Canine Distemper Virus Infection in the Free-Living Wild Canines, the Red Fox (Vulpes vulpes) and Jackal (Canis aureus moreoticus), in Croatia.

The canine distemper virus (CDV), a paramyxovirus that is closely related to the human measles virus and rinderpest virus of cattle, is a highly contagious viral disease in dogs and wild carnivores worldwide. CDV represents a serious threat to domestic and wild animals, especially to the conservation of endangered wild carnivores. Our study aims to investigate the occurrence of CDV in free-living wild canines in Croatia. For this purpose, 176 red foxes and 24 jackal brain samples collected in the frame of the active surveillance of rabies during winter 2021/2022 were tested. This study provided the first comprehensive overview of the prevalence and spatial distribution of CDV in the wildlife of Croatia, including the molecular phylogenetic analysis of the H gene sequence of field CDV strains circulating in red fox and jackal populations of Croatia. The molecular characterization of hemagglutinin gene genomic regions confirmed the phylogenetic clustering of obtained sequences into the Europa 1 genotype. The obtained CDV red fox sequences were mutually very similar (97.60%). This study indicates the high genetic similarity of Croatian CDV red fox sequences and CDV red fox sequences from Italy and Germany, badger sequences from Germany, polecat sequences from Hungary, and dog sequences from Hungary and Germany.

Absence of Hepatitis E Virus (HEV) Circulation in the Most Widespread Wild Croatian Canine Species, the Red Fox (Vulpes vulpes) and Jackal (Canis aureus moreoticus).

Hepatitis E virus (HEV) can infect a wide range of domestic and wild animals, and the identification of new host species is reported successively worldwide. Nevertheless, its zoonotic potential and natural transmission, especially in wildlife remains unclear, primarily due to the discrete nature of HEV infections. Since the red fox (Vulpus vulpus) is the most widespread carnivore worldwide, and has been recognized as a potential HEV reservoir, its role as a potent host species is of increasing interest. Another wild canine species, the jackal （Canis aureus moreoticus）, is becoming more important within the same habitat as that of the red fox since its number and geographical distribution have been rapidly growing. Therefore, we have chosen these wild species to determine their potential role in the epidemiology and persistence of HEV in the wilderness. The main reason for this is the finding of HEV and a rather high HEV seroprevalence in wild boars sharing the same ecological niche as the wild canine species, as well as the risk of the spread of HEV through red foxes into the outskirts of cities, where possible indirect and even direct contact with people are not excluded. Therefore, our study aimed to investigate the possibility of natural HEV infection of free-living wild canines, by testing samples for the presence of HEV RNA and anti-HEV antibodies to gain better epidemiological knowledge of the disease. For this purpose, 692 red fox and 171 jackal muscle extracts and feces samples were tested. Neither HEV RNA nor anti-HEV antibodies were detected. Although HEV circulation was not detected in the tested samples, to our knowledge, these are the first results that include jackals as a growing and important omnivore wildlife species for the presence of HEV infection in Europe.

Circulation of SARS-CoV-Related Coronaviruses and Alphacoronaviruses in Bats from Croatia.

Bats are natural hosts of various coronaviruses (CoVs), including human CoVs, via an assumed direct zoonotic spillover or intermediate animal host. The present study aimed to investigate the circulation of CoVs in a bat colony in the Mediterranean region of Croatia. Guano and individual droppings from four bat species were sampled and tested with the E-gene sarbecovirus RT-qPCR, the pan-CoV semi-nested RT-PCR targeting the RdRp gene and NGS. Furthermore, bat blood samples were investigated for the presence of sarbecovirus-specific antibodies with the surrogate virus neutralization test (sVNT). The initial testing showed E-gene Sarebeco RT-qPCR reactivity in 26% of guano samples while the bat droppings tested negative. The application of RdRp semi-nested RT-PCR and NGS revealed the circulation of bat alpha- and betaCoVs. Phylogenetic analysis confirmed the clustering of betaCoV sequence with SARS-CoV-related bat sarbecoviruses and alpha-CoV sequences with representatives of the Minunacovirus subgenus. The results of sVNT show that 29% of bat sera originated from all four species that tested positive. Our results are the first evidence of the circulation of SARS-CoV-related coronaviruses in bats from Croatia.

SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish.

The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals.

Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT-PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

First evidence of hepatitis E virus infection in a small mammal (yellow-necked mouse) from Croatia.

