Molecular evidence of Ehrlichia canis and Rickettsia massiliae in ixodid ticks of carnivores from South Hungary.

To monitor the emergence of thermophilic, Mediterranean ixodid tick species and tick-borne pathogens in southern Hungary, 348 ticks were collected from shepherd dogs, red foxes and golden jackals during the summer of 2011. Golden jackals shared tick species with both the dog and the red fox in the region. Dermacentor nymphs were collected exclusively from dogs, and the sequence identification of these ticks indicated that dogs are preferred hosts of both D. reticulatus and D. marginatus nymphs, unlike previously reported. Subadults of three ixodid species were selected for reverse line blot hybridisation (RLB) analysis to screen their vector potential for 40 pathogens/groups. Results were negative for Anaplasma, Babesia and Theileria spp. Investigation of D. marginatus nymphs revealed the presence of Ehrlichia canis, Rickettsia massiliae and Borrelia afzelii for the first time in this tick species. These findings broaden the range of those tick-borne agents, which are typically transmitted by Rhipicephalus sanguineus, but may also have Dermacentor spp. as potential or alternative vectors. Ehrlichiacanis was also newly detected in Ixodes canisuga larvae from red foxes. In absence of transovarial transmission in ticks this implies that Eurasian red foxes may play a reservoir role in the epidemiology of canine ehrlichiosis.

Postmortem small babesia-like morphology of Babesia canis - short communication.

Here we report a case of canine babesiosis with unusual morphology of the causative agent. A male, seven-week-old Labrador retriever puppy, exhibiting severe anaemia and haemoglobinuria, was presented at the Clinic of Internal Medicine in February 2011. The puppy was euthanised. The most relevant pathological changes were icterus, severe splenomegaly, generalised lymphadenopathy and haemoglobin nephrosis. Samples were collected from various organs for histology within one hour post mortem. Impression smears were also prepared from the spleen after overnight storage at 4 °C. Tissue sections and smears showed the presence of multiple, coccoid intraerythrocytic bodies that measured 1-2 µm and resembled small babesiae. No large piroplasms were seen. DNA was extracted from the spleen, and a conventional PCR was performed for the amplification of a 450-bp region of the 18S rRNA gene of piroplasms. The causative agent was identified as Babesia canis canis, with 99% sequence identity to other European isolates. Sequence identity to B. gibsoni was only 91%. This is the first account to verify that the morphology of the large canine piroplasm, B. canis, can be uniformly small babesia-like post mortem or following the storage of tissue samples.

Human dirofilariosis in Hungary: an emerging zoonosis in central Europe.

INTRODUCTION: In the temperate regions of Europe, mainly Italy, France, Greece, and in recent years Hungary, a specific filarioid worm, Dirofilaria (Nochtiella) repens, a parasite of dogs, cats and some other carnivores, transmitted by mosquitoes, has occasionally been contracted, causing dirofilariosis in humans. We report a case of dirofilariosis and review all previously observed cases in Hungary. MATERIALS, METHODS AND RESULTS: The authors present a case of human subconjunctival dirofilariosis in a 56-year-old man with mild itching and a burning sensation in his right eye. A slow-moving, whitish, thread-like worm was detected by slit-lamp examination. The worm was surgically removed and parasitological examination identified it as an immature male Dirofilaria repens. A review of Hungarian cases from 1880 to 2000 is presented, with updates of earlier reports. In addition, 16 new cases (8 men; 8 women) diagnosed between 2001 and 2006 are reviewed. The mean age of these 16 patients was 60 years; eight cases had ocular localization, seven were subcutaneous and one was diagnosed in a histopathological section of removed axillary lymph node in a patient with lymphoid leukemia. Twelve of the 16 identified Dirofilariae were females, four were males. According to their recorded histories the patients had not traveled internationally. CONCLUSION: Most of the above cases of dirofilariosis are certainly autochthonous infections. The increasing number of diagnosed cases suggests that this zoonosis is becoming more important in central Europe and that the incidence may increase with the improvement of clinical diagnosis.

