Mirabela Oana Dumitrache

Synopsis of the hard ticks (Acari: Ixodidae) of Romania with update on host associations and geographical distribution

The current paper is a synoptic review of the distribution and host associations of the 25 species of hard tick fauna (family Ixodidae) in Romania. In addition to a full literature survey, original data is presented, based on eight years of occasional or targeted sample collection. The literature data on geographical distribution was transposed digitally to the decimal degree coordinate system. For each species, an updated distribution map is given together with all historical data and new host associations. Overall, our paper records 58 new tick-host associations for Romania: 20 for Ixodes ricinus, 1 for I. apronophorus, 6 for I. arboricola, 2 for I. hexagonus, 9 for I. redikorzevi, 1 for I. trianguliceps, 2 for I. vespertilionis, 2 for Haemaphysalis punctata, 1 for H. sulcata, 2 for H. concinna, 1 for D. marginatus, 4 for Rhipicephalus sanguineus sensu lato, 1 for R. bursa and 6 for Hyalomma marginatum.

Seroprevalence and Geographic Distribution of Dirofilaria immitis and Tick-Borne Infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in Dogs from Romania

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(®) 4Dx(®) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constanţa County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.

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.

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.

Zoonotic pathogens associated with Hyalomma aegyptium in endangered tortoises: evidence for host-switching behaviour in ticks?

BackgroundHyalomma aegyptium is a hard-tick with a typical three-host life cycle. The main hosts are Palearctic tortoises of genus Testudo. However, other hosts can be used by immature ticks for feeding in natural conditions. Given this complex ecology and multiple host use, the circulation of pathogens by H. aegyptium between various hosts can be important from epidemiological point of view. The aim of this study was to evaluate the role of H. aegyptium as natural carrier of four important zoonotic pathogens.MethodsFrom 2008 to 2011, 448 H. aegyptium ticks were collected from 45 Spur-thighed tortoises, Testudo graeca in Romania. DNA was extracted individually from each tick using a commercial kit. DNA was examined for the presence of specific sequences of Borrelia burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia canis and Coxiella burnetii by PCR, according to previously described protocols.ResultsPCR analysis of H. aegyptium revealed the presence of A. phagocytophilum (18.8%), E. canis (14.1%) and C. burnetii (10%). 32.4% of the ticks were infected with at least one pathogen and 9.8% had co-infections. The stages most frequently infected were nymphs (50%) followed by males (33.9%) and females (27%). The number of tortoises which harboured infected ticks was 27/45 examined (60%). From all tested T. graeca, 40% harboured ticks infected with A. phagocytophilum, 46.7% had ticks infected with E. canis and 33.3% had ticks with C. burnetii. This study reports for the first time the presence of A. phagocytophilum and E. canis in H. aegyptium.ConclusionsThe presence and relatively high prevalence of three important zoonotic pathogens in H. aegyptium raises the question of their epidemiologic importance in disease ecology. As tortoises are unlikely to be reservoir hosts for A. phagocytophilum and E. canis and both these pathogens are common in H. aegyptium, this is an important indication for (1) a possible increased host-switching behaviour of these ticks to competent reservoir hosts (i.e. hedgehogs) and (2) transstadial transmission. Furthermore, if we consider also the presence of C. burnetii, we conclude that T. graeca and its ticks should be evaluated more seriously when assessing the eco-epidemiology of zoonotic diseases.

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.

Do the Ticks of Birds at an Important Migratory Hotspot Reflect the Seasonal Dynamics of Ixodes ricinus at the Migration Initiation Site? A Case Study in the Danube Delta

