Current Status of Rabies and Its Eradication in Eastern and Southeastern Europe.

The objective of this paper is to provide an overview of the current status of rabies in Europe, with special emphasis on Croatia and Southeast and East Europe. Due to the systematic implementation of a rabies eradication program by oral vaccination of wild animals, by the end of the 20th century, most West and Central European countries were rabies-free. The EU goal was to eradicate rabies in wildlife and domestic animals by 2020. No matter how achievable the goal seemed to be, the disease is still present in the eastern part of the EU, as was notified in 2020 by two member states-Poland and Romania. Croatia has been rabies-free for the last seven years but given that it borders a non-EU country in which a case of rabies was confirmed in 2020, it will continue to contribute to the maintenance of the rabies-free region. A rabies-free EU can only be achieved by continuous oral vaccination, coordination and a regional approach. The prevention of reintroductions from bordering countries in which rabies has not been eradicated yet, and the support for the eradication efforts made by these countries, are goals still pending.

Viral Metagenomic Profiling of Croatian Bat Population Reveals Sample and Habitat Dependent Diversity.

To date, the microbiome, as well as the virome of the Croatian populations of bats, was unknown. Here, we present the results of the first viral metagenomic analysis of guano, feces and saliva (oral swabs) of seven bat species (Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum, Eptesicus serotinus, Myotis blythii, Myotis nattereri and Myotis emarginatus) conducted in Mediterranean and continental Croatia. Viral nucleic acids were extracted from sample pools, and analyzed using Illumina sequencing. The presence of 63 different viral families representing all seven Baltimore groups were confirmed, most commonly insect viruses likely reflecting the diet of insectivorous bats. Virome compositions of our samples were largely impacted by the sample type: invertebrate-infecting viruses were most frequently found in feces, bacterial viruses in guano, whereas vertebrate-infecting viruses were most common in swabs. Most vertebrate-infecting virus sequences were assigned to retroviruses, parvoviruses, iridoviruses, and poxviruses. We further report the complete genome sequence of a novel adeno-associated virus, densovirus and a near complete length genome sequence of a novel iflavirus. Additionally, one of the most interesting findings in this study was the difference in viromes between two contrasting habitats, the continental and Mediterranean Croatia.

Evaluation of Serological Tests for Detection of Antibodies against Lumpy Skin Disease Virus.

Lumpy skin disease (LSD) is an emerging, transboundary viral pox disease affecting cattle of all ages and breeds. The serological assay for monitoring immunity following vaccination is a virus neutralization test (VNT/OIE) that determines the neutralization index (NI). The first validated enzyme-linked immunosorbent assay (ELISA; IDVet) has become commercially available, facilitating large-scale serosurveillance for LSD. Although the VNT is labor intensive and time consuming, it is still the recommended test by the OIE. Thus, in this study, we modified the virus neutralization test by employing Madin-Darby bovine kidney (MDBK) cells. The qualitative results obtained with the modified method were compared to the qualitative results obtained by VNT/OIE and ELISA. We used blood sera received within a surveillance program for LSD in 2018. In total, 291 serum samples were tested using VNT/MDBK and ELISA. Of 291 samples, 80 samples were tested by VNT/OIE and used for comparison of the performances between VNT/MDBK and VNT/OIE. The compatibility of results obtained by VNT/MDBK and VNT/OIE resulted in a kappa index of 0.9 with overall proportion agreement of 0.96. Agreement between VNT/MDBK and VNT/OIE was achieved in 56 positive and 21 negative samples. The compatibility of results obtained by ELISA and VNT/MDBK were compared on 291 samples in total and resulted in a kappa index 0.834 with overall proportion agreement of 0.955. Agreement between ELISA and VNT/MDBK was achieved in 238 positive and 40 negative samples. The results obtained demonstrated a strong correlation between VNT/MDBK and the other two methods, indicating the suitability of VNT/MDBK for the detection of the LSD virus-specific neutralizing antibodies.

Influence of different factors on the costs and benefits of oral vaccination of foxes against rabies.

The oral rabies vaccination (ORV) programme is the most widespread vaccination programme that is implemented in the European Union and targets wildlife diseases. The size of the project requires significant financial resources, so a cost-benefit analysis is necessary to establish the economic value of the project. We summarized all ORV costs during the period 2011-2016 in Croatia, calculated all established benefits and presented the cost-benefit ratio (CBR). Additionally, we analysed all components included in the ORV and rabies control programme (surveillance, preventive human treatment and vaccination of dogs) to find possibilities to increase the benefits. According to our results, in the period 2011-2016, the CBR was only 0.05, and the majority of the cost was derived from the preventive vaccination of dogs (72.3%). With the implementation of 2- or 3-year vaccination intervals, the CBR can be increased to 1.46 or 1.92, respectively, confirming positive economic value. This study shows the importance of analysing all rabies control and eradication components based on the specific characteristics of a particular country to determine the factors that can be modified to potentially improve the benefits of ORV.

