Jelena Prpić

Porcine astrovirus viremia and high genetic variability in pigs on large holdings in Croatia.

Astroviruses are emerging viral agents, primarily enteropathogenic in mammals, but recently have been acknowledged to have extra-intestinal implications in humans and mink. Porcine astrovirus is thought to be widely distributed and highly prevalent among pigs, nevertheless its clinical significance remains doubtful as it can be detected in diarrheic as well as in healthy pigs. Recent reports imply the immense genetic variability among porcine astrovirus strains with five distinct lineages being characterized so far. Herein, we report porcine astrovirus circulation in the blood of healthy pigs in different age categories bred on two large industrial holdings in Croatia, with viral RNA seroprevalence of 3.89%. These are the first extra-intestinal findings of astrovirus in pigs, indicating a more complex pathogenesis than previously thought. Partial polymerase sequences of serum-derived strains provisionally clustered into porcine astrovirus lineages 2 and 4, sharing high genetic identity with previously described porcine astrovirus strains. The results were supported by detecting porcine astrovirus strains in composite fecal samples, regardless of pig category or holding tested. Phylogenetic analysis of derived strains suggested the presence of porcine astrovirus lineages previously detected in pig sera with an additional highly genetically divergent lineage 5, reported for the first time in Europe. Moreover, the existence of possible sub lineages should not be excluded. The results obtained in the present study, contribute to knowledge of porcine astrovirus pathogenesis; even though its possible clinical significance remains unclear. High fecal prevalence accompanied with vast genetic diversity on a relatively confined area, underscores the importance of pigs as porcine astrovirus reservoirs with eventual recombination events as a possible outcome.

Detection and genetic characterization of tick-borne encephalitis virus (TBEV) derived from ticks removed from red foxes (Vulpes vulpes) and isolated from spleen samples of red deer (Cervus elaphus) in Croatia.

Tick-borne encephalitis (TBE) is a growing public health concern in central and northern European countries. Even though TBE is a notifiable disease in Croatia, there is a significant lack of information in regard to vector tick identification, distribution as well as TBE virus prevalence in ticks or animals. The aim of our study was to identify and to investigate the viral prevalence of TBE virus in ticks removed from red fox (Vulpes vulpes) carcasses hunted in endemic areas in northern Croatia and to gain a better insight in the role of wild ungulates, especially red deer (Cervus elaphus) in the maintenance of the TBE virus in the natural cycle. We identified 5 tick species (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis punctata, Dermacentor reticulatus, Rhipicephalus sanguineus) removed from 40 red foxes. However, TBE virus was isolated only from adult I. ricinus and I. hexagonus ticks showing a viral prevalence (1.6%) similar to or higher than reported in endemic areas of other European countries. Furthermore, 2 positive spleen samples from 182 red deer (1.1%) were found. Croatian TBE virus isolates were genetically analyzed, and they were shown to be closely related, all belonging to the European TBE virus subgroup. However, on the basis of nucleotide and amino acid sequence analysis, 2 clusters were identified. Our results show that further investigation is needed to understand the clustering of isolates and to identify the most common TBE virus reservoir hosts in Croatia. Sentinel surveys based on wild animal species would give a better insight in defining TBE virus-endemic and possible risk areas in Croatia.

High prevalence and genetic heterogeneity of porcine astroviruses in domestic pigs

Porcine astroviruses (PAstVs) are common and genetically diverse viruses of domestic pigs. In the present study, the prevalence of PAstV in healthy domestic pigs in Croatia was 89%. Phylogenetic analysis revealed genetic heterogeneity among PAstV sequences; five lineages were detected, with PAstV-2 being predominant, while PAstV-3 was detected for the first time outside North America.

Analytical methods used for the authentication of food of animal origin

Since adulteration can have serious consequences on human health, it affects market growth by destroying consumer confidence. Therefore, authentication of food is important for food processors, retailers and consumers, but also for regulatory authorities. However, a complex nature of food and an increase in types of adulterants make their detection difficult, so that food authentication often poses a challenge. This review focuses on analytical approaches to authentication of food of animal origin, with an emphasis put on determination of specific ingredients, geographical origin and adulteration by virtue of substitution. This review highlights a current overview of the application of target approaches in cases when the compound of interest is known and non-target approaches for screening issues. Papers cited herein mainly concern milk, cheese, meat and honey. Moreover, advantages, disadvantages as well as challenges regarding the use of both approaches in official food control but also in food industry are investigated.

Characterisation of pseudorabies virus in domestic pigs and wild boars in Croatia.

Serological data imply that pseudorabies (Aujeszkys disease) is present in domestic pigs and wild boars in Croatia. Therefore, this study included testing of brain tissue samples collected from 200 domestic pigs and 105 wild boars originating from seventeen districts of Croatia. The presence of pseudorabies virus (PrV) DNA was confirmed in samples originating from six domestic pigs (3%) and one wild boar (0.95%). Positive samples were sequenced and analysed on the basis of a gC genome fragment. PrV strains have shown to be genetically identical and they are strongly related to some representative strains in the relatively heterogeneous Clade A. The results clearly show that PrV is still circulating among the domestic pig population in Croatia. Furthermore, the presence of pseudorabies virus in wild boars underlines the importance of this species as a PrV reservoir. Continued surveillance is necessary to track the viral spread in order to achieve final eradication of the disease.

