Prevalence and genetic characterization of Echinococcus spp. in cattle, sheep, and swine in Hungary.

A study was conducted to investigate genetic diversity of Echinococcus isolates collected in Hungarian slaughterhouses between 2015 and 2018. Organs of 219 animals with suspected hydatidosis were collected during routine meat inspection and sent to our laboratory. Echinococcus infection was confirmed in 65 animals. These results indicate that prevalence data based on reporting of slaughterhouses are not reliable without the appropriate training of the people responsible for meat inspection. The genetic diversity was evaluated by the DNA sequence analysis of the cytochrome c oxydase subunit 1 (cox1) mitochondrial gene. Echinococcus intermedius (n = 31), Echinococcus granulosus s.s. (n = 2), and Echinococcus multilocularis (n = 3) was identified in swine. In cattle, only E. granulosus s.s. (n = 20) was detected. E. granulosus s.s. (n = 7) was the dominant species in sheep; nevertheless, E. intermedius was also identified in two animals. E. granulosus s.s. (n = 29) and E. intermedius (n = 33) were classified in 13 and three haplotypes, respectively. The genetic diversity and haplotype network of E. granulosus s.s. were similar to that observed in some other countries of Eastern Europe. The genetic diversity of E. intermedius was low with a single dominant haplotype. Cysts were fertile in nine sheep (100%), 22 swine (61%), and three cattle (15%) indicating that all three species play a role in some extent in the epidemiology of cystic echinococcosis in Hungary. Based on the number of animals killed in the slaughterhouses involved in the present study, the rate of infection was 0.013% in sheep, 0.007% in cattle, and 0.001% in swine. As animals with hydatidosis originated from family farms, control programs should mainly focus on these facilities.

Retrospective study on the distribution of Dirofilaria immitis in dogs in Hungary.

Europe has experienced the spreading of vector-borne helminths including heartworms (Dirofilaria immitis) from the Mediterranean countries towards the northern ones in the past decades. Recently, the establishment of D. immitis was confirmed in Hungary on the basis of period prevalence studies involving dogs, red foxes (Vulpes vulpes) and golden jackals (Canis aureus). The aim of our retrospective study was to describe the spatial distribution of the parasite and the time course of spreading of D. immitis in Hungary. Necropsy records of 2622 dogs received at our laboratories from 2001 to 2015 were reviewed for heartworm infections. The locality of origin of animals was recorded in a geographic information system database and compared to the results of the period prevalence study involving wild canids. Autochthonous heartworm infection was detected in 27 dogs. The time course analysis indicates that the parasite established in Hungary in 2007. As temperature is one of the most important determinants of the distribution of D. immitis, the climate of the Great Hungarian Plain is the most suitable region for the establishment of D. immitis in Hungary. Our studies revealed that the Great Hungarian Plain became a D. immitis endemic region for 2015. Nevertheless, sporadic cases in wild canids and dogs also occur in other regions of the country.

Environmental determinants of the spatial distribution of Mesocestoides spp. and sensitivity of flotation method for the diagnosis of mesocestoidosis.

Mesocestoides spp. are zoonotic cestodes of wild and domesticated carnivores. Although the adult stages are relatively harmless intestinal parasites, the metacestode stages (tetrathyridia) can be responsible for life-threatening peritonitis and pleuritis in several species including dogs, cats, non-human primates and probably man. The aim of the present study was to reveal the spatial distribution pattern of Mesocestoides spp. in the most important final hosts, red foxes (Vulpes vulpes), to analyse the relationship of these patterns with landscape and climate by geographical information systems and to evaluate faecal flotation method for the detection of infection in the final host. Fox carcasses, representing 0.5% of the total fox population were randomly selected out of all the foxes of Hungary. The intestinal tract was examined by sedimentation and counting technique. The sensitivity of the flotation method was evaluated by the testing of the faecal samples of 180 foxes infected with Mesocestoides spp. The prevalence of infection was high in foxes (45.8%; 95% CI=41.0-50.6%), and the parasite was detected in all areas of Hungary. The high prevalence of the parasite in foxes suggests that the infection might also be common in outdoor dogs and cats. Mesocestoides infection could not be detected in any of the foxes by flotation method indicating that the sensitivity of the method is less than 0.6%. Therefore, almost all canine and feline infections remain undetected in the veterinary practice. Based on the statistical analysis, the altitude was the only determinant of the spatial distribution of Mesocestoides spp. indicating that infections in carnivores including dogs and cats can be expected mainly in midland regions (150-750 m above sea level). It might be attributed to the altitude-dependent species richness and abundance of the intermediate and final hosts of the parasite.

