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Salmonellaenterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Campylobacter jejuni is an emerging food-borne pathogen that poses a high risk to human health. Knowledge of the strain source can contribute significantly to an understanding of this pathogen, and can lead to improved control measures in the food-processing industry. In this study, slaughterhouse and surface-water isolates of C. jejuni were characterized and compared in terms of their antimicrobial resistance profiles and adhesion to stainless steel and chicken skin. Resistance of C. jejuni biofilm cells to benzalkonium chloride and Satureja montana ethanolic extract was also tested. The data show that the slaughterhouse isolates are more resistant to ciprofloxacin, and adhere better to stainless steel at 42 &deg, C, and at 37 &deg, C in 50% chicken juice. Additionally, biofilm cells of the isolate with the greatest adhesion potential (C. jejuni S6) were harvested and tested for resistance to S. montana ethanolic extract, benzalkonium chloride, and erythromycin, and for efflux-pump activity, as compared to their planktonic cells. The biofilm cells showed increased resistance to both S. montana ethanolic extract and erythromycin, and increased efflux-pump activity. These data indicate adaptation of C. jejuni slaughterhouse isolates to the chicken host, as well as increased biofilm cell resistance due to increased efflux-pump activity.

Newcastle disease virus (NDV), also designated avian paramyxovirus type 1 (APMV-1), is a serious pathogen of poultry, causing highly contagious Newcastle disease (ND), with high morbidity or mortality, depending on the strain. Accordingly, rapid and reliable detection of APMV-1 and differentiation between vaccine and virulent strains is of crucial importance for ND diagnosis and plays an important role in effective control of the disease. In this study, two real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays using minor groove-binding (MGB) probes were developed for broad range detection and simultaneous pathotyping of APMV-1. The two assays were evaluated for their ability to detect in allantoic fluids viral RNA of all known APMV-1 lineages. Additionally, the applicability of the developed assays was assessed by the detection and pathotype prediction of APMV-1 in swabs and organs. The assays demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation ranging from 0.2% to 3.9% and from 0.6% to 7.2% for intra-assay and inter-assay variability, respectively. The results indicated the suitability of both assays as a complementary method for rapid screening and typing of APMV-1.

A total of 670 cloacal swabs were taken from 37 species of wild-living passerine birds in years 2004, 2005, and 2006. The cloacal swabs were pooled into pools of three swabs for analysis. Isolation of avian influenza virus (AIV) and avian paramyxoviruses (APMV) was done on chicken embryos. One-step reverse-transcription polymerase chain reaction (RT-PCR) was used to detect AIV RNA. AIV nucleic acid was detected by RT-PCR in a sample of one common starling (Sturnus vulgaris). All attempts of AIV isolation from wild-living passerine birds’ samples were negative. APMV 2 was isolated in one robin (Erithacus rubecula) sample. In light of the presented results and literature data, passerines appear to play a minor role as potential disseminators of AIV and APMV; however, it seems that some wild-living passerine species could act as occasional carriers.

Rotaviral RNA was detected in the stool sample of an asymptomatic fattening pig at a Slovenian pig farm. To characterize the rotavirus, RT-PCR was used, employing primers specific for the VP7, VP4 and NSP4 genes. Specific products were purified and the sequencing reaction was performed for the molecular analysis of amplified genes. Nucleotide and amino acid sequences of the VP7 gene were found highly identical (85.3–88.1% and 90.7–91.6%) to G1 genotype strains. Phylogenetic and molecular analyses of the VP7 antigen regions revealed the sample to be from a new lineage of G1 genotype. In the molecular analysis of the VP4 gene, only 70.9% nucleotide (76.2% amino acid) identity was found with the most related rotavirus VP4 gene from GenBank. Following this, the NSP4 gene was also analyzed. After the phylogenetic analysis, it clustered with the NSP4 B genotype, but also seemed to represent a new lineage of this genotype. This new rotavirus strain, named P21-5, differed greatly from all rotaviruses characterized so far in all three genes analyzed. The virulence of this strain is not clear yet and has to be investigated.

Background: Rotavirus G9 genotype was thought to be the fifth most common genotype circulating amongst the population. In previous studies in Slovenia, only G1, G3 and G4 genotypes were detected. Objectives: To determine G and P genotypes of rotaviruses causing dehydrating gastroenteritis in children hospitalised at the University Medical Centre Ljubljana during the winter season 2001–2002. Some data obtained in previous years are included, too. Study design: For the G and P genotypes determination, we selected 99 of the total of 565 rotavirus positive samples. RT-PCR was carried out for G gene or partial P gene amplification. The RT-PCR product was used as a template for multiplex nested PCR using genotype-specific primers. In untypable samples, a sequence analysis of a short segment of G or P gene was performed. From the period before July 2001, 183 stool samples were examined using the same methods. Results: Genotype G1 was determined in 37, G4 in 6, and G9 in 28 samples out of 99. Only one sample showed a mixed infection with G1G4 genotype specifics. Following the sequence analysis of the short segment of G gene in 11 G9 genotypes, 2 different clusters of G9 genotype were determined. All samples had the same P genotype—P[8]. G9 genotype had not been detected prior to July 2001. Conclusion: Rotavirus G9 genotype emerged in Slovenia in the year 2001. Two different clusters were determined which have to be further characterised in detail.

