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Bovine viral diarrhea virus (BVDV) subtypes 1f and 1d were isolated for the first time in Slovenia in 1999 and detected later in a majority of BVDV-infected cattle herds. Here, we report the first nearly complete genome sequences of noncytopathogenic BVDV-1f strain SLO/1170/2000 and cytopathogenic BVDV-1d strain SLO/2416/2002, isolated in Slovenia.

The programme of oral vaccination of wildlife started in 1988 in Slovenia and is based on our and the experiences of other countries. Red foxes are the main reservoir of rabies in Slovenia. When the oral vaccination programme started in whole territory of Slovenia in the year 1995, 1089 (28.75%) of tested animals were detected positive among wild and domestic animals. Four years later only 6 (0.5%) positive cases were detected among 1195 tested animals. The number of positive cases been increased again in 2001 to 135 cases. Between 2002 and 2008 the vaccination was done only in the protection zone, a 30 to 50 km wide belt along the southern border with Croatia because no new rabies cases were found in the north-west region of the country. When rabies was reintroduced in Italy in 2008 the vaccination was carried out again in the whole territory of Slovenia. In order to improve the vaccination campaign the stability of two vaccines was measured over 8 weeks. In both vaccines the drop of the virus titre was the highest when baits were placed in the sunlight, but, in the shadow, the virus was detected until day 53 of observation. The aim of this study is to summarise the current status of rabies and to look for the best solutions in the next vaccination campaign.

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 program of oral vaccination of wildlife started in 1988 in Slovenia and is based on our own, as well as experiences of other countries. Red foxes are the main reservoir of rabies in Slovenia. When the oral vaccination program had started on the whole territory of Slovenia in the year 1995, 1089 (28.75%) of tested animals were detected positive among wild and domestic animals. Four years later only 6 (0.5%) positive cases were detected among 1195 tested animals. The number of positive cases has been increasing again in 2001 to 135 cases. Between 2002 and 2008 the vaccination was been done only in the protection zone, i.e. 30 to 50 km wide belt along southern border with Croatia because no new rabies cases were found in the north-west region of Slovenia. When rabies was reintroduced in Italy in 2008 the vaccination is carried out again on the whole territory of Slovenia. To improve the vaccination campaign the stability of two vaccines was measured over 8 weeks. In both vaccines the drop of the virus titre was highest when baits were placed on sunlight, but in the shadow the virus was detected until 53 days of observation. The aim of this study is to summarise the current rabies status and to look for the best solutions in the vaccination campaign to come.

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

Red foxes (Vulpes vulpes) are the main reservoir of rabies in Slovenia, whereas cases of rabies in other wildlife species occur sporadically. In 1995, a program of oral vaccination of wildlife in Slovenia was initiated; baits with oral vaccine were distributed by air at a density of 20 baits/km(2). During 1995, when the oral vaccination program was started, 1,089 cases of rabies (including both wild and domestic animals) were reported. Five years later (1999), only six positive animals were detected among 1,195 tested (0.5%). Despite an increase in bait density (25 baits/km(2)) during the years 2000 and 2001, reported rabies cases increased to 115 and 135, respectively. In 2003, following initiation of a new bait-dropping strategy, which incorporated perpendicular rather than parallel flight lines, the number of rabies cases decreased to eight.

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.

The results of an investigation on bovine viral diarrhoea virus (BVDV) in fetal calf serum (FCS), whole blood and pathological material obtained from sick or dead cattle in Serbia are presented. Whole blood and FCS from sick animals were screened for BVDV antigen (Erns) by ELISA. ELISA positive samples and pathological material from dead animals were inoculated into primary cell cultures of fetal calf testis (FTTe). After threefold passage in FTTe cells, BVDV was detected by direct immunofluorescence and indirect immunoperoxidase tests and by reverse transcriptase-polymerase chain reaction (RT-PCR). Among 64 individual samples of FCS, two were positive for noncytopathogenic BVDV. One cytopathogenic BVDV was isolated from a whole blood sample from a heifer with clinical signs of mucosal disease. The 5'-untranslated region (5'-UTR) of three Serbian BVDV isolates was amplified by RT-PCR, sequenced and, together with 15 recent Slovenian BVDV isolates characterized by phylogenetic analysis. All isolates were classified as BVDV genotype 1 viruses. The majority of the BVDV isolates were of the 1f (Serbia - 2 isolates, Slovenia - 7 isolates) and 1d subtypes (Slovenia - 7 isolates) whilst one Serbian and one Slovenian isolate were genotyped as BVDV 1b.

