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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.

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.