Novel Circo-Like Virus Detected in a Croatian Bat Population.

Viral metagenomics analysis of samples from bats has been carried out as part of bat rabies surveillance in Croatia. Here, we report the complete genome sequence of a novel circo-like virus isolated from a sample of Miniopterus schreibersii bat guano determined by Illumina next-generation sequencing.

Genetic and spatial characterization of the red fox (Vulpes vulpes) population in the area stretching between the Eastern and Dinaric Alps and its relationship with rabies and canine distemper dynamics.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

Control and elimination of rabies in Croatia.

Despite the implementation of control measures (preventive dog vaccination), rabies has become endemic in Croatia, with red foxes being the main reservoir species. Oral rabies vaccination (ORV) campaigns supported by the European Commission have been conducted twice a year since the spring of 2011. The first campaigns were limited to the northern and eastern parts of the country, and from the autumn of 2012, the program was extended to the entire country. The Lysvulpen vaccine containing the SAD Bern strain was used for ORV. Following the vaccination campaigns, the number of rabies cases decreased, and the last positive case was recorded in February 2014. The bait uptake ranged from 24.86% to 84.62% and the immunisation rate from 11.24% to 35.64%.

Genetic analysis of bovine respiratory syncytial virus in Croatia.

Bovine respiratory syncytial virus (BRSV) represents an important causative agent of respiratory tract disease in cattle. This study describes the genetic diversity of BRSV strains detected in beef cattle herds in Croatia during four consecutive years, from the end of 2011 to April 2016. Genetic diversity of circulating Croatian strains is reflected in their clustering within three different genetic subgroups. Analysis of representative BRSV G gene sequences revealed that infections in Croatia were caused by BRSV strains belonging to two new subgroups (VII and VIII identified herein for the first time). In 2014-2016, the subgroup VII strains were replaced with BRSV strains clustered in the previously unidentified subgroup VIII. Furthermore, co-circulation of subgroup II and new subgroup VIII strains in Croatia was recorded in the same time period. Sequences of Croatian BRSV strains within subgroups II and VII revealed unique mutations within an essential immunodominant region, demonstrating continuous evolution of viral mechanisms for immune escape.

Molecular and serological survey of lyssaviruses in Croatian bat populations.

BACKGROUND: Rabies is the only known zoonotic disease of bat origin in Europe. The disease is caused by species belonging to the genus Lyssavirus. Five Lyssavirus species, i.e., European bat lyssavirus (EBLV)-1, EBLV-2, Bokeloh bat lyssavirus, Lleida bat lyssavirus, and West Caucasian bat virus, have been identified in European bats. More recently, a proposed sixth species, Kotalahti bat lyssavirus, was detected. Thus, in this study, active surveillance was initiated in order to obtain insights into the prevalence of lyssaviruses in Croatian bat populations and to improve our understanding of the public health threat of infected bats. RESULTS: In total, 455 bats were caught throughout Continental and Mediterranean Croatia. Antibodies were found in 20 of 350 bats (5.71%, 95% confidence interval 3.73-8.66). The majority of seropositive bats were found in Trbusnjak cave (Continental Croatia, Eastern part), and most seropositive bats belonged to Myotis myotis (13/20). All oropharyngeal swabs were negative for the presence of Lyssavirus. CONCLUSIONS: The presence of lyssaviruses in bat populations was confirmed for the first time in Croatia and Southeastern Europe. The results of this study suggest the need for further comprehensive analyses of lyssaviruses in bats in this part of Europe.

Complete Genome Sequence of a Lumpy Skin Disease Virus Strain Isolated from the Skin of a Vaccinated Animal.

Vaccination of cattle against lumpy skin disease (LSD) can cause adverse reactions. Here, we report the first complete genome sequence of an LSD virus strain isolated from the skin of a vaccinated animal. We confirmed that the sequence of the virus used for vaccination remains stable.

Canine Adipose Derived Mesenchymal Stem Cells Transcriptome Composition Alterations: A Step towards Standardizing Therapeutic.

