Molecular-phylogenetic investigation of trichomonads in dogs and cats reveals a novel Tritrichomonas species.

BACKGROUND: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. METHODS: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. RESULTS: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. CONCLUSIONS: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator-prey link in the evolution of this trichomonad.

Parasitological and molecular investigation of consequences of raw meat feeding (BARF) in dogs and cats: implications for other pets living nearby.

Considering the original nature of carnivorism among dogs and cats, nowadays these pets are increasingly fed BARF (Biologically Appropriate Raw Food/Bone And Raw Food) diet to improve their health conditions. However, this diet may also carry health risks, such as vitamin or mineral deficiencies, and infection with pathogens including parasites. In our study, fecal samples of 89 pet animals kept on BARF diet were subjected to coprological examination followed by molecular analyses. Six of them shed eggs of Dicrocoelium dendriticum. This result was confirmed with PCR and sequencing, and in one case, the DNA of Fasciola hepatica was also demonstrated. In addition, oocysts of Cystoisospora canis, a Cystoisospora ohioensis-like sp. and Eimeria stiedai, as well as sporocysts of a Sarcocystis sp. were also detected. All samples were negative for Neospora caninum and Toxoplasma gondii. In conclusion, no evidence was found for the infection of BARF-fed dogs and cats with parasites that are usually associated with this diet and considered as clinico-pathological risk factors for these pets themselves (e.g., N. caninum, T. gondii). However, fluke eggs (probably originating from ruminants) and oocysts of E. stiedai (from rabbit liver in the food) were demonstrated as pseudoparasites. These species are usually not considered among parasite-associated risks of BARF-feeding, implying that other animals living near BARF-fed pets are neglected in this context. However, where intermediate hosts of D. dendriticum occur in urban areas, BARF-feeding may indirectly affect later other dogs and cats. It was also shown here that BARF-feeding may contribute to the contamination of the environment with E. stiedai oocysts, thus increasing the risks of biliary coccidiosis in nearby living pet rabbits that would otherwise not have access to oocysts of E. stiedai.

Screening of Domestic Cats from North-Eastern Hungary for Hepatozoon felis and Cytauxzoon europaeus That Cause Infections in Local Wildcat Populations.

Among vector-borne protozoa Hepatozoon felis and Cytauxzoon europaeus are considered emerging species in felids in Europe. To investigate the presence of these two protozoa 127 domestic cats and 4 wildcats were screened by PCRs targeting the 18S rRNA gene of Hepatozoon spp. and piroplasms, as well as the cytb gene of Cytauxzoon spp. The samples were collected inside and outside a region of Hungary, where both protozoan groups are endemic in wildcats. Among domestic cats, one proved to be infected with H. felis. Furthermore, spleen samples of four wildcats were also examined, among which three tested positive for H. felis, and one had co-infection with C. europaeus. Importantly, H. felis from the co-infected wildcat belonged to genogroup II, similarly to H. felis from the positive domestic cat. Based on phylogenetic evidence, this genogroup probably represents a separate species from genogroup I of H. felis, which was hitherto reported from Mediterranean countries in Europe. The two other wildcats also harbored H. felis from genogroup I. Neither Hepatozoon nor Cytauxzoon infections were detected outside the recently discovered endemic region. In conclusion, this study demonstrates for the first time in Europe that H. felis from genogroup II may emerge in free-roaming domestic cats in regions where this protozoan parasite is endemic in wildcats.

Molecular evidence of Monocercomonas and Acanthamoeba in the feces of captive reptiles.

Reptiles are frequently kept as pet animals. They are considered as important reservoirs of protozoa with veterinary-medical significance. At a reptile farm in Ireland, fecal samples were collected from 98 captive reptiles, representing 43 species of three orders (Squamata, Testudines, and Crocodylia). After DNA extraction, all samples were screened by conventional PCRs, targeting the ribosomal small subunit (SSU) RNA and alpha-tubulin genes of trichomonads and SSU RNA gene of Acanthamoeba spp. One leopard gecko (Eublepharis macularius) was positive for a not yet reported species/genotype of the genus Monocercomonas, different from M. colubrorum. Various Acanthamoeba genotypes were detected in six reptilian species, i.e., Acanthamoeba genotype T11 in Eunectes notaeus and Heloderma suspectum/horridum; genotype T4 in Varanus exanthematicus, Chlamydosaurus kingii, and Macrochelys temminckii; and the genotype T13 in Iguana iguana. Some of these amoeba species might have clinicopathological significance in both humans and animals. Our findings highlight the importance to monitor pathogenic protozoa in pet as well as wildlife reptiles, as a source of possible infection for animals and humans living nearby.