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.

During a period of five years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5' non-translated region (5' NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.

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

SUMMARY. Clinical outbreaks of severe acute infectious bursal disease (IBD) were recorded since the mid- and late 1990s in several countries in the southeastern part of Europe. Epidemiologic data showed that both infectious bursal disease virus (IBDV)-vaccinated and IBDV-nonvaccinated chickens were affected with acute IBD and mortality up to 50% independent of the IBDV vaccination status of the appropriate parent flocks. For investigation of the causative agent of acute IBD, the variable region of VP2 was amplified, cloned, and sequenced. Nucleotide sequence analysis of polymerase chain reaction fragments showed several silent nucleotide exchanges in comparison with the sequence of the very virulent (vv) IBDV strain UK661. Also, restriction enzyme cleavage sites proposed specific for vvIBDV were present in all investigated strains. On the basis of clinical signs in affected flocks, recorded epidemiologic data, and sequence analysis, it is very likely the IBD-causing strains were of the vv phenotype.

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.

Fifteen infectious bronchitis viruses (IBV) isolated from broiler and broiler breeder flocks in Slovenia between 1990 and 2005 were molecularly characterised. IBV strains were divided into four genotypes by the analysis of the S1 gene region. Four strains belonged to the Massachusetts genotype, one strain was placed into the QX genotype, one strain formed a cluster together with the B1648 strain and nine strains were classified into the 624/I genotype. Nine Slovenian strains of the 624/I genotype formed two subgroups independently of the time of isolation and the geographical origin. Phylogenetic analysis of the partial N gene sequences revealed lower sequence variability and different clustering of the Slovenian IBV. Fourteen strains were grouped together with the strains H120 and D1466. One strain formed a cluster with the strain 793/B.

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.

Acute infectious caliciviral gastroenteritis is a common illness in people all over the world. Two genera of the Caliciviridae family, Norovirus and Sapovirus, which usually cause disease in humans, can also be found in animals where they do not always cause clinical signs of gastroenteritis. To investigate the presence of norovirus (NoV) and sapovirus (SaV) strains in asymptomatic swine and cattle, a total of 525 faecal (406 pigs and 119 cattle) specimens were collected during 2004 and 2005 from 8 pig and 4 cattle farms geographically dispersed across Slovenia. RT-PCRs and sequencing were carried out using primers targeting RdRp and capsid regions of both NoVs and SaVs. NoV positivity was detected in both bovine (2/108 [1.9%]) and porcine (5/406 [1.2%]) faecal specimens while SaV positivity was present only in porcine (29/406 [7.1%]) specimens. All porcine NoV strains (n=5) detected were attributed to a single farm, while the porcine SaV strains (n=29) detected came from 5 different farms. Phylogenetic analysis of nucleotide sequences of partial RdRp fragments placed two of the bovine NoV strains in genogroup GIII. Of the 5 porcine NoV strains, 4 clustered with GII.11, while 1 strain showed the presence of GII.18. The majority [24/29, 82.7%] of the porcine SaV strains clustered in GIII within two separate lineages, while 5 strains clustered into recently identified genetic clusters GVII (3 strains), GVIII (1 strain) and unknown (1 strain), respectively. Although NoV and SaV strains in asymptomatic swine and cattle were detected at low levels, they were still phylogenetically placed in a common pattern within both genera showing great genetic variability. There were no detected human-like strains in this study.

In 2004 and then in 2006 several outbreaks of infectious bursal disease (IBD) were reported in broiler and broiler breeder flocks in Slovenia. In this report ten recently emerged IBD viruses (IBDV) were characterised by sequence analysis of the VP2 hypervariable region and compared to previous Slovene IBDV strains from 1995/1996 and to some representative serotype 1 IBDV strains of different pathotypes. On the basis of nucleotide and amino acid identities, phylogenetic analyses and the presence of very virulent IBDV (vvIBDV) conserved amino acid substitutions, all Slovene isolates from recent outbreaks were identified as vvIBDV. Although some unique nucleotide exchanges and amino acid substitutions have been observed, the results of this study indicated that recent vvIBDV isolates are closely related with those from outbreaks in the 1990s. However, acute IBD has not been reported in commercial flocks in Slovenia for some years. This could lead to the conclusion that poor biosecurity and relaxed vaccination could be responsible for the re-emergence of vvIBDV.

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

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.

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.

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.

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.

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

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.