Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

First report of molecular taxonomic analyses of European beaver metazoan parasites from Hungary.

European beaver (Castor fiber L. 1758) is the biggest rodent species living in Europe. Beavers are semi-aquatic animals; they are defecating directly into the water; thus, they have an important role in spreading parasites related to water (e.g., protozoa and flukes). The first specimens of this once extinct rodent species in Hungary turned up in Szigetköz (upper flow of the Hungarian Danube) in 1991 dispersed from Austria. The reintroduction to Hungary started in 1996, and the population slowly increased in number up to around 4000 individuals, but the knowledge about their parasites is lacking. This is the first report on the metazoan parasites of beavers in Hungary and their molecular taxonomy. In the 5-year study, 47 beavers were trapped in four locations and euthanized with permission. Three different metazoan parasites were collected: larvae and adults of Platypsyllus castoris beetles, nymphs and adults of Schizocarpus sp. mites and eggs and adults of Stichorchis subtriquetrus flukes. From these three parasite species, molecular taxonomic studies were also carried out. The low number of metazoan parasites species detected in Hungarian beavers compared to other European countries (e.g., Poland) might be attributed to host population bottleneck effect during reintroduction. As parasites represent a significant component of the biodiversity and ecosystem, the conservation efforts should focus not only on host species but also on their parasites.

Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

Emerging human alveolar echinococcosis in Hungary (2003-2018): a retrospective case series analysis from a multi-centre study.

BACKGROUND: Human alveolar echinococcosis (AE) caused by Echinococcus multilocularis is an underreported, often misdiagnosed and mistreated parasitic disease mainly due to its low incidence. The aim of this study was to describe the epidemiological and clinical characteristics of human AE patients in Hungary for the first time. METHOD: Between 2003 and 2018, epidemiological and clinical data of suspected AE patients were collected retrospectively from health database management systems. RESULTS: This case series included a total of 16 AE patients. The mean age of patients was 53 years (range: 24-78 years). The sex ratio was 1:1. Four patients (25%) revealed no recurrence after radical surgery and adjuvant albendazole (ABZ) therapy. For five patients (31.3%) with unresectable lesions, a stabilization of lesions with ABZ treatment was achieved. In seven patients (43.8%), progression of AE was documented. The mean diagnostic delay was 33 months (range: 1-122 months). Three AE related deaths (fatality rate 18.8%) were recorded. CONCLUSIONS: AE is an emerging infectious disease in Hungary with a high fatality rate since based on our results, almost every fifth AE patient died in the study period. Differential diagnosis and appropriate surgical and medical therapy for AE is an urging challenge for clinicians in Hungary, as well as in some other European countries where E. multilocularis is prevalent.

Rapid spread and emergence of heartworm resulting from climate and climate-driven ecological changes in Hungary.

In Europe, Dirofilaria immitis persists mainly in the southern countries with a Mediterranean climate. Because spreading of heartworms from these countries towards the northern ones could be observed in the past decades, necropsy records of 4076 Hungarian dogs were reviewed for heartworm infections. The first autochthonous canine D. immitis case was detected on the Great Hungarian Plain in 2007. Until 2011, the number of heartworm infection cases was low, and these cases were restricted to a small part of the Great Hungarian Plain. Since 2012, the number of cases has increased considerably, and the rapid expansion of the parasite's geographic range could also be observed. Our retrospective study has revealed that most of the Hungarian territory became a heartworm endemic region, and the prevalence of infection greatly multiplied over the past 12 years. The establishment, rapid spread, and emergence of D. immitis may be mainly explained by the warming climate in Hungary. However, the partly climate-driven spread of the most important reservoir host in wildlife, the golden jackal (Canis aureus) from the Mediterranean Balkan Peninsula might have also played a significant role. This study is an example of the rapid spread and emergence of pathogens resulting from climate and climate-driven ecological changes. Because a continuous increase in the temperature and further dispersal of golden jackals in Europe are projected, further spread and emergence of heartworm can be expected. Similar spread and emergence of D. immitis could be observed in North America. It cannot be excluded that similar reasons (global warming and rapid dispersal and population growth of the most important wild canine reservoir host) are in the background on both continents.

Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016.