Spatial distribution of Anaplasma phagocytophilum and Hepatozoon canis in red foxes (Vulpes vulpes) in Hungary.

In recent years, Ehrlichia canis and Hepatozoon canis transmitted by Rhipicephalus sanguineus were reported from Hungary. The aim of the present study was to reveal the spatial distribution pattern of pathogens transmitted by R. sanguineus in a sentinel species, red fox (Vulpes vulpes) in Hungary and to analyse the relationship of these patterns with landscape and climate by geographical information systems. Fox carcasses, representing 0.5% of the total fox population were randomly selected out of all the foxes of Hungary. The spleen samples of the animals were tested by real-time PCR for Anaplasma platys, Babesia vogeli, E. canis and H. canis infection. Positive results were confirmed by conventional PCR followed by sequencing. The prevalence of H. canis infection was 22.2% (95% CI=18.4-26.4%), and this parasite was detected in all areas including the mountain regions of Hungary. These findings indicate that other tick species or other transmission routes (oral and transplacental) might be in the background of the countrywide distribution of H. canis. Anaplasma platys was not found; nevertheless, the prevalence of Anaplasma phagocytophilum infection transmitted by Ixodes ricinus was 12.5% (95% CI=9.7-16.1%) in foxes. B. vogeli and E. canis infection was not detected. There was no correlation between environmental parameter values in the home range of foxes and A. phagocytophilum or H. canis infection, which is in line with that observed in the case of tick species infesting foxes in Hungary. The results of this study indicate that R. sanguineus, if present, might be rare in Hungary. Our baseline study can be used for future evaluation of the effect of climate change on the spreading and emergence of R. sanguineus transmitted pathogens in Hungary.

Environmental determinants of the spatial distribution of Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus in Hungary.

Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus (syn. Capillaria aerophila) are the most important lungworm species infecting wild and domesticated canids in Europe. To investigate the spatial distribution of these parasites and the factors influencing their circulation in the fox populations, 937 red foxes (Vulpes vulpes) were tested for lungworm infection in Hungary. The prevalence of A. vasorum, C. vulpis and E. aerophilus infection was high (17.9, 24.6 and 61.7%). The distribution pattern of infection in foxes and the relationship of this pattern with landscape and climate was analyzed by geographic information system. Based on the analysis, the annual precipitation was the major determinant of the spatial distribution of A. vasorum and C. vulpis and E. aerophilus. Nevertheless, the mean annual temperature also influenced the distribution of A. vasorum and E. aerophilus. The positive relationship with annual precipitation and the negative relationship with mean annual temperature can be attributed to the sensitivity of larvae, eggs and intermediate hosts (snails and slugs) of lungworms for desiccation. Based on the highly clumped distribution of A. vasorum and C. vulpis, the indirect life cycle (larvae, slugs and snails) of these parasites seems to be particularly sensitive for environmental effects. The distribution of E. aerophilus was considerably less clumped indicating a lower sensitivity of the direct life cycle (eggs) of this parasite for environmental factors. Based on these results, lungworm infections in canids including dogs can be expected mainly in relatively wet and cool areas.

Dirofilaria immitis: an emerging parasite in dogs, red foxes and golden jackals in Hungary.

