TRICHINELLA INFECTIONS IN RED FOXES (VULPES VULPES) AND GOLDEN JACKALS (CANIS AUREUS) IN SIX DISTRICTS OF SERBIA.

Wild animals, including red foxes (Vulpes vulpes) and golden jackals (Canis aureus), are the most important reservoirs of Trichinella spp. Although the red fox is considered one of the main reservoirs of Trichinella spp. in Europe, only a few animals have been examined in Serbia. The present study assessed Trichinella spp. infection in red foxes and golden jackals from the six districts in Serbia. Thirty-seven carcasses of red foxes and 13 carcasses of golden jackals shot during the official hunting season were examined. Larvae of Trichinella spp. were detected in 13 (35%) of 37 red foxes and in 8 (61%) of 13 golden jackals. Trichinella spiralis and Trichinella britovi were the only two species identified after a multiplex polymerase chain reaction analysis. Trichinella britovi infection was detected in 85% of red foxes and in 38% of golden jackals, and T. spiralis was detected in 15% of red foxes and in 63% of golden jackals. The findings emphasize the need for an active surveillance program for Trichinella spp. infection in wildlife in Serbia and the whole of the Balkans, with special attention on the red fox because it is widespread and occurs in high densities.

American Foulbrood-Old and Always New Challenge.

American foulbrood (AFB) is exclusively an infectious disease of honey bee larvae (Apis mellifera) and their subspecies that is spread easily and rapidly and is often present in apiaries. Due to the resistance and pathogenicity of the bacterial causative agent of the disease, which has considerable epizootiological and economic significance for beekeeping, AFB was classified as a highly dangerous, infectious animal disease by the World Organization for Animal Health (WOAH). Considering the severity of the infection, a frequent occurrence, rapid and easy spread, epizooty and enzooty are common. We tried to present an overview of the latest information related to AFB through several chapters. In addition to the latest data on the etiology of the causative agent, the most important elements of the clinical signs of the disease are also listed. Along with an overview of classic microbiological and the latest molecular methods of diagnosis, we also discuss AFB treatment from its differential diagnostic aspect. We hope that through demonstrating the mentioned preventive measures and measures of good beekeeping practice, the review will contribute to the preservation of the health of bees and therefore the overall biodiversity of the planet.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Whole-genome sequencing of African swine fever virus from wild boars in the Kaliningrad region reveals unique and distinguishing genomic mutations.

Introduction: Since the first report of outbreaks of African swine fever (ASF) in Georgia in 2007, the disease has expanded into Europe, Russia, and Asia, spreading rapidly via contact with infected animals including domestic pigs and wild boars. The vast expansion of this Genotype II African swine fever virus (ASFV) across wide-ranging territories and hosts inevitably led to the acquisition of novel mutations. These mutations could be used to track the molecular epidemiology of ASFV, provided that they are unique to strains restricted within a certain area. Whilst whole-genome sequencing remains the gold standard for examining evolutionary changes, sequencing of a single locus with significant variation and resolution power could be used as a rapid and cost-effective alternative to characterize multiple isolates from a single or related outbreak. Material and methods: ASFVs obtained during active ASF outbreaks in the Russian region of Kaliningrad between 2017 and 2019 were examined. Since all of the viruses belonged to Genotype II and no clear differentiation based on central variable region (CVR) sequencing was observed, the whole-genome sequences of nine ASFV isolates from this region were determined. To obtain insights into the molecular evolution of these isolates, their sequences were compared to isolates from Europe, Asia, and Africa. Results: Phylogenetic analysis based on the whole-genome sequences clustered the new isolates as a sister lineage to isolates from Poland and Germany. This suggests a possible shared origin followed by the addition of novel mutations restricted to isolates from this region. This status as a sister lineage was mirrored when analyzing polymorphisms in MGF-505-5R and MGF-110-7L, whilst a polymorphism unique to sequences from Kaliningrad was identified at locus K145R. This newly identified mutation was able to distinguish the isolates obtained from Kaliningrad with sequences of Genotype II ASFVs available on GenBank. Discussion: The findings of this study suggest that ASFVs circulating in Kaliningrad have recently obtained this mutation providing an additional marker to the mutations previously described.

Validation of TaqMan-Based Assays for Specific Detection and Differentiation of Wild-Type and Neethling Vaccine Strains of LSDV.

