Lumpy skin di sease (LSD) is an important disease of cattle which is included in the OIE list of notifi able terrestrial animal diseases because of its great economic importance. The etiological agent is the Lumpy skin disease virus (LSDV).In the control of LSD attenuated strains of LSDV and SPPV are successfully used as vaccine strains in infected areas. In the case of vaccination policy, due to the possibility of mild or systemic post-vaccination reactions in vaccinated animals, the application of diagnostic procedures that will rapidly and specifi cally differentiate LSDV fi eld strains from LSD vaccine virus strains are extremely important. Rapidity in diagnostics and disposal of infected animals is one of the key factors in the prevention of spreading the disease.In the presented study we have described the development and validation of two realtime TaqMan-PCR assays for a rapid, sensitive and specifi c detection of the virulent fi eld LSDV strain currently circulating in the Balkan Peninsula. Specifi city for the fi eld strain and exclusivity for vaccine strains was tested on 171 samples from naturally infected and vaccinated animals.The results of this study show that both developed real-time PCR assays are more sensitive than the conventional nested PCR in detecting fi eld LSDV strains thus enabling rapid and high-throughput detection of animals infected with fi eld strains of LSDV.In conclusion, both KV-2 and FLI real-time PCR assays described in this study are simple, rapid, sensitive and suitable for routine use in a diagnostic laboratory and have the potential to replace conventional nested gel-based PCR assays as the standard procedure for the detection of fi eld strains of LSDV in clinical samples.

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.

The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.

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

Total fungal count, incidence of fungi and aflatoxin B1 (AFB1) concentration were studied in 33 samples of bee pollen randomly collected from beekeepers in Serbia. The total number of fungi was determined by dilution method whereas AFB1 was detected using the Enzyme-Linked Immuno-Sorbent Assay (ELISA). The mycological estimation showed the presence of nine genera of fungi as followed: Acremonium, Alternaria, Aspergillus, Cladosporium, Epiccocum, Fusarium, Mucor, Penicillium and Rhizopus, with total number ranging from 1 x 103 to 1 x 105 CFU g-1. The results have shown the predominance of the fungi from the genera Aspergillus and Alternaria. Among Aspergillus species it was observed that the most frequent species was A. flavus with incidence of 27.27 %. Mycotoxin AFB1 was detected as 100% positive in all samples (100%) with an average concentration of 8.61 ?g kg-1. The obtained results indicated that honey bee pollen must be strictly controlled during its manipulation in the harvesting and manufacturing. Therefore, the implementation of good manufacturing (beekeeping) practice to define procedures for honeybee products could be crucial to reduce the risk of possible contamination and provide natural and safety product without risk on the human health. [Projekat Ministarstva nauke Republike Srbije, br. 46008, br. 46009 i br. 46010]

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.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004–2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Brucellosis is a zoonotic disease caused by Brucella spp. and a major concern for livestock. Most human cases are caused by B. melitensis and clinical presentation is usually a mild febrile illness. However, treatment failure is frequent and more severe complications can occur. In Austria, every human brucellosis is investigated to determine whether it was imported from endemic areas or is the sign of an undetected autochthonous transmission. For this study, 21 B. melitensis strains isolated in Austria between 2005 and 2019 were collected, 17 strains from 15 different patients and four strains from cattle. Whole genome sequencing combined with core-genome MLST analysis was used to characterize these strains. A cluster of seven isolates from 2018 (three human and four cattle isolates) was identified, with fewer than two allelic differences. They corresponded to the only Austrian B. melitensis outbreak that happened over the past 15 years. The other 12 Austrian brucellosis cases were single cases, and geographical origins were available for 8/12. Genomic data was used to locate probable geographical origins and compared with the results of the epidemiological investigations. Austrian strains were compared with 67 published B. melitensis sequences available on NCBI. The result of genomic analysis matched for 7/8 cases with documented conclusion of the epidemiological investigation. Genome analysis also pointed to the geographical origin for three of the four cases with missing epidemiological data. Strains from six cases were grouped together (<40 allelic differences) with 4/6 cases imported from the Balkans. Additional B. melitensis isolates from Serbian animals were analyzed and grouped with this branch, suggesting frequent importation from Balkan countries to Austria. Overall, this study highlights the specificities of human brucellosis in Austria. It also underlines the value of whole genome sequencing as a tool to investigate brucellosis cases, allowing to identify and investigate outbreaks but also to support epidemiological investigation of imported cases. However, the reliability of such methods depends on the number of strains for comparison, which can be challenging in low incidence countries. Increasing the availability of published sequences with documented geographical origins would help establishing genomic-based methods for investigating brucellosis cases.