Environmental factors influencing the distribution of "Theileria annae" in red foxes, Vulpes vulpes in Romania.

Red foxes, Vulpes vulpes are among the most widely spread carnivores in the world, invading also urban areas and are often parasitized by various ticks and directly exposed to several vector-borne pathogens, including the commonly present "Theileria annae". Considering the paucity of data on the possible vectors of this pathogen and the presence of the infection in various locations across the globe, the aim of our study was to understand the potential role of various environmental factors on the distribution of "T. annae" in red foxes from a well-defined region within the Carpathians, Romania. Between July 2016 and April 2017, a total of 347 blood samples originating from red foxes from 13 counties were tested using a PCR specifically designed for "T. annae". In order to assess the potential distribution of "T. annae" based on niche modelling, we used presence-only data and 15 ecological variables. The probability of presence models was built using MaxEnt software. Of all sampled foxes, 20.1% (66 unique locations in 8 counties) were positive for "T. annae" DNA. There was no significant difference between the prevalence in males and females, nor between juveniles and adults. The sequences were all identical to each other and showed 100% identity to other sequences deposited in GenBank. The highest contribution to the spatial model was represented by the agricultural land coverage. This is the first study to demonstrate the presence of "T. annae" in foxes in Romania and the first spatial analysis for "T. annae" highlighting the importance of the environmental factors on its distribution.

Molecular confirmation of Hepatozoon canis in Mauritius.

In this study, Hepatozoon species was molecularly identified and characterized for the first time on the Indian Ocean island of Mauritius. Partial sequences of the 18S rRNA gene of the Hepatozoon isolates were analysed from three naturally infected dogs. The sequences of H. canis were similar to the 18S rRNA partial sequences (JX112783, AB365071 99%) from dog blood samples from West Indies and Nigeria. Our sequences were deposited in the GenBank database.

Filarioid infections in wild carnivores: a multispecies survey in Romania.

BACKGROUND: Filarioids are vector-borne parasitic nematodes of vertebrates. In Europe, eight species of filarioids, including zoonotic species, have been reported mainly in domestic dogs, and occasionally in wild carnivores. In Romania, infections with Dirofilaria spp. and Acanthocheilonema reconditum are endemic in domestic dogs. Despite the abundant populations of wild carnivores in the country, their role in the epidemiology of filarioid parasites remains largely unknown. The aim of the present study was to assess the host range, prevalence and distribution of filarioid infections in wild carnivores present in Romania. METHODS: Between May 2014 and February 2016, 432 spleen samples originating from 14 species of wild carnivores have been tested for the presence of DNA of three species of filarioids (D. immitis, D. repens and A. reconditum). RESULTS: Overall 14 samples (3.24%) were molecularly positive. The most prevalent species was D. immitis (1.62%), accounting for 50% (n = 7) of the positive animals. The prevalence of D. repens was 1.39%, while that of A. reconditum was 0.23%. No co-infections were detected. Dirofilaria immitis DNA was detected in five golden jackals, Canis aureus (7.58%), one red fox, Vulpes vulpes (0.33%), and one wildcat, Felis silvestris (10%). The presence of D. repens DNA was detected in two red foxes (0.66%), two golden jackals (3.03%), one grey wolf (7.14%), and one least weasel, Mustela nivalis (33.33%). Acanthocheilonema reconditum DNA was found only in one red fox (0.33%). CONCLUSION: The present study provides molecular evidence of filarial infections in wild carnivore species in Romania, suggesting their potential epidemiological role and reports a new host species for D. repens.

Eurasian golden jackal as host of canine vector-borne protists.