Although canine adipose derived stem cells (cASCs) morphology characteristics and differentiation ability are well documented, transcriptome alterations of undifferentiated cASCs during ex vivo cultivation remain unknown. Here we demonstrate, for the first time, the transcriptome composition of isolated cASCs in undifferentiated state originating from six donors. Transcriptome changes were monitored during ex vivo cultivation between passage 3 (P3) and P5, which are mostly used in therapy. Influence of donors' age in given passage number on transcriptome composition was also investigated. Cultivation from P3 to P5 resulted in 16 differentially expressed genes with cooverexpression of pluripotency and self-renewal transcription factors genes SOX2 and POU5F1 dominant in old donors' cells. Furthermore, cASCs demonstrated upregulation of IL-6 in young and old donors' cells. In addition, ex vivo cultivation of cASCs revealed well-known morphological alterations accompanied with decrease in expression of CD90 and CD44 markers in P4 and higher monitored by flow cytometry and successful osteo- and chondrodifferentiation but inefficient adipodifferentiation in P3. Our results revealed the impact of ex vivo cultivation on nature of cells. Correlation of transcriptome changes with secretome composition is needed and its further impact on therapeutic potential of cASCs remains to be evaluated in clinical trials.

Evaluation of ELISA for the detection of rabies virus antibodies from the thoracic liquid and muscle extract samples in the monitoring of fox oral vaccination campaigns.

BACKGROUND: The main goal of oral vaccination of foxes is eradication of rabies in the red fox population as rabies reservoirs. To evaluate the success of vaccination a serological testing is conducted as a part of monitoring program. Two different methods are used regarding rabies serology: virus neutralisation test and ELISA. METHODS: In this study the reliability of BioPro ELISA was evaluated for testing haemolytic thoracic liquids and muscle extracts originated from 147 foxes in comparison to mFAVN. Also, the influence of heat treatment of samples on test results was investigated. RESULTS: The specificity of the test for not-heat treated samples was 92.98% and sensitivity 79.20%. Diagnostic validity of the ELISA compared to the mFAVN test when not-heat treated samples were used was 89.16%. The specificity of the test for heat treated samples was 79.10% and sensitivity 96.36%. Diagnostic validity of the BioPro ELISA compared to the mFAVN test for heat treated samples was 94.30%. CONCLUSION: According to this study, the BioPro ELISA is reliable tool for detection of rabies specific antibodies in the context of evaluation of oral vaccination of foxes from poor quality samples as a substitution for virus neutralisation tests.

Faecal virome of red foxes from peri-urban areas.

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.

Detection and molecular characterisation of bovine corona and toroviruses from Croatian cattle.

BACKGROUND: Bovine coronavirus (BCoV) together with bovine torovirus (BToV), both members of the Coronaviridae family, order Nidovirales are the most common viral enteric pathogens. Although studied separately, their joint occurrence and the molecular diversity in cattle in Croatia have not been investigated. METHODS: A survey is carried out on 101 fecal samples from diarrheic young and adult cattle during the 3-year period from i) one large dairy herd, ii) four small herds and iii) three nasal and paired fecal samples from calves with symptoms of respiratory disease. Samples were submitted to RT-PCR and sequencing for BCoV Nucleocapsid gene, BCoV Spike gene and BToV Spike gene. RESULTS: BCoV was detected in 78.8 % of fecal samples from symptomatic cattle and three nasal and paired fecal samples from calves with respiratory symptoms. BToV was detected in 43.2 % of fecal samples from symptomatic cattle and a fecal sample from calves with respiratory symptoms. Molecular characterisation of those viruses revealed some nucleotide and aminoacid differences in relation to reference strains. CONCLUSIONS: BToV should be regarded as a relevant pathogen for cattle that plays a synergistic role in mixed enteric infections.

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.

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.

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.

Detection of Bovine viral diarrhea virus-specific neutralizing antibodies in fresh colostrum: a modification of the virus neutralization test.

To eliminate cytotoxic effects of colostrum on cells, a modified virus neutralization test (VNT) for the detection of Bovine viral diarrhea virus-specific neutralizing antibodies in colostrum was developed. The new test was compared to the World Organization for Animal Health-recommended VNT and the results evaluated. The agreement of the new test compared to the standard VNT was determined to be 98%, whereas sensitivity and specificity of the modified VNT compared to the standard VNT were 100%. Bovine viral diarrhea virus-specific antibodies were detected in 42 sera samples and 38 colostrum samples. The antibody titers in serum and colostrum showed a high correlation (n = 56, r = 0.9719, P < 0.001). The modified virus neutralization technique described herein succeeds in eliminating cytotoxic effects and can be readily applied for the detection of specific antibodies against other infectious agents in colostrum.

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.