Since the role of wild rodents/small mammals in hepatitis E virus (HEV) epidemiology has been a subject of considerable debate, this study was conducted to investigate the potential presence of HEV RNA in small rodents collected within their natural habitats and to detect if they can be potential reservoirs of the virus. A total of 483 small rodents were captured using snap traps placed at 11 regions in Croatia. Sampling was undertaken in 2008 and repeated from 2010 to 2014. Liver samples were tested for the presence of HEV RNA. HEV RNA was detected in only one liver sample (0.21%) originated from Apodemus flavicollis from the location Medvednica, nearby Zagreb collected in 2014. According to the sequence analysis, the isolate has shown to be a member of Orthohepevirus A species, genotype HEV-3. The genotyping results confirmed grouping into subtype 3a, general cluster 3abchij.The detected HEV strain showed to be genetically highly related to strains found in humans and/or domestic pigs and wild boars from Croatia. Our finding indicates that wild small mammals could play a role in the epidemiology of HEV-3 infection and therefore should be taken under consideration as potential reservoirs or/and transmitters of the disease. However, further investigation is needed to recognize their potential for maintaining the infection in natural conditions.

Genetic diversity of hepatitis E virus (HEV) strains derived from humans, swine and wild boars in Croatia from 2010 to 2017.

BACKGROUND: To fulfill epidemiological data and investigate possible interspecies transmission, this study shall attempt to sequence representative HEV strains of human, swine and wild boar origin collected from 2010 to 2017 in Croatia. METHODS: In total, 174 anti-HEV antibody positive human sera samples; 1419 blood or faeces samples of swine, as well as 720 tissue and/or blood samples of wild boar originating from different counties (18 in total) in Croatia were tested for the presence of HEV RNA. RESULTS: HEV RNA was detected in 26 human sera samples (14.9%; 95% CI 10.4-21.0%). HEV RNA was detected in 216 tested swine (15.2%; 95% CI 13.5-17.1%), regardless of age, farm breeding system or geographical origin. Viral RNA was also detectable in faeces samples which prove that swine actively participate in shedding HEV into the environment. Of the total of 720 tested wild boar samples, 83 were HEV RNA positive (11.5, 95% CI 9.4-14.1%) originating from six counties. According to the sequence analysis all strains have shown to be members of Orthohepevirus A genotype HEV-3, regardless of host. The genotyping results confirm grouping of sequences into four subtypes of HEV strains of which subtypes 3a and 3c belong to the general cluster 3abchij, and were predominately detected during the study, while subtypes 3e and 3f fall within cluster 3efg. Strains within subtypes 3a and 3e were found in humans, swine and wild boars; subtype 3c strains were derived from humans and swine, whereas subtype 3f strains were found only in humans. Strains belonging to subtypes 3a and 3c were derived during the entire investigated period and may be considered endemic in Croatia, whereas strains within subtypes 3e and 3f were detected sporadically indicating the possibility of newly imported infections. CONCLUSIONS: All detected strains show to be genetically highly related to strains found in humans and/or animals from other European Countries, indicating that trade of live animals or wild boar movement increases the risk of HEV infection spread. Furthermore, homologous strains found in different investigated species within this study indicate interspecies transmission of HEV and/or an existence of an accessible mutual source of infection.

The silent spread of Porcine Bocavirus in Croatian pigs: should we be concerned?

A survey was conducted to evaluate the presence and prevalence of Porcine Bocavirus (PBoV) in Croatian domestic pigs by means of PCR targeting the NS1 gene fragment of PBoV. This study included testing of faecal samples collected from 10 small commercial farms and 11 small backyard holdings in Croatia. The presence of PBoV was confirmed by PCR in 24 out of 57 composite faecal samples from small commercial farms and in 12 out of 43 composite faecal samples from small backyard holdings. The PCR products of 18 positive samples were sequenced for genotyping. PBoV sequences grouped into the PBoV-a, PBoV-b and PBoV-c groups with 90.81% to 99.25% nucleotide identity. All Croatian PBoV sequences showed a high nucleotide and amino acid identity with PBoV sequences from China and Hong Kong, the United States, Sweden, and Slovenia. These results clearly show that PBoV is circulating among the domestic pig population in Croatia.

First recorded case of paramyxovirus infection introduced into a healthy snake collection in Croatia.