Hostnik P., D. Barlia-Maganja, I. Toplak, J. Grom: Persistence of Rabies Virus Antibodies in the Sera of Fox Cubs Vaccinated with the Vaccine Lysvulpen . Acta Vet. Brno 2003, 72: 207-212. Persistence of maternal antibodies transfer from rabies-immune vixens to their cubs was studied. Eight vixens (Vulpes vulpes) were vaccinated one month before pregnancy with Lysvulpen, vaccine for oral vaccination of foxes. Twenty-one cubs born in the first half of April were divided in two groups. One group (n = 18) of cubs was vaccinated and the control group (n = 3) was not vaccinated. The sera of adult foxes and their cubs were collected periodically and rabies neutralising antibody titre was measured by fluorescent antibody virus neutralisation (FAVN) test. Rabies neutralising antibodies were detected in all vaccinated vixens. The level of rabies neutralising antibody titre was not changed one year after oral vaccination. The geometric mean titre of rabies neutralising 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. We found that the persistence of rabies maternal antibodies in fox cubs was limited to two months after birth. Hence, the oral vaccination campaign in April or at the beginning of May is too early for southern part of Europe because juvenile foxes have still maternal antibodies that can prevent active immunisation. Moreover, they are too small to take the baits. Maternal antibodies, lyssavirus, baits, flurescent antibody virus neutralization test, vaccination campaigns, timing

Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT-PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT-PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT-PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT-PCR. Because of the possibility of template carry-over contamination, a closed one step RT-PCR method was tested. This technically simplified approach was then combined with the PCR-enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR-ELISA method for the detection of RT-PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work.

A Slovenian BVD control and eradication programme was initiated in 1994, and the results from testing of bovine herds for antigen and antibodies in 1996 are presented. Samples originating from breeding herds, breeding herds for young bulls, and insemination stations were tested by antigen or antibody ELISA, or by PCR. Out of 7968 samples from 354 herds we found 18% of the animals antibody-positive. In one region situated in the north-east of Slovenia we found the herds to be almost nearly free of BVDV infections (5% prevalence). No positive antigen ELISA findings were done in 374 blood samples from recruitment herds for young bulls, whereas two out of 206 sera were investigated by PCR-reacted positive. The differences in seroprevalence found between regions is thought to be caused by differences in summer pasturing and husbandry practices.

Classical swine fever (CSF) is a highly contagious multi-systemic haemorrhagic viral disease of pigs. Not only domestic pigs, but also wild boar appear to play a crucial role in the epidemiology of CSF. Spleen (n = 739) and blood coagulum (n = 562) sampled from wild boars (Sus scrofa) shot in 2002, and serum samples from 746 wild boar shot in 2003 and 2004, were tested throughout Slovenia. In 2002, 17 samples were positive on enzyme-linked immunosorbent assay (ELISA) test for antibodies against classical swine fever virus (CSFV). Positive ELISA test was confirmed by a virus neutralization test. All other samples were negative. This is the first report that describes the epidemiology of CSFV from 2002 on, and the monitoring of the wild boar population in Slovenia at present.

Background Small ruminant lentiviruses (SRLV) are members of the Retroviridae family and infect goats and sheep worldwide. Detection of specific antibodies using AGID and ELISA is the most commonly used means of diagnosing SRLV infection. The most frequent molecular method for detecting the provirus genome is PCR, using peripheral blood leucocytes as target cells. Real time PCR has also recently been used. The aim of this study was to develop a real time PCR for detection of SRLV in order to improve molecular diagnostics of SRLV infections in sheep and goats. Results Two new real time PCR assays using TaqMan probes for the specific detection of genotype A (MVV assay) and genoptype B (CAEV assay) SRLV strains and differentiation between them were developed and validated at both analytical and diagnostic levels following MIQE guidelines. The validation results showed that the new real time PCR is 100% specific, with a reliable limit of detection of 26 (CAEV assay) and 72 (MVV assay) plasmid DNA copies, while compared to ELISA the diagnostic sensitivity of both assays was 79% when tested with Slovenian SRLV field samples. Intra-assay and inter-assay coefficients of variation showed overall good repeatability and reproducibility of the new real time PCR assays, except for the highest dilutions. Conclusions Two new TaqMan probe based real time PCR assays for the specific detection of genotype A and B SRLV strains and differentiation between them were developed and validated. They can serve as an additional tool for confirming infection with SRLV and may also be useful for early detection of infected animals prior to seroconversion.

