Long Time No Hear, Magnificent Wohlfahrtia! Morphological and Molecular Evidence of Almost Forgotten Flesh Fly in Serbia and Western Balkans.

The "beautiful viviparous fly", Wohlfahrtia magnifica, may have a magnificent appearance due to its striking morphology; however, it is a potentially deadly agent of obligate traumatic myiasis in humans and animals, with a serious impact on welfare and economics. The fly is found across the Palearctic realm, including the Western Balkan region, with reports from former Yugoslavian countries from the first half of the 20th century. In this paper, a recent case of wohlfahrtiosis recorded in Northern Serbia is evidenced using morphological and molecular techniques. Larvae were collected from two adult sheep with severe hoof myiasis and two young sheep with genital and interdigital myiasis. Morphological identification was performed for adults bred from the infested vulva and third-stage larvae (L3) collected from the hoof wounds, supported with barcoding sequences of the COI gene obtained from larval pairs from the hoof wounds of older and the genitalia of younger sheep. W. magnifica was identified according to the appearance of male fly terminalia and the morphology of L3, which was confirmed after the comparison of representative sequences of the COI gene (deposited in GenBank™ under accession numbers MT027108-MT027114) to those available in GenBank™. This finding represents the first reported case of wohlfahrtiosis in the Western Balkans in 80 years, highlighting the need to re-inform relevant stakeholders to achieve adequate disease control.

Host-pathogen interaction in arthropod vectors: Lessons from viral infections.

Haematophagous arthropods can harbor various pathogens including viruses, bacteria, protozoa, and nematodes. Insects possess an innate immune system comprising of both cellular and humoral components to fight against various infections. Haemocytes, the cellular components of haemolymph, are central to the insect immune system as their primary functions include phagocytosis, encapsulation, coagulation, detoxification, and storage and distribution of nutritive materials. Plasmatocytes and granulocytes are also involved in cellular defense responses. Blood-feeding arthropods, such as mosquitoes and ticks, can harbour a variety of viral pathogens that can cause infectious diseases in both human and animal hosts. Therefore, it is imperative to study the virus-vector-host relationships since arthropod vectors are important constituents of the ecosystem. Regardless of the complex immune response of these arthropod vectors, the viruses usually manage to survive and are transmitted to the eventual host. A multidisciplinary approach utilizing novel and strategic interventions is required to control ectoparasite infestations and block vector-borne transmission of viral pathogens to humans and animals. In this review, we discuss the arthropod immune response to viral infections with a primary focus on the innate immune responses of ticks and mosquitoes. We aim to summarize critically the vector immune system and their infection transmission strategies to mammalian hosts to foster debate that could help in developing new therapeutic strategies to protect human and animal hosts against arthropod-borne viral infections.

Gastrointestinal Helminths in Horses in Serbia and Various Factors Affecting the Prevalence.

PURPOSE: Data on gastrointestinal infections in horses in Southeastern Europe are limited; thus, this study aimed to add to the existing knowledge on this topic by reporting on the prevalence of intestinal parasites of horses in the territory of the Republic of Serbia. METHODS: In the period from April 2017 to December 2018, parasitological examination of 548 samples from horses of different breed, age and sex from four regions and 18 districts of the country was performed. Coprological diagnostic was done by using qualitative methods without concentration and qualitative methods with concentration of parasitic elements. Quantification of the obtained results was performed using semi-qualitative faecal egg count. RESULTS: Four helminthoses were detected in the examined samples: P. equorum (8.57%), O. equi (3.65%), strongylid eggs (71.17%) and Anoplocephala spp. (0.91%). The total prevalence of helminthoses was 77.19%. Monoinfections were significantly more present 70.07% compared to coinfections (7.12%). The highest prevalence of helminthoses was detected in free-ranging horses (93.10%-27/29), in autumn 86.67% (117/135) and winter 79.71% (165/207), in Sumadija and West Serbia region (100%), and in the youngest category (100%). Significant difference (p < 0.001) was detected in the prevalence of monoinfections by strongylids and O. equi and also coinfections by strongylid/P. equorum between horses of different age categories. CONCLUSION: Obtained results are of great contribution to clinical parasitology and pathology, especially from the aspect of animal health, welfare and preservation of horse population.

Molecular Survey of Dirofilaria and Leishmania Species in Dogs from Central Balkan.

Dirofilariosis and leishmaniosis are severe parasitic diseases in dogs, and their causative agents can also be pathogenic to humans. In this study, we conducted a multicentric survey in the regions of Serbia and North Macedonia with the goal to establish an epidemiological scenario of dirofilariosis and leishmaniosis in the territory of Central Balkan. Using molecular analyses, a total of 535 dogs from Northern Serbia (NS), Southern Serbia (SS) and North Macedonia (NM) were screened for the presence of Dirofilaria spp. and Leishmania spp. We confirmed that Central Balkan is an endemic region for Dirofilaria (D.) immitis, as it was found to be the dominant species in this area, with the highest prevalence of 8.75% in NM, followed by NS (6.68%) and a significantly lower prevalence in SS (1.51%). Two dogs (2.5%) from NM were positive for Leishmania (L.) infantum infection. None of the dogs from Serbia tested positive for Leishmania spp. High prevalence and dominance of D. immitis species, and the rising threat of L. infantum spread to the territory of Serbia, suggest that preventive measures are of a great necessity to combat the spread of these vector-borne zoonoses.