IntroductionThis paper reviews the current knowledge and understanding of Cryptosporidium spp. and Giardia spp. in humans, animals and the environment in 10 countries in the eastern part of Europe: Bosnia and Herzegovina, Croatia, Czech Republic, Estonia, Hungary, Latvia, Poland, Romania, Serbia and Slovenia. Methods: Published scientific papers and conference proceedings from the international and local literature, official national health service reports, national databases and doctoral theses in local languages were reviewed to provide an extensive overview on the epidemiology, diagnostics and research on these pathogens, as well as analyse knowledge gaps and areas for further research. Results:Cryptosporidium spp. and Giardia spp. were found to be common in eastern Europe, but the results from different countries are difficult to compare because of variations in reporting practices and detection methodologies used. Conclusion: Upgrading and making the diagnosis/detection procedures more uniform is recommended throughout the region. Public health authorities should actively work towards increasing reporting and standardising reporting practices as these prerequisites for the reported data to be valid and therefore necessary for appropriate control plans.

Evaluation of faecal flotation methods followed by species-specific PCR for detection of Echinococcus multilocularis in the definitive hosts.

Echinococcus multilocularis is one of the most pathogenic zoonotic parasites in the temperate and arctic region of the Northern Hemisphere. For estimating the potential risk of human infection in endemic areas, reliable antemortem methods are needed to detect the parasite in carnivore definitive hosts. The sensitivity of routine flotation techniques for detection of E. multilocularis eggs was found to be low (3-33%) depending on the flotation solution used (specific gravities = 1.3-1.4). An improved faecal flotation followed by a species-specific PCR is described with a sensitivity of 74% (95% CI = 62-84%) and a specificity of 100% (95% CI = 94-100%). These parameters are similar to those of the intestinal scraping technique (sensitivity = 78%, specificity = 100%). The sensitivity of the improved flotation was significantly higher (P < 0.0001) than that of routine flotation techniques. The costs of the method are similar or lower than those of other antemortem diagnostic methods. Based on these data, the method is suitable for surveys of domesticated and wild carnivores.

Seasonal activity and tick-borne pathogen infection rates of Ixodes ricinus ticks in Hungary.

Ixodes ricinus is the most important tick species in Europe as it is most widely distributed and transmits the majority of tick-borne zoonotic pathogens. As limited data are available for Hungary, the aim of the present study was to investigate the seasonal timing of questing by I. ricinus and the infection rate of this tick species with all major tick-borne zoonotic pathogens. Monthly collections of I. ricinus were carried out over 3 consecutive years by dragging a blanket in 6 biotopes representing different areas of Hungary. Altogether, 1800 nymphs (300 per collection point) were screened as pooled samples (each of 5 specimens) by PCR-based methods for tick-borne pathogens. I. ricinus larvae, nymphs, and adults had bimodal activity patterns with a major peak in the spring. As newly moulted ticks of all stages are thought to emerge in the autumn of each year, it appears that most newly emerged ticks delayed their questing until the following spring. The minimum prevalence of Borrelia burgdorferi sensu lato was 2.5%. Borr. afzelii, Borr. burgdorferi sensu stricto, Borr. garinii, Borr. lusitaniae, and Borr. valaisiana were identified by hybridization. The minimum infection rate with spotted fever group rickettsiae was 1.9%. Rickettsia helvetica was identified in all biotopes. The minimum prevalence of Anaplasma phagocytophilum, Babesia divergens and Bab. microti was low (0.3-0.5%). Bartonella spp.-, Francisella tularensis-, and TBE virus-specific amplification products were not detected. Relative to the results of comparable studies carried out in the Carpathian Basin, the prevalence of tick-borne pathogens was low in Hungary. This might be attributed to the climatic difference between the lowland areas of Hungary and submountain areas of the surrounding countries involved in the studies.

Genetic variability of Echinococcus granulosus sensu stricto in Europe inferred by mitochondrial DNA sequences.