Small ruminant lentiviruses (SRLV) are spread throughout the world, including Slovenia, where the first evidence of caprine arthritis encephalitis virus (CAEV) infection was found in 1996. This study was conducted to investigate the molecular and genetic characteristics of SRLV infection in Slovenia in order to classify our strains in relation to other known SRLV strains worldwide as well as to establish molecular techniques in concordance with serology. In this study, 340 goats and sheep were tested. Serological examination revealed that 57% of the goats and only 14% of the sheep were seropositive. The results of this study also show that the polymerase chain reaction (PCR) used in this study is less reliable than ELISA, with only 60.6% of the seropositive animals being PCR positive. Thirty-eight nucleotide sequences of the gag region encoding the matrix protein were determined and compared to sequences derived from the GenBank, revealing that Slovenian SRLV strains belong to sequence groups A and B, being maedivisna virus (MVV) and CAEV-like, respectively. In one goat herd, the presence of more than one genotype was confirmed and the majority of goat SRLV sequences were more closely related to MVV than to CAEV prototype strains.

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.

An epidemiology of infection with equine arteritis virus (EAV) on one stud farm with approximately 350 horses in the period from 1995 to 2008 was studied. Infection was detected by virological methods, using a virus neutralisation test (VNT) for EAV antibody detection in serum samples, and virus isolation and RT-PCR test for virus detection in semen. No clinical picture of the disease was observed. The highest seroprevalence (nearly 100%) was among stallions and old mares, while seroprevalence among young fi llies, before mating, was lower than 9%. A high incidence for seroconversion was detected among fi llies after mating. Virus was detected by RT-PCR and by a virus isolation test in the semen of 40.7% of 76 seropositive stallions. The 8 stallions, which were shedding EAV, were infected within the period of the first three years after birth, but the other 12 seropositive stallions, which were negative for EAV in semen samples, became firstly seropositive 5 years after birth. In this study we confirmed that the major transmission of EAV on the stud farm occurred from shedding stallions to fillies during the mating time, but an important role of virus transmission to other horses is also played by contact between different groups of animals. Virus positive stallions were castrated and a new breeding unit for young foals was established. EAV negative foals were vaccinated and were bred outside the farm up to 3 years of age., Prikazana je epizootiologija arteritisa konja na jednoj ergeli s 350 konja u razdoblju od 1995. do 2008. godine. Zaraza je bila dokazana na osnovi serološke pretrage virus neutralizacijskim testom (VNT), izdvajanja i identifikacije virusa te RT-PCR-om u sjemenu pastuha. Klinički znakovi bolesti nisu bili primijećeni. Najveća seroprevalencija (gotovo 100%) bila je dokazana u pastuha i starih kobila dok je seroprevalencija u ždrjebica prije pripusta bila manja od 9%. Visoka incidencija serokonverzije bila je dokazana u ždrjebica nakon pripusta. Virus je bio dokazan RT-PCR-om i izdvojen iz sjemena 40,7% od 76 serološki pozitivnih pastuha. Osam pastuha koji su izlučivali virus arteritisa bilo je zaraženo u prvim trima godinama života, a ostalih 12 serološki pozitivnih u kojih virus nije bio izdvojen iz sjemena postali su prvi put serološki pozitivni pet godina nakon ždrijebljenja. Potvrđeno je da se virus u najvećoj mjeri prenosio s pastuha koji su izlučivali virus na ždrjebice za vrijeme pripusta. Za prijenos virusa važan je bio i izravan dodir među različitim skupinama životinja. Pastusi pozitivni na virus bili su kastrirani te je osnovana nova uzgojna jedinica za ždrebad. Ždrebad negativna na virus arteritisa bila je cijepljena, a do treće godine držana izvan ergele.

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.

Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.

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.

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.

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.

Summary 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.

A selection of 43 bovine viral diarrhoea viruses isolated from mainly persistently infected cattle on 23 Slovenian farms between 1997 and 2001 were characterised genetically. Viral RNA was extracted from infected cell cultures, reverse transcribed and amplified by PCR with primers targeting the 5'-UTR and the N(pro) gene, followed by direct sequencing of purified PCR products obtained for both genomic regions. The N(pro) sequences provided the best genetic resolution, and gave also higher statistical support for phylogenetic classification of the viruses. Thirty-eight of the Slovenian isolates were of genetic subtypes 1d and 1f, four were 1b, and one subtype 1g. No BVDV type 2 viruses were found. This genetic prevalence matched those previously reported for neighbouring countries, as opposed to findings reported for more distant European countries, e.g. France, Spain and the UK. From eight cattle herds several virus isolates were analysed; with one exception all isolates from each herd were of the same genetic group. Extended sequencing of the N(pro) and part of the C gene of virus isolates with identical 5'-UTR sequences allowed differentiation between isolates obtained at different times from one herd.

