Sándor Hornok

Molecular Investigations of Rickettsia helvetica Infection in Dogs, Foxes, Humans, and Ixodes Ticks

ABSTRACT Rickettsia helvetica, a tick-borne member of the spotted-fever-group rickettsiae, is a suspected pathogen in humans; however, its role in animals is unknown. The aims of this study were to establish a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (to determine potential exposure risk) and blood samples from Canidae and humans (to determine prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase gene (gltA) assay for several rickettsiae. Blood samples from 884 dogs, 58 foxes, and 214 human patients and 2,073 ticks (Ixodes spp.) collected from either vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR positive. Ticks from cats were more frequently PCR positive than ticks from dogs. Sequencing of the 23S rRNA and/or the gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, Rickettsia monacensis, which has not been previously found in Switzerland, was identified. In conclusion, R. helvetica was frequently detected in the tick population but not in blood samples. Nevertheless, due to the broad host range of Ixodes ticks and the high rate of infestation with this agent (i.e., R. helvetica was 13 times more frequent in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The PCR assay described here represents an important tool for studying this topic.

Birds as potential reservoirs of tick-borne pathogens: First evidence of bacteraemia with Rickettsia helvetica

BackgroundBirds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne disease-causing agents. Therefore, the aim of this study was to evaluate the presence of three emerging, zoonotic tick-borne pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation.MethodsBlood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.ResultsDuring the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host.ConclusionBased on the present results birds are potential reservoirs of both I. ricinus transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter, at least in Central Europe.

Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae)

In an attempt to identify the main vector and possible transmission routes of Anaplasma spp. in a region of Hungary with high prevalence of ovine and bovine anaplasmosis, DNA was extracted from 316 haematophagous arthropods (individually or in pools), including 4 species of ixodid ticks, 6 species of tabanid flies and hornflies. Midichloria-like organisms were identified with PCR (amplifying a portion of the 16S rRNA gene) and sequencing from Dermacentor marginatus and Ixodes ricinus. Significantly higher 16S positive D. marginatus individuals were collected in March than in April, suggesting earlier questing of ticks that contain rickettsial agents (thus endosymbionts). Midichloria- and Wolbachia-like organisms were also found in randomly caught horse flies (Tabanus bovinus and T. tergestinus) as well as hornflies (Haematobia irritans), respectively, with 97-99% similarity to sequences deposited in the GenBank. Although all ticks were negative in the Anaplasma spp.-specific msp4 PCR, four individuals of T. bovinus collected near to grazing cattle were positive for Anaplasma marginale. The results of the present study provide the first molecular evidence for the potential mechanical vector role of T. bovinus in the transmission of A. marginale, and broaden the range of haematophagous arthropods harbouring Midichloria-like bacteria, for the first time in any Dermacentor or Tabanus species.

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.

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary.

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.

Bovine besnoitiosis emerging in Central-Eastern Europe, Hungary.

BackgroundBesnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries.FindingsClinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses.ConclusionsThis is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region represents a further example, when human activity (i.e. cattle trading) is the main factor involved in the geographical spread of an infectious disease.

Survey on blood-sucking lice (Phthiraptera: Anoplura) of ruminants and pigs with molecular detection of Anaplasma and Rickettsia spp.

Lice may serve as biological or mechanical vectors for various infectious agents. To investigate louse infestation of ruminants and pigs, and pathogens potentially transmitted by them, anopluran lice (n=1182) were collected in Hungary, and evaluated for the presence of anaplasma, rickettsia and haemotropic mycoplasma DNA. On cattle the following species were found: Linognathus vituli (57%), Haematopinus eurysternus (38%) and Solenopotes capillatus (5%). L. vituli had a lower mean individual count/host when compared to H. eurysternus. On calves only L. vituli was observed, with a higher louse burden than on full-grown cattle. H. eurysternus and S. capillatus were more likely to occur simultaneously with another species on the same host, than L. vituli. Goats infested with Linognathus stenopsis had the overall highest prevalence (68%), while pigs harbouring Haematopinus suis showed the lowest (<1%). Anaplasma DNA was detected in 50% of pools analysed. In L. vituli Anaplasma ovis (or a closely related novel Anaplasma marginale genotype) was identified. Anaplasma-positivity of H. suis suggests that pigs may extend the reservoir and/or host spectrum of relevant species. Anaplasma-infected L. stenopsis pools show for the first time that caprine anaplasmosis is endemic in Hungary. Rickettsia spp. were demonstrated from Linognathus spp. and H. eurysternus. No haemotropic mycoplasmas were detected in any samples. In conclusion, this is the first molecularly confirmed report of bovine and ovine Anaplasma spp. in L. vituli, L. stenopsis and H. suis. The present results suggest that phthirapterosis of domestic animals deserves more attention, and lice should be evaluated among the broad range of potential vectors of arthropod-borne pathogens.

Seroprevalence of canine babesiosis in Hungary suggesting breed predisposition

Six hundred fifty-one blood samples were collected from urban and rural dogs in various parts of Hungary to measure antibody levels to Babesia canis with indirect fluorescent antibody test. Thirty-seven (5.7%) of the sera showed positivity with titers between 1:80 and 1:10,240. Seroconverted dogs were found in 13 locations of the country. It is concluded that canine babesiosis is becoming more prevalent in Eastern Hungary. Seropositivity increased then declined with age, reaching a maximum in case of 3.1- to 5-year-old dogs. Prevalence of antibodies to B. canis was significantly higher among german shepherds and komondors. This suggests a genetic predisposition of german shepherd dogs to chronic babesiosis (carrier status) with long-term maintenance of their seropositivity. On the other hand, heavy-coated komondors are phenotypically more suitable for repeated exposure to ticks, potentially infected with B. canis. This is the first report on the seroprevalence of canine babesiosis in Hungary.

Synanthropic Birds Associated with High Prevalence of Tick-Borne Rickettsiae and with the First Detection of Rickettsia aeschlimannii in Hungary

The aim of this study was to analyze synanthropic birds as risk factors for introducing ticks and tick-borne pathogens into human settlements, with an emphasis on rickettsiae. Altogether 184 subadult ticks were found on 5846 birds. Tick infestation was most prevalent during the spring. In this sample group the majority of ticks were molecularly identified as Ixodes ricinus, and three individuals collected from the European robin as Hyalomma marginatum marginatum. The latter is the first molecularly confirmed occurrence of this species in Hungary. Rickettsia aeschlimannii was detected in H. marginatum, also for the first time in Hungary, and in ticks from an urbanized bird species north of the Mediterranean countries. The overall prevalence range of rickettsiae (including R. helvetica and R. monacensis) in ticks of synanthropic birds was 29-40%, exceeding that in questing ticks of relevant species reported earlier. Additionally, in specimens of I. ricinus, the presence of Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, and a new Francisella-like genotype was also verified. Thus, it can be concluded that birds with urban or periurban habitats pose a high risk as tick carriers and reservoirs of zoonotic agents, especially of rickettsiae.

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.