Hungary was not considered to be a heartworm (Dirofilaria immitis) endemic country until 2007, when the first autochthonous canine infection was described. Herein we report additional autochthonous heartworm infections in two dogs (Canis familiaris), twenty red foxes (Vulpes vulpes) (n=534; prevalence: 3.7%; 95% CI=2.4-5.7%) and two golden jackals (Canis aureus) (n=27; prevalence: 7.4%; 95% CI=2.1-23.4%) coming from eight counties. The identification of the parasite was based on morphology, morphometrics and amplification of 12S rDNA followed by sequencing in all cases. Our results indicate that Hungary became a D. immitis endemic country in the past decade. The prevalence and intensity of heartworm infection in wild canids is similar to or lower than that observed in the Mediterranean countries of Europe (3.7-7.4% vs. 0.4-12.7% and 1.5 vs. 2.9-4.4 worms/animal). These findings are in line with the results of the recently developed climate based forecast model to predict the establishment of D. immitis in Hungary.

Environmental determinants of the spatial distribution of Trichinella britovi and Trichinella spiralis in Hungary.

Trichinella spiralis and Trichinella britovi are the two most common species of the genus Trichinella persisting in the European wildlife. To investigate the spatial distribution of these Trichinella spp. and the factors influencing their circulation in Hungary, 3304 red foxes (Vulpes vulpes) and 0.29 million wild boars (Sus scrofa) were tested for Trichinella sp. infection in Hungary from 2006 to 2013. Trichinella spp. larvae from 68 (2.06%) foxes and 44 (0.015%) wild boars were identified by a multiplex PCR as T. britovi or T. spiralis. The locality of origin of foxes and wild boars were recorded in a geographic information system database. There was no correlation between environmental parameters in the home range of foxes and wild boars and the T. spiralis larval counts, but there was a positive correlation between the boundary zone of Hungary and T. spiralis infection (P<0.0001; odds ratio: 24.1). These results indicate that the distribution of T. spiralis in the Hungarian wildlife is determined by the transborder transmission of the parasite from the surrounding endemic countries. Multiple regression analysis was performed with environmental parameter values and T. britovi larval counts. Based on the statistical analysis, non-agricultural areas (forests, scrubs, herbaceous vegetation and pastures) and the mean annual temperature (P<0.0001; odds ratios: 9.53 and 0.61) were the major determinants of the spatial distribution of T. britovi in Hungary. The positive relationship with non-agricultural areas can be explained by the generalist feeding behaviour including scavenging of foxes in these areas. The negative relationship with the mean annual temperature can be attributed to the slower decomposition of wildlife carcasses favouring a longer survival of T. britovi larvae in the host carrion and to the increase of scavenging of foxes.

Environmental determinants of the spatial distribution of Echinococcus multilocularis in Hungary.

Human alveolar echinococcosis, caused by the metacestode stage of Echinococcus multilocularis, is one of the most pathogenic zoonoses in the temperate and arctic region of the Northern Hemisphere. To investigate the spatial distribution of E. multilocularis and the factors influencing this distribution in the recently identified endemic area of Hungary, 1612 red fox (Vulpes vulpes) carcasses were randomly collected from the whole Hungarian territory from November 2008 to February 2009 and from November 2012 to February 2013. The topographic positions of foxes were recorded in geographic information system database. The digitized home ranges and the vector data were used to calculate the altitude, mean annual temperature, annual precipitation, soil water retention, soil permeability, areas of land cover types and the presence and buffer zone of permanent water bodies within the fox territories. The intestinal mucosa from all the foxes was tested by sedimentation and counting technique. Multiple regression analysis was performed with environmental parameter values and E. multilocularis counts. The spatial distribution of the parasite was clumped. Based on statistical analysis, mean annual temperature and annual precipitation were the major determinants of the spatial distribution of E. multilocularis in Hungary. It can be attributed to the sensitivity of E. multilocularis eggs to high temperatures and desiccation. Although spreading and emergence of the parasite was observed in Hungary before 2009, the prevalence and intensity of infection did not change significantly between the two collection periods. It can be explained by the considerably lower annual precipitation before the second collection period.

Environmental determinants of the spatial distribution of Alaria alata in Hungary.

