Risk of rabies introduction by non-commercial movement of pets.

In response to a Commission request, EFSA has carried out a quantitative assessment of the risk of rabies introduction into the UK, Ireland, Sweden, and Malta due to the movement of pets incubating rabies at the time of movement. The risk that a pet is incubating rabies at the time of first vaccination is equal to the prevalence of rabies-incubating pets in the population of origin. Following induction of protective immunity by vaccination, animals already incubating rabies will still develop clinical disease as a function of time after vaccination (termed type A risk). A waiting period will reduce this risk. Afew animals may not be protected after single-shot primary vaccination. Such animals may become infected during the waiting period after vaccination. The risk of becoming infected after the first vaccination (termed type B risk) depends on the prevalence and efficiency of vaccination. Serological testing can be used to identify non-immune pets (depending on test specificity) and will therefore reduce this risk accordingly. The type A and B risks were modelled as a function of the waiting period after vaccination and fitted to a non-linear model incorporating vaccination efficiency and test specificity. The model can be used to quantify the risk of moving pets from rabies infected areas and also to investigate the effect of different control measures. In quantitative terms, the type A risk constitutes by far the major risk. Therefore, a waiting period (defined as the time spent between vaccination and pet movement to the destined country) is the major effective measure to mitigate the risk of rabies introduction due to an animal being infected before primo-vaccination. Serological testing will only add significantly to risk reduction when waiting periods exceed 100 days. Within the EU, the rabies prevalence in most countries is so low that the risk can be considered negligible. However, for some countries the risk is non-negligible.

Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.

In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log(10) more sensitive than the classical RT-PCR and at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.

First international collaborative study to evaluate rabies antibody detection method for use in monitoring the effectiveness of oral vaccination programmes in fox and raccoon dog in Europe.

The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97.2% and 97.5%, respectively. The overall agreement values obtained for the four marketed oral vaccines used in Europe were all equal to or greater than 95%. The coefficients of concordance obtained by laboratories ranged from 87.2% to 100%. The results of this collaborative study show good robustness and reproducibility of the BioPro ELISA Rabies Ab kit.

Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2).

The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.

Comparison of infectious haematopoietic necrosis virus (IHNV) isolation on monolayers and in suspended cells.

A cell culture virus isolation procedure for infectious haematopoietic necrosis virus (IHNV) in the epithelioma papulosum cyprini cell line (EPC) is described. Ovarian fluid samples were collected from fish and tested for IHNV at 9 farms. The samples were inoculated in parallel on 24 h old EPC cell monolayers and in freshly trypsinized cells. The titre of the initial virus isolation and of first passages were compared using the 2 methods for each sample. Titres were consistently higher in suspended cells and this method also proved more sensitive for isolation of IHN virus from ovarian fluids of infected fish.

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia.

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.

An indirect immunofluorescent test for detection of rabies virus antibodies in foxes.

The blood-containing fluids in the thoracic cavity or blood from the heart from 177 red foxes (Vulpes vulpes) in Slovenia were evaluated for rabies antibodies by rapid fluorescent focus inhibition test (RFFIT) and an adapted indirect immunofluorescent test (IIF) in 1994. We evaluated the usefulness of anti-dog fluorescein-isothiocyanate (FITC) conjugate instead of anti-fox FITC conjugate in detection of antibodies against rabies virus in fox sera. In the RFFIT test, 92 (52%) of the fox samples were positive and 70 (40%) samples were negative for rabies antibodies; 15 (8.5%) samples were not suitable for examination in this test. In the IIF test, 98 (55%) fox samples were positive and 79 (45%) sera were negative. The IIF test was suitable for the rapid detection of antibodies against rabies virus in foxes, as often required for vaccine efficacy trials.

Detection of foot and mouth disease virus by RT-PCR and microplate hydridization assay using inactivated viral antigens.

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

Identification of a genetically diverse sequence of porcine reproductive and respiratory syndrome virus in Slovenia and the impact on the sensitivity of four molecular tests.

A total 91 serum samples and 51 pig tissue samples were collected between October 2009 and June 2010 from 30 herds, where a clinical picture of infection or/and porcine reproductive and respiratory syndrome (PRRS) antibody-positive pigs were detected. Of the 142 samples tested, 65 (45.8%) were identified as porcine reproductive and respiratory syndrome virus (PRRSV) positive by a one-step reverse transcription and polymerase chain reaction (RT-PCR). The sequencing results of 258 nucleotides in ORF7 from 30 herds with PRRSV-positive samples revealed the circulation of six genetically different strains of PRRSV, all belonging to the Subtype 1 (Type I). Twenty-three (76.6%) of the thirty positive herds were infected with a genetically identical cluster, with 98.9-100% nucleotide identity between the herds, representing the detection of a new strain of PRRSV in Europe, not published previously. From these 23 herds, positive PRRSV samples were detected with gel-based RT-PCR, but all gave false-negative results with two commercial real-time kits. When using a third commercial real-time kit, 28 (93.3%) of 30 positive samples in gel-based RT-PCR were detected as the Type I, confirming that the sensitivity of this real-time kit is much greater than the sensitivity of the previous two. The influence of new genetic variants of PRRSV circulating in Slovenia on molecular diagnosis and the control of the infection is discussed.

Molecular epidemiology of the rabies virus in Slovenia 1994-2010.

A molecular epidemiology study was performed on a selection of 30 rabies-positive brain samples collected between 1994 and 2010 in Slovenia and originating from the red fox (n=19), badger (n=3), cattle (n=3), dog (n=2), cat (n=1), marten (n=1) and horse (n=1). Based on the comparison of 1092 and 672 nucleotide sequences of nucleoprotein (N) and partial glycoprotein (G) gene regions, a low genetic diversity of the circulating strains was detected, but both phylogenetic trees were consistent with the topology where partial nucleoprotein or glycoprotein genes were used. A high sequence identity in the N and G gene to rabies virus isolates from neighbouring countries was found. The Slovenian strains were clearly different from the vaccine strains SAD B19 and SAD Bern, which have been used in Slovenia since 1988.

The modification of fluorescent antibody virus neutralization (FAVN) test for the detection of antibodies to rabies virus.

The fluorescent antibody virus neutralization test (FAVN) for the detection of antibodies against rabies virus was modified by using a monoclonal anti-rabies antibodies and peroxidase anti-mouse conjugate instead of a fluorescent anti-rabies conjugate. The results were read on an automatic multi-channel spectrophotometer. A total of 182 serum samples from dogs were tested by both the original and modified FAVN methods and the results were compared. Good correlation was found between the two tests. Practically, the modified FAVN test was quicker and could be used for a larger number of samples.

The persistence of rabies virus antibodies in the sera of fox cubs.

The persistence of maternal antibodies transfer from rabies-immune vixens to their fox cubs was studied. Eight vixens (Vulpes vulpes) were vaccinated 1 month before pregnancy with Lysvulpen vaccine for oral vaccination of foxes. Twenty-one were foxes born at the first half of April. The geometrical mean titre of rabies neutralizing antibodies of fox cubs sampled in May was 1.31 IU/ml and has dropped successively to 0.54 IU/ml in June samples and to 0.18 IU/ml in July samples. It has been proven that the duration of rabies maternal antibodies in fox cubs was limited to 2 months after birth.

Rabies: doubtful and discordant results in fluorescent antibody test.

By a fluorescent antibody test animal brain samples were examined for the rabies antigen. Two observers read the results. Doubtful, suspicious or weak results in the test were reanalyzed in a virus isolation test on the murine neuroblastoma cell line. From 37 samples estimated doubtful by the fluorescent antibody test, 17 were positive in the virus isolation test.