BACKGROUND: Jackals are medium-sized canids from the wolf-like clade, exhibiting a unique combination of ancestral morphotypes, broad trophic niches, and close phylogenetic relationships with the wolf and dog. Thus, they represent a potential host of several pathogens with diverse transmission routes. Recently, populations of the Eurasian golden jackal Canis aureus have expanded into the Western Palaearctic, including most of Europe. The aim of our study was to examine Eurasian golden jackals from Romania, Czech Republic and Austria for a wide spectrum of vector-borne protists and to evaluate the role of this species as a reservoir of disease for domestic dogs and/or humans. RESULTS: Diagnostic polymerase chain reaction (PCR) DNA amplifications revealed 70% of jackals to be positive for Hepatozoon, 12.5% positive for piroplasms, and one individual positive for Leishmania infantum. Phylogenetic analyses of partial 18S rDNA sequences invariably placed sequenced isolates of Hepatozoon into the H. canis clade. For piroplasms, both the 18S and cox1 sequences obtained confirmed the presence of Babesia canis and "Theileria annae" in 5 and 2 individuals, respectively, providing the first records of these two piroplasmids in Eurasian golden jackals. A single animal from Dolj County (Romania) was PCR-positive for L. infantum, as confirmed also by sequencing of ITS1-5.8S. CONCLUSIONS: Apparently, expanding populations of jackals can play a significant role in spreading and maintaining new Babesia canis foci in Central Europe. The role of jackals in the epidemiology of "Theileria annae" and H. canis is probably similar to that of red foxes and should be taken into account in further research on these parasites. Also the presence of L. infantum deserves attention. Our study confirms that once established, the populations of Eurasian golden jackals constitute natural reservoirs for many canine vector-borne diseases, analogous to the role of the coyotes in North America.

Use of a commercial serologic test for Angiostrongylus vasorum for the detection of A. chabaudi in wildcats and A. daskalovi in badgers.

Three species of the genus Angiostrongylus are known to infect European carnivores: A. vasorum (mainly in canids but also in other carnivores), A. chabaudi (in felids) and A. daskalovi (in mustelids). A. vasorum is responsible for clinically severe disease in domestic dogs, most commonly diagnosed based on fecal examination and serological detection of circulating antigens. Considering the poorly known host range and the challenging larval differentiation in the feces between the three species of Angiostrongylus infecting European carnivores, our aim was to evaluate the cross-reactivity of A. chabaudi and A. daskalovi with A. vasorum using a commercial serologic test developed for domestic dogs. Badgers (Meles meles) (n=10) and wildcats (Felis silvestris) (n=8) were examined between 2015 and 2016 by full parasitological necropsy with subsequent morphological and molecular identification of nematodes and by serology, using IDEXX Angio Detect™ tests. Five out of the ten badgers and two out of the eight wildcats were harboring nematodes in the pulmonary arteries. All nematodes were identified morphologically as A. daskalovi in badgers and A. chabaudi in wildcats, respectively. Serological examination of the plasma samples revealed the positivity of the same animals as found in necropsy. None of the animals negative at necropsy was positive at serology. The 100% correlation between the necropsy results and the serologic positivity to IDEXX Angio Detect™ in badgers infected with A. daskalovi and wildcats infected with A. chabaudi suggest that these rapid tests are able to identify circulating antigens of all species of Angiostrongylus found in European carnivores: A. vasorum, A. daskalovi and A. chabaudi. The possibility for future in-clinic use of this test in domestic cats should be further investigated.

Altitude-Dependent Prevalence of Canine Granulocytic Anaplasmosis in Romania.

Canine granulocytic anaplasmosis (CGA) is an important tick-borne disease with worldwide distribution. The importance of this disease resides in the ability of Anaplasma phagocytophilum to infect humans and several animal species. The aim of the study was to evaluate the prevalence rate of CGA in different altitudinal areas of Romania. A total of 357 canine blood samples were collected during 2010-2013 from eight counties. To assess the influence of the altitude on A. phagocytophilum prevalence, the samples were collected from four different altitude areas (coastal 0-5 meters; lowland 6-100 meters; hilly areas 200-300 meters; low mountain areas >500 meters). These samples were evaluated for the presence of A. phagocytophilum DNA by amplifying part of the Ankyrin repeat protein (AnkA) gene. A higher prevalence was obtained for coastal compared with remaining areas, suggesting an influence of altitude on the CGA. Moreover, the results suggest an influence of climate and rainfall. In the present research work, we highlight the risk of granulocytic anaplasmosis in Central and Southern Romania, with a greater risk associated to Southern lowland region, especially in coastal areas. The importance of these results resides in the zoonotic potential of the canine A. phagocytophilum strains. In conclusion, the altitude and precipitation level may be risk factors for A. phagocytophilum infection in dogs and other hosts, including humans.

Thelazia callipaeda in wild carnivores from Romania: new host and geographical records.