Zoonotic ecotype-I of Anaplasma phagocytophilum in sympatric wildcat, pine marten and red squirrel - Short communication.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, dogs, cats, horses and tick-borne fever in ruminants. In Europe, its main vector is the tick species Ixodes ricinus. In this study, spleen and liver samples, as well as ticks from 18 wild-living mammals (belonging to seven species) were analysed for the presence of A. phagocytophilum with molecular methods. The zoonotic ecotype-I of A. phagocytophilum was identified in a European wildcat (Felis silvestris) and its tick, a European pine marten (Martes martes) and a Eurasian red squirrel (Sciurus vulgaris). All PCR-positive samples were collected in 2019 and originated in the same geographic area. These results indicate that taxonomically diverse mammalian species can maintain the local enzootic cycle of the same genotype of A. phagocytophilum. To the best of our knowledge, this is the first report of the zoonotic variant of A. phagocytophilum in the wildcat and in the European pine marten in a broad geographical context, as well as in the red squirrel in Hungary. Since all these host species are well known for their urban and peri-urban presence, the results of this study verify their role in the synanthropic enzootic cycle of granulocytic anaplasmosis and tick-borne fever.

Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary.

BACKGROUND: Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. METHODS: DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. RESULTS: One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. CONCLUSIONS: This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick-host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively.

Investigation on haplotypes of ixodid ticks and retrospective finding of Borrelia miyamotoi in bank vole (Myodes glareolus) in Switzerland.

The current status of tick species, important tick-borne bacteria and protozoan parasites is well-documented in Switzerland. However, reports on the genetic diversity and geographical relationships of tick species in this country appear to be in part lacking or outdated. Thus, the aim of this study was to collect ticks from various host species in southern Switzerland, to compare them in a geographical context and to screen in these samples rare tick-borne pathogens hitherto not reported or having low prevalence in Switzerland. In 2019-2020 altogether 177 ixodid ticks were collected from the vegetation, as well as from humans (n = 17), dogs (n = 23), cats (n = 41), red deer (n = 8), a European rabbit and a European hedgehog at 25 locations in three cantons of south Switzerland. Tick species were identified morphologically, followed by DNA extraction and comparison of mitochondrial haplotypes with molecular-phylogenetic methods. Tick DNA extracts, as well as sixty-two rodent liver or spleen tissue DNA extracts (representing six species) available from 2005 to 2006 were screened for trypanosomes, Occidentia massiliensis and Borrelia miyamotoi. Morphologically, three tick species were identified: Ixodes ricinus (n = 170), Rhipicephalus sanguineus sensu lato (n = 6) and I. hexagonus (n = 1). In contrast to companion animals (dogs, cats) immature ticks (larvae and nymphs) predominated on humans, which was a highly significant association (P < 0.0001). Molecular comparison of the cytochrome c oxidase subunit I (cox1) gene with GenBank data established the species as R. sanguineus sensu stricto and confirmed I. hexagonus, both showing 99.8-100% sequence identity to conspecific ticks from northern Italy. Seventy-nine specimens morphologically identified as I. ricinus revealed high 16S rRNA gene haplotype diversity and represented two phylogenetic groups. Two I. ricinus haplotypes from Switzerland belonged to the same haplogroup with I. inopinatus from Spain, Germany and Austria as well as with I. ricinus reported from a broad geographical range of Europe (including Italy, the Netherlands, Poland, Latvia and Sweden). All 141 tick DNA extracts (from five R. sanguineus s.l., 135 I. ricinus and one I. hexagonus) and 62 rodent tissue DNA extracts were negative for trypanosomes and O. massiliensis. However, B. miyamotoi was identified in a bank vole (Myodes glareolus) and three ticks by sequencing. From Switzerland, this is the first report of tick haplotypes that are phylogenetically closely related to I. inopinatus. However, based on their morphology, both specimens are considered as I. ricinus. These results highlight the importance that the identification of I. inopinatus should be based on coherent morphologic and molecular properties. This is also the first report of rodent-borne B. miyamotoi in Switzerland. Taking into account the year of collection (2005), in a chronological order this might be the first indication of B. miyamotoi in any rodent species in Europe.

Molecular epidemiological study of Trichomonas gallinae focusing on central and southeastern Europe.

