Angela Monica Ionică

Ixodid ticks in red foxes (Vulpes vulpes) from Romania

Among wildlife, the red foxes (Vulpes vulpes) are one of the most adapted wild species to anthropic ecosystems. They are well recognized as important reservoirs for a large number of zoonotic agents in Europe, including ticks and tick-borne pathogens. Currently, there are few available data on the importance of red foxes in the ecoepidemiology of vector-borne diseases. Therefore, the aim of this study was to screen the dynamics of tick infestation in 357 red foxes from 12 Romanian counties. Tick identification was performed using the morphological keys. The overall prevalence of tick infestation was 43.7%. The 5753 collected ticks belonged to five species: Ixodes hexagonus (on 113 out of 156 foxes; prevalence 72.44%), I. ricinus (28.84%), I. crenulatus (7.7%), Dermacentor marginatus (7.05%) and Haemaphysalis punctata (0.64%). Coinfestation occurred in 24 foxes (22 with 2 tick species; 2 with 3 tick species) with the following associations: I. ricinus + I. hexagonus (n = 10), I. hexagonus + D. marginatus (n = 5), I. ricinus + I. crenulatus (n = 4), I. ricinus + D. marginatus (n = 2), I. hexagonus + I. crenulatus (n = 1), D. marginatus + I. hexagonus + I. ricinus (n = 1), and H. punctata + I. hexagonus + I. ricinus (n = 1). This study indicates that foxes are hosts to a relevant number of tick species with recognized vectorial role. The provided information can facilitate the understanding of the ecology of ticks and can be the basis for studies on the epidemiology of tick-borne diseases. The high prevalence of tick infestation in red foxes, coupled with the increasing presence of this species in synanthropic environments and a more nature-oriented lifestyle of people, can pose a significant threat to human health, through the emergence of tick-borne diseases. Moreover, foxes represent a good model of sentinel species. This research was performed as part of project IDEI PCE 236/2011.

Further spreading of canine oriental eyeworm in Europe: first report of Thelazia callipaeda in Romania

BackgroundDespite the increasing number of reports of autochthonous cases of ocular thelaziosis in dogs in several European countries, and the evident emergence of human cases, the distribution and spreading potential of this parasite is far for being fully known. In Romania, despite intensive surveillance performed over recent years on the typical hosts of T. callipaeda, the parasite has not been found until now.MethodsIn October 2014 a German Shepherd was presented for consultation to a private veterinary practice from western Romania with a history of unilateral chronic conjunctivitis. Following a close examination of the affected eye, nematodes were noticed in the conjunctival sac. The specimens collected were used for morphological examination (light microscopy) and molecular analysis (amplification of the cytochrome c oxidase subunit 1 gene, followed by sequencing).ResultsThirteen nematodes were collected, all identified morphologically as T. callipaeda. The history of the dog revealed no travel outside Romania, and during the last year, not even outside the home locality. The BLAST analysis of our sequence showed a 100% similarity T. callipaeda haplotype h1.ConclusionsThis is the first report of T. callipaeda in Romania, which we consider to be with autochthonous transmission. These findings confirm the spreading trend of T callipaeda and the increased risk of emerging vector-borne zoonoses.

Tick parasites of rodents in Romania: host preferences, community structure and geographical distribution

BackgroundTicks are among the most important vectors of zoonotic diseases in temperate regions of Europe, with widespread distribution and high densities, posing an important medical risk. Most ticks feed on a variety of progressively larger hosts, with a large number of small mammal species typically harbouring primarily the immature stages. However, there are certain Ixodidae that characteristically attack micromammals also during their adult stage. Rodents are widespread hosts of ticks, important vectors and competent reservoirs of tick-borne pathogens. Micromammal-tick associations have been poorly studied in Romania, and our manuscript shows the results of a large scale study on tick infestation epidemiology in rodents from Romania.MethodsRodents were caught using snap-traps in a variety of habitats in Romania, between May 2010 and November 2011. Ticks were individually collected from these rodents and identified to species and development stage. Frequency, mean intensity, prevalence and its 95% confidence intervals were calculated using the EpiInfo 2000 software. A p value of <0.05 was considered statistically significant.ResultsWe examined 423 rodents (12 species) collected from six counties in Romania for the presence of ticks. Each collected tick was identified to species level and the following epidemiological parameters were calculated: prevalence, mean intensity and mean abundance. The total number of ticks collected from rodents was 483, with eight species identified: Ixodes ricinus, I. redikorzevi, I. apronophorus, I. trianguliceps, I. laguri, Dermacentor marginatus, Rhipicephalus sanguineus and Haemaphysalis sulcata. The overall prevalence of tick infestation was 29.55%, with a mean intensity of 3.86 and a mean abundance of 1.14. Only two polyspecific infestations were found: I. ricinus + I. redikorzevi and I. ricinus + D. marginatus.ConclusionsOur study showed a relatively high diversity of ticks parasitizing rodents in Romania. The most common tick in rodents was I. ricinus, followed by I. redikorzevi. Certain rodents seem to host a significantly higher number of tick species than others, the most important within this view being Apodemus flavicollis and Microtus arvalis. The same applies for the overall prevalence of tick parasitism, with some species more commonly infected (M. arvalis, A. uralensis, A. flavicollis and M. glareolus) than others. Two rodent species (Mus musculus, Rattus norvegicus) did not harbour ticks at all. Based on our results we may assert that rodents generally can act as good indicators for assessing the distribution of certain tick species.

