Molecular identification of Taenia hydatigena from domestic and free-living animals in Slovakia, Central Europe.

Taenia hydatigena is a cosmopolitan tapeworm that uses canids or felines as definitive hosts, while the larval stage (metacestode), formerly referred to as cysticercus tenuicollis, infects a wide variety of intermediate hosts, in particular ruminants. In the present study, we used partial nucleotide sequences of the cox1 and nad1 genes of T. hydatigena from different animal species to analyse the intraspecies genetic diversity of this economically important parasite. Twenty-four samples of metacestodes or adults of T. hydatigena from infected sheep, chamois, roe deer, fallow deer, wild boar, and dogs from Slovakia were collected and further analysed. Several haplotypes of T. hydatigena were identified with unique mutations that have not been previously recorded in Slovakia. Analysis of nucleotide polymorphism revealed the existence of 9 and 13 haplotypes, with relatively low nucleotide pairwise divergence ranging between 0.3-1.3 and 0.2-1.8% for the Hcox and Hnad haplotypes, respectively. In general, low nucleotide and high haplotype diversities in the overall population of T. hydatigena from the study indicate a high number of closely related haplotypes within the explored population; nucleotide diversity per site was low for cox1 (Pi = 0.00540) and slightly higher for nad1 (Pi = 0.00898). A molecular study confirmed the existence of genetic variation within T. hydatigena isolates from Slovakia. However, further investigations with more samples collected from different intermediate and definitive hosts are required in order to investigate the epidemiological significance of the apparent genetic differences observed in this study.

First molecular detection of Onchocerca flexuosa (Wedl, 1856) in red deer in Slovakia.

The present paper deals with the post-mortem diagnostics of onchocerciasis and the molecular detection of causative agents of this disease in wild ruminant ungulates (Cervus elaphus, Dama dama and Capreolus capreolus). The animals were shot in hunting seasons 2017 and 2018, in two regions of the Eastern Slovakia. The total number of examined skins was fifty-eight. The presence of subcutaneous nodules was confirmed in 27.59% (95% CI 16-39) of animals. All positive skins belonged to red deer individuals (47.06%; 95% CI 30-64). The nodules were present mainly in the back area and in the lumbar area, and their sizes ranged from 2.9 to 24.1 mm, with the average count of 10 nodules per animal. Thirteen worms, isolated from the nodules collected from 13 animals, were subjected to molecular identification. Applying the standard PCR method, targeting the mitochondrial 12S rRNA, 16S rRNA and NADH-dehydrogenase gene, and subsequent sequencing, all the worms were identified as Onchocerca flexuosa Wedl, 1856. The sequences were submitted to GenBank under specific accession numbers. Two samples were identified as Onchocerca flexuosa haplotype B, in which T176A and A177T were present. Despite the presence of mutations in the 12S rRNA of the Onchocerca flexuosa, the standardized PCR remains to be a very specific and sensitive method that uses this fragment as a selectable marker for the detection of the studied parasite.

Canine dirofilariosis endemic in Central Europe-10 years of epidemiological study in Slovakia.

The study presents the comprehensive results of a detailed epidemiological study on canine dirofilariosis in Slovakia, Central Europe. More than 4000 dogs were investigated, and several epidemiological factors were considered. The mean prevalence in individual regions ranged from 2.0 % in northern Slovakia to more than 25.0 % in the south-western part of the country, with the nematode Dirofilaria repens confirmed as the dominant causative agent. Canine dirofilariosis occurred more often in animals more than 3 years old and in dogs of large and giant breed sizes. Short-haired animals were infected more often than dogs with a long coat. Also, the infection was significantly more prevalent in animals kept in rural areas in comparison with urban environments.Counts of microfilariae (mf) in peripheral blood reached their highest levels in May and August and corresponded to activity peaks and population maximums of potential vectors, the mosquito species Aedes vexans and Culex pipiens, the two most prevalent species in Slovakia. Moreover, two dogs naturally infested with D. repens were included in the experiment in order to monitor daily microfilarial periodicity. This fluctuation showed the same tendency in both animals, with a peak of circulating mf recorded at 4 a.m. and minimal mf counts at 4 p.m.

