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Bovine viral diarrhea virus (BVDV) subgenotype 1e was isolated for the first time in Slovenia in 2006. Here, we report the complete genome sequence of BVDV-1e, strain SLO/2407/2006. The published genome will increase our understanding of the molecular characteristics of the BVDV-1e strains circulating in Europe.

Porcine epidemic diarrhea virus (PEDV) was detected for the first time in Slovenia in January 2015. The complete genome sequence of PEDV strain SLO/JH-11/2015, obtained from a fecal sample of a fattening pig with diarrhea in September 2015, is closely related to recently detected European strains.

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 aim of this study was to determine the prevalence ofVibrio parahaemolyticusin shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Seča, Piran, Strunjan and Debeli Rtič) between 2006 and 2008. Samples were examined and analysed for the presence ofV. parahaemolyticusby conventional and molecular methods. The presence ofVibrioin the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection ofV. parahaemolyticus-specifictoxRandtlhgenes and of the virulence-associatedtdhandtrhgenes. Out of 168 samples examined, 24 were positive fortoxRandtlhgenes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies ofV. parahaemolyticuswere isolated from only one sample positive forV. parahaemolyticusby PCR.

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.

The large heterogeneity among porcine reproductive and respiratory syndrome virus (PRRSV) isolates is probably the main obstacle to its effective control using current commercial vaccines. Intentionally exposing all breeding pigs to PRRSV circulating on the farm could eliminate porcine reproductive and respiratory syndrome (PRRS) from the herd. The objective of this study was to eliminate PRRS from a farrow-to-finish pig farm by serum inoculation. The owner was acquainted with the strict biosecurity measures. Breeding pigs were immunised with serum, which was obtained from PRRSV-positive weaners from the same farm. The percent of antibody high positive breeding pigs decreased six months after serum inoculation, while 34 months after serum inoculation no more antibody high positive pigs were detected and 56.8% of breeding pigs and all other categories were free of antibodies. In the breeding herd no virus was detected during all testing while PRRSV circulated in 2-month-old weaners until 12 months after serum inoculation. Later all tested samples from weaners, growers and fatteners were negative. Herd closure and the adoption of strict biosecurity measures are essential. Serum inoculation of the breeding herd proved to be a successful measure for eliminating PRRS from this farrow-to-finish farm.

Noroviruses are a leading cause of viral gastroenteritis in humans and are responsible for many outbreaks worldwide. Mussels are one of the most important foodstuffs connected with norovirus outbreaks, also resulting in multinational dimensions. Two hundred and thirty-eight (238) samples of mussels (Mytilus galloprovincialis) were collected in periods between the years 2006-2008 and 2010-2012 to study the prevalence of noroviruses (NoVs) from harvesting areas along the Adriatic coast of Slovenia. Between 2006 and 2008, 9.1% to 24.6% of mussel samples tested by specific GI and/or GII real-time RT-PCR methods were found to be positive for NoVs while between 2010 and 2012 the percentage of NoV positive samples varied from 12.5% to 22.2%. At the nucleotide level within the RdRp gene fragment the genetic diversity of NoVs detected in mussels ranged between 78.8-81.0% nucleotide identity among GII strains (92.1-99.6% within the GII.P4 genotype), 100% nucleotide identity among GI and 58.4-60.2% among GI and GII strains. Nine of the NoV strains detected from mussels were genotyped as GII.4, while two samples were within GI.P2 and one was a positive sample within genotype GII.P21. This study confirmed that mussels are a potential source of the NoV infection. The detected NoVs share the same topology on the phylogenetic tree within the NoV strains detected in water samples and human patients, not only from Slovenia but also from many different countries worldwide. We can assume that mussels in harvesting areas are not only contaminated from the surrounding area but also by contaminated water and sewage from large transport ships, which are regularly present in the area.

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.

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 μg of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (104±0.15 TCID50/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.

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

The genetic diversity of acute bee paralysis virus (ABPV) in honeybees was studied in Slovenia. A total of 248 honeybee samples obtained from 134 different apiaries in Slovenia were tested for the presence of ABPV by RT-PCR. Specific 398-base pair (bp) products were generated with primers amplifying the ORF2 region and 452-base pair (bp) products with primers amplifying the ORF1 region of the viral genome. To characterise the overall nucleotide diversity among the ABPV sequences, phylogenetic trees with 54 and 29 samples were constructed from 357 nucleotides from ORF2 and 408 nucleotides from ORF1, respectively. The nucleotide comparison of Slovenian ABPV strains revealed two distinct clusters in ORF2 and ORF1, showing 91.2–92.5% and 96.7–97.2% nucleotide identity, respectively. Comparison of data regarding the geographical location of the ABPV-positive samples with the constructed phylogenetic trees revealed the random distribution of the two clusters throughout Slovenia.

Real-time polymerase chain reaction (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. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment. A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction −3.479 ± 0.19 and an average slope of standard curve made of ten-fold dilutions of RNA −3.409 ± 0.18.

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.

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 November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were con- firmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides (nt)) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Geno- type I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, ampli- fied from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

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 presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.

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.

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.

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.

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.

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.

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.