**2. Materials and methods**

Materials for the research were samples from dogs and samples of vectors. The research was planed as serological examination of dog blood samples for dirofilariosis and leishmaniasis. The vectors (mosquitoes) were collected, identified, and analyzed for the presence of causative agents of dirofilariosis in the northern part of Serbia.

During spring and summer of 2014 (May–September), 292 samples of mosquitoes were collected and identified. Collecting was done with lamps with dry ice. The identification of mosquitoes (for gender and species) was done at Faculty of Agriculture, University of Novi Sad. Vector identification was done with microscopic observation. The analysis of vectors for the presence of causative agent for dirofilariosis was done by a molecular method (PCR). PCR analysis were performed at the Scientific Veterinary Institute of "Novi Sad." The samples were pooled as 20 mosquitoes into one pool. In collected mosquitoes, a molecular method of PCR was performed. DNA extraction was done with commercial kits from Quiagen (QIAmp), during a 2-day protocol. PCR was done according to the prescription of Rishniw et al. [36]. Primers used for PCR analysis were primers 5′–3′, forward: DIDR-F1\_for AGTGCGAATTG‐ CAGACGCATTGAG and reverse: DIDR-R1\_rev AGCGGGTAATCACGACTGAGTTGA. Determination was done based on 542 bp for *D. immitis*.

In total, 170 of blood samples from dogs were examined for dirofilariosis and leishmaniasis. Serological analysis for dirofilariosis and leishmaniasis were done from blood samples of dogs obtained by venous punctation. The blood samples were divided into three groups, according to the way of life of the dogs:

**•** Group of hunting and military dogs (79 samples)—in this group, samples were analyzed from dogs that are actively used for hunting. They had their owners, and they mostly did not receive any preventive treatment against parasites. Not one of these dogs has ever left Serbia.


Methods used in the study were the following: modified Knott test and PCR for dirofilariosis and ELISA test for leishmaniasis.

Analysis for dirofilariosis was done with the modified Knott test for the detection of microfi‐ laria in circulation. Analysis for all the samples was done on the same day of sampling or the next day. Samples were taken with anticoagulant. The procedure of the modified Knott test was performed according to the instructions of Genchi et al. [35]. The modified Knott test was done in all the samples—from dogs with clinical symptoms as well as from dogs without any clinical symptoms for dirofilariosis. Positive samples found by the modified Knott test were then selected for molecular analysis to be done by PCR. DNA extraction was done with QIAmp commercial kits for DNA extraction (Quiagen, by the instructions of the producer). After that, a PCR was performed according to the protocol from Rishniw et al. [36]. The same primers were used in the protocol of *Dirofilaria* DNA detection in blood samples as in mosquito samples (primers 5′–3′):

Forward: DIDR-F1\_for AGTGCGAATTGCAGACGCATTGAG and

#### Reverse: DIDR-R1\_rev AGCGGGTAATCACGACTGAGTTGA.

Determination was done based on 542 bp for *Dirofilaria immitis*.

For diagnostics of leishmaniasis, same blood samples were used taken from the same dogs. Analysis for leishmaniasis was done by ELISA method (commercial kit by Ingenaza, done by the prescription of the producer). From blood samples, sera samples were obtained by centrifugation. After that, blood sera samples were kept on –20°C until ELISA was performed.