Integrated morphological and molecular identification of cat fleas (Ctenocephalides felis) and dog fleas (Ctenocephalides canis) vectoring Rickettsia felis in central Europe.

Fleas of the genus Ctenocephalides are the most common ectoparasites infesting dogs and cats world-wide. The species Ctenocephalides felis and Ctenocephalides canis are competent vectors for zoonotic pathogens such as Rickettsia felis and Bartonella spp. Improved knowledge on the diversity and phylogenetics of fleas is important for understanding flea-borne pathogen transmission cycles. Fleas infesting privately owned dogs and cats from the Czech Republic (n=97) and Romania (n=66) were subjected to morphological and molecular identification and phylogenetic analysis. There were a total of 59 (60.82%) cat fleas (Ctenocephalides felis felis), 30 (30.93%) dog fleas (Ctenocephalides canis), 7 (7.22%) European chicken fleas (Ceratophyllus gallinae) and 1 (1.03%) northern rat flea (Nosopsyllus fasciatus) collected in the Czech Republic. Both C. canis and C. felis felis were identified in Romania. Mitochondrial DNA sequencing at the cox1 gene on a cohort of 40 fleas revealed the cosmopolitan C. felis felis clade represented by cox1 haplotype 1 is present in the Czech Republic. A new C. felis felis clade from both the Czech Republic and Romania is also reported. A high proportion of C. canis was observed from dogs and cats in the current study and phylogeny revealed that C. canis forms a sister clade to the oriental cat flea Ctenocephalides orientis (syn. C. felis orientis). Out of 33 fleas tested, representing C. felis felis, C. canis and Ce. gallinae, 7 (21.2%) were positive for R. felis using diagnostic real-time PCR targeting the gltA gene and a conventional PCR targeting the ompB gene. No samples tested positive for Bartonella spp. using a diagnostic real-time PCR assay targeting ssrA gene. This study confirms high genetic diversity of C. felis felis globally and serves as a foundation to understand the implication for zoonotic disease carriage and transmission by the flea genus Ctenocephalides.

Geographical distribution and prevalence of Borrelia burgdorferi genospecies in questing Ixodes ricinus from Romania: a countrywide study.

The paper reports the prevalence and geographical distribution of Borrelia burgdorferi sensu lato (s.l.) and its genospecies in 12,221 questing Ixodes ricinus ticks collected at 183 locations from all the 41 counties of Romania. The unfed ticks were examined for the presence of B. burgdorferi s.l. by PCR targeting the intergenic spacer 5S-23S. Reverse line blot hybridization (RLB) and restriction fragment length polymorphism (RFLP) analysis were performed for identification of B. burgdorferi genospecies. The overall prevalence of infection was 1.4%, with an average local prevalence between 0.75% and 18.8%. B. burgdorferi s.l. was found in ticks of 55 of the 183 localities. The overall prevalence B. burgdorferi s.l. in ticks in the infected localities was 3.8%. The total infection prevalence was higher in female ticks than in other developmental stages. Three Borrelia genospecies were detected. The most widely distributed genospecies was B. afzelii, followed by B. garinii and B. burgdorferi sensu stricto (s.s.). The study is the first countrywide study and the first report of B. burgdorferi s.s. in Romania. The distribution maps show that higher prevalences were recorded in hilly areas, but Lyme borreliosis spirochetes were also present in forested lowlands, albeit with a lower prevalence.