Trichomonas gallinae is a geographically widespread protozoan parasite of birds. In this study, oropharyngeal swab samples were collected in Hungary and Romania from 99 columbiform birds, including 76 feral pigeons (Columba livia domestica: 42 kept for racing, 32 with urban and two with rural habitat), four common wood pigeons (C. palumbus), 16 ring doves (Streptopelia risoria) and three Eurasian collared doves (S. decaocto). These samples were analyzed for the presence of T. gallinae using molecular methods. Racing feral pigeons had significantly higher prevalence of T. gallinae infection than urban feral pigeons. The rate of PCR-positivity was the highest among wood pigeons and ring doves. Based on 18S rRNA gene, T. gallinae was the most heterogenous among racing feral pigeons sampled in a trading-breeding place. Clinical signs were associated with only one 18S rRNA gene subtype. The most divergent 18S rRNA gene subtype, Trichomonas sp. Hu-TG37 clustered with T. canistomae and T. tenax and represents probably a new species. To our knowledge, this is the first report on the genetic diversity of T. gallinae in the southeastern European region. The results suggest that most detected T. gallinae 18S rRNA gene subtypes are not host-specific and do not cause clinical signs. The highest number of 18S rRNA gene subtypes was demonstrated among racing feral pigeons. Significantly more captive than free-living columbiform birds had T. gallinae infection. These data highlight the importance of epizootic monitoring of the genetic diversity and presence of T. gallinae in trading-breeding places of pigeons and doves.

Dogs are final hosts of Sarcocystis morae (Apicomplexa: Sarcocystidae): First report of this species in Hungary and its region - Short communication.

In this study, faecal samples of four American Staffordshire terrier dogs (used for illegal fighting) were analysed by DNA extraction, molecular-phylogenetic and parasitological methods, in order to examine the occurrence of protozoan, apicomplexan parasites. In one sample, the DNA of Sarcocystis morae was shown to be present. This species was identified based on 100% identity with already reported sequences of S. morae from cervids in Lithuania and Spain. The result was also confirmed by phylogenetic analysis. The sporocysts of the canine S. morae isolate measured 14.95 × 9.75 µm on average. This is the first molecular evidence in support of the final host role of domestic dogs in the life cycle of S. morae. The most likely source of the infection was raw meat given to the examined dog to increase its physical achievement. In conclusion, under similar circumstances dogs may participate in the life cycle of S. morae in a 'natural way', shedding sporocysts/oocysts when used for hunting or taken to walks in forested areas.

Babesia gibsoni emerging with high prevalence and co-infections in "fighting dogs" in Hungary.

Babesia gibsoni is considered as an emerging protozoan parasite of dogs in North America and Europe. However, no data have been published on its prevalence, molecular-phylogenetic characteristics and associated co-infections in dogs used for illegal fighting (i.e. predisposed to acquiring this piroplasm via biting) in Europe. In this study, blood samples from 79 American Staffordshire Terrier dogs, confiscated for illegal dog fights, were molecularly analyzed for tick-borne pathogens. B abesia gibsoni was detected in 32 dogs, i.e. with a prevalence of 40.5%. In addition, Babesia vulpes was found in 8 samples (prevalence of 10.1%), for the first time in dogs in Hungary. Canine hemoplasmas were also identified in 49 samples (62%): only Mycoplasma haemocanis in 32 (40.5%) dogs, only "Candidatus Mycoplasma haematoparvum" in 9 (11.4%) dogs, and both hemoplasmas in 8 (10.1%) dogs. Thus, hemoplasma infections also showed a particularly high prevalence in this dog population. Based on a partial fragment of the 18S rRNA gene, B. gibsoni from Hungary exhibited complete sequence identity with conspecific strains reported from Europe and Asia. The cytochrome c oxidase subunit 1 (cox1) gene sequence of this isolate showed the closest identity with B. gibsoni reported from Japan but had a nonsynonymous mutation (M33I). Furthermore, the 11 B. gibsoni-positive samples analyzed for sequence variants of the cytochrome b (cytb) gene showed the presence of a common mutation (P310S). Most importantly, B. gibsoni had two further nonsynonymous mutations, M121I and F258L, in a dog with severe and relapsing anemia following atovaquone treatment. Phylogenetically, both cytb sequence variants clustered together, with a clear geographical pattern showing the closest relationship of both haplotypes identified in Hungary with those from China and Japan. To the best of our knowledge, this is the first cox1 and cytb characterization of B. gibsoni in Europe, as well as the first report on the emergence of this piroplasm and hemoplasmas with high prevalence among "fighting dogs" north of the Mediterranean Basin.