Istituto Superiore di Sanitàpage 2 of 8 Aim and field of applicationTo identify the species or genotype of single Trichinella larvae preserved in ethanol by a multiplex PCR analysis. This method can be applied to larvae collected from human biopsies or from muscle tissues of animal origin. Principle of the methodThe PCR is a molecular biology technique that allows for the amplification of specific nucleic acid fragments, of which the initial and terminal nucleotide sequences are known (oligonucleotide pair). If a species (or genotype) has its own characteristic DNA portion, due to its composition and/or dimension, it is possible to choose an oligonucleotide pair allowing for its amplification. The PCR amplification is characterized by a high sensitivity and specificity.A modification of the "standard PCR" is the multiplex-PCR, where two or more oligonucleotide pairs are used. In this case, it is possible to amplify with a single PCR analysis more than one sequence at the same time.Today, 8 sibling species, namely T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae, and T. zimbabwensis and 3 genotypes, Trichinella-T6, Trichinella-T8 and Trichinella-T9, have been identified in the genus Trichinella. All of the Trichinella species and genotypes differ among them by the composition and/or dimension of the nucleotide sequences of different loci; consequently, the comparative analysis of three nucleotide sequences belonging to the ITS1, ITS2 and ESV, allows the univocal identification of most of the epidemiologically relevant taxa: T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae, T. zimbabwensis and Trichinella-T6.The size of the ITS1, ITS2 and ESV fragments produced by the amplification with nucleotide pairs are shown in Table A Using the multiple-PCR technique with 5 oligonucleotide pairs, it is possible to identify single larvae with only one amplification assay at the species or genotype level. References European Union Reference Laboratory for Parasites

Rabies is one of the oldest known zoonotic diseases that has significant impact on public health, but still remains neglected in Serbia. Rabies virus can infect humans and other mammals and causes inflammation of the brain associated with encephalomyelitis and neurological symptoms. In 2010, Veterinary Directorate (national Competent Authority for animal health in Serbia) has started multi-annual project of oral rabies vaccination of foxes and other wild carnivores (e.g. jackals), as support of long-term programme of eradication of rabies in Serbia, co-funded by EU (financed by Instrument for Pre-Accession Assistance). Monitoring of the effectiveness of oral vaccination campaigns has been carried out in continuation from 2011 and was based on: (i) post-mortem laboratory examination of brain tissue of target animals (foxes, jackals and other carnivores) by fluorescent antibody test (FAT), (ii) detection of antibodies against rabies virus in serum samples by ELISA and (iii) detection of tetracycline biomarker in the mandibles for the evaluation of vaccine bait uptake. From September 2011 to May 2014, the total number of 4943 brain tissue samples, 4241 sera and 4971 mandibles were analysed. Confirmed rabies-positive brains decreased from 10 in 2011/2012 to 6 in 2012/2013 and eventually to 1 positive case in 2013/2014. The seroconversion rate increased from 10.48% (133/1269) in 2011/2012 to 20.11% (362/1800) in 2012/2013 and 42.23% (495/1172) in 2013/2014. Along with the seroconversion, the number of detected tetracycline-positive mandibles demonstrated an increasing tendency in the same period, being 49.67% (682/1373) in 2011/2012, 62.60% (1294/2067) in 2012/2013 and 90.33% (1383/1531) in the monitoring programme carried out in 2013/2014. Presented results confirmed that ORV of foxes and other wildlife in Serbia against rabies was successful and characterized by steady increase of vaccine baits uptake and immunization of animals.

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.

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.

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