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.

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.

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.

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.

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.

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.

The heritability and specificity of responsiveness to infection with Haemonchus contortus in sheep.

High responder (HR) and low responder (LR) lines of sheep of the Hungarian Merino breed were selected by using faecal egg counts (epg) as parameter of responsiveness after double artificial Haemonchus contortus test infections. A total of 81 lambs born to assortative matings of HR rams with HR ewes and LR rams with LR ewes, as well as 28 control lambs from unselected parent sheep, were studied to estimate the heritability of responsiveness. A relatively high level of heritability (h2 = 0.49 +/- 0.17) was observed. A comparative assessment of responder classification carried out by two subsequent and distinct H. contortus and Trichostrongylus colubriformis infections revealed a remarkable degree of agreement: identical results in 69.7% and marginal differences in 23.2% of 99 animals. These results suggest that by selecting sheep for high responsiveness to a certain species of nematodes, one can also achieve a substantial improvement of resistance against a wide range of other nematode species.

The distribution of nematode egg counts and larval counts in grazing sheep and their implications for parasite control.

The frequency distribution of gastrointestinal nematode egg and lungworm larval counts was examined in 101 and 87 naturally infected ewes on two farms. The egg and larval outputs of the two flocks followed the negative binomial pattern of distribution (with k values below unity) at each time of sampling, which suggests highly overdispersed worm burdens. The results of the statistical analysis indicated that a relatively small part of the two flocks was responsible for the excretion of the majority of both gastrointestinal nematode eggs and Dictyocaulus filaria larvae. It is concluded that by eliminating "wormy" individuals of flocks either by selective breeding or by their selective anthelmintic treatment, effective control of parasite transmission can be achieved. Because of the phenomenon of nematode "clumping" it seems necessary to expand new methods for estimation of flock productivity caused by nematodes in livestock and to incorporate negative binomial parameter (k) in mathematical models of nematode population dynamics.

Potentiation of ionophorous anticoccidials with dihydroquinolines: compatibility of lasalocid and semduramicin with duokvin.

Three battery tests were conducted to reveal whether or not there is an interaction between the new dihydroquinoline antioxidant, duokvin and lasalocid or the new anticoccidial, semduramicin, similar to that observed with some other ionophorous anticoccidials. In terms of body weight gain, no significant difference due to toxic interaction between duokvin and any dose of lasalocid or semduramicin was detected in chickens experimentally infected with oocysts of Eimeria tenella and E. mitis. Anticoccidial efficacy at reduced doses of both lasalocid and semduramicin in combination with duokvin showed numerical improvement; however, this again proved to be insignificant. The lack of incompatibility of this antioxidant with lasalocid or semduramicin allows their simultaneous administration on the one hand, but it fails to enable a substantial reduction of the chemoprophylactic concentration of anticoccidials in the broiler ration on the other.

Cryptosporidiosis in birds--a review.

The morphology, life cycle, maintenance, host specificity, incidence of Cryptosporidium species infecting birds, as well as the epidemiology, clinical signs, pathology, immunology, diagnosis, therapy, and control of avian cryptosporidiosis are reviewed. Based on the accepted criteria used for differentiation of Cryptosporidium isolates into valid species, this review places the validity of C. meleagridis in doubt and suggests that C. meleagridis isolated from birds is very closely related to, or identical with C. parvum infecting more than 100 species of mammals.

Ectoparasite infestations of red foxes (Vulpes vulpes) in Hungary.

A survey was carried out to investigate the ectoparasite infestations of 100 red foxes (Vulpes vulpes) in Hungary. The overall prevalence of flea and tick infestation was high (62 and 86% with the dominance of the anthropophil Pulex irritans and Ixodes ricinus), but the number of parasites was low to moderate. Felicola vulpis was not found in the present study, and the prevalence of Otodectes cynotis was only 2%. Based on prevalence (21%), mange lesion scores, and the negative correlation between lesion scores and condition of foxes, Sarcoptes infestation should be considered as the most important parasitosis of foxes in Hungary. Besides the ecological significance of these parasites, the high overall prevalence of mange and anthropophil flea and tick infestations of foxes and the appearance of these animals in the synanthropic environment as a result of the increasing population size, may result in the increasing incidence of flea, tick and accidental mite infestation of man and domestic animals, and may enhance the transmission rate of some vector-borne diseases.

Characterization of Cryptosporidium spp.--recent developments and future needs.

Cryptosporidia, widely distributed protozoan parasites of vertebrates have recently attracted increasing interest due to several serious waterborne outbreaks, the life-threatening nature of infection in immunocompromised patients, and the realization of economic losses caused by these pathogens in livestock. Genetic polymorphism within Cryptosporidium spp. is being detected at a continuously growing rate, owing to the widespread use of modern molecular techniques. The aim of this paper is to review the current status of taxonomy, genotyping, molecular phylogeny, and characterization of cryptosporidia, and to highlight the need for polyphasic typing, i.e. an integrated approach comprising standardized morphologic, biologic, and molecular methods for describing Cryptosporidium species and isolates, and for establishing "virtual" reference strains.

