Brigita Slavec

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR.

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.

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.

A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.

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.

Variation of vlhA gene in Mycoplasma synoviae clones isolated from chickens

Mycoplasma synoviae synthesizes haemagglutinin VlhA, which cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA. Previous studies have shown that the 3′-end of the expressed vlhA gene can recombine with vlhA pseudogenes in a process called gene conversion, but there have been no data about diversification of the expressed vlhA gene in M. synoviae populations replicating in chickens. Following intratracheal inoculation with the M. synoviae strain ULB 02/T6, which showed only minor vlhA gene variation prior to inoculation, we investigated temporal changes in MSPB epitopes defined by monoclonal antibodies (mAbs) 3B4 and 50, as well as diversification of the vlhA gene sequence in M. synoviae populations recovered from chicken tracheas. In cultures isolated 8 and 18 days post inoculation (p.i.), most colonies showed variation of MSPB epitopes for mAbs 3B4 and 50. They also changed 3′-end vlhA gene sequences. Further diversity of the vlhA gene occurred in cultures isolated 8 weeks and 5 months p.i. The vlhA gene sequences from isolated cultures shared only 65 to 80% sequence identity with vlhA gene of the inoculated ULB 02/T6 culture. Notably, in most of those cultures their vlhA gene sequences contained stop codons potentially causing premature terminations of translation. Interestingly, in one culture isolated 8 weeks p.i. (clone T6-8W/IT2A) the 3′-vlhA gene sequence was identical in the last 1140 bases to that of the first vlhA pseudogene positioned the most far (upstream) of the expressed vlhA gene. This is the first demonstration of temporal diversity of the vlhA gene in M. synoviae populations isolated from chicken tracheas.

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.