A rapid and sensitive method for the detection of infectious bursal disease virus (IBDV) RNA in different chicken lymphoid organs was developed. The method is based on a single-step reverse transcription polymerase chain reaction (RT-PCR) procedure and the enzyme-linked immunosorbent assay (ELISA) detection method of amplified products. Vaccinal IBDV strain and field isolates were used for the optimization of RT-PCR and for the determination of conditions for microplate hybridization and colorimetric detection of the amplicons. With this method, viral RNA could be detected in various stages of infection in samples of different lymphoid organs. Bursas and cecal tonsils were suitable organs for viral RNA detection at different times during IBDV infection. The RT-PCR/ELISA method can be applied for IBDV detection in routine diagnostic tests, which are not usually carried out because of the difficulties involved in isolating the virus.

Rapid, sensitive and specific laboratory diagnostic methods are necessary to confirm outbreaks of classical swine fever. The detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation on cell culture, antigen detection, or molecular methods. To reduce the time and the number of steps in the diagnostic procedure a sensitive and rapid detection method based on specific amplification of the pestiviral RNA by one-step reverse transcription-polymerase chain reaction (RT-PCR) followed by detection and differentiation of the amplification products by pestivirus-, bovine viral diarrhoea virus- (BVDV-) and CSFV-specific capture probe hybridisation and colorimetric assay in microwell plates (enzyme liked immunosorbent assay (ELISA)) was developed. Two different methods using two gene regions for pestivirus RT-PCR amplification were carried out. One pair of primers was selected from the 5'-UTR region and the second one from the gene region coding for N(pro), C and E0 proteins. The designed oligonucleotide primers were used for several pestivirus reference strains as well as for some field isolates detection in cell culture supernatants and in clinical specimens. The specificity and sensitivity of both methods were compared using EZ rTth RNA PCR kit and ACCESS RT-PCR system for combined RT-PCR assay. The use of one-step RT-PCR eliminates the additional manipulations that are generally required for a two reaction system and limits the risk of carry-over contamination. Labelling of PCR products with digoxigenin (DIG) during the amplification reaction enables colorimetric assessment of hybridisation reactions. For solution hybridisation pestivirus-, BVDV- and CSFV-specific biotin-labelled capture probes were used. By serial dilutions of DIG-labelled PCR products the RT-PCR-ELISA was found to be 100-times more sensitive than the conventional agarose gel electrophoresis. Higher sensitivity of RT-PCR-ELISA detection using specific biotin-labelled probes offers the opportunity to eliminate strain specific nested PCR and to overcome the problems with contamination and false positive results.

A total of 7339 fox, dog, cat, cattle, sheep and other mammalian brains were tested to rabies virus antigen. Results of fluorescent antibody test (FAT) were evaluated by two microscopists. Doubtful and discordant results in FAT were analyzed again in virus isolation test (VIT) using mouse neuroblastoma (NA) cell line. Twenty-eight brain samples were determined as doubtful, while 9 brain samples were determined to be positive by one microscopist and negative by the other. Samples which were shown as doubtful and discordant in FAT were retested in VIT. Seventeen of these were positive in the VIT., Na prisutnost antigena virusa bjesnoće bilo je pretraženo ukupno 7339 uzoraka mozgova lisica, mačaka, goveda, ovaca i drugih vrsta životinja. Rezultate izravnog imunofluorescentnog testa vrednovala su dva stručnjaka. Uzorci koji su u testu imunofluorescencije davali sumnjive rezultate, ili se rezultati stručnjaka nisu slagali bili su pretraženi izdvajanjem virusa na kulturi stanica. Rabljena je stanična linija neuroblastoma miševa. Dvadeset osam uzoraka bilo je proglašeno sumnjivim na bjesnoću. Rezultat prvog mikroskopskog pregleda u devet uzoraka bio je pozitivan, a drugog negativan. Od ukupno 37 uzoraka, koji su bili ponovno pretraženi izdvajanjem virusa, 17 je bilo pozitivnih na bjesnoću.

Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E(RNS) and protein NS2-3 using commercially available ELISA kits. E(RNS) and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E(RNS) and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5' non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.

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.

0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved doi:10.1016/j.vetmic.2004.10.003 Page 182, Section 4, paragraph 3, lines 5–6 should read Luzzago et al. (2001). Page 184, References section, reference Lugazzo et al. should read: Luzzago, C., Bandi, C., Bronzo, V., Ruffo, G., Zecconi, A., 2001. Distribution pattern of bovine viral diarrhoea virus strains in intensive cattle herds in Italy. Vet. Microbiol. 83, 265–274.