Prevalence of intestinal parasites in dogs from public shelters in Serbia.

Data on endoparasitic infections in dogs from dog shelters in Southeastern Europe are limited; thus, this study aimed to add to the existing knowledge on this topic by reporting on the prevalence of intestinal parasites in dogs from public dog shelters in the Republic of Serbia. In 2017 and 2018, individual and pooled fecal samples, were collected from 1267 dogs from six shelters. All samples were qualitatively examined for parasites using flotation tests. Seven taxa of intestinal parasites were identified: Cystoisospora spp., ascarids: Toxocara canis and Toxascaris leonina, hookworms, Trichuris vulpis, taeniids and Dipylidium caninum. The overall prevalence of intestinal parasites was 58. 3 % (78. 1 % in young dogs and 53.1 % in adult dogs). The parasites detected in both young (<1 year old) and adult dogs (>1 year old) were Cystoisospora spp. (20 % and 4.9 %), T. canis (33.5 % and 14.7 %), T. leonina (7.7 % and 2.3 %), and hookworms (16.9 % and 15 %), respectively. However, T. vulpis (9.6 %), taeniids (1.3 %), and D. caninum (5.4 %) were detected only in adult dogs. In the Belgrade shelter, young dogs had a higher prevalence of endoparasitic infections (18.9 %, 49/260) than adult dogs (14.8 %, 149/1007). In the Subotica, Jagodina and Nis shelters, young dogs had significantly higher (p < 0.001 and p < 0.05, respectively) prevalence of endoparasitic infections (10 %, 12.3 % and 14.6 %) than adult dogs (5.3 %, 8 % and 7.2 %). These results will be useful for establishing health care programs in dog shelters and implementing effective strategies for the control of intestinal parasites.

Capillaria plica in Red Foxes (Vulpes vulpes) from Serbia: Epidemiology and Diagnostic Approaches to Urinary Capillariosis in Domestic Carnivores.

PURPOSE: This study aimed to determine the presence of C. plica in red foxes and to point out the possibilities of different diagnostic methods, that could be used for diagnosis of urinary capillariosis in domestic carnivores. METHODS: Seventeen red foxes from hunting grounds in the Kolubara District, Republic of Serbia, were examined in February 2019. Animals to be shot were selected based on the criteria of gamekeepers and the permission of the competent Ministry. After hunting, the foxes were examined by ultrasound and autopsied. After the necropsy, biochemical and sediment analysis of urine morphological identification of isolated adult parasites, and macroscopic/microscopic examination of the urinary bladder and kidney tissue were performed. RESULTS: Adults of C. plica were detected in 3/17 foxes (17.6%) by ultrasound imaging and in 6/17 foxes (35.3%) by necropsy examination. Parasite eggs were found in the urinary sediment of 9/17 foxes (52.9%). The predominant histopathologic changes were amyloid degeneration of the renal glomeruli and proximal tubules (8/17 foxes-47%) as well as acute cystitis (7/17 foxes-41.2%). The occurrence of C. plica was determined in 12/17 (70.6%) of the examined foxes. DISCUSSION: This study is the first record of the C. plica in red foxes in the Republic of Serbia. Diagnostic methods used in this study could make possible the early revealing of capillariosis in domestic carnivores and could provide reliable clinical and parasitological screening of suspect animals. CONCLUSION: The study presents the first report of urinary capillariosis in a fox population in Serbia. The established high prevalence of C. plica in foxes could presume its higher prevalence in domestic and wild carnivores in the future.

Coexistence of genetically different Varroa destructor in Apis mellifera colonies.

The aim of this study was to investigate the genetic diversity of Varroa destructor parasitizing Apis mellifera colonies and to test for possible host-parasite association at the mitochondrial DNA (mtDNA) level. Six A. mellifera haplotypes (including a novel C2aa) and five haplotypes of V. destructor were detected in 29 analyzed colonies from eight sampling sites in Serbia. We revealed the presence of the K and S1 haplotypes as well as KS1 and KP1 heteroplasmic mite individuals in all localities, while the P1 haplotype was only found in four sampling sites. Significant differences in V. destructor genetic diversity were found at both apiary and colony levels, with mite haplotypes coexisting in almost all tested colonies. In addition, a significant correlation between the number of analyzed mites per colony and the number of identified V. destructor haplotypes was observed. However, no significant host-parasite relationship was found, suggesting that mites bearing different haplotypes as well as those heteroplasmic individuals are well adapted to the host, A. mellifera, independently of the identified haplotype present in each colony. Our results will contribute to future population and biogeographic studies concerning V. destructor infesting A. mellifera, as well as to better understanding their host-parasite relationship.

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, Palic 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% (Palic) 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 Palic 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.

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.

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.

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. Varroa destructor (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.

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.