Danijela Rihtarič

University of Ljubljana, Lubliano, Ljubljana, Slovenia

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Publications (18)11.8 Total impact

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    DESCRIPTION: This research describes the first detection of honeybee-pathogenic viruses in samples of clinically affected Carniolan gray bee collected between 2007 and 2009 on whole territory of Slovenia.
    Full-text · Research · Dec 2015
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    Ivan Toplak · Danijela Rihtaric · Peter Hostnik · Janko Mrkun
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    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.
    Full-text · Article · Jan 2015 · Slovenian Veterinary Research
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    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.
    Full-text · Article · Jan 2014 · Journal of wildlife diseases
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    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.
    Full-text · Article · Jan 2014 · Slovenian Veterinary Research
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    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.
    Full-text · Article · Jan 2013 · Slovenian Veterinary Research
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    I Toplak · D Rihtarič · P Hostnik · J Grom · M Stukelj · Z Valenčak
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    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.
    Full-text · Article · Jan 2012 · Journal of virological methods
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    ABSTRACT: This research describes the detection of six honeybee viruses in samples of clinically affected Carniolan gray bee collected between 2007 and 2009 on the territory of Slovenia. Using one-step reverse transcription-PCR (RT-PCR), 60 bee samples originated from 45 apiaries were screened for the presence of six honeybee viruses. Samples were found positive for acute bee paralysis virus (ABPV = 40%), black queen cell virus (BQCV = 83,3%), chronic bee paralysis virus (CBPV = 18,3%), deformed wing virus (DWV = 70%), Kashmir bee virus (KBV = 1,7%) and sacbrood bee virus (SBV = 8,3 %). Mortality and paralysis were often evident in the apiaries and could be connected with ABPV and/or CBPV infections. Both viruses were detected in clinically affected apiaries with high bee mortality and with paralysis symptoms showing flightless bees, trembling and crawling at the hive entrance. The severity of clinical manifestation with high bee losses were associated with higher number of viruses detected in the samples. Among virus positive samples, 27% of them were infected with one virus, 30% with two viruses, 25% with three viruses and 15% of samples contained four viruses simultaneously. The results of this study provide data about the detection of several bee viruses in affected bee colonies and viral infections in Carniolan bee have to be investigated by further research.
    Full-text · Article · Jan 2012 · Slovenian Veterinary Research
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    P. Hostnik · Maurer Wernig · Danijela Rihtaric · J. Grom · T. Malovrh · I. Toplak

    Full-text · Article · Jan 2012
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    D Rihtarič · P Hostnik · J Grom · I Toplak
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    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.
    Full-text · Article · Apr 2011 · Veterinary Microbiology
  • D. Rihtarič · P. Hostnik · J. Grom · A.F. Steyer · A. Steyer · I. Toplak
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    ABSTRACT: Over 950 bat species have been described, representing 20 percent of living mammal species. In Slovenia we can find up to 30 different insectivore bat species. Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including astroviruses. In this preliminary study 100 samples of seven different bat species from 27 different geographic locations in Slovenia were tested for the presence of astrovirus nucleic acid. Astrovirus nucleic acid was found in eight out of 100 examined samples in two bat species, where astroviruses have not yet been described. These were horseshoe bat (Rhinolophus hipposideros) and Daubenton's bat (Myotis daubentonii). Nucleotide sequences alignment of strains SLO1A 0055 and SLO1A 0067 revealed only one nucleotide difference, the nearest sequence in the GenBank database was bat astrovirus strain Rp/Jiangxi/A860/2005 with 76% nucleotide identity. Nucleotide sequences of the astrovirus strains with the names SLO1A 0058 and SLO1A 0066 are distinguished by one nucleotide and are related to strain Bat astrovirus Rf/Shandong/A977/2006 with 78% nucleotide identity. Nucleotide sequence of strain SLO1A 2172 is the most closely related to Bat astrovirus N58-49/Germany/2008 with 81% nucleotide identity. According to phylogenetic analysis these Slovenian bat astroviruses are related to other published bat astroviruses from China and Germany.
    No preview · Article · Jan 2011 · Slovenian Veterinary Research
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    P. Hostnik · Danijela Rihtaric · J. Grom · T. Malovrh · I. Toplak
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    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.
    Full-text · Article · Jan 2011 · Acta veterinaria
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    ABSTRACT: In all of the European Union countries where implementing fox oral vaccination is carried out rabies incidence has drastically changed. Some of them have already gained rabies free country status, while the others are getting close to this goal. In Slovenia the vaccine containing atenuated rabies virus, strain SAD B19 has been used since last five years. Foxes captured in the vaccination area, are tested for rabies incidence, bait withdrawal percentage and the presence of the anti-rabies antibodies. In the year 1995 the number of affected animals was 1089, this is 36.5 % of tested animals. In four years of oral vaccination this number has fallen to 6 cases (0,6 %). In period from 2007-2010, 1956 foxes older than 1 year were tested to biomarker presence and 1512 samples were found positive to the biomarker, which accounts for 77,7 % of bait uptaking fox. This result is the proof that the vaccination model used in Slovenia is a success.
    No preview · Article · Jan 2011 · Slovenian Veterinary Research
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    ABSTRACT: Molecular epidemiology studies allow us insight into the genetic diversity of the viruses detected in different farms and can be tool for tracking transmission of the virus between infected farms. Slovenia was free of PRRS disease until 2004. PRRS virus was detected for the first time in Slovenia in 2009. The molecular epidemiology study was performed on 60 PRRS viruses, which were detected in 48 different pig farms between 2009 and 2011, using the direct sequencing method and comparison of 258 nucleotides in a part of the ORF7 region of PRRS virus genome. High genetic diversity of PRRS viruses detected in infected herds was found. PRRS viruses from individual pig farms were classified into one of the nine identified genetic subgroups of genotype I (a, b, c, d, e, f, g, h) with 83.3 to 100% nucleotide identity to each other. According to the results of this study we can conclude that, by imports of live pigs from other countries, PRRS virus was often introduced in recent years. In 33 (68.7%) infected pig farms, almost genetically identical isolates of PRRS virus were found, but which was different from published strains in GenBank (with only 92% nucleotide identity to the closely related strain). Comparison of the nucleotide sequence of the PRRS viruses from 33 farms showed 96.9 to 100% identity to each other, confirming the successful transmission of this PRRS virus between infected farms. PRRS viruses, which have been demonstrated in our farms, are quite different (89.1 to 96.1% identity) compared to the reference strain of PRRS Lelystad (genotype I) and we suspect that after vaccination with commercial vaccines based on Lelystad virus the protection could be very poor.
    Full-text · Article · Jan 2011 · Slovenian Veterinary Research
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    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.
    Full-text · Article · Oct 2010 · Diseases of Aquatic Organisms
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    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 different 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 specific 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 specific 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.
    Full-text · Article · Mar 2010 · Zdravniški vestnik
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    Danijela Rihtaric · Peter Hostnik · Andrej Steyer · Joze Grom · Ivan Toplak
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    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.
    Full-text · Article · Mar 2010 · Archives of Virology
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    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.
    Full-text · Conference Paper · Feb 2010
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    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 .
    Full-text · Article · Jan 2010 · Zdravstveni vestnik