Molecular analysis of Anaplasma phagocytophilum and Babesia divergens in red deer (Cervus elaphus) in Western Austria.

Wild ungulates may act as reservoirs of various vector borne pathogens that can infect humans and domestic animals. In the present study, blood samples from 196 red deer (Cervus elaphus) from Western Austria (Vorarlberg, Tyrol and Salzburg) were collected on filter paper and tested for Anaplasmataceae, Piroplasmida, Rickettsia and filarioid helminths using molecular tools. Babesia divergens was detected in ten (5.1%) and Anaplasma phagocytophilum in three (1.5%) of the 196 samples. Filarioid helminths, Rickettsia spp. and Theileria spp. were not detected. These findings indicate that red deer may serve as reservoirs of Babesia divergens and Anaplasma phagocytophilum in Western Austria. Further investigations are needed to assess the presence of these pathogens in ticks in this geographical region, and the significance of these pathogens in both animals and humans.

First record of Orthopodomyia pulcripalpis (Rondani, 1872) (Diptera: Culicidae) in Austria.

During a three-year mosquito monitoring from 2014 to 2016, the strictly ornithophilic, originally Mediterranean species Orthopodomyia pulcripalpis (Rondani, 1872) was collected as single specimen for the first time in Austria in the district of Penzing in Vienna. Morphological species determination was confirmed by analysis of the mitochondrial cytochrome c oxidase subunit I gene. We thus not only confirm the existence of another mosquito species in Austria, but also add a new genus to the Austrian Culicidae taxa list.

Morphological and molecular identification of nasopharyngeal bot fly larvae infesting red deer (Cervus elaphus) in Austria.

Nasopharyngeal myiases are caused by larvae of bot flies (Diptera: Oestridae), which have evolved a high specificity for their hosts. Bot flies (n = 916) were collected from 137 (57.6 %) out of 238 red deer (Cervus elaphus) hunted in Vorarlberg and Tyrol (Western Austria). After being stored in 75 % ethanol, larvae were identified to species level and developmental stage using morphological and morphometric keys. Larvae were also molecularly characterized by polymerase chain reaction (PCR) amplification and partial sequencing of the mitochondrial cytochrome oxidase subunit I gene. Morphological and molecular analysis allowed identification of larvae as Cephenemyia auribarbis and Pharyngomyia picta. Genetic variations were also examined within the specimens collected in both geographical locations.

Molecular identification and phylogenetic analysis of Dipetalonema evansi (LEWIS, 1882) in camels (Camelus dromedarius) of Iran.

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far and molecular studies are scarce compared to other livestock. In this study, we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa-stained blood smears, and blood was also spotted on Whatman FTA(®) cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8 %) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however, D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s), have become feasible with the provision of the first specific COI sequences in this study.

Phylogenetic relationships and new genetic tools for the detection and discrimination of the three feline Demodex mites.

Two feline Demodex mite species have been described as causative agents of feline demodicosis, until recently a third species was detected. We provide an updated analysis on the phylogenetic relationship of Demodex mites. In addition, we present the first qPCR assay for the detection and differentiation of all three feline mite species in a single reaction. Specimen of Demodex cati, Demodex gatoi, and the recently discovered third species were collected from skin scrapings and fecal flotation for DNA extraction, conventional PCR, sequencing, and alignment. A total of 24 sequences of the partial 16S rRNA gene were used to estimate the evolutionary divergence in a p-distance model and a maximum likelihood phylogenetic tree. For the qPCR assay, new primers and fluorescent probes for the simultaneous detection of all three feline Demodex mites were designed. A consensus fragment of 351 bp was phylogenetically analyzed. The third species sequence of our study shares 98.6 % similarity to the available sequence in GenBank®. It is most similar to D. gatoi (82.41 %) and most distant to the canine Demodex injai (78.28 %). In contrast, D. gatoi is most similar to human Demodex brevis (87.01 %). The multiplex qPCR detected and discriminated the three different mite species in one reaction. The detection limit is ≤1.4 ng of mite DNA. The three feline Demodex species have distinct genotypes and did not cluster in one genetic clade. The species differentiation and assessment of evolutionary relationships will ultimately support correct diagnostics and treatment approaches.

