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A comparison between a rare old breed and a common breed of ovine prion protein (PrP) genotype profiles was performed in 3 Romanian sheep flocks. The total number of animals used in the research was 698 sheep, of which 53 were Merino breed (MB) rams, 12 were Tigaie with Black Head breed (TBHB) rams, 340 were MB ewes, and 293 were TBHB ewes. The PrP genotype was investigated by identification of codons 136, 154, and 171 and by gene sequencing between codons 101 and 227. All sheep groups contained the ARR, AHQ, ARH, ARQ, and VRQ haplotypes, with frequencies ranging from 1.37% (ARH in TBHB ewes) to 51.71% (ARQ in TBHB ewes). TBHB had a low prevalence of AHQ (2.05% in ewes, 4.17% in rams), ARH (1.37% in ewes, 4.17% in rams), and VRQ (1.88% in ewes, 8.33% in rams). The highest haplotype in TBHB was ARQ (51.71% in ewes, 37.50% in rams). ARQ was the most frequent haplotype in the MB (49.41% in ewes, 42.45% in rams). MB had a low prevalence of AHQ (3.24% in ewes, 6.60% in rams), ARH (1.47% in ewes, 5.66% in rams), and VRQ (3.53% in ewes, 9.43% in rams). The results indicated that 66.66% of TBHB rams and 68.26% of TBHB ewes have scrapie-resistant genotypes, despite the absence of a genetic selection program for scrapie. The proportion of scrapie resistant alleles was higher in the TBHB rams than in the MB rams.

Onchocerca lupi is a filarial nematode, which infects the scleral conjunctival tissue of dogs, wolves and cats. Whilst adult nematodes localize in the conjunctive tissue of sclera or in the retrobulbar, microfilariae are found in the skin, and they are rarely diagnosed in asymptomatic animals. Since the first report of human ocular infection 5 years ago, up to 10 zoonotic cases have been identified in patients worldwide. We report, for the first time in Romania, three cases of canine ocular onchocercosis in dogs. Fragments of the harvested worms were characterized morphologically and molecularly. This article expands knowledge on the distribution of this parasite in Eastern Europe and sounds an alarm bell for ophthalmologists about the possible occurrence of human cases of O. lupi infection.

Rabies has been present in Romania since antiquity and remains a continual threat to veterinary and public health. The current epizootic of rabies in Romania is fox mediated. However, little is known about the genetic diversity among rabies virus isolates from this part of the Balkan region. In this study, we analyzed a cohort of 64 rabies viruses isolated from infected animals in Romania collected between 2005 and 2008. Partial nucleoprotein sequence data (322 bp) were obtained and compared to a panel of available sequence data of Eurasian classical rabies virus (genotype 1) isolates. The Romanian sequences were identified as belonging to the cosmopolitan lineage of rabies viruses that have been reported throughout the world. The sequences showed a high degree of heterogeneity and six different lineages were identified. Single isolates clustered with the formerly established Eastern European group, the North-Eastern European group or with isolates from the Southwest of Russia. However two lineages were unique to Romania. This sequence diversity is unprecedented in European countries and could be a result of trans-border movement or remnants of previous epidemic waves.

Anaplasma platys was first identified and described in North America as a Rickettsia-like, platelet-specific organism in dogs with infectious canine cyclic thrombocytopenia. In Europe, A. platys has so far mainly been described for some Mediterranean countries. Here, we describe a case of A. platys infection in a dog from Romania, confirmed by PCR. Additionally, the dog had a co-infection with Hepatozoon canis. To the best of our knowledge, this is the first case of A. platys infection in Romania and the first case of a co-infection with A. platys and H. canis altogether. Both pathogens should be considered as possible disease agents in dogs suffering from disease associated with tick bite in south-eastern Europe.

This study reports the first incursion into European poultry of H5N1 highly pathogenic notifiable avian influenza A (HPNAI) viruses from clade 2.3.2 that affected domestic poultry and wild birds in Romania and Bulgaria, respectively. Previous occurrences in Europe of HPNAI H5N1 in these avian populations have involved exclusively viruses from clade 2.2. This represents the most westerly spread of clade 2.3.2 viruses, which have shown an apparently expanding range of geographical dispersal since mid-2009 following confirmation of infections in wild waterfowl species in Mongolia and Eastern Russia. During March 2010, AI infection was suspected at post-mortem examination of two hens from two backyard flocks in Tulcea Country, Romania. HPNAI of H5N1 subtype was confirmed by reverse transcription polymerase chain reaction (RT-PCR). A second outbreak was confirmed 2 weeks later by RT-PCR, affecting all hens from another flock located 55 km east of the first cluster. On the same day, an H5N1 HPNAI virus was detected from a pooled tissue sample collected from a dead Common Buzzard found on the Black Sea coast in Bulgaria. Detailed genetic characterization of the haemagglutinin gene revealed the cleavage site of the isolates to be consistent with viruses of high pathogenicity belonging to clade 2.3.2 of the contemporary Eurasian H5N1 lineage. Viruses from a clade other than 2.2 have apparently spread to wild birds, with potential maintenance and spread through such populations. Whilst the scale of threat posed by the apparent westward spread of the clade 2.3.2 viruses remains uncertain, ongoing vigilance for clinical signs of disease as part of existing passive surveillance frameworks for AI, and the prompt reporting of suspect cases in poultry is advised.

Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs' greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic.

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing. This work was performed in the frame of EU-funded Network of Excellence for Epizootic Disease Diagnosis and Control (EPIZONE, contract FOOD- CT-2006-016236, www.epizone-eu.net) and partially through the German federal ministry for education and research (BMBF, grant 01KI1016A, www.bmbf.de). Gratefully acknowledged is the support through the OIE funded laboratory twinning project on rabies between Friedrich-Loeffler-Institute and EVCCRI (File Ref: GKB/KH2009/22, wwwoie.int). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.