First detection of Toxoplasma gondii Africa 4 lineage in a population of carnivores from South Africa.

Introduction: There have only been a few molecular studies conducted on the detection of T. gondii in tissues of carnivores in South Africa, with no data on the genetic diversity of this parasite. That is why the aim of this study was to detect and genotype T. gondii DNA in tissues of selected wild and domestic carnivores in South Africa. Methods: Samples were collected from 80 animals of 20 species (mainly road-killed) in the four provinces of Limpopo (n=57), Mpumalanga (n=21), Gauteng (n=1) and Free State (n=1) during the period 2014-2018. Samples of brain (n=31), heart (n=4), liver (n=40), spleen (n=2) and lung (n=3) were used to detect T. gondii by real-time PCR targeting a 529 bp repeating fragment of T. gondii DNA. Samples that were positive in real-time PCR were genotyped using 15 microsatellite markers. Results: T. gondii DNA was detected in 4 (5 %) samples: in the brain from a Black-backed Jackal (Canis mesomelas), in the liver from a African Wildcat (Felis silvestris lybica) and in the liver and heart of two Rusty-spotted Genets (Genetta maculata) respectively. The DNA sample from Black-backed Jackal was genotyped and characterized as belonging to the type Africa 4 lineage (equivalent to RFLP genotype ToxoDB#20), that is a widespread lineage in Africa. Discussion: This is the first genetic characterization of T. gondii isolated from a wild carnivore on the African continent and the first report of T. gondii in Black-backed Jackal. The Africa 4 lineage was also confirmed in the region of Southern Africa for the first time.

Detection of antibodies to Toxoplasma gondii and Neospora caninum in animals from three zoos in Slovakia.

The aim of this study was to detect antibodies to Toxoplasma gondii and Neospora caninum in exotic animal species kept in three zoos in Slovakia. Antibodies to T. gondii and N. caninum were detected by commercial Enzyme-linked immunosorbent assay (ELISA, ID Screen Toxoplasmosis Indirect Multispecies and ID Screen Neospora caninum Indirect Multispecies, ID Vet, Montpellier, France). Antibodies to T. gondii and N. caninum were detected in 43% (24/56) and 5% (3/55) of animals, respectively. The three animals with N. caninum antibodies: two wolves (Canis lupus) and one Hartmann's mountain Zebra (Equus zebra hartmannae), were clinically healthy, and both wolves simultaneously had antibodies to T. gondii. The results of our study provide a picture of the recent circulation of T. gondii in three Slovakian zoos with the S/P (ratio of antibodies in the sample to antibodies in positive control) value higher than 200%, found in five animals (9%) indicating acute toxoplasmosis. The highest S/P value (296%) was detected in a Roloway monkey (Cercopithecus roloway), which was healthy without clinical signs, presuming that Roloway monkey is a species less susceptible to T. gondii infection. Results of our study showed the presence of T. gondii and N. caninum in Slovakian zoos, confirming recent T. gondii infections according to the high level of antibodies detected in five animals, referring to acute toxoplasmosis.

Blood meal analysis: host-feeding patterns of biting midges (Diptera, Ceratopogonidae, Culicoides Latreille) in Slovakia.

Biting midges of the genus Culicoides are vectors of important pathogens affecting domestic and wild animals and have played a major role in the re-emergence of new outbreaks of bluetongue (BTV) and Schmallenberg (SBV) viruses in Europe. To determine vector-host specificity, trophic preference from blood meal analysis is of major importance in the surveillance of arthropod-borne diseases. Of 28,752 specimens collected, we identified 17 Culicoides species and investigated a total of 48 host sequences from the blood meals. Culicoides obsoletus/C. scoticus, C. dewulfi, C. pulicaris, C. lupicaris, C. punctatus, C. newsteadi, C. riethi, and C. furcillatus were found to feed on mammals (cattle, horses, and humans), birds (domestic chickens), small rodents (Apodemus flavicollis), and hares (Lepus europaeus). To our knowledge, this is the first study investigating trophic preferences of Culicoides spp. in Slovakia. This study demonstrated that Culicoides species are able to feed on domesticated host vertebrates as well as birds, rodents, and humans.