The detection of different Dirofilaria species using direct PCR technique.

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 µl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.

First Nationwide Monitoring Program for the Detection of Potentially Invasive Mosquito Species in Austria.

In Austria, only fragmented information on the occurrence of alien and potentially invasive mosquito species exists. The aim of this study is a nationwide overview on the situation of those mosquitoes in Austria. Using a nationwide uniform protocol for the first time, mosquito eggs were sampled with ovitraps at 45 locations in Austria at weekly intervals from May to October 2020. The sampled eggs were counted and the species were identified by genetic analysis. The Asian tiger mosquito Aedes albopictus was found at two sites, once in Tyrol, where this species has been reported before, and for the first time in the province of Lower Austria, at a motorway rest stop. The Asian bush mosquito Aedes japonicus was widespread in Austria. It was found in all provinces and was the most abundant species in the ovitraps by far. Aedes japonicus was more abundant in the South than in the North and more eggs were found in habitats with artificial surfaces than in (semi-) natural areas. Further, the number of Ae. japonicus eggs increased with higher ambient temperature and decreased with higher wind speed. The results of this study will contribute to a better estimation of the risk of mosquito-borne disease in Austria and will be a useful baseline for a future documentation of changes in the distribution of those species.

Vector-borne bacteria in blood of camels in Iran: New data and literature review.

Despite close association between camels and humans, molecular based studies on vector-borne pathogens infecting camels are scarce compared to other animals in Iran. The current study was carried out to investigate the occurrence of vector-borne bacteria in the blood of dromedaries by molecular tools. A total of 200 peripheral blood samples were collected from apparently healthy animals. Microscopic examination was performed on Giemsa-stained blood smears, and drops of blood were spotted on Whatman FTA® cards for molecular analyses. Genomic DNA was extracted from the cards, and PCR amplification followed by sequencing of positive samples was carried out for the detection of Anaplasmataceae, spotted fever group (SFG) rickettsiae, Bartonella spp. and Borrelia spp. Intra-cytic forms of any blood pathogens could not be detected by light microscopy. PCR results revealed 30 animals (15%) to be infected with Anaplasmataceae bacteria. Analyses of sequences revealed a strain of Anaplasma sp. identical to Candidatus Anaplasma camelii isolated from camels, cattle and deer in Asia and Africa. Neither SFG rickettsiae, nor Borrelia or Bartonella species were found. Further studies for determining epidemiological role of camels and its zoonotic potential are recommended. This paper reviews the current knowledge on camels' tickborne bacteria including microscopy, serology and molecular studies.

Landscape structure affects distribution of potential disease vectors (Diptera: Culicidae).

BACKGROUND: Vector-pathogen dynamics are controlled by fluctuations of potential vector communities, such as the Culicidae. Assessment of mosquito community diversity and, in particular, identification of environmental parameters shaping these communities is therefore of key importance for the design of adequate surveillance approaches. In this study, we assess effects of climatic parameters and habitat structure on mosquito communities in eastern Austria to deliver these highly relevant baseline data. METHODS: Female mosquitoes were sampled twice a month from April to October 2014 and 2015 at 35 permanent and 23 non-permanent trapping sites using carbon dioxide-baited traps. Differences in spatial and seasonal abundance patterns of Culicidae taxa were identified using likelihood ratio tests; possible effects of environmental parameters on seasonal and spatial mosquito distribution were analysed using multivariate statistical methods. We assessed community responses to environmental parameters based on 14-day-average values that affect ontogenesis. RESULTS: Altogether 29,734 female mosquitoes were collected, and 21 of 42 native as well as two of four non-native mosquito species were reconfirmed in eastern Austria. Statistical analyses revealed significant differences in mosquito abundance between sampling years and provinces. Incidence and abundance patterns were found to be linked to 14-day mean sunshine duration, humidity, water-level maxima and the amount of precipitation. However, land cover classes were found to be the most important factor, effectively assigning both indigenous and non-native mosquito species to various communities, which responded differentially to environmental variables. CONCLUSIONS: These findings thus underline the significance of non-climatic variables for future mosquito prediction models and the necessity to consider these in mosquito surveillance programmes.