BACKGROUND: Thelazia callipaeda is a vector-borne zoonotic nematode parasitizing the conjunctival sac of domestic and wild carnivores, rabbits and humans, with a vast distribution in Asia and the former Soviet Union. In Europe, the nematode has an emerging trend, being reported in Italy, France, Switzerland, Germany, Spain, Portugal, Bosnia and Herzegovina, Croatia, Romania, Greece and Serbia, with human cases known in Italy, France, Spain, Serbia and Croatia. In Romania, the infection was so far reported only in dogs, whereas there are no reports in wildlife despite the large numbers of wild carnivores in the country. The aim of this study was to evaluate the role of wild carnivores in the natural cycle of T. callipaeda in Romania. METHODS: Between 2014 and 2016, 89 wild carnivores (64 golden jackals, Canis aureus, 13 grey wolves, Canis lupus, nine wildcats, Felis silvestris and three Eurasian lynxes, Lynx lynx) have been examined. During the necropsy, both eyes of all the examined animals have been thoroughly inspected for the presence of parasites. If present, all nematodes were collected in absolute ethanol (for molecular analysis of the partial cox1 gene) or in 4 % formalin (for morphological identification). RESULTS: In total, three animals were found to be infected with T. callipaeda: a grey wolf, a golden jackal and a wildcat. The BLAST analysis of all the sequences showed a 100 % similarity to T. callipaeda haplotype h1. To the best of our knowledge, this study represents the first report of T. callipaeda in golden jackals, and the first study on T. callipaeda in wildlife from Romania. CONCLUSION: Our data broaden the host spectrum and geographical distribution of T. callipaeda, highlighting the role of wild carnivores as natural reservoirs for the infection and confirming the ongoing expanding trend of this zoonotic nematode in Europe.

Role of golden jackals (Canis aureus) as natural reservoirs of Dirofilaria spp. in Romania.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-transmitted zoonotic nematodes, causing heartworm disease and skin lesions, respectively, in carnivores. In Europe, the domestic dog is apparently the main definitive host, but patent infections occur also in other species of carnivores. The rapid spread of the golden jackals (Canis aureus) throughout Europe opens a question of involvement of this species in the sylvatic cycle of pathogens in the colonised territories, including Dirofilaria spp. METHODS: Between January 2014 and May 2015, 54 golden jackals from 18 localities in Romania were examined by full necropsy for the presence of adult filarioid nematodes and blood samples from all animals were screened for the presence of microfilariae of D. immitis, D. repens and Acanthocheilonema reconditum by multiplex PCR DNA amplification. RESULTS: Nematodes morphologically identified as D. immitis were found in 18.52% of the animals, originating from the southern part of Romania. No D. repens or A. reconditum were found at necropsy. The molecular prevalence in blood samples from the same animals was 9.26% for D. immitis and 1.85% for D. repens. All samples were negative by PCR for A. reconditum. CONCLUSION: The relatively high prevalence of Dirofilaria spp. infections in golden jackals from Romania together with the increasing density of the jackal populations highlight their potential role in the transmission of these zoonotic parasites and in the maintenance of natural disease foci.

Molecular detection of Anaplasma platys infection in free-roaming dogs and ticks from Kenya and Ivory Coast.

BACKGROUND: Anaplasma platys is a bacterium parasitic in the canine platelets, representing the causative agent of canine cyclic thrombocytopenia, with a worldwide distribution, mainly in tropical countries. The agent has zoonotic potential, being reported in several human clinical cases. The suspected vector is the tick Rhipicephalus sanguineus (sensu lato), widely distributed in animals and humans in the tropical regions of South America, Africa, Asia and Australia, but also in southern Europe. Only few scattered data concerning the epidemiology of A. platys in sub-Saharan Africa are available. The aim of our study was to evaluate the epidemiological status of A. platys in dogs and cats from rural communities in eastern and western Africa, where dogs and their ticks live in close contact with humans. METHODS: A total of 216 canine and 22 feline blood samples and ticks feeding on them were collected in 2013 and 2014 from eight localities in Ivory Coast and four localities in Kenya. PCR was performed using specific primers targeting a portion of the 16S rRNA gene, followed by sequencing. RESULTS: The main results highlight the high prevalence of A. platys infection in dogs in both Eastern and Western Africa and report it for the first time in Eastern Africa and in Rhipicephalus camicasi. CONCLUSIONS: The presence of A. platys DNA in R. camicasi feeding on negative hosts together with the relatively high prevalence of A. platys, correlated with the absence of the probable vector Rhipicephalus sanguineus (s.l.) in Kenyan Island suggest the involvement of other tick species in the transmission of A. platys.