Uroš Krapež

Circulation of Infectious Bronchitis Virus Strains from Italy 02 and QX Genotypes in Slovenia Between 2007 and 2009

SUMMARY. Fourteen infectious bronchitis viruses (IBVs) detected in broiler, broiler breeder, and layer flocks in Slovenia between 2007 and 2009 were molecularly analyzed by reverse transcription–PCR and direct sequencing of the S1 gene. Phylogenetic analyses based on partial S1 gene sequences revealed that seven strains were classified as the Italy 02 genotype, a genetic group of IBV that has not previously been detected in Slovenia. Another seven strains were classified as the QX genotype. The results of phylogenetic analyses and epidemiologic investigations revealed that the genetic classification correlates with the geographic origins of the IBV strains. Greater genetic variability (maximum nucleotide and amino acid divergences were 4.8% and 9.9%, respectively) was observed within the Slovenian strains from the Italy 02 genotype detected between 2007 and 2009 than within strains from the QX genotype isolated in 2008 and 2009 (1.2% and 1.3%, respectively). The Slovenian strains classified as the Italy 02 genotype form a separate genetic cluster. These strains shared five specific amino acid substitutions that became fixed during the period of surveillance. Strains from the QX genotype that shared one specific amino acid substitution also form a separate genetic cluster.

Field efficacy of different vaccines against infectious bursal disease in broiler flocks.

A field study was performed to determine the efficacy of three commercially available vaccines against infectious bursal disease (IBD) in commercial broilers raised in a high IBD virus (IBDV) risk area. Live attenuated intermediate and intermediate plus vaccines were used in four flocks. Birds were vaccinated orally at the estimated vaccination time. Three broiler flocks were vaccinated subcutaneously with a turkey herpesvirus (HVT)-IBD vector vaccine at one day old. Evaluation of the efficacy of different vaccines was focused on humoral immune response, bursa/body weight (B/Bw) ratio, molecular detection of IBDV in ileocaecal tonsils and bursa of Fabricius, and production parameters. The serological results showed that although the uptake of all three vaccine strains was confirmed in the lymphoid organs, no significant antibody response to vaccination was detected in flocks vaccinated with intermediate and intermediate plus vaccines. A significant increase in antibody titres detected in flocks vaccinated with the vector vaccine indicated its ability to induce an immune response in birds with a high level of maternally derived antibodies. Observations obtained in this field trial did not confirm the expected reduction of the B/Bw ratio in flocks vaccinated with less attenuated vaccines. No significant differences were observed between birds vaccinated with the vector vaccine and those immunised with the intermediate plus vaccine. Very virulent IBDV was confirmed in the flock vaccinated with the intermediate vaccine. The infection induced reduced B/Bw and moderate mortality but did not affect the production parameters. Field infection was not detected in broilers vaccinated with the intermediate plus vaccine and the vector vaccine.

A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1.

Newcastle disease virus (NDV), also designated avian paramyxovirus type 1 (APMV-1), is a serious pathogen of poultry, causing highly contagious Newcastle disease (ND), with high morbidity or mortality, depending on the strain. Accordingly, rapid and reliable detection of APMV-1 and differentiation between vaccine and virulent strains is of crucial importance for ND diagnosis and plays an important role in effective control of the disease. In this study, two real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays using minor groove-binding (MGB) probes were developed for broad range detection and simultaneous pathotyping of APMV-1. The two assays were evaluated for their ability to detect in allantoic fluids viral RNA of all known APMV-1 lineages. Additionally, the applicability of the developed assays was assessed by the detection and pathotype prediction of APMV-1 in swabs and organs. The assays demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation ranging from 0.2% to 3.9% and from 0.6% to 7.2% for intra-assay and inter-assay variability, respectively. The results indicated the suitability of both assays as a complementary method for rapid screening and typing of APMV-1.

Surveillance of Influenza A Viruses in Wild Birds in Slovenia from 2006 to 2010

SUMMARY. Within the framework of the surveillance program for the early detection of H5 and H7 subtypes of avian influenza (AI) viruses, samples from 2547 wild birds of different species that were collected between 2006 and 2010 were examined by PCR-based methods. AI viruses of various subtypes were detected in 4.4% of birds from four different orders: Anseriformes, Ciconiiformes, Charadriiformes, and Pelecaniformes. Highly pathogenic avian influenza (HPAI) H5N1 viruses were detected only in 2006. HPAI H5N1 virus was confirmed in 1.9% of birds from four different species. Comparison of nucleotide sequences of the H5N1 hemagglutinin gene indicated that two different HPAI H5N1 viruses from the European–Middle Eastern–African clade 1 had been introduced into Slovenia, despite the relatively short duration of the HPAI outbreak. Low pathogenic avian influenza (LPAI) viruses were detected in 2.5% of birds during a 5-yr period. The subtypes H1, H2, H3, H4, H5, H7N7, H8, H10, H11, and H13N6 were determined in 18 out of 64 cases. The highest prevalence (81%) of LPAI viruses, including the H5 subtype, were found in birds sampled as a part of the “active” surveillance system.