Molecular Identification of Hemoprotozoan Parasites in Camels (Camelus dromedarius) of Iran.

BACKGROUND: Although camels represent a valuable source of food, wool and hide in many countries, in-depth information about their vector-borne pathogens is scarce compared to other animals. The aim of the current study was to characterize vector-borne protozoa in the blood of dromedaries from Iran by molecular tools. METHODS: From June to July 2014, 200 peripheral blood samples were collected from asymptomatic one-humped camels in two provinces of Kerman and Sistan- va-Baloochestan in central and southeastern Iran. Microscopic examination was performed on Giemsa-stained blood smears, and drops of blood were spotted on Whatman FTA® cards for further analyses. Genomic DNA was extracted from the cards, and PCR was carried out for the detection of piroplasms and trypanosomes, followed by sequence analysis of positive samples. RESULTS: One sample was positive Trypanosoma spp. trypomastigotes in light microscopy. PCR results revealed one positive sample each with Theileria annulata and Trypanosoma evansi. CONCLUSION: Camels were identified as hosts for bovine Mediterranean theileriosis in the investigated area. The presence of Tr. evansi, the causative agent of surra disease, was also confirmed in camels of Iran. Further studies are recommended in order to investigate their impact on the health and productivity of camels and other livestock in this region.

Screening blood-fed mosquitoes for the diagnosis of filarioid helminths and avian malaria.

BACKGROUND: Both Dirofilaria repens and recently D. immitis are known to be endemic in Hungary. As one of several recent cases, the fatal case of a dog infested with D. immitis in Szeged, Southern Hungary, received attention from the media. Hence it was decided to catch mosquitoes in the garden where the dog lived to screen for filarioid helminths and Plasmodium spp. using molecular tools. METHODS: Mosquitoes were caught in Szeged, in the garden where the infected dog was kept, in July 2013 with M-360 electric mosquito traps and were stored in ethanol until further procedure. Female mosquitoes were classified to genus level by morphology. Each mosquito was homogenized and analyzed for filarioid helminths and avian malaria using standardized PCR techniques. Positive mosquito samples were further identified to species level by comparing a section of the mitochondrial COI gene to GenBank® entries. RESULTS: In this study, 267 blood-fed mosquitoes were caught in July 2013 in Szeged. Subsequent molecular screening revealed that not only D. immitis was present in the analyzed specimens but also DNA of D. repens, Setaria tundra and Plasmodium spp. was confirmed. CONCLUSIONS: The analysis of blood-fed mosquitoes for the diagnosis of Dirofilaria spp. and other mosquito-borne pathogens seems to be an adequate technique to evaluate if filarioid helminths are present in a certain area. Usually only unfed female mosquitoes are analyzed for epidemiological studies. However, blood-fed mosquitoes can only be used for screening if a pathogen is present because the role of the mosquito as vector cannot be classified (blood of bitten host). Furthermore, Setaria tundra was confirmed for the first time in Hungary.

Autochthonous Dirofilaria repens in Austria.

BACKGROUND: In Europe animal and human infections due to Dirofilaria repens are increasing. FINDINGS: In a nationwide screening for filarioid parasites in Austria, 7,632 mosquitoes were collected from June till October 2012 and divided into 437 pools according to same trapping date and sight and mosquito species. For the molecular detection, a real-time PCR approach was followed by conventional PCR. D. repens was detected in the villages Moerbisch and Rust, Burgenland in one Anopheles maculipennis group and one Anopheles algeriensis species pool, respectively. CONCLUSIONS: The geographical distribution of the two positive pools points to the invasion of D. repens from Eastern neighboring countries. The finding of D. repens in mosquito vectors suggests the occurrence of the causative agent for cutaneous dirofilariosis in Austria.