BACKGROUND: In the present study, we describe the first paramyxovirus infection in a snake collection in Croatia caused by an introduction of new snakes that were not previously tested and didn't show any signs of disease. CASE PRESENTATION: In less than a month after introduction into a healthy colony, new snakes began to show respiratory symptoms (i.e. mouth opening, wheezing, etc.) and died within a month and a half after antibiotic therapy was applied. The same symptoms and a high mortality rate were then observed in in-contact snakes from other collections belonging to different snake families. CONCLUSIONS: Two entries of new snakes in different time periods were recorded and recognized as possible sources of infection. We stress the need for veterinary health control and monitoring of snakes prior to transportation as well as implementing obligatory quarantine measures to minimize the risk of infection among newly established snake groups.

Distribution and Molecular Characterization of Hepatitis E virus in Domestic Animals and Wildlife in Croatia.

Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5%) and wild boars (12.3%), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production.

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

Serological data imply that pseudorabies (Aujeszky's 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.

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.

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.

BACKGROUND: Porcine 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. RESULTS: PCV2 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. CONCLUSION: The 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.

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.

The wild boar attack--a case report of a wild boar inflicted injury and treatment.

Croatia is a relatively safe country in regards to wild animal attacks and trauma to humans, even though there are a few reported cases of wild animal attacks on humans almost every year. As a biodiversity hot-spot it is inhabited by a few wild animal species that are known to attack humans and cause serious, sometimes even fatal injuriCs to humans throughout the world, such as wolves, brown bears and wild boars. We present a case of a recent wild boar attack on a human--a hunter that occurred in central Croatia in the year 2012. The injured person was a part of a group of hunters involved in a drive hunt on wild boars. He sustained a 4 cm long laceration of the left knee by a wounded male wild boar. After the surgical and antibiotic treatment he recovered completely and without any complications.

Wildlife-vehicle collisions in Croatia--a hazard for humans and animals.

Wildlife-vehicle collisions (WVC) have increased and now there is a world-wide problem related to significant mortality of wildlife, habitat fragmentation, change in behavior and even disappearance of local endangered populations. Along with these deleterious effects on wildlife, WVC can also result in injuries and deaths of humans. During the three-year monitoring, a total of 7,495 wildlife-vehicle collisions were recorded, including mainly roe deer (73%), while other species were less frequently affected (wild boar--9%; brown hare--5%; and red deer and pheasant each with 4%). Incidence of wildlife-vehicle collisions were observed according to territorial distribution, seasonal and daily occurrence and type of road (total and per 1 km).

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 it's 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.

Prevalence of antibodies to selected viral pathogens in wild boars (Sus scrofa) in Croatia in 2005-06 and 2009-10.

We determined prevalence of antibody to selected viral pathogens important for domestic pigs and livestock in 556 wild boar (Sus scrofa) sera collected during 2005-06 and 2009-10 in four counties in Croatia. These counties account for an important part of the Croatian commercial pig production and have a high density of wild boars. Samples were tested for antibodies to porcine parvovirus (PPV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2), swine influenza virus, porcine respiratory and reproductive syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, and swine vesicular disease virus (SVDV). Antibodies to all of the infectious pathogens except SVDV were detected. There was a statistically significant difference in prevalence between the two periods for PPV, ADV, PCV2, PRRSV, and PRCV, with a higher prevalence of PPV and ADV in the 2009-10 period (P<0.05). During the same period, the prevalence of PCV2, PRRSV, and PRCV was lower (P<0.05). Our results provide information on the current disease exposure and health status of wild boars in Croatia and suggest that wild boars may act as a reservoir for several pathogens and a source of infection for domestic pigs and other livestock as well as humans, especially for ADV.

The centenary progress of molecular genetics. A 100th anniversary of T. H. Morgan's discoveries.

A century ago, Thomas Hunt Morgan, the American scientist, studied the cytogenetic changes of drosophila and came to cytogenetic explanation of Mendel's basic laws of genetic heredity. These studies resulted in today's Mendel-Morgan chromosomal theory of heredity. On the occasion of the hundredth anniversary of this important discovery the authors have decided to give a review of the most significant achievements in the field of molecular genetics until the completion of the Human Genome Project. The most important points concerning the technology of DNA recombination and genetic engineering are also presented. The final section discusses the significance of previous achievements of molecular genetics in biomedicine and other related fields. There is also a tabular presentation of the sequence of the most important findings in the field of molecular genetics through time.

Malformation of the permanent maxillary canine after dental infraction in a wild boar (Sus scrofa L.).

Intra-alveolar infraction and malformation of the apical part of the permanent left maxillary canine in an adult male wild boar (Sus scrofa L.) is documented. The infraction and its sequelae produced an irregular mass of reparative tissue that led to the formation of two growing ends of the tooth.