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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.

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.

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri.

Reports of Sarcocystis rileyi -like protozoa ('rice breast disease') from anseriform birds had been rare in Europe until the last two decades, when S. rileyi was identified in northern Europe and the UK. However, despite the economic losses resulting from S. rileyi infection, no recent accounts are available on its presence (which can be suspected) in most parts of central, western, southern and eastern Europe. Between 2014 and 2019, twelve mallards ( Anas platyrhynchos ) were observed to have rice breast disease in Hungary, and the last one of these 12 cases allowed molecular identification of S. rileyi , as reported here. In addition, S. rileyi was molecularly identified in the faeces of one red fox ( Vulpes vulpes ). The hunting season for mallards in Hungary lasts from mid-August to January, which in Europe coincides with the wintering migration of anseriform birds towards the south. Based on this, as well as bird ringing data, it is reasonable to suppose that the first S. rileyi -infected mallards arrived in Hungary from the north. on the other hand, red foxes ( Vulpes vulpes ), which are final hosts of S. rileyi , are ubiquitous in Hungary, and our molecular finding confirms an already established autochthonous life cycle of S. rileyi in the region. Taken together, this is the first evidence for the occurrence of S. rileyi in Hungary and its region.

Cimex lectularius, the common bedbug is an important, emerging pest of both veterinary and medical importance. Here a recently discovered, genetically distinct new species of the C. lectularius group is described morphologically, as Cimex pulveratus Hornok sp. nov.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.

Kinetoplastids are flagellated protozoa, including principally free-living bodonids and exclusively parasitic trypanosomatids. In the most species-rich genus, Trypanosoma, more than thirty species were found to infect bats worldwide. Bat trypanosomes are also known to have played a significant role in the evolution of T. cruzi, a species with high veterinary medical significance. Although preliminary data attested the occurrence of bat trypanosomes in Hungary, these were never sought for with molecular methods. Therefore, amplification of an approx. 900-bp fragment of the 18S rRNA gene of kinetoplastids was attempted from 307 ixodid and 299 argasid ticks collected from bats, and from 207 cimicid bugs collected from or near bats in Hungary and Romania. Three samples, one per each bat ectoparasite group, were PCR positive. Sequencing revealed the presence of DNA from free-living bodonids (Bodo saltans and neobodonids), but no trypanosomes were detected. The most likely source of bodonid DNA detected here in engorged bat ectoparasites is the blood of their bat hosts. However, how bodonids were acquired by bats, can only be speculated. Bats are known to drink from freshwater bodies, i.e. the natural habitats of B. saltans and related species, allowing bats to ingest bodonids. Consequently, these results suggest that at least the DNA of bodonids might pass through the alimentary mucosa of bats into their circulation. The above findings highlight the importance of studying bats and other mammals for the occurrence of bodonids in their blood and excreta, with potential relevance to the evolution of free-living kinetoplastids towards parasitism.

Man-made barriers are well known for their effects on ecosystems. Habitat fragmentation, for instance, is a recognised consequence of modern-day infrastructure. The aim of the present study was to investigate the diversity and abundance of tick species, as well as the risks of acquiring tick-borne infections in habitats adjacent to a freeway. Therefore, ixodid ticks were collected from the vegetation at two-week intervals (in the main tick season, from March to June) in eight habitats of different types (forest, grove, grassland) along both sides of a freeway. Ixodes ricinus females were molecularly screened for three species of tick-borne bacteria. In the study period, 887 ixodid ticks were collected. These included 704 I. ricinus (79.4%), 51 Dermacentor reticulatus (5.7%), 78 D. marginatus (8.8%), 35 Haemaphysalis inermis (3.9%) and 19 H. concinna (2.1%). There was no significant difference in the abundance of tick species between similar habitats separated by the freeway, except for the absence of Dermacentor spp. on one side. In I. ricinus females, the overall prevalence of Anaplasma phagocytophilum was low, and (in part due to this low rate) did not show significant difference between the two sides of the freeway. Rickettsia helvetica had significantly different overall prevalence between two distant habitats along the same side of the freeway (12.3% vs. 31.4%), but not between habitats on the opposite sides. Borrelia burgdorferi s.l. showed significantly different overall prevalence between habitats both on the same and on the opposite sides of the freeway (8.6-35.9%), and the difference was higher if relevant habitats were also separated by the freeway. Importantly, the prevalence rate of the Lyme disease agent was highest in a forested resting area of the freeway, and was significantly inversely proportional to the prevalence of A. phagocytophilum (taking into account all evaluated habitats), apparently related to deer population density. Prevalence rates of these bacteria also differed significantly on single sampling occasions between: (1) closely situated habitats of different types; (2) distant and either similar or different habitat types; and (3) habitats on the opposite sides of the freeway. In conclusion, the findings of the present study show that a fenced freeway may contribute to differences in tick species diversity and tick-borne pathogen prevalence along its two sides, and this effect is most likely a consequence of its barrier role preventing deer movements.

