Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

Cross-sectional serosurvey of selected infectious diseases in wild ruminants in Serbia.

The role of wildlife in maintaining infectious diseases in veterinary medicine is often neglected, although the disease eradication process in domestic animals is continuously affected by the risk of pathogens transmission from wildlife as a primary source. The main aim of this paper was to estimate the prevalence and distribution of selected infectious diseases in wild ruminants in Serbia. In total, 259 sera from wild ruminants were tested for specific antibodies to bluetongue virus, Schmallenberg virus, Bovine viral diarrhea/border disease virus, Capripox virus, West Nile fever virus, Bovine herpes virus-1, Coxiella burnetii, Brucella spp., and Leptospira spp. Specific Capripox virus and Leptospira spp. antibodies were not detected in any of the 259 wild ruminant samples. Although one animal was detected positive for BVDV/BDV specific antibodies, with 99.8% confidence, the prevalence of BVD within this population could be very low i.e. essentially free from BVD infection. One and three positive animals were detected for Brucella spp. and Coxiella burnetii antibodies, respectively. Bovine herpes virus-1 specific antibodies were detected in 20.85% of the samples. The estimated seroprevalence of vector-borne diseases was 20.5% for Schmallenberg disease, 34.3% for West Nile fever, and 38.6% for Bluetongue. Considering the reported results, wildlife health status is a result of different factors in complex relation, such as the presence of disease in domestic animals, disease nature, pathogen characteristics, environmental factors, presence, and vector competence. Wildlife should be considered not only as a risk but as a source of important information on disease distribution and its indicators.

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

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.

Avian tuberculosis in a free-living Eurasian griffon vulture.

Although Mycobacterium spp. often cause disease in domestic birds (chickens and companion birds), there are few data on avian tuberculosis in wild populations, especially in birds of prey. We describe here a case of a young adult female, free-living Eurasian griffon vulture (Gyps fulvus) that was found dead. Granulomas were grossly evident in the lungs at autopsy, and tuberculosis was suspected. Ziehl-Neelsen staining revealed large numbers of intracellular acid-fast-positive bacteria within granulomas. Examination on Löwenstein-Jensen medium was negative, but mycobacteria growth indicator tube medium results were positive. For the molecular detection of Mycobacterium spp., the primer set IS901F and IS901R was used. Positive results were observed on gel electrophoresis, indicating the presence of Mycobacterium avium subsp. avium DNA. Although tuberculosis is not considered to be a common cause of death in wild birds, it undoubtedly deserves special attention because vultures are generally considered to be a species resistant to a large number of pathogens. Determination of the cause of death of griffon vultures is important for future conservation measures for this sensitive wild species.

First Report of Alveolar Hydatid Disease (Echinococcus multilocularis) in a Golden Jackal (Canis aureus).

BACKGROUND: Alveolar hydatid disease caused by the tapeworm Echinococcus multilocularis is a parasitic disease present in the northern hemisphere. Echinococcus multilocularis is a parasite of canid and felid carnivores as definitive hosts, and small mammals, particularly rodents as intermediate hosts. Other animal species and humans can be aberrant intermediate hosts for this parasite. It is known that besides acting as definitive hosts, domestic dogs can rarely become infected with the larval form of E. multilocularis and develop alveolar echinococcosis; however, a role of wild canids as aberrant intermediate hosts has not been documented until now. To the best of our knowledge the present paper provides the first description of alveolar hydatid disease in a golden jackal (Canis aureus). CASE PRESENTATION: Necropsy of the yearling female animal found a large, round, tumor-like mass, 20 cm in diameter, with a rough, multilobulated surface in the abdominal cavity, connected to the liver and omentum. On the cut surface this tumor-like lesion was multicystic, with a number of locular cavities filled with a clear yellowish to orange watery fluid and a large area of necrosis in the central part of the mass. Histopathology revealed multiple cystic spaces separated by fibrous sheaths and inflammatory cells-lymphocytes, plasma cells, neutrophil and eosinophil granulocytes. The cysts contained either pale, hyaline, eosinophilic laminar and occasionally amorphous, acellular, PAS-positive structures, or metacestodes with invaginated protoscolices. In several cysts round calcified bodies (calcareous corpuscles) were noted. Microscopic examination showed everted and inverted protoscolices which were attached to fragments of the brood capsule or free in hydatid fluid. By comparing consensus nucleotide sequence of 457 bp obtained by PCR reaction with sequences deposited in NCBI GenBank it is determined that it was 100% identical with E. multilocularis sequences under accession numbers MH259778.1, MH259776.1, AB668376.1, EU704124.1 and AB018440.2. CONCLUSIONS: The present paper provides a proof that the golden jackal, besides being a definitive host, can also serve as the aberrant intermediate host for E. multilocularis.