Transstadial Transmission of Borrelia turcica in Hyalomma aegyptium Ticks

Borrelia turcica comprises the third major group of arthropod-transmitted borreliae and is phylogenetically divergent from other Borrelia groups. The novel group of Borrelia was initially isolated from Hyalomma aegyptium ticks in Turkey and it was recently found in blood and multiple organs of tortoises exported from Jordan to Japan. However, the ecology of these spirochetes and their development in ticks or the vertebrate hosts were not investigated in detail; our aims were to isolate the pathogen and to evaluate the possibility of transstadial transmission of Borrelia turcica by H. aegyptium ticks. Ticks were collected from Testudo graeca tortoises during the summer of 2013 from southeastern Romania. Engorged nymphs were successfully molted to the adult stage. Alive B. turcica was isolated from molted ticks by using Barbour-Stoenner-Kelly (BSK) II medium. Four pure cultures of spirochetes were obtained and analyzed by PCR and sequencing. Sequence analysis of glpQ, gyrB and flaB revealed 98%–100% similarities with B. turcica. H. aegyptium ticks collected from T. graeca tortoises were able to pass the infection with B. turcica via transstadial route, suggesting its vectorial capacity.

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

BackgroundAnaplasma 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.MethodsA 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.ResultsThe 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.ConclusionsThe 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.

Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania

Granulocytic anaplasmosis is a common vector-borne disease of humans and animals with natural transmission cycle that involves tick vectors, among which Ixodes ricinus is the most important. The present paper reports the prevalence and geographical distribution of A. phagocytophilum in 10,438 questing Ixodes ricinus ticks collected at 113 locations from 40 counties of Romania. The unfed ticks were examined for the presence of A. phagocytophilum by PCR targeting a portion of ankA gene. The overall prevalence of infection was 3.42%, with local prevalences ranging between 0.29% and 22.45%, with an average prevalence of 5.39% in the infected localities. The infection with A. phagocytophilum was detected in 72 out of 113 localities and in 34 out of 40 counties. The highest prevalence was recorded in females followed by males and nymphs. The results and the distribution model have shown a large distribution of A. phagocytophilum, covering Romanias entire territory. This study is the first large scale survey of the presence of A. phagocytophilum in questing I. ricinus ticks from Romania.

Eurasian golden jackal as host of canine vector-borne protists

BackgroundJackals 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.ResultsDiagnostic 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.ConclusionsApparently, 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.

Mosquitoes in the Danube Delta: searching for vectors of filarioid helminths and avian malaria

BackgroundMosquitoes are arthropods of major importance to animal and human health because they are able to transmit pathogenic agents such as filarioids (Spirurida), vector-borne nematodes, which reside in the tissues of vertebrates. In Europe, recent research has mostly focused on mosquito-borne zoonotic species, while others remain neglected. Mosquitoes are also vectors of avian malaria, which has an almost worldwide distribution, and is caused by several Plasmodium species and lineages, the most common being P. relictum. The Danube Delta region of Romania is one of the most important stopover sites for migratory birds. The local mosquito fauna is diverse and well represented, while filarial infections are known to be endemic in domestic dogs in this area. The aim of the present study was thus to assess the potential vector capacity for various filarial helminths and avian malaria of mosquitoes trapped in the Danube Delta.MethodsIn July 2015, mosquitoes were collected at seven sites located in and around a rural locality in the Danube Delta region of Romania, using CO2-baited traps and hand aspirators. Additionally, a trap was placed next to a microfilaremic dog co-infected with Dirofilaria repens and D. immitis. All randomly trapped mosquitoes were identified to the species level and pooled according to date, sampling site, and taxon. Three hundred individual mosquitoes sampled next to the microfilaremic dog were processed individually and divided into abdomen and thorax/head. Following DNA extraction, all samples were screened for the presence of DNA of filarioid helminths and avian malaria agents by PCR techniques.ResultsAll 284 pools (a total of 5855 mosquitoes) were negative for filarioid DNA. One pool of Culex modestus mosquitoes was positive for Plasmodium sp. lineage Donana03. In the individually extracted mosquitoes, one abdomen of Aedes vexans was positive for D. repens DNA, one thorax/head of Ae. vexans was positive for DNA of Setaria labiatopapillosa, and two thorax/head of Cx. pipiens f. pipiens were positive for P. relictum lineage pSGS1.ConclusionThe present study suggests the vector competence of Cx. modestus and Cx. pipiens for avian Plasmodium including pathogenic species P. relictum and Ae. vexans for mammalian filarioids. Moreover, it indicates the role of Cx. pipiens f. pipiens as a potential natural vector of P. relictum lineage pSGS1 in nature.