The genetic diversity of Echinococcus granulosus sensu stricto (s.s.) metacestodes from four European countries was evaluated by the DNA sequence analysis of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Of the 312 organisms investigated, 132 were from Bulgaria, 35 from Hungary, 89 from Italy and 56 from Romania. Considerable intraspecific variation was observed in the mitochondrial cox1 sequences: 24 haplotypes were detected in the Eastern European population and seven in the Italian population. The Eastern European population parsimony network displayed a star-like features consisting of the most common haplotype EG1 (G1 genotype) and the three major haplotypes: EG2, EG3 and EG4. The EG1 was also the major haplotype in the Italian population network, though with a higher prevalence (73%) compared to the Eastern European network. The percentage of the population constituted by the G1 genotype was used as an indirect index to evaluate the genetic diversity within E. granulosus s.s. populations of Eurasia. A clinal correlation between the percentage of the G1 genotype and the geographical regions of Eurasia was observed: the G1 genotype is highly represented in the Mediterranean Basin; it decreases in Eastern Europe and South-West Asia and increases in China. This clinal correlation could reflect the spreading of livestock domestication from Southern-Western Asia during the Neolithic period, beginning around 12,000 BC.

Detection of a novel Francisella in Dermacentor reticulatus: a need for careful evaluation of PCR-based identification of Francisella tularensis in Eurasian ticks.

Francisella tularensis, the causative agent of tularemia, has been detected in ixodid ticks in some regions of North America, Europe, and Asia. In the present study, 245 Dermacentor reticulatus, 211 Ixodes ricinus, and 194 Haemaphysalis concinna adults from Hungary were tested for the presence of F. tularensis by polymerase chain reaction (PCR) assays based on 16S ribosomal RNA (16S rDNA) and T-cell epitope of a Francisella membrane protein (TUL4). No Francisella-specific amplification products were detected in I. ricinus and H. concinna ticks. Francisella DNA was identified using PCR assays based on 16S rDNA and TUL4 gene in D. reticulatus with similar prevalence (minimum 1.2%) as demonstrated in earlier European and Asian studies detecting F. tularensis in D. reticulatus. However, the 16S rDNA and TUL4 gene sequences of the Francisella-like agent occurring in D. reticulatus differed from the homologous sequences of Francisella spp. deposited in GenBank. Phylogenetic reconstructions showed that the new genotype detected in D. reticulatus was closely related to Francisella-like endosymbionts of North American Dermacentor ticks. Although further studies are needed on the relationship of this bacterium with ticks, the results highlight the need for careful evaluation of PCR-based identification in European and Asian laboratories that screen ixodid ticks for F. tularensis.

Onchocercosis: a newly recognized disease in dogs.

In the past 15 years, onchocercosis has been reported with increasing frequency in dogs in Europe and the United States, and 64 cases have been described so far. According to some authors, the Onchocerca sp. responsible for canine cases spills over from domestic or wild ungulates into dogs. However, canine Onchocerca does not match any of the descriptions for species of Onchocerca reported from domesticated and wild animals in Europe or North America. The nucleotide sequences of canine Onchocerca are also unique within the genus. Moreover, patent Onchocerca infections can be seen only in accidental hosts closely related to the natural hosts. In canine onchocercosis cases, high microfilarial load could be observed indicating that canids might be the definitive hosts of the parasite. Therefore, others suggested that Onchocerca lupi Rodonaja, 1967 originally described from a wolf (Canis lupus) can be responsible for these infections, which is a typical example for host switch and site shift, the dominant modes of speciation of the genus Onchocerca. The morphology, molecular characteristics, phylogeny, life cycle, host specificity, geographical distribution of Onchocerca sp. infecting dogs, as well as the clinical signs, pathology, laboratory diagnosis, therapy and possible zoonotic significance of canine onchocercosis are reviewed. Research into human onchocercosis has been hampered by the lack of analogous models. As infections in dogs may provide a practical experimental system, further studies should be encouraged to try to establish experimental Onchocerca infections in dogs.

Bartonella infections in fleas (Siphonaptera: Pulicidae) and lack of bartonellae in ticks (Acari: Ixodidae) from Hungary.

Fleas (95 Pulex irritans, 50 Ctenocephalides felis, 45 Ctenocephalides canis) and ixodid ticks (223 ixodes ricinus, 231 Dermacentor reticulatus, 204 Haemaphysalis concinna) were collected in Hungary and tested, in assays based on PCR, for Bartonella infection. Low percentages of P. irritans (4.2%) and C. felis (4.0%) were found to be infected. The groEL sequences of the four isolates from P. irritans were different from all the homologous sequences for bartonellae previously stored in GenBank but closest to those of Bartonella sp. SE-Bart-B (sharing 96% identities). The groEL sequences of the two isolates from C. felis were identical with those of the causative agents of cat scratch disease, Bartonella henselae and Bartonella clarridgeiae, respectively. The pap31 sequences of B. henselae amplified from Hungarian fleas were identical with that of Marseille strain. No Bartonella-specific amplification products were detected in C. canis, I. ricinus, D. reticulatus and H. concinna pools.