Decoupling of active and passive reasons for the invasion dynamics of Aedes albopictus Skuse (Diptera: Culicidae): Comparisons of dispersal history in the Apennine and Florida peninsulas.

Aedes albopictus is an important vector of several diseases including dengue- and Chikungunya fever and is a potential vector of Zika-fever. The invasion dynamics of Aedes albopictus was reconsidered by comparing the temperature-related development of the mosquito with the observed real geographical distribution in Florida and in Italy. The potential number of generations and the annual dispersal distances of the mosquito were calculated for the estimates. The estimated total dispersals are 3.6-4.6 km/year/generation in Italy and 4.6-5.3 km/year/generation in Florida, values that are at least five to six times higher than those derived from release and recapture studies and from the previously measured flying distances of female Asian tiger mosquitoes. Subtracting the calculated dispersal distances with the known active dispersal of female Ae. albopictus, the passive dispersal component of the total dispersal distances was found to be 2.8-4.1 km/year/generation in Italy and 3.8-4.8 km/year/generation in Florida. Our results confirm that the active dispersal of female mosquitoes plays a secondary role in determining the rate of areal expansion and, in contrast, passive factors may play a primary role. It was concluded, based on similar average values of the passive dispersal distances of the mosquito in Florida and Italy, that at large spatial scales the anthropogenic component can be well estimated.

Influence of the biotope on the tick infestation of cattle and on the tick-borne pathogen repertoire of cattle ticks in Ethiopia.

BACKGROUND: The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens. METHODS: In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens. RESULTS: Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type. CONCLUSIONS: The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.

High prevalence of Hepatozoon-infection among shepherd dogs in a region considered to be free of Rhipicephalus sanguineus.

Blood samples and ticks were collected from 100 shepherd dogs, 12 hunting dogs and 14 stray dogs in southern Hungary, in order to screen them for the presence of Hepatozoon spp. by PCR. Out of 126 blood samples, 33 were positive (26%). Significantly more shepherd dogs (31%) were infected, than hunting (8%) and stray dogs (7%). Three genotypes of Hepatozoon canis were identified by sequencing, differing from each other in up to six nucleotides in the amplified portion of their 18S rRNA gene. In Dermacentor marginatus larvae/nymphs and Dermacentor reticulatus nymphs, H. canis was present only if they had been collected from PCR-positive dogs, and the genotypes were identical in the ticks and their hosts. However, two Haemaphysalis concinna nymphs removed from a PCR-negative dog were found positive for H. canis, and the genotype detected in specimens of this tick species differed from that in the blood of their respective hosts. These results indicate that canine hepatozoonosis may be highly prevalent in regions where Rhipicephalus sanguineus is considered to be non-endemic. In addition, H. canis was identified for the first time in Hungary, as well as in D. marginatus, D. reticulatus and Ha. concinna ticks. Canine hepatozoonosis was significantly more prevalent west of the Danube river (where higher densities of red fox and golden jackal populations occur), suggesting a role of wild carnivores in its epidemiology.

Non-pet dogs as sentinels and potential synanthropic reservoirs of tick-borne and zoonotic bacteria.

Blood samples were collected from 100 shepherd dogs, 12 hunting dogs and 14 stray dogs (apparently healthy) in southern Hungary to screen for the presence of emerging tick-borne pathogens. Based on real-time PCR results, 14 dogs (11%) had single or dual haemoplasma infection, and a same number of samples were positive for Anaplasma phagocytophilum. In one sample Coxiella burnetii was molecularly identified, and 20.3% of dogs seroconverted to the Q fever agent. Rickettsaemia (sensu stricto) was also detected in one animal. This is the first molecular evidence of autochthonous infection of dogs with the above pathogens in Hungary. The relatively high prevalence of haemoplasma and anaplasma infection among non-pet dogs is suggestive of a prolonged carrier status and bacteraemia of these animals rendering them epidemiologically significant as potential reservoirs and sentinels for tick-borne infections.

First record of autochthonous canine leishmaniasis in Hungary.

