[Show abstract][Hide abstract] ABSTRACT: Serum and oral swab samples were collected from a persistently infected cow and her calf in a two-month period to test usefulness of oral swab samples for the detection of bovine viral diarrhea virus. Nucleic acids of the virus were detected by two molecular methods: conventional gel-based RT-PCR and commercial real-time RT-PCR. The bovine viral diarrhea virus genome was detected in serum and oral swab samples on days 0, 7, 15, 22, 23, 29, 36, 37, 43, 44, 46, 51, 52, 53, and 57. The dry cotton swabs showed a reduction of diagnostic sensitivity after three days when samples were stored at room temperature (+ 21 degrees C), but storage of oral swab samples at +4 degrees C or in a freezer (< - 15 degrees C) for at least 10 days had no negative impact on the detection of the virus. No reduction of diagnostic sensitivity was observed when oral swab samples were collected in tubes with a liquid virus transport medium. Oral swabs provide an easy, reliable and cost-effective sampling tool for identification of PI animals, together with RT-PCR methods. The oral swab sampling could be especially useful for screening newborn calves during testing and removing PI animals from bovine viral diarrhea virus-infected herds.
Slovenian Veterinary Research 01/2015; 52(1):23-30. · 0.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Oral vaccination campaigns to eliminate fox rabies were initiated in Slovenia in 1995. In May 2012, a young fox (Vulpes vulpes) with typical rabies signs was captured. Its brain and salivary gland tissues were found to contain vaccine strain SAD B19. The Basic Logical Alignment Search Tool alignment of 589 nucleotides determined from the N gene of the virus isolated from the brain and salivary glands of the affected was 100% identical to the GenBank reference SAD B19 strain. Sequence analysis of the N and M genes (4,351 nucleotides) showed two nucleotide modifications at position 1335 (N gene) and 3114 (M gene) in the KC522613 isolate identified in the fox compared to SAD B19.
[Show abstract][Hide abstract] ABSTRACT: In late summer 2011, Germany and the Netherlands reported the first cases of acute infection in cattle caused by a novel Orthobunyavirus, named the Schmallenberg virus (SBV). The first malformations due to SBV were observed in December 2011 in the Netherlands, Belgium and Germany. SBV was first identified in Slovenia in a flock of 23 sheep where nine aborted foetuses with malformations were found on a farm. Viral nucleic acid of SBV was detected by real-time polymerase chain reaction (RT-qPCR) from the brain and spleen samples with a protocol developed by the Friedrich-Loeffler Institute (FLI), Germany. Between January and April 2013 a total of 77 malformed calves were tested and 25 calves identified as SBV
positive by RT-qPCR. The majority of malformed animals had one or more of the following pathological lesions: arthrogryposis, brachygnathia, torticollis, scoliosis, hydranencephaly and brain and spinal cord malformations. Additionally, two archive samples collected in September 2012 were identified as SBV positive, confirming that SBV infection was already present in Slovenia in 2012. The sequencing analysis of the partial L-segment confirmed that the strain detected in Slovenia was 100% identical to the chmallenberg virus isolate Germany (JX853179), identified in 2011. SBV-positive herds have been located throughout Slovenia.
Slovenian Veterinary Research 01/2014; 51(1):43-51. · 0.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In late summer 2011, Germany and the Netherlands reported the first cases of acute infection in cattle caused
by a novel Orthobunyavirus, named the Schmallenberg virus (SBV). The first malformations due to SBV were observed in December 2011 in the Netherlands, Belgium and Germany. SBV was first identified in Slovenia in a flock of 23 sheep where nine aborted foetuses with malformations were found on a farm. Viral nucleic acid of BV was detected by real-time polymerase chain reaction (RT-qPCR) from the brain and spleen samples with a rotocol developed by the Friedrich-Loeffler Institute (FLI), Germany. Between January and April 2013 a total of 7 malformed calves were tested and 25 calves identified as SBV positive by RT-qPCR. The majority of malformed animals had one or more of the following pathological lesions: arthrogryposis, brachygnathia, torticollis, coliosis, hydranencephaly and brain and spinal cord malformations. Additionally, two archive samples collected in September 2012 were identified as SBV positive, confirming that SBV infection was already present in Slovenia in 2012. The sequencing analysis of the partial L-segment confirmed that the strain detected in Slovenia was 100% identical to the Schmallenberg virus isolate Germany (JX853179), identified in 2011. SBV-positive herds have been located throughout Slovenia.