Prevalence of Pigeon Circovirus Infections in Feral Pigeons in Ljubljana, Slovenia

SUMMARY. Pigeon circovirus (PiCV) was detected by real-time PCR in cloacal swabs, pharyngeal swabs, and serum samples taken from 74 feral pigeons (Columba livia var. domestica) that were caught at various locations in the city of Ljubljana, Slovenia. PiCV infections were detected in the majority of the tested birds. The highest (74.3%) detection rate was observed in the cloacal swabs and the lowest (31.1%) in serum samples. PiCV DNA was more readily detected in the cloacal swabs, pharyngeal swabs, and serum samples of birds younger than 1 yr. Molecular analysis of partial open reading frame V1 sequences showed that PiCV strains detected in feral pigeons share high nucleotide and amino acid sequence identities with PiCV strains detected in ornamental, racing, meat, and feral pigeons. RESUMEN. Reporte de Caso—Prevalencia de las infecciones asociadas con circovirus de las paloma en las palomas en libertad en Ljubljana, Eslovenia. El circovirus de las palomas (PiCV) fue detectado por PCR en tiempo real en hisopos cloacales, hisopos faríngeos y en muestras de suero tomadas de 74 palomas en libertad (Columba livia var. domestica) que fueron capturadas en distintos lugares de la ciudad de Ljubljana, Eslovenia. Se detectó la infección por el circovirus de las palomas en la mayoría de las aves analizadas. El porcentaje de detección más alto (74.3%) se observó en los hisopos cloacales y la más baja (31.1%) en muestras de suero. El ADN del circovirus de las palomas fue detectado más fácilmente en los hisopos cloacales, hisopos faríngeos y en muestras de suero de las aves menores de un año. El análisis molecular de las secuencias parciales del marco de lectura continua V1 mostró que las cepas de circovirus detectadas en las palomas en libertad comparten altas identidades en las secuencias de nucleótidos y de aminoácidos con las cepas de circovirus detectadas en palomas ornamentales, de competencia, productoras de carne y salvajes.

Inclusion body hepatitis (IBH) outbreak associated with fowl adenovirus type 8b in broilers

The causative agent of inclusion body hepatitis (IBH) was identified as fowl adenovirus (FAdV) type 8b, a member of the Fowl adenovirus E species, based on PCR results of adenoviral polymerase and the hexon gene in an outbreak of acute mortality that affected a broiler flock of 12,000 animals. In two waves of elevated mortality rate, a total of 264 chickens were found dead. Affected birds showed ruffled feathers, depression, watery droppings and limping. The most common pathological lesions seen on necropsy were pale, swollen and friable livers. On histological examination, acute hepatitis characterized by necrosis of hepatocytes, with large basophilic intranuclear inclusion bodies, were observed. In addition, infectious bursal disease virus and infectious bronchitis virus were detected in the same flock.

Molecular analysis of infectious bronchitis viruses isolated in Slovenia between 1990 and 2005: a retrospective study

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.

Evidence of avian influenza virus and paramyxovirus subtype 2 in wild-living passerine birds in Slovenia

A total of 670 cloacal swabs were taken from 37 species of wild-living passerine birds in years 2004, 2005, and 2006. The cloacal swabs were pooled into pools of three swabs for analysis. Isolation of avian influenza virus (AIV) and avian paramyxoviruses (APMV) was done on chicken embryos. One-step reverse-transcription polymerase chain reaction (RT-PCR) was used to detect AIV RNA. AIV nucleic acid was detected by RT-PCR in a sample of one common starling (Sturnus vulgaris). All attempts of AIV isolation from wild-living passerine birds’ samples were negative. APMV 2 was isolated in one robin (Erithacus rubecula) sample. In light of the presented results and literature data, passerines appear to play a minor role as potential disseminators of AIV and APMV; however, it seems that some wild-living passerine species could act as occasional carriers.

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

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.

Molecular Characterization of Avian Paramyxovirus Type 1 (Newcastle Disease) Viruses Isolated from Pigeons Between 2000 and 2008 in Slovenia

Abstract 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.