Bojan Gajić

Sex Determination in 58 Bird Species and Evaluation of CHD Gene as a Universal Molecular Marker in Bird Sexing

The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim.

Eyeworm infections in dogs and in a human patient in Serbia: A One Health approach is needed.

Thelazia callipaeda eyeworm has been frequently reported parasitizing humans in Asia. In Europe, the parasite is endemic in wild and domestic carnivores and only eight cases have been reported in humans so far. We describe the first case of human thelaziosis in Serbia, along with two cases in dogs from the same area. A One Health approach, based on cooperation amongst veterinarians and physicians, is strongly advised for this emerging infection in order to assess the risk for and prevent of the zoonotic infection.

Haplotype identification and detection of mitochondrial DNA heteroplasmy in Varroa destructor mites using ARMS and PCR–RFLP methods

In the present study, amplification refractory mutation system (ARMS) and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) methods were used for identification of recently described Serbia 1 (S1) and Peshter 1 (P1) mitochondrial haplotypes of Varroa destructor. Based on single nucleotide polymorphisms (SNPs) within cytochrome oxidase 1 (cox1) and cytochrome b (cytb) gene sequences, a total of 64 adult V. destructor females were analyzed from locations where the S1 and P1 haplotypes had been detected previously. Results of haplotype identification obtained by ARMS and PCR–RFLP methods were completely consistent with the sequencing data. Furthermore, in some analyzed samples the occurrence of site heteroplasmy at haplotype-defining sites was detected, as it was confirmed by double peaks in the sequence chromatograms. Neither mites with simultaneous nucleotide variability, nor those with combined SNP and heteroplasmy in cox1 and cytb were found. Given that this is the first occurrence of site heteroplasmy in V. destructor, the origin of this phenomenon and possible specific traits of heteroplasmic mites have yet to be determined.

Variability of the honey bee mite Varroa destructor in Serbia, based on mtDNA analysis

Only two mitochondrial haplotypes (Korea and Japan) of Varroa destructor, the ectoparasitic honey bee mite, are known to be capable of infesting and successfully reproducing in Apis mellifera colonies worldwide. Varroadestructor (then called Varroa jacobsoni) was observed in Serbia for the first time in 1976. In order to obtain insight into the genetic variability of the mites parasitizing A. mellifera we analyzed 45 adult female mites sampled from nine localities dispersed throughout Serbia. Four fragments within cox1, atp6, cox3 and cytb mtDNA genes were sequenced. The Korea haplotype of V. destructor was found to be present at all localities, but also two new haplotypes (Serbia 1 and Peshter 1) were revealed, based on cox1 and cytb sequence variability. The simultaneous occurrence of Korea and Serbia 1 haplotypes was observed at five localities, whereas Peshter 1 haplotype was identifed at only one place.

Cytological And Molecular Identification Of Haemogregarina Stepanowi In Blood Samples Of The European Pond Turtle (Emys Orbicularis) From Quarantine At Belgrade Zoo

Abstract Blood smears stained with Diff Quick are the initial tool for cytological diagnosis of Haemogregarina spp. However, the development of sensitive and specific molecular methods enabled the detection and identification of parasites in the sample and to clarify the evolutionary relationships of adeleorinid parasites within the Apicomplexa. The current study was attempted in order to perform cytological investigation and molecular identification of the hemoparasites in thirty European pond turtles (Emys orbicularis) from the quarantine section at Belgrade Zoo, which have been found in poor health condition with massive skin hemorrhages, based on intraerythrocytic parasitic forms on hematological smears and 18S rDNA sequence, respectively. Different life cycle stages of the Haemogregarina sp. were noticed within the erythrocytes in the peripheral blood. Biochemical analysis indicated lower values of AST and iron in most of the infected turtles while hematological analysis showed a changed hematocrit value, a decrease in the number of red blood cells and low hemoglobin levels. Amplifications of the 18S rDNA sequence of Haemogregarina were detected in 30/30 (100%) turtles with clinical symptoms. The identity of PCR products was confirmed by direct DNA sequencing. Future research concerning H. stepanowi in Serbia should be applied to its definitive host-the leech.

