Milos Vucicevic

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.

Clinical babesiosis and molecular identification of Babesia canis and Babesia gibsoni infections in dogs from Serbia

Canine babesiosis is a frequent and clinically significant tick-borne disease. Sixty symptomatic dogs with clinical findings compatible with babesiosis were included in this study conducted in Serbia. After clinical examination, blood samples were taken for microscopic examination, complete blood count (CBC), Canine SNAP 4Dx Test, DNA analyses and sequencing. The main clinical signs included apathy, anorexia, fever, brown/red discoloration of urine, pale mucous membranes, icterus, splenomegaly, and vomiting. The main clinicopathological findings in Babesia infections were a slight to severe thrombocytopenia and a mild to very severe normocytic normochromic anaemia. Microscopic evaluation revealed 58 positive samples with the presence of large and small intraerythrocytic piroplasms in 57 and 1 sample(s), respectively. No co-infections were found using SNAP test. Two Babesia species, B. canis (58/60) and B. gibsoni (2/60), were differentiated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Species identification was further confirmed by sequencing PCR products of B. gibsoni samples and six randomly selected B. canis samples. All dogs were treated with imidocarb dipropionate (6.6 mg/kg of body weight), given intramuscularly twice at an interval of 14 days. This report presents the first molecular evidence of the occurrence of B. gibsoni and B. canis, confirmed by DNA sequencing, in sick dogs from Serbia.

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.

MOLECULAR SEX DETERMINATION OF 20 BIRD SPECIES PROTECTED IN THE REPUBLIC OF SERBIA

Considering that more than 50% of bird species are monomorphic, the identification of gender based on phenotypic characteristics is extremely difficult. The aim of this study is sex determination in species inhabiting the Republic of Serbia, all under the state protection and declared by IUCN as endangered. DNA was isolated from feathers. Sex determination was based on sex-specific CHD gene amplified by 2550F/2718R primer set. Sexing gave good results in all 91 samples from 20 species including 6 species where molecular sexing has not previously been successful.

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.

Improved DNA-Based Identification of Cervidae Species in Forensic Investigations

Abstract The main reasons for wildlife forensic research are animal poaching, illegal trade, and falsified game meat products. Small trace amounts, old and degraded materials present the most common samples in revealing criminal activities in this field. This is the reason why it is crucial to use adequate and reliable methods and samples to identify animal species killed outside the hunting season or species protected by law. In this study, different endpoint PCR and real-time PCR protocols were compared in the identification of three Cervidae species (Capreolus capreolus, Cervus elaphus, Dama dama) from old and damaged material found in an enclosed area where the animals were kept. From a total of 129 samples, end point PCR provided results for 119 samples, while real-time PCR was successful in all cases. Also, we created and tested a protocol for simultaneous analyses of different types of samples, which is of great importance as when the amplification is carried out simultaneously it is more cost efficient and speeds up the process.

A molecular and haematological study of Theileria equi in Balkan donkeys

Equine piroplasmosis in donkeys has been recognised as a serious problem of major economic importance. The present molecular study is the first investigation of the presence of Theileria equi and Babesia caballi in Balkan donkeys and of the possible haematological alterations related to it. A total of 70 apparently healthy donkeys from Serbia were included in this study. The overall prevalence of T. equi infection in donkeys tested with multiplex PCR was 50%. There was no B. caballi-positive sample. Infections in donkeys included in this study seem to be associated with decreased red blood cell count, haemoglobin concentration, haematocrit and platelet count, and with increased white blood cell count, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Altered haematological parameters in donkeys can lead to a decrease in working capacity and production performance. Further molecular research and long-term monitoring of equine piroplasmosis is needed in Serbia and throughout Europe.

Historical overview of methods for sex determination in birds

Determinacija pola ptica je komplikovana, prevashodno jer je preko 50% vrsta monomorfno (nema morfoloških razlika između polova). Pre primene molekularno genetičkih metoda, koristile su se brojne metode čija je glavna karakteristika da su nepouzdane. Zbog značaja analiza, one moraju biti pouzdane, ekonomične, bezbedne i brze. Visokokonzervativni CHD gen je definisan 1995. godine na W hromozomu ptica, dok je na Z hromozomu definisan dve godine kasnije. Razlika u dužini intronskih sekvenci CHD gena Z i W hromozoma, omogućava razlikovanje polova nakon amplifikacije specifičnih fragmenata primenom specifičnih prajmera. Molekularno genetičke metode imaju primat nad svim ostalim metodama jer osim toga što su bezbedne za izvođenje (i po pticu i po čoveka), daju pouzdane rezultate, a mogu se koristiti kod svih vrsta ptica.