Ocular onchocercosis in dogs: aberrant infection in an accidental host or lupi onchocercosis?

Four adult dogs that had spent their entire life in Hungary, were found to be infected with filaroid nematodes of the genus Onchocerca. The morphology and location of the parasites as well as pathological lesions were similar to those described earlier in the one Hungarian and five US dogs. Only moderate morphological differences were noted between the adults of Onchocerca sp. infecting dogs and O. volvulus of man or O. lienalis of cattle. Nevertheless, the morphology of microfilariae of Onchocerca from dogs is unique within the genus. Their length was less than half the length of microfilariae of other Onchocerca spp. known so far. In addition to size differences, several characteristic morphological features were observed. The unsuccessful attempt to infect dogs with O. lienalis, the absence of O. volvulus and O. lienalis in endemic regions of canine onchocercosis, the different size, morphology, and location of the adults in dogs and cattle, the exceptionally small size and unique morphology of microfilariae of Onchocerca of canids indicate that a distinct species might be responsible for canine onchocercosis. Since the larval concentration in the skin was high (50-3600 microfilariae g(-1)) in all affected dogs, the diagnosis prior to surgical removal of worm nodules can be based on the examination of a small skin snip collected from the head or abdominal region. Infections in dogs may provide a model to study human onchocercosis, therefore, further studies are encouraged on the feasibility of experimental infection of dogs with this Onchocerca species.

Influence of in ovo administered Cryptosporidium baileyi oocyst extract on the course of homologous infection.

In order to evaluate the effect of in ovo vaccination on avian cryptosporidiosis, two doses (1 and 10microg) of Cryptosporidium baileyi oocyst extract (OE) were injected into the amnionic sac of embryonated, specific pathogen-free chicken eggs. After hatching these birds as well as infected controls (IC) were inoculated with 8x10(5) C. baileyi oocysts at 10 days of age. Another group of chickens remained uninfected (UC). Faecal oocyst shedding was measured every second day, and weekly ELISAs were performed to monitor seroconversion. Those chickens that received OE during embryogenesis showed dose-dependent shift in their oocyst shedding, with higher oocyst output of OE1 and OE10 birds compared to IC ones. The patency was significantly longer in the OE10 group than in IC or OE1. ELISA results showed low seroconversion of OE1 and OE10 chickens prior to homologous challenge. Challenge infection resulted in antibody levels without significant difference between IC, OE1 and OE10 groups. These data suggest that in ovo vaccination with C. baileyi oocyst extract does not promote immune response, moreover, it may impair immunity and thus delay the clearance of cryptosporidia from chickens.

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.

Extraintestinal nematode infections of red foxes (Vulpes vulpes) in Hungary.

A survey was carried out to investigate the prevalence and worm burden of extraintestinal nematodes in 100 red foxes (Vulpes vulpes) of Hungary. The overall prevalence of nematode infections of the respiratory tract was 76%. Eucoleus aerophilus (Capillaria aerophila) was the predominant species (66%), followed by Crenosoma vulpis (24%), Eucoleus (Capillaria) böhmi (8%) and Angiostrongylus vasorum (5%). Pearsonema (Capillaria) plica was found in 52% of the urinary bladders. In 3% of the foxes, Trichinella britovi was present in muscle samples. The high prevalence of lungworms and P. plica and the fox colonisation in urban areas may enhance the prevalence of these nematode infections in domestic dogs and cats, and the flow of T. britovi from the sylvatic cycle to the domestic cycle, enhancing the risk of infections in humans.

Canine ocular onchocercosis in Hungary.

An adult male mongrel dog that had spent its entire life in Hungary, was found to have infection with filaroid nematodes of the genus Onchocerca. The gravid male and female parasites were embedded in bean-sized granulomatous masses on the conjunctiva and the sclera of both eyes. The cuticle of females consisted of two separated layers in longitudinal sections, the external layer bearing ridges and the internal layer showing striations. The ridges were marked, rounded in shape, and the ratio of body diameter to the distance between ridges varied between 7:1 and 10:1. At midbody of the worms, two striations could be seen between each pair of ridges: one under every ridge and one between neighbouring ridges. Numerous exceptionally small (96.4 microm x 6.4 microm) microfilariae were seen in the uteri of females and the surrounding tissues and isolated from skin biopsy materials. The morphology and location of the parasite and histopathological lesions of the Hungarian case were similar to that described in dogs in the United States. This case is the first documented ocular Onchocerca infection in dogs outside the western United States. Thus, onchocercosis should be considered in the differential diagnosis of ocular and periocular nodules in dogs also in Europe.