Alaria alata is a potential zoonotic parasite, which is widely distributed in Eurasia. To assess the risk of human infection, it is important to know the spatial distribution pattern of the parasite and factors influencing this pattern. To investigate these relationships, 1612 red fox (Vulpes vulpes) carcasses were randomly collected from the whole Hungarian territory, and the intestines were examined by sedimentation and counting technique. The spatial distribution of the parasite was highly clumped. The topographic positions where the foxes had been shot and the intensity of infections were recorded in geographic information system database. Digitized home ranges of infected and uninfected foxes were analysed on the background of geographic vector data of altitude, land cover types, permanent waters, mean annual temperature, annual precipitation and soil permeability. Multiple regression analysis was performed with environmental parameter values and A. alata scores. Based on the statistical analysis, lack of permanent waters, mean annual temperature, annual precipitation and soil permeability were the major determinants of the spatial distribution of A. alata. It can be explained by the use of biotopes by the intermediate hosts. The lack of permanent waters results in the use of temporary waters by the second intermediate hosts, frogs. The higher temperature, the lower precipitation and the higher soil permeability lead to earlier desiccation of temporary waters, and tadpoles and frogs infected with mesocercariae can be more easily predated by the final hosts (e.g., red foxes). Moreover, temporary waters are more easily contaminated with the faeces of the final hosts containing eggs than permanent waters. Therefore, high infection rate with A. alata can be expected mainly in lowland areas, where the hydrogeography of permanent waters is less complex, the precipitation is lower, the mean temperature and the soil permeability are higher than in highland areas.

Echinococcus multilocularis and Trichinella spiralis in golden jackals (Canis aureus) of Hungary.

Over the last decades the distribution area of the golden jackal (Canis aureus) has increased significantly in Europe, particularly in the Balkan Peninsula and in Central Europe. Vagrant individuals were described in many European countries. Herein, we report Echinococcus multilocularis (total worm count: 412) and Trichinella spiralis (101 larvae/g for muscles of the lower forelimb) infections in two golden jackals shot in Hungary. It is a new host record of E. multilocularis and T. spiralis in Europe and Hungary, respectively. As jackals migrate for long distances through natural ecological corridors (e.g., river valleys), they may play a significant role in the long distance spread of zoonotic parasites into non-endemic areas of Europe. Therefore, monitoring zoonotic parasites in this host species can be recommended in the European Union.

Spatial distribution of Trichinella britovi, T. spiralis and T. pseudospiralis of domestic pigs and wild boars (Sus scrofa) in Hungary.

Trichinellosis is a foodborne disease caused by the consumption of raw meat and raw meat-derived products from swine, horse and some game animals infected with nematode worms of the genus Trichinella. Between June 2006 and February 2011, 16 million domestic pigs and 0.22 million wild boars (Sus scrofa) were tested for Trichinella sp. in Hungary. Trichinella infection was not found in any pigs slaughtered for public consumption. Nevertheless, Trichinella spiralis was detected in four backyard pigs when trace back was done following a family outbreak. Trichinella infection was demonstrated in 17 wild boars (0.0077%). Larvae from wild boars were identified as Trichinella britovi (64.7%), T. spiralis (29.4%) and Trichinella pseudospiralis (5.9%). Although the prevalence of Trichinella sp. infection in wild boars and domestic pigs is very low, the spatial analysis reveals that the level of risk differs by region in Hungary. Most of the T. britovi infected wild boars (63.6%) were shot in the north-eastern mountain area of Hungary; whereas domestic pigs and wild boars infected with T. spiralis were detected only in the southern counties bordering Croatia and Romania. In the north-western and central counties, the prevalence of Trichinella infection seems to be negligible.

Spatial distribution and genetic diversity of Echinococcus multilocularis in Hungary.