TITLE: Analyse des repas sanguins : modes d'alimentation des hôtes des ceratopogonidés (Diptera, Ceratopogonidae, Culicoides Latreille) en Slovaquie. ABSTRACT: Les moucherons piqueurs du genre Culicoides sont des vecteurs d'agents pathogènes importants affectant les animaux domestiques et sauvages et ont joué un rôle majeur dans la réémergence de nouvelles épidémies de la fièvre catarrhale ovine (FCO) et de la maladie à virus Schmallenberg (SBV) en Europe. Pour déterminer la spécificité vecteur-hôte, la préférence trophique issue de l'analyse des repas sanguins est d'une importance majeure dans la surveillance des maladies transmises par les arthropodes. À partir de 28 752 spécimens collectés, nous avons identifié 17 espèces de Culicoides et étudié un total de 48 séquences d'hôtes à partir des repas de sang. Nous avons trouvé que Culicoides obsoletus / C. scoticus, C. dewulfi, C. pulicaris, C. lupicaris, C. punctatus, C. newsteadi, C. riethi et C. furcillatus se nourrissent à partir de mammifères (bovins, chevaux et humains), oiseaux (poulets domestiques), petits rongeurs (Apodemus flavicollis) et lièvres (Lepus europaeus). À notre connaissance, il s'agit de la première étude portant sur les préférences trophiques de Culicoides spp. en Slovaquie. Cette étude a démontré que des espèces de Culicoides sont capables de se nourrir d'hôtes vertébrés domestiqués ainsi que d'oiseaux, de rongeurs et d'humains.

Fatal toxoplasmosis in wild European brown hares (Lepus europaeus) in tularaemia endemic areas of the Czech Republic: Poses risk of infection for humans?

Toxoplasma gondii may cause fatal infection in European brown hares (Lepus europaeus). However, the role of this parasite in terms of mortality rate in tularaemia endemic areas, amount of parasites in affected organs and circulating genotypes, is still unknown. In total, 36 hares (killed or found dead) were submitted for pathomorphological examination as a part of the national tularaemia and brucellosis monitoring. Tissue samples (lung, heart, liver, spleen and kidney) were tested by quantitative real-time PCR targeting 529 bp region of T. gondii. Genotyping was performed by a 15 microsatellite markers method in a single multiplex PCR assay. The same tissues of hares were simultaneously used for the bacteriological cultivation. Toxoplasma gondii was detected by qPCR in the tissues of two hares. Spleen and lungs of one infected hare have been found harbouring up to ~7 millions of T. gondii parasites per gram of tissue. Both positive samples were characterized as T. gondii type II, one archetypal clonal type II and the other one a type II variant (W35 = 244). Bacteria Francisella tularensis was proved in pooled samples of three hares but without coinfection with T. gondii; all hares were negative for Brucella suis. Toxoplasma gondii has significant impact on mortality of European brown hares in tularaemia endemic areas and parasite load within the animal tissues may present high risk of human infection.

Description of Culicoides (Culicoides) bysta n. sp., a new member of the Pulicaris group (Diptera: Ceratopogonidae) from Slovakia.

BACKGROUND: Species of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are mainly known as vectors of arboviruses such as bluetongue (BTV) and Schmallenberg (SBV). Among the known vectors, few species within the subgenus Culicoides Latreille, 1809 have been implicated in the transmission of BTV and SBV. Nevertheless, phylogenetic studies had revealed the presence of cryptic and undescribed species in Europe, raising questions about their vectorial role. A previous integrative study, associating morphology and barcode data, raised the hypothesis of the presence of undescribed species in Slovakia. The present study, combining morphological and molecular approaches, is aimed to support the hypothesis and a description of Culicoides bysta n. sp. is provided. METHODS: Series of male and female specimens were dissected and several of them were sequenced for the barcode region of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Bayesian inference phylogenetic analyses based on 72 cox1 sequences of the species belonging to the Pulicaris group of the subgenus Culicoides, were carried out and the frequencies of intra/interspecific variations were analyzed. The morphology of abundant material of the new species (31 females and 12 males) was examined and compared with the paratypes of Culicoides boyi Nielsen, Kristensen & Pape, 2015 and with specimens of Culicoides pulicaris Linnaeus, 1758. For females, suture distances on the eyes were newly evaluated as a diagnostic character and for males we assessed a new measurement on the ninth tergite and on the apicolateral processes. RESULTS: Both phylogenetic analysis and barcode distances supported the distinct status of the new species, Culicoides bysta n. sp. described as a member of the Pulicaris group based on the morphology of males and females. The new species is closely related to C. boyi and C. pulicaris but can be distinguished on the basis of the wing pattern and the ratio between the two eye sutures. Both newly evaluated characters, i.e. eyes in females and male genitalia appeared to be diagnostic for distinguishing the new species described herein. CONCLUSIONS: The vector potential of the recently described species C. boyi and C. bysta n. sp. to transmit arboviruses, such as BTV and SBV, is unknown. When considering these two species as being close to C. pulicaris, the previous data, such as the vector implication for C. pulicaris in BTV transmission, should be revaluated in future.