Lumpy skin disease (LSD) is an important animal disease with significant health and economic impacts. It is considered a notifiable disease by the OIE. Attenuated strains of LSDV have been successfully used as vaccines (LAV) but can also produce mild or systemic reactions. Vaccination campaigns using LAVs are therefore only viable if accompanying DIVA assays are available. Two DIVA qPCR assays able to distinguish Neethling-based LAVs and wild-type LSDV were developed. Upon validation, both assays were shown to have high sensitivity and specificity with a diagnostic performance comparable to other published DIVA assays. This confirmed their potential as reliable tools to confirm infection in animals during vaccination campaigns based on Neethling vaccine strains.

Intensive West Nile Virus Circulation in Serbia in 2018-Results of Integrated Surveillance Program.

The results of the Serbian national integrated West Nile virus (WNV) surveillance program conducted in 2018 and funded by the Serbian Veterinary Directorate are presented. The WNV surveillance program encompassed the entire territory of Serbia and was conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the program was early detection of WNV circulation in the environment and timely reporting to the public health service and local authorities to increase clinical and mosquito control preparedness. The program was based on the detection of WNV presence in wild birds (natural hosts) and mosquitoes (virus vectors) and on serological testing of sentinel horses (WNV-specific IgM antibodies). The season 2018 was confirmed to be the season of the most intensive WNV circulation with the highest number and severity of human cases in Serbia ever reported. The most intense WNV circulation was observed in the northern and central parts of Serbia including Vojvodina Province, the Belgrade City area, and surrounding districts, where most positive samples were detected among sentinel animals, mosquitoes and wild birds. The majority of human cases were preceded by the detection of WNV circulation during the surveillance. The WNV surveillance program in 2018 showed satisfactory results in the capacity to indicate the spatial distribution of the risk for humans and sensitivity to early detection of WNV circulation in the environment.

Twenty-five-year study of Nosema spp. in honey bees (Apis mellifera) in Serbia.

A total of 7386 samples of adult honey bees from different areas of Serbia (fifteen regions and 79 municipalities) were selected for light microscopy analysis for Nosema species during 1992-2017. A selection of honey bee samples from colonies positive for microsporidian spores during 2009-2011, 2015 and 2017 were then subjected to molecular diagnosis by multiplex PCR using specific primers for a region of the 16S rRNA gene of Nosema species. The prevalence of microsporidian spore-positive bee colonies ranged between 14.4% in 2013 and 65.4% in 1992. PCR results show that Nosema ceranae is not the only Nosema species to infect honey bees in Serbia. Mixed N. apis/N. ceranae infections were detected in the two honey bee samples examined by mPCR during 2017. The beekeeping management of disease prevention, such as replacement of combs and queens and hygienic handling of colonies are useful in the prevention of Nosema infection.

Neethling vaccine proved highly effective in controlling lumpy skin disease epidemics in the Balkans.

Despite the wide use of the live attenuated Neethling lumpy skin disease (LSD) vaccine, only limited data existed on its efficacy and effectiveness prior to the large LSD epidemic in the Balkans, which took place during 2016-2017. In addition, analysis of risk factors for the disease was hardly performed with proper control for vaccination effects and potential differences in exposure to the virus. Data from the LSD epidemics in six Balkan countries (Bulgaria, Greece, Serbia, Montenegro, Former Yugoslav Republic of Macedonia (FYROM) and Albania) affected during 2016 were analyzed to determine vaccine effectiveness (VE) and risk factors for LSD infection at the farm level. Vaccination was performed along the occurrence of the epidemics and thus vaccination status of some of the farms changed during the epidemic. To allow for this, left truncated and right censored survival analysis was used in a mixed effects Cox proportional hazard regression model to calculate VE and risk factors for LSD. The results indicated of an average VE of 79.8% (95% CI: 73.2-84.7)) in the six countries, with the lowest VE of 62.5% documented in Albania and up to VE of more than 97% as documented in Bulgaria and Serbia. Analysis of time from vaccination to development of protective immunity showed that VE mostly developed during the first 14 days after vaccination. Data from Greece showed that the vaccination adjusted hazard ratio for LSD was 5.7 higher in grazing farms compared to non-grazing farms. However, due to a difference in geographical location of grazing and non-grazing farms and higher vaccination rate in non-grazing farms, this effect can be at least partly attributed to indirect protection due to herd immunity provided by surrounding vaccinated farms.

Clinical, epidemiological and epizootic features of a Q fever outbreak in the border region between Serbia and Montenegro.