Prevalence of endoparasitic infections of birds in zoo gardens in Serbia

This report represents the prevalence of endoparasitic infections of birds in zoological gardens in Serbia. Coprological examination conducted during 2015 and 2016, involved samples from 318 individual birds belonging to 112 species, in three zoo gardens (Belgrade, Palić and Bor). Individual faeces samples were analysed by the flotation method in salt solution. The presence of endoparasites was established in 51.96% (Belgrade), 46.16% (Palić) and 16.66% (Bor) of the tested birds. We diagnosed coccidian oocysts, eggs of Capillaria spp, Heterakis spp, Trichostrongylus spp, Ascaridia spp, Syngamus trachea and eggs of yet unidentified trematode. There were no detection of parasites from the class of cestodes. Prevalence of infection with coccidia, depending on location, was in the ranges between 8.33% and 32.84%, capillariosis 2.77% and 29.41%, heterakiosis 2.56% and 12.74%, trichostrongylidosis 5.55% and 25.64%, askaridiosis 10.25% and 10.78%, syngamosis 2.94% and trematodes of unknown etiology 2.45%. Coinfections with two or three parasitic species were detected in Belgrade and Palić Zoo Gardens, while quadruple infection was established in birds of Belgrade Zoo Garden. Obtained results can be used in establishing programs for health protection of endangered bird species.

ZASTUPLJENOST DIROFILARIA IMMITIS, BORRELIA BURGDORFERI, ANAPLASMA SPP. I EHRLICHIA SPP. KOD NEVLASNIČKIH PASA NA PODRUČJU BEOGRADA

Bolesti pasa koje se prenose vektorima predstavljaju oboljenja virusne, bakterijske ili parazitske etiologije, koja se najcesce prenose hematofagnim artropodama, komarcima i krpeljima. Cilj ovog rada bio je da se ispita prisustvo infekcija ciji se uzrocnici prenose komarcima (DiroGilaria immitis) i krpeljima (Borrelia burgdorferi, Anaplasma phagocytophilum/A. platys, Ehrlichia canis/E. ewingii) kod nevlasnickih pasa na teritoriji Beograda. U periodu od septembra do decembra 2015. godine sakupljeno je 86 uzoraka krvi od klinicki zdravih nevlasnickih pasa iz razlicitih beogradskih opstina. Svi uzorci testirani su modiNikovanim Knott-ovim testom i komercijalnim SNAP® 4Dx Plus® Test-om. Infekcija izazvana barem jednim od ispitivanih uzrocnika ustanovljena je kod 27,91% pasa. Najzastupljeniji uzrocnik bila je D. immitis, koja je dijagnostikovana kod 17,44% životinja. Na prisustvo antitela protiv Anaplasma spp. pozitivno je bilo 9,30% pasa, dok je 2,33% jedinki bilo seropozitivno na B. burgdorferi, odnosno na Ehrlichia spp. Koinfekcije sa dva patogena zabeležene su kod 3,49% pasa. Rezultati ovog istraživanja pokazuju da su nevlasnicki psi na teritoriji grada Beograda izloženi delovanju razlicitih vrsta zoonoznih uzrocnika koji se prenose komarcima i krpeljima, zbog cega treba preduzeti odgovarajuce mere za suzbijanje ovih artropoda.

Prevalence and intensity of infection with gastrointestinal nematodes in sheep in eastern Serbia

A coprological examination of 680 grazing sheep was performed in Eastern Serbia from March 2011 to November 2012 in order to determine the presence of gastrointestinal (GI) nematode parasites. Fecal samples were randomly collected and examined by using qualitative and quantitative coprological techniques. It was found that 74.56% sheep were infected. Samples that contained nematode eggs were processed for larval development and eleven nematode genera were identified: Haemonchus (46.91%), Ostertagia (25.88%), Marshallagia (21.91%), Cooperia (14.12%), Trichostrongylus (39.85%), Nematodirus (35.88%), Bunostomum (23.97%), Strongyloides (17.06%) Oesophagostomum (40.73%), Chabertia (32.79%) and Trichuris (10.88%). Higher prevalence of infection was observed in females (p<0.01), as well as in adults (p<0.001). Regarding the intensity of infection, in 40.63% sheep it was low, in 51.87% moderate and in 7.50% high. There was no difference in intensity of infection considering sex and age of animals. Moreover, simultaneous infection with different number of nematode genera was dependent on sheep’s age (p<0.001). These results suggest that GI nematodes are a conspicuous problem of grazing sheep in the study area. [Projekat Ministarstva nauke Republike Srbije, br. III 046002]