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Examines the genetic characteristics of type 2 porcine circoviruses (PCV-2) in wild boar in Slovenia. Use of polymerase chain reaction to study PCV-2; DNA isolation; DNA sequence analysis.

Abstract: 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. [Copyright &y& Elsevier]

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.

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in 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 of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in 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 of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes 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 of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.

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.

Fourteen avian paramyxovirus type 1 (APMV-1; Newcastle disease) viruses isolated from dead free-living and domestic pigeons in Slovenia between 2000 and 2008 were analyzed by a molecular characterization of a part of the fusion protein gene, which included the region encoding the fusion protein cleavage site. Phylogenetic analysis indicated that the Slovene pigeon paramyxovirus type 1 (PPMV-1) viruses do not cluster together but instead are divided into two groups--4bi and 4bii--of sublineage 4b. Nine Slovenian strains were placed in group 4bii. Five other strains clustered together with PPMV-1 from group 4bi. The sequence of the fusion protein cleavage site of all Slovenian strains was typical for pathogenic APMV-1. The 112RRQKRF117 motif was present in the strains from group 4bii, whereas strains from group 4bi displayed the 112GRQKRF117 motif.

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., (2009 Elsevier B.V. All rights reserved.)

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 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.

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.

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.

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.

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.

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 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.