Zdenko Biđin

Detection and characterization of avian nephritis virus in ducklings

The first evidence of avian nephritis virus (ANV) in ducks is described. A diagnostic investigation was performed on three duck farms in Croatia. Samples from dead-in-shell ducklings and ducklings aged 30 days were collected and prepared for molecular and histopathological examination. Intestinal and liver samples were tested by polymerase chain reaction (PCR) for the presence of ANV, duck enteritis virus, duck hepatitis virus 1 and Derzsys disease virus. Multiple tissues were collected for histological examination and lesions were found to be confined to the kidney and intestine. Moderate focal interstitial and periglomerular mononuclear cell infiltrates (mostly lymphocytes and plasma cells) were detected in the kidney. The duodenum showed rather diffuse pericryptal mononuclear cell hyperplasia (lymphocytes) and fibroplasia. ANV was detected by PCR in all the intestinal samples, while no other viruses were found. Sequence comparisons of the portion of the open reading frame 1b encoding the RNA-dependent RNA polymerase gene confirmed that the virus detected and sequenced from ducklings shared high nucleotide and amino acid identities with ANV-1. Additional work is required to determine the clinicopathological significance of ANV infection in ducks.

Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos

The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsys disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.

Identification and Phylogenetic Diversity of Parvovirus Circulating in Commercial Chicken and Turkey Flocks in Croatia

SUMMARY. Phylogenetic diversity of parvovirus detected in commercial chicken and turkey flocks is described. Nine chicken and six turkey flocks from Croatian farms were tested for parvovirus presence. Intestinal samples from one turkey and seven chicken flocks were found positive, and were sequenced. Natural parvovirus infection was more frequently detected in chickens than in turkeys examined in this study. Sequence analysis of 400 nucleotide fragments of the nonstructural gene (NS) showed that our sequences had more similarity with chicken parvovirus (ChPV) (92.3%–99.7%) than turkey parvovirus (TuPV) (89.5%–98.9%) strains. Phylogenetic analysis grouped our sequences in two clades. Also, the higher prevalence of ChPV than TuPV in tested flocks was defined. The necropsy findings suggested a malabsorption syndrome followed by a preascitic condition. Further research of parvovirus infection, pathogenesis, and the possibility of its association with poult enteritis and mortality syndrome (PEMS) and runting and stunting syndrome (RSS) is needed to clarify its significance as an agent of enteric disease.

Circulation and phylogenetic relationship of chicken and turkey-origin astroviruses detected in domestic ducks (Anas platyrhynchos domesticus)

The natural occurrence of chicken and turkey-origin astroviruses in domestic ducks (Anas platyrhynchos domesticus) is described. Twenty-two duck flocks were covered by this research. The liver, spleen, kidney and intestines were sampled and tested by reverse-transcription polymerase chain reaction for the presence of avian nephritis virus (ANV), chicken astrovirus (CAstV), turkey astrovirus (TAstV)-1, TAstV-2 and duck astrovirus. The astrovirus infection was confirmed in multiple organ samples from 59.1% of tested flocks. CAstV was detected in one flock, TAstV-2 in three flocks and ANV in 10 flocks. The molecular and phylogenetic analysis of the small open reading frame (ORF) 1b fragment (130 nucleotides) of all chicken and turkey-origin astroviruses detected in ducks showed that ANV-sequence group was more distant from CastV, TAstV-1 and TAstV-2 sequences, which formed a separate, more related group. ANV sequences were divided into three subgroups, suggesting that several types of ANV were circulating in Croatian duck flocks. The comparison of the partial ORF 1b (254 nucleotides) duck ANV sequences with 21 ANVs detected in various avian species (chickens, turkeys, geese, guinea fowl and pigeons) revealed they shared the higher nucleotide (95.6 to 97.2%) and amino acid (98.8 to 100%) identity with two ANV-2-like sequences from chickens (GA-SEP-A451-05 and GA-CK-SEP ANV-364-2005). Phylogenetic neighbour-joining tree analysis based on the same nucleotide alignment, and performed using the Jukes-Cantor method, clustered the compared sequences into three groups. All analysed duck ANV sequences showed a close phylogenetic relationship with chicken-origin ANVs. Additional work is required to determine the significance and pathogenicity of chicken and turkey-origin astroviruses in domestic ducks.

Influence of environmental and nutritional stressors on yolk sac utilization, development of chicken gastrointestinal system and its immune status

The impact of practically all the everyday stress factors occurring in the first days of the chick life on the rate of the utilisation of the yolk sac content, on the chicken immune status and on the growth of the animals body and intestinal masses was investigated. The impact of the stress was investigated during the first five days of chicks life. The chicks were divided into five groups, each comprising 44 birds. The first chick group was exposed to moderate cold (2–3°C below the optimal temperature for the chick age). The second group was exposed to moderate heat (2–3 °C above the optimal temperature for the age). The third and fourth group were deprived of feed and drinking water for 12 hours and 24 hour, respectively. The fifth chick group was the control group: the birds in this group had optimal environmental conditions and received feed and drinking water immediately upon entering the trial. Ten chicks were taken every day and weighed. Their blood samples were obtained for the assessment of infectious bursal disease virus antibody titres using ELISA method. After that, the birds were sacrificed, their intestinal masses weighed and the quantity of resorbed yolk measured. The greatest body and intestinal masses were found in the control animals. The body and intestinal masses were the lowest in the group which was denied water and food for 24 hours. The resorption of the yolk sac content was approximately equal in all the chick groups, but the resorption rate varied by days. The highest resorption was observed in the chick groups on the day when feed was denied to them, so that this high resorption could be associated with the birds body energy requirements. The resorption of the remaining yolk is associated with feed intake: in chicks to which feed is withheld the yolk is resorbed through the yolk sac wall directly into the blood, which increased its usability. When energy requirements are not satisfied, the chicks body probably utilises resorbed antibodies from the yolk remnants, as their glycoprotein composition is suitable for this purpose.

Differentiation of Infectious Bursal Disease Viruses Isolated in Croatia

SUMMARY. Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996–2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732–1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI− CfoI−, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI− restrictions. Two SacI+ StuI+ CfoI+ TaqI− SspI− field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI− SspI− restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL® IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL® SPF vaccine could belong to the mild strains.