Human alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is the most pathogenic helminthic zoonosis in the temperate and arctic region of Europe. Between November 2008 and February 2009, 840 red fox (Vulpes vulpes) carcasses, were randomly collected from the whole Hungarian territory. The intestinal mucosa from all the foxes was tested by sedimentation and counting technique. E. multilocularis adult worms were detected in foxes of 16 out of the 19 Hungarian counties and in the suburban areas of the capital, Budapest. The prevalence and abundance of infection was significantly (P<0.001) higher in the north-western half (16.2%, CI=14.5-17.9; m ± SE=165.5 ± 112.4) than in the south-eastern half of the country (4.2%, CI=3.2-5.2; m ± SE=3.6 ± 2.1). The highest prevalence (26.6%, CI=22.5-30.8%) and abundance (m ± SE=614.2 ± 469.3) was observed in the Northern Mountain Region bordering Slovakia. The multi-locus microsatellite analysis of 81 worms showed the presence of four out of the five main European profiles. The H profile was the most common profile (55.5%) with nine genotypes, followed by the G (18.5%) with two genotypes, E (13.6%) with one genotype and D (12.4%) with two genotypes. The genetic distance was not statistically correlated with the geographical distance of the samples, supporting the hypothesis that the geographical distance is only a minor factor among those involved in the genetic distribution of this parasite in Europe. These data indicate that Hungary should be considered as a peripheral area of a single European focus, where the dispersal movement of foxes resulted in the spreading of the parasite from one county to another within a time period short enough to avoid a substantial genetic drift.

Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralis in red foxes (Vulpes vulpes) in Hungary.

The red fox (Vulpes vulpes) is considered one of the main reservoir of Trichinella spp. in Europe. As limited information on Trichinella infection in wildlife of Hungary is available, 2116 red foxes, representing more than 3% of the estimated fox population of the country, were screened to detect Trichinella larvae by a digestion method. Trichinella larvae from the 35 positive foxes were identified by a multiplex PCR as Trichinella britovi (30 isolates, 85.7%), Trichinella spiralis (4 isolates, 11.4%), and Trichinella pseudospiralis (1 isolate, 2.9%). The true mean intensity of T. britovi, T. spiralis and T. pseudospiralis larvae in lower forelimb muscles was 23.6, 3.5 and 13.5larvae/g, respectively. T. spiralis was detected only in the southern and eastern regions. The non-encapsulated T. pseudospiralis was recorded for the first time in Hungary. Although the overall true prevalence of Trichinella infection in foxes was only 1.8% (95% confidence interval, CI=1.5-2.1%), the spatial analysis reveals different risk regions. In the north-eastern counties bordering Slovakia and Ukraine (21% of the Hungarian territory), the true prevalence of Trichinella infection is significantly higher than that observed in other regions (6.0%, CI=4.8-7.1%). In the southern counties bordering Croatia, Serbia and Romania (41% of the Hungarian territory), the true prevalence of Trichinella infection is moderate (1.4%, CI=1.0-1.8%). In the north-western and central counties (38% of Hungarian territory), the prevalence of Trichinella infection is significantly lower (0.2%, CI=0.1-0.4%) than that of the other regions. Based on the statistical analysis and the evaluation of epidemiological data, none of the counties can be considered free of Trichinella infection. In the past decade, Trichinella infection has been detected only in few backyard pigs, and only few wild boar-related autochthonous infections in humans were described. Nevertheless, these results highlight the need of the maintenance of a strict monitoring and control programmes on Trichinella infection in farmed and hunted animals of Hungary.

Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis.

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.

Temporal distribution of Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary.

A survey was carried out over a 4-year period to describe the temporal distribution of three 'anthropophilic' tick species, Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary. Altogether 4658 adult ticks belonging to the three species were collected from 1931 red foxes (Vulpes vulpes) killed in an area of about 70,000 km(2) representing all major climatic areas of the country. The seasonal activity of the three species was different. I. ricinus ticks were most active between April and June with an activity peak in May. A less marked increase of activity was also observed in September and October. The highest activity of D. reticulatus ticks was seen between September and November with an activity peak in October, nevertheless, a marked increase of activity could also be observed in April. Small number of I. ricinus and D. reticulatus were collected in all other months. H. concinna ticks were active from May to July with an activity peak in June and completely disappeared between October and March. The temporal distribution of the three tick species might be used for predictions on the seasonality of tick-borne diseases in Hungary.

Rickettsiae of the spotted-fever group in ixodid ticks from Hungary: identification of a new genotype ('Candidatus Rickettsia kotlanii').