First record of Phlebotomus (Transphlebotomus) mascittii in Slovakia.

A large-scale entomological survey was carried out in summer 2016 in the Czech Republic and Slovakia. It revealed, for the first time, the presence of the phlebotomine sand fly Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Phlebotominae) in south-western Slovakia. Species identification of a captured female was confirmed by both morphological and sequencing (COI) analyses.

First insights into indoor and outdoor Culicoides activity related to the risk period for Bluetongue virus transmission in Eastern Slovakia.

The epizootic of Bluetongue virus (BTV) throughout Europe in 2006 revealed insufficient knowledge regarding seasonal activities, endo/exophilic and endo/exophagic behaviour of the species of the Culicoides genus (Diptera: Ceratopogonidae). Although several studies have been carried out in Western Europe, bringing new knowledge of the above mentioned topics, there was still a gap to fill in Central Europe. Therefore, this study investigated seasonal variations in the indoor/outdoor Culicoides activity observed in the south-eastern Slovakia from 2012 to 2014, using a light-trapping method. In total, 52,741 Culicoides were collected and identified; majority of them were caught outdoors (82.6%), with the highest activity period being mid-June. The C. obsoletus/C. scoticus and C.punctatus species predominated, altogether representing 88.8 and 94.1% of the total indoor and outdoor collections, respectively. Positive correlation was observed between the temperature and the abundance, whereas no correlation with other studied factors was detected. In autumn, Culicoides activity was observed outdoors at temperatures ranging between-1.5°C and 9.3°C, whereas the indoor activity was detected at temperatures ranging between-3.9°C and 0°C. This demonstrated that the most significant BTV vectors in the studied area are C. obsoletus/C. scoticus. The C. dewulfi and C. chiopterus vectors appeared to be of lower significance. The period with the highest risk of the BTV transmission in Eastern Slovakia appeared to be mid-June. The autumnal Culicoides activity inside the cowsheds may be important for the assessment of virus overwintering. This study is the first to provide the data on indoor/outdoor behaviour of biting midges in Slovakia.

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).

Morphological and molecular analysis of the genus Culicoides (Diptera: Ceratopogonidae) in Slovakia with five new records.

The biodiversity of Culicoides from eastern Slovakia was investigated by light trapping. An integrative taxonomy approach combining DNA barcode sequence and morphological analyses was used to accurately identify specimens. Five species were newly recorded from Slovakia: Culicoides picturatus Kremer & Deduit, C. gejgelensis Dzhafarov, C. clastrieri Callot et al., C. griseidorsum Kieffer and C. odiatus Austen. The checklist of the Culicoides species recorded from SK has been updated to 63 species and barcode sequence data is provided for 8 species not previously available on GenBank. Conflict between results from molecular and morphological analyses resulted in the discovery of some potentially new cryptic species and the inability of DNA barcodes to distinguish C. festivipennis Kieffer from C. clastrieri, C. salinarius Kieffer from C. manchuriensis Tokunaga and C. pallidicornis Kieffer from C. subfasciipennis Kieffer. These conflicts suggest further study is required to clarify the status of these species. 

Culicoides midges (diptera: ceratopogonidae) as vectors of orbiviruses in Slovakia.