Migratory birds play important roles as distributors of ticks within and between continents. In the Old World, the most important migratory route of birds links Asia, Europe and Africa. During their migration, birds use various stopover sites, where they feed and rest and where ticks may attach or detach, creating new natural foci for vector-borne diseases. Danube Delta is one of the most important migration hotspots and so far no studies were focused on ticks of migratory birds herein. The aim of the present study was to assess the species diversity and seasonal dynamics of ticks parasitizing migratory birds in Danube Delta Biosphere Reserve. Migratory birds were trapped on Grindul Lupilor (44°41′N; 28°56′E) using mist nets during 4 migratory seasons (2 spring and 2 autumn) in 2011 and 2012. From each bird, all the ticks were collected and identified based on morphological features. Epidemiological parameters (prevalence, mean abundance, mean intensity) were calculated and all data were analysed statistically based on the season (spring and autumn), regional status of birds (migrants and breeding) and foraging behaviour (ground feeders, reed-bed feeders, foliage feeders). A total of 1434 birds (46 species) were captured. Ticks were found on 94 birds (10 species). Significantly more migratory birds hosted ticks, compared to resident birds. The 400 collected ticks belonged to four species: Ixodes ricinus (92.25%), I. arboricola (6.25%), I. redikorzevi (1.00%) and Haemaphysalis punctata (0.50%). A higher prevalence was found for I. ricinus in spring, with higher prevalence of nymphs in this season, while larvae occurred with the same prevalence in both seasons. Larval intensity was higher during spring and nymphs were more abundant during autumn. The seasonal differences in our study may be related not to the local seasonal dynamics of ticks, but on the seasonal dynamics at the site of migration initiation.

Autochthonous canine leishmaniasis in Romania: neglected or (re)emerging?

Canine leishmaniasis is a vector-borne zoonotic disease caused by the protozoan parasite Leishmania infantum. In Romania between 1955 and 2013, no cases of human autochthonous visceral leishmaniasis were reported. Data regarding canine leishmaniasis is similarly scarce. Since the first report of clinical autochthonous canine leishmaniasis in 1935, there were only three sporadic reports of positive dogs all without any clinical signs. Our study reports the first clinical case of autochthonous canine leishmaniasis in the last 80 years, stressing the importance of a targeted surveillance of Leishmania infection, as infected dogs act as the primary reservoir for zoonotic visceral leishmaniasis.

Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Romania.

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are two tick-borne pathogens of medical and/or veterinary importance which are distributed worldwide. Erinaceus roumanicus, the Northern white-breasted hedgehog, is a common synanthropic species that is known to carry not only the hedgehog tick, Ixodes hexagonus, but also I. ricinus, the most common European tick species. I. ricinus is the main vector of both mentioned pathogens. Within this framework and because only limited information is available on the role of E. roumanicus in the ecology of B. burgdorferi s.l. and Anaplasma phagocytophilum in Europe, we carried out an epidemiological surveillance on this species in Romania. From the 57 examined hedgehogs collected in 12 different counties, 24 presented tick infestation. Most ticks (n=959) were morphologically identified as larvae, nymphs, or adults of I. ricinus. The prevalence of B. burgdorferi s.l. was 0.4%, and that of A. phagocytophilum 12%. In all positive cases for B. burgdorferi s.l., restriction fragment length polymorphism revealed the genospecies B. afzelii. In Romania, only limited information is available on the epidemiology of B. burgdorferi s.l. and A. phagocytophilum. As hedgehogs commonly share the same environment with humans and other potential reservoir hosts for tick-borne pathogens, our study provides new epidemiological data of public health importance.

CO2 flagging - an improved method for the collection of questing ticks

BackgroundMost epidemiological studies on tick-borne pathogens involve collection of ticks from the environment. An efficient collection method is essential for large sample pools. Our main aim was to evaluate the efficacy of a new method, where traditional flagging was enhanced by the use of CO2 dispersed into the white flannel. The CO2 was spread through a rubber hose network inserted into the flag blanket. The research was conducted in spring, in March-April 2011 in two locations from Cluj County, Romania.MethodsThe research was conducted in March-April 2011 in two locations from Cluj County, Romania. The flag to be tested contained a fine silicone rubber hose network which dispersed the CO2 in the shaft. On each collection site n=30 samplings were performed. Each sampling consisted in the simultaneous use of both flags (with and without CO2) by two persons. The CO2 concentration level on the flag canvas surface was measured. The efficacy of the method was determined by counting comparatively the total number of ticks and separate developmental stage count.ResultsUsing the CO2 improved flag, 2411 (59%) Ixodes ricinus and 100 (53.8%) Dermacentor marginatus ticks were captured, while the CO2-free flag accounted for the collection of 1670 I. ricinus (41%) and 86 (46.2%) D. marginatus ticks. The addition of CO2 prompted a concentration difference on the surface of the flag ranging between 756.5 and 1135.0 ppm with a mean value of 848.9 ppm.ConclusionThe study showed that the CO2 enhanced sweep flag increased the ability of I. ricinus (p < 0001) but not of D. marginatus to be attracted to the flag blanket.