[Babesia microti and Anaplasma phagocytophilum: two emerging zoonotic pathogens in Europe and Hungary]. / Babesia microti és Anaplasma phagocytophilum: két növekvo jelentoségu kórokozó Európában és hazánkban.

Babesia microti and Anaplasma phagocytophilum was recently reported with a minimum prevalence of 0.9 and 1.3% in Hungary based on the PCR-sequencing analysis of 452 European sheep ticks (Ixodes ricinus). These results and the epidemiological data of the neighbouring countries indicate that human cases caused by these pathogens may occur in the country. The aim of the present paper is to summarise the current knowledge on the morphology, life cycle and distribution of B. microti and A. phagocytophilum, and the epidemiology, clinical features, diagnosis, treatment and control of babesiosis and granulocytic anaplasmosis.

[Current knowledge on human trichinellosis]. / Az ember trichinellosisával kapcsolatos aktuális ismeretek.

Until the beginning of the nineties, the control of trichinellosis was thought to be a solved problem in Europe. However, as a consequence of social, economical and ecological changes in Central Eastern and Eastern Europe in the past decade, trichinellosis became a re-emerging infectious disease in the region. Molecular genetic studies demonstrated that at least two Trichinella species can be responsible for trichinellosis outbreaks in the region, and the epidemiology of trichinellosis is more complex than previously thought. Although Trichinella spiralis has been successfully eradicated in Hungary, it was recently demonstrated that Trichinella britovi persists in the sylvatic cycle. In view of the considerable epidemiological changes in the region, the aim of this review is to summarise the current knowledge on the clinical aspects of human trichinellosis.

[Rickettsia helvetica: an emerging tick-borne pathogen in Hungary and Europe]. / Rickettsia helvetica: egy növekvo jelentoségu kullancs terjesztette kórokozó hazánkban es Európában.

Rickettsia helvetica belonging to spotted fever group rickettsiae was recently detected by polymerase chain reaction followed by sequencing in European sheep ticks (Ixodes ricinus) from Hungary. Current knowledge on these rickettsiae and the clinical and diagnostic aspects of R. helvetica infection is summarized. In acute cases, R. helvetica is generally responsible for flu-like symptoms. Nevertheless, recent data indicate that in chronic cases, these rickettsiae can be responsible for perimyocarditis resulting sudden cardiac death and might play a role in the pathogenesis of aortic valve disease. The diagnosis can be based on serological, molecular and histological methods. A summary of the information available from Hungary and neighbouring countries on the prevalence of tick-borne encephalitis virus, Anaplasma, Borrelia, Francisella, Rickettsia and Babesia infections in I. ricinus is also presented.

[Human alveolar echinococcosis: an emerging zoonosis in Hungary and Europe]. / Humán alveolaris echinococcosis: egy növekvo jelentoségu zoonosis hazánkban és Európában.

Human alveolar echinococcosis, caused by the metacestode stage of Echinococcus multilocularis, is one of the most dangerous zoonoses in the temperate and arctic areas of northern hemisphere. The mortality of the disease exceeds 95% in untreated or inadequately treated patients. In the past three decades, the spread of this parasite was observed in Europe as a consequence of human interventions resulting in the population size increase of foxes. The authors demonstrated the presence of E. multilocularis in foxes in all northern counties of Hungary and the existence of hyperendemic regions in the Counties Nógrád and Gyor-Moson-Sopron. The aim of the present paper is to summarize the current knowledge on the life cycle and distribution of E. multilocularis, and the epidemiology, clinical features, diagnosis, treatment and control of human alveolar echinococcosis.

Echinococcus multilocularis: an emerging pathogen in Hungary and Central Eastern Europe?

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is reported for the first time in Red Foxes (Vulpes vulpes) in Hungary. This parasite may be spreading eastward because the population of foxes has increased as a consequence of human interventions, and this spread may result in the emergence of alveolar echinococcosis in Central Eastern Europe.