Potential mosquito (Diptera:Culicidae) vector of Dirofilaria repens and Dirofilaria immitis in urban areas of Eastern Slovakia.

This paper follows the study from 2013 focused on the molecular screening of mosquitoes as vectors of Dirofilaria spp. which provided the information on Aedes vexans as a potential vector of Dirofilaria repens in Slovakia. Current entomological and molecular research indicates that Ae. vexans can participate also in the transmission of Dirofilaria immitis within the region. Using the standard PCR method, we examined 10,500 mosquitoes (Ae. vexans, Ae. rossicus, Anopheles maculipennis s.l., Ochlerotatus sticticus, Ochlerotatus cantans, Ochlerotatus caspius, Culex pipiens/Culex torrentium, Coquillettidia richiardii), collected using CO2-baited traps at six locations in the Eastern Slovakia. Out of 105 pools, 6 pools of mosquitoes Ae. vexans were positive for D. repens DNA (minimum infective rate in Ae. vexas was 6:6.900, i.e. 0.8 per 1.000 mosquitoes), within which 4 were concurrently positive for D. immitis (minimum infective rate in Ae. vexans was 4:6.900 i.e. 0.5 per 1.000 mosquitoes).

Molecular detection of co-infections with Anaplasma phagocytophilum and/or Babesia canis canis in Dirofilaria-positive dogs from Slovakia.

Recently, several arthropod-borne infections have been introduced into previously non-endemic regions in Europe as the result of various global changes. At the same time, endemic regions are expanding and the risk of co-infections is rising, due to climate change that allows vectors to move and spread infectious diseases into new areas. The aim of the current study was to confirm simultaneous infections with Anaplasma phagocytophilum and/or Babesia canis canis in Dirofilaria-infected dogs from Slovakia, central Europe. Genomic DNA was isolated from 366 blood samples of microfilaraemic dogs without clinical signs of infection. Samples were further screened for the presence of canine tick-borne pathogens using PCR and sequencing. This survey revealed co-infection with four arthropod-borne pathogens, in particular, Dirofilaria repens, Dirofilaria immitis, A. phagocytophilum, and B. canis canis. While D. repens, responsible for canine subcutaneous dirofilariosis, is scattered through the whole territory of the country, D. immitis occurs only in endemic areas of southeastern and southwestern Slovakia in mixed infection with D. repens. Co-infection with A. phagocytophilum was reported in 3.27% of the dogs positive for D. repens; mixed infection with D. repens and B. canis canis was detected in 3.55% of the tested blood samples. Eastern Slovak Lowland represents a natural focus of B. canis canis and is a highly endemic area for canine dirofilariosis. The presence of triple infection with D. repens, A. phagocytophilum, and B. canis canis was detected in one dog originating from the eastern lowland region of Slovakia. This study highlights the importance of co-infected, clinically healthy dogs in the spreading of several different arthropod-borne pathogens and the necessity for detailed epidemiological surveys, especially in newly infested areas.

Canine dirofilariosis under specific environmental conditions of the Eastern Slovak Lowland.

The aim of the present study was to collect data from Eastern Slovak Lowland, southern Slovakia, to assess risk of the spread of canine dirofilariosis. Climate and environmental conditions in the Eastern Slovak Lowland are ideally suitable for the occurrence of vector-borne diseases. In the past, an endemic locality of dangerous mosquito transmitted malaria was found in this area. Today, another zoonotic parasitic disease threatens--dirofilariosis. The results of the first detailed study revealed a 34.44% prevalence in dogs harbouring dirofilariae. D. repens was diagnosed in all infected specimen, with 2 individuals being co-infected also with D. immitis.