Hungary is traditionally regarded as a leishmaniasis-free country, and human or canine cases diagnosed locally have been recorded as imported. However, recent entomological surveys have verified the presence in Hungary of Phlebotomus neglectus and Phlebotomus perfiliewi perfiliewi, which have been incriminated as competent vectors of Leishmania infantum elsewhere in Europe. Following the occurrence in October 2007 of an undisputable clinical case of L. infantum canine leishmaniasis (CanL) in a 4-year-old female pug in a kennel of 20 dogs in Tolna province, an investigation was performed to assess the infection status in that canine population and to search for putative phlebotomine vectors. Another female pug became sick during the study period (May-November 2008) and L. infantum was confirmed as the causative agent. The other animals appeared clinically healthy; however, 4 additional dogs were found positive by indirect fluorescent antibody test (2 dogs), or by buffy-coat PCR (1 dog), or by both methods (1 dog). Hence the overall Leishmania infection prevalence in the kennel was 30% (6/20). All dogs were born in the same place and had been always kept outdoors. They had neither been abroad nor received a blood transfusion. No sand flies were collected with CDC Standard Miniature Light traps, Mosquito Magnet(®) X (MMX) dry ice-baited traps, or sticky traps placed either in or around the kennel and at nearby chicken yards during July and August of 2008 and 2009. Considering the dogs' historical background and the failure to trap any sand fly vectors in the kennel area, the origin of CanL in this site remains unexplained.

First surveys to investigate the presence of canine leishmaniasis and its phlebotomine vectors in Hungary.

Hungary is regarded as free of leishmaniasis because only a few imported cases have been reported. However, southern Hungary has a sub-Mediterranean climate, and so it was included in the EU FP6 EDEN project, which aimed to map the northern limits of canine leishmaniasis (CanL) in Europe. The numbers of traveling and imported dogs have increased in the last decade, raising concerns about the introduction of CanL caused by Leishmania infantum. Serum samples were collected from 725 dogs (22 localities, 6 counties) that had never traveled to endemic countries, as well as from other potential reservoir hosts (185 red foxes and 13 golden jackals). All sera were tested by the indirect fluorescent antibody test, but they were sero-negative using the OIE cut-off of 1:80 serum dilution except for those of two dogs resident since birth in southern Hungary. These had not received a blood transfusion, but the mode of transmission is unclear because no sandfly vectors were caught locally. From 2006 to 2009, phlebotomine sandflies were sampled in the summer months at 47 localities of 8 counties. They were trapped with castor-oil-impregnated sticky-paper, light, and CO(2)-baited traps. Small numbers of two vectors of Leishmania infantum were found. Phlebotomus neglectus occurred in three villages near to Croatia and one in north Hungary at latitude 47 °N, and Phlebotomus perfiliewi perfiliewi was trapped at two sites in a southeastern county close to the sites where it was first found in 1931-1932. Our report provides baseline data for future investigations into the northward spread of CanL into Hungary, which we conclude has yet to occur.

First molecular evidence of Anaplasma ovis and Rickettsia spp. in keds (Diptera: Hippoboscidae) of sheep and wild ruminants.

To evaluate the presence of rickettsial agents in hippoboscid flies with molecular methods, 81 sheep keds (Melophagus ovinus) were collected from 23 sheep, 144 deer keds (Lipoptena cervi) were caught in the environment, and a further 463 and 59 individuals of the latter species were obtained from fresh carcasses of 29 red deer and 17 roe deer, respectively. DNA was extracted individually or in pools. Anaplasma ovis was demonstrated in all examined sheep keds, and from one pool of free-living deer keds. Rickettsia helvetica or other, unidentified rickettsiae were also present in one pool of sheep keds, and in four pools of deer keds from both red deer and roe deer. This is the first account of polymerase chain reaction positivity of hippoboscid flies for A. ovis and rickettsiae. These results raise the possibility that-apart from cattle and roe deer as already reported-sheep and red deer might also play a reservoir role in the epidemiology of rickettsioses.