In recent years, rapid spread of Culicoides-borne pathogens such as bluetongue (BT) and Schmallenberg viruses have been reported in Europe. In this study we examined the Culicoides populations in farms with wild and domestic ruminants in Eastern Slovakia with the aim to confirm the presence of biting midges serving as potential vectors of important pathogens. The main vector complexes were the Obsoletus complex (54%; n=4,209) and the Pulicaris complex (23%; n=1,796). To estimate the relative abundance of the cryptic species of the Obsoletus complex (Culicoides obsoletus, Culicoides scoticus and Culicoides montanus), we performed the multiplex polymerase chain reaction (PCR) based on ITS-2 and ITS-1 segments, on 125 midges randomly sampled. The relative abundance of C. obsoletus ranged from 5.26% in the farm with wild ruminants to 85.71% in another farm with cattle and sheep. A total of 112 pools of parous and gravid females belonging to the Obsoletus and Pulicaris complexes were tested for virus detection by the real-time reverse transcription polymerase chain reaction (RT-PCR) for BT virus, as well as for the Epizootic Hemorrhagic Disease Virus (EHDV), with negative results.

First record of Aedes albopictus in Slovakia.

During a routine inspection of the mosquito fauna in the Kosická Basin (Eastern Slovakia), in one of the monitored locations we have caught 4 females of the invasive mosquito Aedes albopictus, using the CO2 baited CDC traps. Occurrence of this particular mosquito has already been reported in many European countries; in Slovakia, however, this is the first finding ever. The finding of Ae. albopictus extends the list of the mosquito fauna in Slovakia to 50 species and Slovakia thus ranks among other 20 European countries where this mosquito was observed. The presence of Ae. albopictus increased the probability of transmission of canine and human dirofilariosis in urban environment.

Dirofilaria repens microfilariae in Aedes vexans mosquitoes in Slovakia.

In this study, we screened field-caught mosquitoes for presence of Dirofilaria spp. by using a polymerase chain reaction (PCR) assay. Potential occurrence of Dirofilaria repens and Dirofilaria immitis microfilariae was examined in 3,600 mosquitoes of eight species (Aedes vexans, Aedes cinereus, Aedes rossicus, Culex pipiens, Culiseta annulata, Ochlerotatus sticticus, Ochlerotatus cantans and Ochlerotatus caspius) collected from five locations in two districts (Kosice and Trebisov) of Eastern Slovakia, endemic region of canine dirofilariasis. Collection of mosquitoes was performed between May and August 2012 in premises known to be inhabited by Dirofilaria-infected dogs. PCR assays were performed on 72 pools, each pool containing 50 mosquitoes of the same species, collected on the same location. Each pool was examined separately for the presence of D. immitis and D. repens, respectively. A positive finding of D. repens was recorded in one pool of A. vexans mosquitoes collected in Kosické Olsany village. Minimum infection rate in A. vexans was 1:1,750, i.e. 0.57 per 1,000 mosquitoes. The identity of D. repens was confirmed by direct sequencing of PCR product which has shown 100% homology with sequence attributed to D. repens (GenBank accession number AJ271614). This study represents the first molecular evidence of D. repens microfilariae in mosquitoes in Slovakia and highlights a need for better surveillance of zoonotic dirofilariasis in central Europe.

Evaluation of bendiocarb cytotoxicity in mammalian and insect cell cultures.

There is an increasing need for rapid and easily interpreted in vitro assays to screen for possible cytotoxicity of pesticides. The objective of this study was to investigate the effect of the carbamate insecticide bendiocarb on mammalian and insect cell cultures. The cytotoxicity of this insecticide was evaluated by cell proliferation and cellular damage was assessed by evaluation of the cytopathic effect and lactate dehydrogenase (LDH) leakage. Cells of insect origin (Sf21) were the most sensitive to bendiocarb with significant (P < 0.01) suppression of their proliferative activity ranging from 10(-1)-10(-5) M. However, significant suppression of proliferative activity was also recorded in rat liver cells (WBF344; 10(-1)-10(-3) M; P < 0.01-0.05) and rabbit kidney cells (RK13; 10(-1) M; P < 0.01). In contrast with the proliferation activity of cells, a cytopathic effect based on cellular damage and LDH leakage into the medium was observed only at the highest concentration (10(-1) M) in RK 13 and WBF344 cells, but not in the Sf21 insect cell line. Our results indicate that bendiocarb exposure caused a cell-type dependent decrease in cell proliferation; however, cell damage and LDH leakage into the medium were not present or were strongly limited, dependent on the cell phenotype. Cell proliferation was shown as a sensitive indicator for evaluation of the cytotoxic effect of bendiocarb in vitro; on the other hand, microscopic signs of cellular damage and LDH leakage were insufficient in vitro markers.