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.

Comparative pathological findings in mute swans (Cygnus olor) naturally infected with highly pathogenic Avian influenza viruses H5N1 and H5N8 in Serbia.

The aim of this study was to compare pathological lesions and viral antigen expression in the organs of mute swans (Cygnus olor) naturally infected with highly pathogenic avian influenza virus subtypes H5N1 and H5N8. The examination was conducted on the carcasses of 22 mute swans which died during the avian influenza outbreaks in Serbia in 2006 and 2016-2017. Avian influenza virus subtype H5N8 isolated from mute swans in 2016-2017 was clustered within the 2.3.4.4 clade group B. After necropsy, lung, liver, spleen, pancreas, kidney and brain tissues were sampled for histopathology and immunohistochemical examination. Avian influenza virus nucleoprotein polyclonal antibodies were used for detecting the viral antigen in the examined tissues. The most significant gross lesions were necrosis and haemorrhages in the pancreas. Major histological lesions were multifocal necroses in the pancreas, spleen and liver, non-purulent encephalitis, lung congestion and oedema. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas and brain was strongly consistent with histological lesions in both infected groups. Our findings showed that pancreas was the most affected organ in all examined mute swans. In addition to increased mortality rate, similar pathological findings were detected in mute swans naturally infected with highly pathogenic avian influenza viruses H5N1 and H5N8.

Cardiopulmonary parasitic nematodes of the red fox (Vulpes vulpes) in Serbia.

Among the wild canids, the red fox (Vulpes vulpes) is recognised as an important reservoir species for a range of parasites, including cardiopulmonary nematodes of public health and veterinary importance. As cross-host transmission between the red fox and domestic carnivores can play an important role in the epizootiology of cardiopulmonary parasitic diseases, the aim of the present investigations was to obtain data on the geographical distribution of cardiopulmonary nematodes of the red fox. The material for examination consisted of 83 foxes which were legally hunted at different locations during a three-month period from December 2017 to February 2018. The presence of four emerging species in Europe (Angiostrongylus vasorum, Crenosoma vulpis, Dirofilaria immitis and Eucoleus aerophilus) was revealed in red foxes of Serbia. Crenosoma vulpis and E. aerophilus were detected in foxes in both the plain and the mountainous areas across the country. Dirofilaria immitis is distributed in red foxes near alluvial rivers in Vojvodina province (northern Serbia). Angiostrongylosis caused by A. vasorum was demonstrated to exist in two enzootic foci with a high percentage of infected foxes in a plain area of northern Serbia. To the best of our knowledge, C. vulpis and A. vasorum were discovered for the first time in red foxes in central Serbia. The results provide strong evidence for veterinarians to take into consideration the parasitic nematodes discovered in red foxes in the differential diagnosis of diseases of companion animals. In the context of the 'One Health' approach the results related to the distribution of the zoonotic species E. aerophilus and D. immitis can be useful for medical epidemiology.

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.

Physicochemical parameters and microbiological status of honey produced in an urban environment in Serbia.

Honey is a natural substance produced by honey bees (the genus Apis) enjoyed by people due to its unique nutritional and medicinal properties. The aim of this study was to determine the physicochemical parameters (moisture, ash, water-insoluble content, reducing sugars, sucrose, free acidity, diastase activity, hydroxymethylfurfural, and electrical conductivity) and microbiological status (total number of aerobic mesophilic bacteria, total number of sulfite-reducing clostridia, the presence of Salmonella spp., total numbers of fungi and yeasts and the presence of Clostridium botulinum) in honey (honeydew, blossom, sunflower, acacia, and linden) produced in an urban environment in Serbia. We analyzed 19 apiary samples of honey, collected during the 2011 harvesting season, by using recommendation methods. Physicochemical parameters of the examined honey produced in the urban environment indicated the honeys were of acceptable quality. Bacillus spp. were detected in four honeys, yeasts were detected in three honeys, and Clostridium botulinum type E was detected in one honey using PCR. The current study also showed the presence of diverse honey varieties in Serbia.

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 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.