Dermacentor reticulatus is a tick species of high medical and veterinary importance, emerging in several parts of Europe. Up to now most studies focusing on zoonotic rickettsiae in D. reticulatus were based on ticks collected in a limited part of the questing period, and did not take into account the potential seasonal variations in the rate of infection with tick-borne rickettsiae. The aim of the present study was to investigate the latter phenomenon, i.e. to screen D. reticulatus adults, collected monthly in two urban habitats of Budapest, for the presence of three zoonotic Rickettsia spp. Altogether 852 D. reticulatus adults were collected, which showed significantly similar seasonal activity in the two evaluated habitats. Among the 413 molecularly analysed ticks, R. helvetica-infected D. reticulatus were only collected during autumn in habitat-1, in contrast to habitat-2. The overall prevalence of R. raoultii in D. reticulatus adults was significantly higher in habitat-1 than in habitat-2. In addition, the seasonal distribution of R. raoultii-infected ticks was different between the two habitats (in habitat-2 significantly more R. raoultii-infected ticks were collected in the autumn, in comparison with winter and spring). Rickettsia slovaca was not detected in any of the molecularly analysed ticks. The results clearly indicate that a single-time or seasonally biased collection of D. reticulatus adults and their subsequent molecular analysis may not be informative on the real prevalence of rickettsiae. This is because the availability/ activity of infected ticks shows significant seasonal fluctuations, both within and between habitats. Instead, for screening D. reticulatus-borne rickettsiae, it is important to collect monthly samples and then to assess seasonal prevalence and actual habitat-associated eco-epidemiological risks.

Fourteen long-legged ixodid ticks (6 nymphs and 8 larvae) were collected from Bechstein's bat (Myotis bechsteinii) in Rochefort, Belgium. All ticks were morphologically identified as Ixodes ariadnae, based on their long legs (Haller's organ longer than maximum diameter of tarsus I), broad palps and posteriorly reverse bell-shaped scutum with wavy surface. The DNA was extracted from these ticks, followed by PCR amplification of part of their cytochrome oxidase subunit I (COI) gene. All obtained sequences were 100% identical with each other, and with the COI sequence of I. ariadnae reported previously from Hungary and Germany. Taking into account that the collection site in the present study is close to the French border of Belgium, and migration of Bechstein's bat is known between Belgium and France, it is reasonable to suppose that I. ariadnae also occurs in France. This is the first record of I. ariadnae in Western Europe, outside its formerly known geographical range (Central Europe).

Hard ticks and tsetse flies are regarded as the most important vectors of disease agents in Sub-Saharan Africa. With the aim of screening these blood-sucking arthropods for vector-borne pathogens belonging to the family Anaplasmataceae in South-Western Ethiopia, four species of tsetse flies (collected by traps) and seven species of ixodid ticks (removed from cattle) were molecularly analysed. DNA was extracted from 296 individual ticks and from 162 individuals or pools of tsetse flies. Besides known vector-pathogen associations, in Amblyomma cohaerens ticks sequences of Anaplasma marginale and A. phagocytophilum were detected, the latter for the first time in any ticks from cattle in Africa. In addition, part of the gltA gene of Ehrlichia ruminantium was successfully amplified from tsetse flies (Glossina pallidipes). First-time identification of sequences of the above pathogens in certain tick or tsetse fly species may serve as the basis of further epidemiological and transmission studies.

A long-legged tick was collected from a hibernating greater mouse-eared bat (Myotis myotis) in Baden-Württemberg, Germany. Based on morphological characteristics as well as on partial COI and 16S rDNA gene sequences the tick was identified as an engorged female of Ixodes ariadnae. The greater mouseeared bat is a new host record for this tick species. Taking into account the geographical position of the collection site and the known migration distance of the greater mouse-eared bat, the present data suggest the autochthonous occurrence of I. ariadnae in Germany. This is the first record of I. ariadnae in Germany, and in any country other than Hungary, where this species has been recently discovered.

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.