Slovenian Veterinary Research 01/2013; 51(1):43-51. · 0.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 confirmed. 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 Genotype 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, amplified 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Archives of Virology 03/2010; 155(4):507-14. DOI:10.1007/s00705-010-0612-5 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Slovenia till 2008 almost no testing for the presence of lyssaviruses in bats had been performed. Following the increased knowledge of the presence of European Bat Lyssaviruses in some Europe bat populations, a question arose regarding the status of EBLV in our country. The Veterinary Administration of the Republic of Slovenia commissioned a two-year active surveillance focussing on the a) Eptesicus serotinus and Myotis daubentonii species known to be reservoirs of EBLV, and b) species which roost in buildings and therefore have a potentially higher risk of coming into contact with humans. Sampling of bat saliva and blood was done during the summers of 2008 and 2009. Bats were sampled from approximately 30 roosts and almost 40 mist netting sessions in foraging habitats or in front of supposed roosts across all Slovenia. We collected 490 saliva samples and approximately 440 blood samples (blood was not extracted from pregnant females and weaker specimens). We sampled 145 Eptesicus serotinus, 61 Myotis daubentonii and 63 Rhinolophus hipposideros, 57 M. myotis, 57 Nyctalus noctula, 50 M. emarginatus and 19 Pipistrellus kuhlii and 15 M. mystacinus s. lat. One to six samples were collected from 9 additional species (R. ferrumequinum, M. bechsteinii, M. nattereri, M. cappacinii, N. leisleri, P. pipistrellus, P. pygmaeus, P. nathusii and Miniopterus schreibersii). During the project, almost 50 bat cadavers in different states, from fresh to mummified, were collected, and brain samples or swabs of their cranial cavities were taken. Saliva and brain samples were tested by RT-PCR test. Total viral RNA was extracted from samples using QIAamp® Viral RNA Mini Kit (Qiagen, Germany). Extracted RNA was stored at -70 °C until analysis. Reverse transcription (RT) with polymerase chain reaction (PCR) was performed in one tube (One-Step RT-PCR Kit, Qiagen, Germany) with primer set N1161P and N1579M to amplify 419 bp PCR product of the nucleo-phosphoprotein (N-P) gene segment. Sera samples were pooled (3-5 samples per pool) and antibodies against Lyssavirus were detected by FAVN test. Analyses for EBLV were all negative, and to date there is no confirmed EBLV presence in Slovenia. We can conclude that for the present, bats in Slovenia do not pose a significant public health risk. However, our recommendations are: a) to start a passive surveillance, b) to start raising the understanding of the real and potential risks involved in handling bats, focussing on medical doctors, veterinarians, biologists and bat volunteers, c) to start a new active surveillance in the case of an increase in EBLV reports in neighbouring countries and central Europe.
2nd Iternational Berlin Bat Meeting: Bat Biology and Infectious Diseases, Berlin, 19-21 February 2010; 02/2010
[Show abstract][Hide abstract] ABSTRACT: Background: To study bats, as a reservoir for European bat lyssavirus (EBLV) in Slovenia, native bat samples were tested in year 2008. Bats were captured from diff erent locations in Slovenia and blood samples, mouth and brain swabs were collected from live and dead bats. 260 samples of oral swabs and 38 brain samples were tested by specifi c RT-PCR assay to detect lyssavirus genome.
Results: 216 blood samples, collected from the same bats, were tested by FAVN (Fluorescent Antibody Virus Neutralization) test to detect the prevalence of lyssavirus antibodies among bats. Virus RNA was not detected in any of the samples, all blood samples werealso negative for specifi c antibodies.
Conclusions: Despite the data from this study, EBL viruses can cause fatal infections in humans and all bats involved in contact incidents with humans should be tested to determine whether the victim was exposed to EBL virus. In order to prevent lyssavirus transmission from bats to humans, all bat handlers and laboratory personnel should be informed about the possible risk of lyssavirus exposure via bats and their vaccination against rabies is strongly recommended .