Faecal virome of red foxes from peri-urban areas

Abstract Red foxes (Vulpes vulpes) are the most abundant carnivore species in the Northern Hemisphere. Since their populations are well established in peri-urban and urban areas, they represent a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. In this study, we evaluated the faecal virome of juvenile and adult foxes from peri-urban areas in central Croatia. The dominating mammalian viruses were fox picobirnavirus and parvovirus. The highest number of viral reads (N =1412) was attributed to a new fox circovirus and complete viral genome was de novo assembled from the high-throughput sequencing data. Fox circovirus is highly similar to dog circoviruses identified in diseased dogs in USA and Italy, and to a recently discovered circovirus of foxes with neurologic disease from the United Kingdom. Our fox picobirnavirus was more closely related to the porcine and human picobirnaviruses than to known fox picobirnaviruses.

The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity.

Genetic heterogeneity of RNA populations influences virus pathogenesis, epidemiology and evolution. Therefore, accurate information regarding virus genetic structure is highly important for both diagnostic and scientific purposes. For the Hepatitis E virus (HEV), the causal agent of hepatitis in humans, the intra-host population structure has been poorly investigated, mainly using the less sensitive RFLP-based approach. The objective of this study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in genetic variation research, for the characterization of HEV quasispecies. The analysis was conducted on 50 clones of five swine isolates and 30 clones of three human HEV isolates. To identify and quantify the sequence variants present in each HEV isolate, 348bp long fragments of the amplified conserved ORF2 region were separated by cloning. Ten clones per isolate were subjected to SSCP and sequenced in a parallel experiment. The results show a high correlation of SSCP haplotype profiling with the sequencing results, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies.

Phylogenetic comparison of porcine circovirus type 2 (PCV2) and porcine reproductive respiratory syndrome virus (PRRSV) strains detected in domestic pigs until 2008 and in 2012 in Croatia

BackgroundPorcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) have been present for the last 2 decades in Croatia, causing large economical losses in the pig production. The clinical features of the infections are mostly manifested by the development of respiratory problems, weight loss and poor growth performance, as well as reproductive failure in pregnant sows. Even though the infections are continuously recognized in some regions in Croatia, the heterogeneity of the detected viral strains from 2012 has not yet been investigated. The objective of this study was to compare virus strains of PCV2 and PRRSV detected until 2008 in Croatia with strains isolated in 2012 to gain a better epidemiological understanding of these two infections.ResultsPCV2 and PRRSV strains detected in 2012 in fattening pigs from regions where these two diseases have been previously described were compared to strains that have been detected in the same regions within the past two decades. The phylogenetic analysis revealed that the circulating PCV2 and PRRSV strains are distantly related to the previously described Croatian viral strains. However, when compared to known isolates from the GenBank a high genetic identity of PRRSV isolates with isolates from Hungary, Denmark and the Netherlands was found.ConclusionThe results of this study reveal that even though PCV2 and PRRSV are constantly present in the investigated regions in Croatia, the viral strains found in 2012 genetically differ from those detected in earlier years. This indicates that new entries into the pig population appeared with regard to both infections, probably as a result of pig trade.

Pseudorabies in hunting dogs in Croatia with phylogenetic analysis of detected strains

Pseudorabies virus (PrV) can persist within the wild boar population allowing them to be potential reservoirs of PrV for domestic pigs but also other animal species including hunting dogs. This article presents a case report of PrV infection in two hunting dogs from different central Croatian districts. Clinically, symptoms regarding the CNS and automutilation predominated in both animals, following their death. Histological evidence of perivascular infiltration of brain tissue, positive cytopathological changes after inoculation into cell culture and PCR confirmed the presence of PrV. Further sequencing of PrV gC genome fragment confirmed the presence of Clade A genotype, similar to strains derived previously from domestic pigs and wild boars in Croatia. The first description of pseudorabies in hunting dogs in Croatia confirming the possibility of viral transmission through contact from wild boars to hunting dogs has been presented.

Preliminary serological and molecular investigation of selected viral pathogens in Croatian cervid species

A total of 131 blood samples and 175 spleen samples were collected from three cervid species: roe deer (Capreolus capreolus), red deer (Cervus elaphus) and fallow deer (Dama dama) inhabiting the continental part of Croatia. Serum samples were tested for antibodies against bovine herpesvirus 1, parainfl uenza-3 virus, bluetongue virus, bovine respiratory syncytial virus, hepatitis E virus, bovine viral diarrhoea virus and enzootic bovine leukosis virus. The tested sera were negative for bovine viral diarrhoea virus, enzootic bovine leukosis virus, bluetongue virus, bovine respiratory syncytial virus and hepatitis E virus antybodies. The antibody prevalence in roe deer and red deer samples was 21.11% for bovine herpesvirus 1 and 75.55% for parainfl uenza-3 virus. Sera from bovine herpesvirus 1 positive animals were subsequenty tested with comparative virus neutralization test and bovine herpesvirus 1 neutralising antibodies were found in 18 (out of 19) sera. In fallow deer, no antibodies against any of the viral pathogens were detected. All spleen samples tested for bovine viral diarrhoea virus and enzootic bovine leukosis virus came back negative, except for one red deer spleen sample found to be weakly diarrhoea virus-positive. Our fi ndings provide the fi rst information on the exposure of Croatia-inhabiting cervid species to viral pathogens, and could serve as valuable baseline data for future investigations regarding deer exposure to various pathogens and the distribution of diseases shared between wildlife and livestock. As of now, the epidemiology of these viruses in the Croatian cervid population has been only poorly understood, so that further research is recommended.