In order to investigate haemotropic Mycoplasma (formerly Eperythrozoon) infection of goats, blood samples and blood-sucking lice (Linognathus stenopsis) were collected in two goat herds. DNA was extracted from 20 blood samples and from 49 lice allocated to six pools according to host individuals. Haemoplasma infection was detected in four goats by real-time PCR. From the sample with the highest bacterial load the simultaneous presence of M. ovis and 'Candidatus M. haemoovis' was demonstrated by cloning and sequencing. Louse pools were haemoplasma negative, including those from bacteraemic animals. However, not only were Anaplasma inclusion bodies seen in blood smears from goats, but relevant PCR-positivity was also detected among lice. This is the first report of a molecular investigation on caprine haemoplasmas, including analysis of their bloodsucking lice. In summary, goats are susceptible to both molecularly characterised ovine haemoplasmas. On the other hand, goat sucking lice (L. stenopsis) do not appear to be potential vectors of these agents.

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.

Although haemobartonellosis was previously reported in Hungary, until now the diagnosis (based on morphological identification in blood smears) has only been suggestive of the occurrence of the large species, recently reclassified as Mycoplasma haemofelis. However, in July 2007 a cat was presented at a small animal clinic with severe haemolytic anaemia, icterus and haemoglobinuria. While biochemical parameters were within the reference range, the cat had leukocytosis and rapidly decreasing haematocrit values, and eventually died 7 days after the sudden onset of aggravating clinical signs. From blood samples of the cat 'Candidatus Mycoplasma haemominutum' was identified by molecular methods, according to its 100% 16S rRNA gene sequence homology with two Swiss isolates and one isolate from the UK. The rapid termination of the disease and the high pathogenicity of the causative agent observed in this case are unusual, taking into account that PCR results were negative for immunosuppressive viruses. This is the first record of this feline haemoplasma species 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.

Previously unpublished data from 1958 to 1967 attest the occurrence of Babesia divergens in cattle in several endemic foci of Northeast Hungary. During that period the number of clinical cases showed fluctuation with intervals of 4-5 years and monophasic seasonality (peaking in June). In order to assess the current status of bovine babesiosis in that region, blood samples were collected from 654 cattle on 44 farms of 36 settlements in or near the endemic area during 2005, and serum levels of IgG antibodies to B. divergens were measured by indirect fluorescent antibody test (IFAT). Only 2 samples (0.3%) showed positivity. In one village clinical babesiosis was observed over the past few years. Animals brought into the endemic area during the spring developed haemoglobinuria in the summer of the same year, but those introduced during the summer or autumn showed clinical signs only after two years. Sampled animals born and raised locally had neither haemoglobinuria nor seroconversion. Reduction in the number of cases during the past decades may have been influenced by the availability of hosts (i.e. decrease of cattle breeding) and the activity of vectors associated with climate-related changes (e.g. increase of annual sunlight hours in the endemic area). This is the first report on the prevalence of antibodies to B. divergens in cattle in Hungary.

In order to assess the seroprevalence of bovine neosporosis with indirect fluorescent antibody test (IFAT), blood samples were collected randomly from 1063 beef and dairy cattle belonging to 12 different breeds in Northeast Hungary. Antibodies to Neospora caninum were detected in 27 (2.5%) of the animals, kept on 19 of the 42 settlements included in this survey. Since samples were collected on 50 farms, herd prevalence amounted to 38%. The percentage of cattle with seroconversion increased with age, suggesting a postnatal source of infection. The highest rate of positivity was detected in Aberdeen Angus (3.3%) and Holstein-Friesian cows (3.2%), and the lowest in Limousine (0.9%), but no breed predisposition was statistically substantiated. Neosporosis was more prevalent in dairy (3.4%) than in beef (1.9%) cattle, although the difference was not significant. Only three out of the seropositive cows, all of them Holstein-Friesians, had a history of abortion.

Since the introduction of Anguillicola crassus into Europe, anguillicolosis has been a considerable problem in several countries. From 1991, periodical eel mortality occurred in Lake Balaton, Hungary. However, eels with a worm burden of 20 to 50 parasites did not show severe swimbladder lesions, which observation cast doubts on the primary aetiological role of the parasite in the eel kill. In order to study the pathology of the infection, from the spring of 1996 until October of the same year, 51 eels were collected from two regions of Lake Balaton and examined for swimbladder changes. To detect humoral antibodies, an enzyme-linked immunosorbent assay (ELISA) was performed, using cuticular-oesophageal worm antigen. The results of the test show the applicability of the method. However, no direct correlation was found between antibody levels or the intensity of infection and the swimbladder lesions. The low level of specific antibodies and the increasing severity of swimbladder changes in the autumn suggest that parasite-induced immunity is insufficient to prevent reinfection.