect and can lead to false-negative results. It is possible instead to estimate the "probability of freedom from infection" and its opposite, the probability of infection despite having a negative test result.

Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands.

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk.

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows.

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested.

Public health risk analysis of European bat lyssavirus infection in The Netherlands.

We present the frequency and the nature of contact incidents of the Serotine bat, Eptesicus serotinus, with humans and with companion animals (specifically cats and dogs), in The Netherlands between 2000 and 2005. Out of 17 bats in bite contact with humans, five tested positive for European bat lyssavirus (EBLV) type 1a. Cats had the most numerous contacts with bats (49 times) but a relatively low number of these bats were EBLV positive (six times).

Public health awareness of emerging zoonotic viruses of bats: a European perspective.

Bats classified in the order Chiroptera are the most abundant and widely distributed non-human mammalian species in the world. Several bat species are reservoir hosts of zoonotic viruses and therefore can be a public health hazard. Lyssaviruses of different genotypes have emerged from bats in America (Genotype 1 rabies virus; RABV), Europe (European bat lyssavirus; EBLV), and Australia (Australian bat lyssavirus; ABLV), whereas Nipah virus is the most important recent zoonosis of bat origin in Asia.

European bat lyssaviruses, The Netherlands.

To study European bat lyssavirus (EBLV) in bat reservoirs in the Netherlands, native bats have been tested for rabies since 1984. For all collected bats, data including species, age, sex, and date and location found were recorded. A total of 1,219 serotine bats, Eptesicus serotinus, were tested, and 251 (21%) were positive for lyssavirus antigen.

Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe.

Sets of serum and milk samples were collected from various countries and prepared, lyophilised and distributed by 1 laboratory to 12 reference laboratories in Europe. The serum sets contained the three European bovine herpesvirus 1 (BHV1) reference serum samples (EU1, EU2 and EU3), serum samples from naturally and experimentally BHV1-infected cattle, from vaccinated, and vaccinated-challenged cattle, from uninfected cattle, and a series of serum dilutions. In addition, sets of milk samples were distributed.

Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein E(rns) for serologic diagnosis of pestivirus infections in swine.

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein E(rns) were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the E(rns) protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed.

Development of a classical swine fever subunit marker vaccine and companion diagnostic test.

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively.

A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle.

Bovine respiratory syncytial virus (BRSV) strains are tentatively divided in subgroups A, AB and B, based on antigenic differences of the G protein. A Dutch BRSV strain (Waiboerhoeve: WBH), could not be assigned to one of the subgroups, because the strain did not react with any monoclonal antibody against the G protein. We describe here that the WBH strain has accumulated critical mutations in subgroup-specific domains of the G protein gene, which also occur but then independently in G protein genes of BRSV subgroup A or B strains.

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus.

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.

Critical factors affecting the diagnostic reliability of enzyme-linked immunosorbent assay formats.

This paper aims to evaluate different formats of the enzyme-linked immunosorbent assays (ELISAs) for detection of virus-specific antibodies and focuses on factors that may influence the diagnostic reliability of such tests. Newly developed and well-established ELISAs for detection of infections of bovine herpesvirus 1 (BHV1), bovine respiratory syncytial virus (BRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV) and bovine viral diarrhoea virus (BVDV) are used as examples. Differences between competitive and non-competitive ELISAs are described, with special reference to the influence of the antigen, the conjugated antibody and the test sample on the test results.

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus.

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected.

Antigenic and molecular analyses of the variability of bovine respiratory syncytial virus G glycoprotein.

Antigenic variation among eight bovine respiratory syncytial virus (BRSV) isolates was determined using monoclonal antibodies (MAbs) specific for the attachment (G) protein. Two major (and one intermediate) subgroups were identified, as well as one strain that did not fit any pattern. The subgroups could also be differentiated on the basis of the Mr of the F protein cleavage product, F2.

Probability of detecting antibodies to bovine herpesvirus 1 in bulk milk after the introduction of a positive animal on to a negative farm.

The purpose of this study was to assess the probability that the introduction of one or more bovine herpesvirus 1 (BHV-1)-seropositive animals would result in the bulk milk of a clean herd becoming BHV-1-positive. Probability calculations (stochastic and deterministic) were based on the distribution of the log(titre) of 828 positive animals and the daily milk production of the herds and of the individual cows. They showed that the probability in average sized herds of 45 dairy cows is only between 10 and 25 per cent and that even in small herds of 25 cows the introduction of a positive animal would go undetected in the majority of cases.

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus.

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells.

Antibody responses against epitopes on the F protein of bovine respiratory syncytial virus differ in infected or vaccinated cattle.

The fusion protein F of bovine respiratory syncytial virus (BRSV) is an important target for humoral and cellular immune responses, and antibodies against the F protein have been associated with protection. However, the F protein can induce antibodies with different biological activity, possibly related to distinct antigenic regions on the protein. Therefore, epitopes were mapped on the F protein using monoclonal antibodies.

Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue.

Bovine respiratory syncytial virus is an important respiratory pathogen in cattle. Recently, subgroups of BRSV have been identified, based on antigenic differences. However, little is known about subgroups of BRSV that circulate in the cattle population.

A European inter-laboratory trial to evaluate the reliability of serological diagnosis of bovine herpesvirus 1 infections.

The detection of cattle latently infected with bovine herpesvirus 1 (BHV1) is of importance in control programs and in international trade activities. Therefore, tests to detect specific antibodies in serum must be highly sensitive. To evaluate the reliability of serological diagnosis of BHV1 infections in Europe, seventeen laboratories in 15 European countries were asked to determine BHV1-specific antibodies in a panel of bovine serum samples using the serological tests available in their laboratory.

Experimental reproduction of respiratory disease in calves with non-cell-culture-passaged bovine respiratory syncytial virus.

To reproduce experimentally clinical bovine respiratory syncytial virus (BRSV) infections in cattle, we isolated BRSV from a calf in the field that suffered from acute respiratory disease. Cell culture passage of the virus was avoided to prevent any modification of the biological properties of the virus. The isolated BRSV was passaged in specific-pathogen-free (SPF) calves.

Antibody responses against the G and F proteins of bovine respiratory syncytial virus after experimental and natural infections.

Antibodies against the two major surface glycoproteins of bovine respiratory syncytial virus (BRSV), G and F, play a role in protection against BRSV-associated disease, but only the antibody response against the F protein has been well described. Therefore, we used a novel peptide-based enzyme-linked immunosorbent assay (G peptide-ELISA) to compare immunoglobulin G (IgG) and IgG subclass antibody responses against the G protein with the antibody response against the F protein, as measured by a conventional BRSV ELISA (F-ELISA). Experimental infection of cattle induced significantly lower antibody titers than did natural infection.

Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G.

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA.

Quantitative investigation of population persistence and recurrent outbreaks of bovine respiratory syncytial virus on dairy farms.

Objective: To determine if transmission of virus among seropositive cattle is a plausible mechanism for the permanent presence of bovine respiratory syncytial virus (BRSV) in dairy herds, and how likely, with the scenario for persistence, there will be only 1 clinical outbreak of BRSV per year.

Design: Build a stochastic model, parameter estimation from serologic data on BRSV, and interpret the estimated parameter values from the model analysis.

Sample Population: Monthly data on the prevalence of antibodies directed against BRSV in sera from all cattle in 6 dairy herds.