Three common European 'anthrophilic' ticks, Ixodes ricinus, Haemaphysalis concinna and Dermacentor reticulatus, were collected in Hungary and tested, in assays based on nested PCR, for rickettsiae of the spotted-fever group. Low percentages of I. ricinus (2.7%) and H. concinna (1.0%) and a high percentage of D. reticulatus (26.8%) were found to be infected. The rickettsiae in the ticks were then identified, by sequencing of the genes coding for 16S ribosomal RNA (16S rDNA), citrate synthase (gltA) and the rOmpA outer-membrane protein (ompA), as Rickettsia helvetica, Rickettsia monacensis, Rickettsia sp. RpA4, or what is probably a newly recognized Rickettsia species ('Candidatus Rickettsia kotlanii'). These results raise the possibility that rickettsiae other than Rickettsia slovaca are involved in human disease in Hungary. Current knowledge on the distributions of the rickettsiae of the spotted-fever group that are emerging in Europe is also summarized.

Spatial distribution of Dermacentor reticulatus and Ixodes ricinus in Hungary: evidence for change?

A survey was conducted to investigate the spatial distribution of Ixodes ricinus and Dermacentor reticulatus in Hungary and to compare these data with the results of a previous large-scale survey. In the survey conducted in the 1950s, D. reticulatus adults were detected in two isolated areas of two counties, and the presence of these ticks in the collection was explained by accidental introduction. In the present survey, D. reticulatus became the second most common species occurring in all 16 counties involved in the monitoring and showed high prevalence. The change in the spatial distribution of this tick species, the increase of incidence of Babesia canis infection in Hungary, and the increasing number of canine babesiosis case reports from other Central and Central Eastern European countries since the 1970s suggest an expansion of the geographic range of D. reticulatus and intensification of the transmission rate of B. canis and probably other D. reticulatus-borne diseases (e.g. tularemia and tick-borne lymphadenopathy) in the region. The spatial distribution of I. ricinus was roughly in line with the results of the earlier survey. I. ricinus was the most common tick species being present in all 16 counties with the highest prevalence. Nevertheless, the comparison of the data of the previous and current survey cannot be used for fine-scale analysis; thus, it cannot be dismissed that the spatial distribution of I. ricinus also changed during the past decades. The spatial distribution patterns of tick-borne encephalitis in Hungary and other Central Eastern European countries may indicate such a change.

Anaplasma phagocytophilum: an emerging tick-borne pathogen in Hungary and Central Eastern Europe.

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, Ehrlichia equi and Anaplasma phagocytophila) is the causative agent of granulocytic ehrlichiosis (anaplasmosis) in humans, horses, sheep, cattle, dogs and cats. In the present study, 452 European sheep ticks (Ixodes ricinus) collected from 100 red foxes (Vulpes vulpes) in Hungary were tested for the pathogen, as 112 pools each containing five or fewer ticks from one fox. Six of the pools, representing ticks from six different foxes, were found infected in the PCR-based test employed. This is the first time that A. phagocytophilum has been reported in Hungary. A summary of the information available from Central Eastern Europe on the prevalence of A. phagocytophilum in ticks, its seroprevalence in humans, and the number of human cases of granulocytic ehrlichiosis known in the region is presented.

Canine onchocercosis in Greece: report of further 20 cases and molecular characterization of the parasite and its Wolbachia endosymbiont.

Recently, sporadic cases of subconjunctival Onchocerca infection have been reported in dogs in Greece and Hungary. Herein we report further cases from Greece and the results of the molecular analysis of Onchocerca sp. removed from Greek dogs and its Wolbachia endosymbionts. Twenty dogs of various breeds, 1-11 years of age with subconjunctival onchocercosis (4 cases each in right or left eye, 12 cases in both eyes) were presented having similar manifestations. Periorbital swelling, exophthalmos, lacrimation, discharge, photophobia, conjunctival congestion, corneal edema, protrusion of the nictitating membrane, and subconjunctival granuloma or cyst formation were the most important clinical signs. After surgical excision of the periocular masses containing the worms, all animals recovered fully from onchocercosis. Based on the similarities of the clinical picture of the Greek and Hungarian cases, the similar morphology of the Greek and Hungarian isolates, and the identical sequences of the cytochrome oxidase gene of the filarial parasites and that of the 16S ribosomal RNA gene from their Wolbachia endosymbionts, the Onchocerca sp. isolated from dogs in Greece and Hungary appears to belong to the same species.