Ivana Lojkić

Two outbreaks of neuropathogenic equine herpesvirus type 1 with breed-dependent clinical signs

Equine herpesvirus type 1 (EHV-1) is a worldwide spread pathogen of horses. It can cause abortion, respiratory and neurological disease and consequentially significant economic losses in equine industries. During 2009, two outbreaks of EHV-1 were confirmed in two stud farms in Eastern Croatia. The first outbreak occurred in February following the import of 12 horses from USA, serologically negative to EHV-1 before transport. Four mares aborted in the late stage of pregnancy and one perinatal death was recorded. Other six mares showed clinical signs of myeloencephalopathy with fatal end in four. One month later, the second EHV-1 outbreak was confirmed in stud farm about 100 km further with 17 abortions, three perinatal deaths and one mild neurological case. Epidemiological data showed that the disease was probably introduced in the first stud farm during international transport. The second outbreak started with the introduction of clinically healthy stallion from the first stud farm. Molecular characterisation and phylogenetic analysis confirmed that, despite different clinical signs, the identical virus caused both outbreaks. Both horse populations were free from EHV-1 infection before the outbreak and had not been vaccinated. Significant difference in clinical signs could be explained by different breed-related risk factors.

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.

Modification of the fluorescent antibody virus neutralisation test--elimination of the cytotoxic effect for the detection of rabies virus neutralising antibodies.

The virus neutralisation test is used for the quantitation of specific antibodies in serum samples. However, the success of the test depends on the quality of samples. In the case of poor quality samples, a cytotoxic effect can be observed and the results of the test can be compromised. Additionally, the cytotoxic effect limits the use of different substances, such as muscle extract or liquid from thoracic cavity (thoracic liquid), as a sample for the detection of rabies virus neutralising antibodies in the follow-up of fox oral vaccination campaigns. To eliminate the cytotoxic effect, a modified fluorescent antibody virus neutralisation (mFAVN) test was developed and evaluated. In the mFAVN test, inocula were removed after a 1h and the cytotoxic effect was prevented. According to the results obtained, the specificity of the mFAVN test compared to the FAVN test was 88.8% and the sensitivity was 94.4%. The diagnostic validity of the test was 0.99 (CI=0.98-1.00). To evaluate the possibility of using muscle extract and thoracic liquid as samples for the virus neutralisation test, 102 sera, muscle extract and thoracic liquid samples of dog origin were tested with the mFAVN test. The correlation between sera and muscle extracts was 87.9% (r=0.88, p<0.001). The correlation between sera and thoracic liquid was 94.2% (r=0.94, p<0.001). These findings indicated that both muscle extract and thoracic liquid could be used as samples for detection of rabies virus neutralising antibodies in the follow-up of oral vaccination campaigns. To evaluate the level of elimination of the cytotoxic effect, the 102 samples of sera, muscle extracts and thoracic liquid of dog origin were also tested in parallel using the mFAVN and FAVN tests. In the mFAVN test, no instance of cytotoxic effect was observed in the cells. In the FAVN test, two sera (1.9%), 35 muscle extracts (34.3%) and 56 thoracic liquid samples (54.9%) showed cytotoxic effect. The results of this study strongly suggest that cytotoxic effect can be eliminated completely from the rabies virus neutralising antibody detection tests used in the follow-up of oral vaccination campaigns and that very poor quality samples, such as muscle extract and thoracic liquid, can be used.

A pathological condition possibly caused by spontaneous trichotecene poisoning in Brahma poultry: first report

Trichotecene poisoning in poultry can cause oral lesions, haemorrhages, depletion and necrosis in the lymphopoetic organs and death. Spontaneous poisonings with these toxins are rarely described. This paper describes the spontaneous poisoning of two Brahma chickens with T-2 toxin, diacetoxyscirpenol and deoxynivalenol. Two out of 10 chickens died under signs of depression and loss of appetite. Histopathological analysis revealed vacuolar dystrophy of the liver, necrosis and depletion of lymphocyte in the bursa of Fabricius as well as multiple necroses in the glandular stomach and gut. Even though quantities of 0.70 mg/kg T-2 in the food together with 0.50 mg/kg diacetoxyscirpenol significantly differ from the median lethal dose for chickens reported in literature (4.97 mg/kg), parasitological, virological and histopathological results indicate trichotecenes as the causative agents of this pathological condition.

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.

Clinical rabies in cattle imported into Croatia

RABIES is a fatal zoonosis caused by the rabies virus (RABV) or rabies-related viruses (genus Lyssavirus ) and transmitted by the bite of a rabid animal (Dietzgen and others 2011). RABV has been eliminated from most of western and central Europe, but geographically associated classification of European RABVs to four main phylogenetic groups (Bourhy and others 1999) is still useful in inferring their phylogeny. In Croatia, rabies has been endemic since 1977, with the red fox ( Vulpes vulpes ) as the main disease reservoir. Although the disease is mostly perpetuated within the fox population, foxes occasionally transmit it to other wild and domestic species. In a previous study (Lojkic and others 2012), we demonstrated that only RABV strains from the eastern European (EE) group and the western European (WE) group are present in Croatia. Currently, a nationwide oral vaccination programme, which started in 2011 with financial help from the EU, is undertaken twice a year. Here, we report cases of clinical rabies on a beef …

Diversity of currently circulating rabies virus strains in Croatia.

Sylvatic rabies has been present in Croatia for more than three decades, with the red fox (Vulpes vulpes) as the main reservoir. The present epidemic of sylvatic rabies in Croatia started already in 1977 and in the past ten years the disease has become enzootic in the entire country and thus represents a considerable veterinary and public health threat. A genetic characterization and phylogenetic analysis of rabies virus isolates (RABV) from Croatia was performed using panel of 32 selected rabies-positive brain samples from domestic and wild animals collected between 2008 and 2010. Based on the comparison of 367-nucleotide sequences of a conserved region of the nucleoprotein (N) gene (nucleotides 75-441), the phylogenetic analysis revealed a low genetic diversity of currently circulating RABV strains in Croatia. 18 RABV isolates mainly originating from Eastern Croatia clustered with the formerly established Eastern European (EE) lineage, and the rest (14) were identical with the West European (WE) group. Both phylogenetic groups seem to coincide in central regions on both sides along the Save River. A high sequence identity in the N gene of the RABV isolates from neighbouring countries was found.