INTRODUCTION: Q fever is a zoonosis which commonly manifests as an acute febrile disease accompanied by pneumonia or hepatitis. The aim of this study was to reveal the reservoirs, sources and routes of infection relevant for the Q fever outbreak that occurred in the border region between Serbia and Montenegro. METHODOLOGY: A prospective study was conducted from 3rd to 23rd March, 2016 in Brodarevo, village near the Serbian-Montenegro border. The EU case definition for Q fever was applied and serological evidence of IgM and/or IgG antibody for phase II antigen Coxiella burnetii used for laboratory confirmation. Animal infection was proven by detection of specific biomarkers for Q fever by ELISA and Real-Time PCR. RESULTS: In total, ten patients were registered with Q fever, giving an attack rate of 0.5% in the village. A severe form of disease with atypical pneumonia ended up with hospitalization of eight patients. Serological surveillance was conducted in 30 herds of the receptive animals in the outbreak area. Overall the anti-Coxiella antibody seroprevalence was 20.6%. Positive molecular findings (68.4%) accompanied with high seroprevalence (63.2%) were identified in a mini-farm of sheep and cattle in the nearby Orasac, these were considered to be active sources of infection. The most probable route of C. burnetii transmission was the inhalation of contaminated aerosols originating from infected animals. CONCLUSION: The main reservoirs for human Q fever at the border region between Serbia and Montenegro are infected cattle and ruminants. Adoption of a comprehensive strategy for disease prevention and control at the intergovernmental level is urgent.

Trichinella britovi in Game Meat Linked to Human Trichinellosis Outbreak in Serbia.

After a human trichinellosis outbreak in Zlatibor District, Serbia, in 2016, Trichinella larvae were found in wild boar ( Sus scrofa) meat products. One hundred and fourteen people were infected during the outbreak. The larvae were determined to be Trichinella britovi using the polymerase chain reaction method. Trichinella britovi has previously been identified in Serbia, but this is the first case of the species being confirmed in food samples linked to human trichinellosis. The results of the study confirmed that the T. britovi is able to affect human health. In addition, this study suggests the role of wild boars as reservoirs of T. britovi in Serbia.

Methodology and results of integrated WNV surveillance programmes in Serbia.

Studies conducted during the past few years have confirmed active West Nile virus (WNV) circulation in Serbia. Based on these studies and the epidemiological situation, the Veterinary Directorate of the Ministry of Agriculture and Environmental Protection launched national WNV surveillance programmes in 2014 and 2015. The programmes encompassed the territory of Serbia and were conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the programmes was early detection of WNV and timely reporting to the public health service and local authorities to increase both clinical and mosquito control preparedness. The WNV surveillance programmes were based on direct and indirect surveillance of the presence of WNV by the serological testing of initially seronegative sentinel horses and chickens as well as through viral detection in pooled mosquito and wild bird samples. The most intense WNV circulation was observed in all seven districts of Vojvodina Province (northern Serbia) and Belgrade City, where most of the positive samples were detected among sentinel animals, mosquitoes and wild birds. The West Nile virus surveillance programmes in 2014 and 2015 showed satisfactory results in their capacity to indicate the spatial distribution of the risk for humans and their sensitivity to early detect viral circulation at the enzootic level. Most of the human cases were preceded by the detection of WNV circulation as part of the surveillance programmes. According to the existing data, it can be reasonably assumed that WNV infection, now an endemic infection in Serbia, will continue to present a significant problem for the veterinary service and public health.

First insights into the genetic diversity of Echinococcus granulosus sensu stricto (s.s.) in Serbia.

Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu stricto (s.s.) is a cosmopolitan zoonotic infection which is endemic in Serbia where it is subject to mandatory reporting. However, information on the incidence of the disease in humans and prevalence of hydatid infection in livestock remains limited. We used sequenced data of the cytochrome c oxidase subunit 1 (cox 1) mitochondrial gene to examine the genetic diversity and population structure of E. granulosus (s.s.) from intermediate hosts from Serbia. We also compared our generated nucleotide sequences with those reported for neighbouring European countries. Echinococcus canadensis was molecularly confirmed from pig and human hydatid isolates. E. granulosus (G1) was confirmed from sheep and cattle hydatid isolates as well as the first molecular confirmation in Serbia of E. granulosus G2 in sheep and E. granulosus G3 in sheep and cattle hydatid isolates. The Serbian E. granulosus (s.s.) parsimony network displayed 2 main haplotypes (SB02 and SB05) which together with the neutrality indices were suggestive of bottleneck and/or balancing selection. Haplotype analysis showed the presence of the common E. granulosus haplotype described from other worldwide regions. Investigation of the pairwise fixation (Fst) index suggested that Serbian populations of E. granulosus (s.s.) from sheep and cattle hosts showed moderate genetic differentiation. Six of the Serbian haplotypes (SB02-SB07) were shared with haplotypes from Bulgaria, Hungary and/or Romania. Further studies using a larger number of hydatid isolates from various locations across Serbia will provide more information on the genetic structure of E. granulosus (s.s.) within this region.