Zuzana Hurníková

Dirofilariosis in Slovakia - a new endemic area in Central Europe

SummaryThe first systematic research on canine dirofilariosis in Slovakia started in February 2007. In total 287 dogs of various age, sex, breeds, and utilization from southern Slovakia were examined within the study until September 2007. Microfilariaemia was detected in 99 (34.5 %) blood samples. Histochemical staining and PCR approach were used for Dirofilaria species identification. Dirofilaria repens was confirmed in all infected dogs; it occurred in 92 (32.1 %) dogs as a single infection and in 6 (2.1 %) dogs in mixed infection with Dirofilaria immitis. For the first time both species were detected in Eastern Slovakia. Outdoor keeping and age higher 3 years become evident as important risk factors. Male individuals and dogs with long hair were significantly more frequently infected when compared with females and short hair breeds.The present study revealed the occurrence of highly endemic area of dirofilariosis in Slovakia and certified that climatic conditions in the country are favourable for development of the parasite and for further spread of this zoonotic infection.

Endoparasites of red fox (Vulpes vulpes) in the Slovak Republic with the emphasis on zoonotic species Echinococcus multilocularis and Trichinella spp.

SummaryDue to specific geographical localization, climatic and geomorphologic conditions, several serious parasitic diseases circulate in the territory of the Slovak Republic that makes this area an ideal model territory of the central European red fox system. The red fox is an important reservoir host of parasites, which can be spread to another animals and humans. Our study was aimed at determining the current prevalence of certain parasites in red foxes from the entire territory of the Slovak Republic and identifies some ecological factors influencing their epidemiology. Within the first systematic investigation of red foxes carried out between the years 2000 and 2006 in total 4026 foxes were examined for Echinococcus multilocularis (prevalence 31.1 %) and 4699 foxes were investigated for the presence of Trichinella spp. larvae (10.4 % infected). The results of the next separate study revealed that 83.3 % of 1198 red foxes in the Slovak Republic had coccidian oocysts and helminth eggs in their faeces. Fifteen helminth species including two trematode, four cestode and nine nematode species were detected by coprological examination. Nine of these parasite taxa have zoonotic potential: Capillaria spp. (prevalence 22.4 %), Ancylostoma caninum (18.1 %), Toxocara canis (12.5 %), Taenia spp. (12.2 %), Mesocestoides spp. (5.8 %), Strongyloides stercoralis (1.6 %), Hymenolepis diminuta (0.6 %), Dipylidium caninum (0.4 %) and Opisthorchis felineus (0.3 %). Toxascaris leonina was the most common helminth species found in this survey (42.9 %).

Long-term survey on Trichinella prevalence in wildlife of Slovakia.

In Slovakia, monitoring the prevalence of Trichinella spp. in wildlife was performed since 2000 in the main reservoir animals, the red fox (Vulpes vulpes) and wild boar (Sus scrofa), using artificial digestion method as recommended by International Commission on Trichinellosis. The results of investigation performed in 5270 red foxes showed that Trichinella infection is widespread across Slovakia and prevalence increased significantly from 4.9% in 2000 to 20.5% in 2007. Recently, a higher Trichinella prevalence (0.11%) in wild boars was also demonstrated. The results indicate that foxes and wild boars are involved in the spread of Trichinella, although the latter host species seems to play a secondary role in the maintenance of the sylvatic cycle in Slovakia. Trichinella britovi is the predominant species circulating in Slovakia, both in foxes and wild boars, and Trichinella spiralis occurs only sporadically. Mixed infections of T. britovi and Trichinella pseudospiralis were recorded in 2005 in one wild boar from Eastern Slovakia and in 2006 in one red fox from the same region. These findings are important with respect to an outbreak caused by T. pseudospiralis in a pig farm in the same district 3 years ago. This study provides a complex picture on Trichinella occurrence in all regions of Slovakia and may be a good basis for evaluating the risk of parasite transmission to the domestic cycle and human beings.

First autochthonous case of canine Angiostrongylus vasorum in Slovakia

Angiostrongylus vasorum is a metastrongyloid nematode that may cause cardiopulmonary disease, neurological signs and coagulopathies in dogs. The parasite has an indirect life cycle with molluscs as intermediate hosts, in which the infective third larval stage develops. Recently, A. vasorum has been repeatedly reported in dogs outside the endemic areas, indicating that this parasite is widely distributed over Europe. This is the first record of an autochthonous infection in a dog from Slovakia that was casually diagnosed during routine preventive parasitological examination. A. vasorum first-stage larvae were recovered using the Baermann technique and identified by length and characteristic tail morphology. The animal originated from Slovakia and had not travelled abroad. The dog had been regularly walked on grass fields with a concentrated presence of common species of Gastropoda and frogs. The owner reported that the dog had been licking and eating grass and it had shown curiosity for molluscs and frogs. The first finding of A. vasorum-infected dog in Slovakia has confirmed that the parasite is spreading beyond the traditional hyperendemic foci, which accentuates the need for monitoring and increasing of disease awareness in primary care clinical practice.

Parasitic infections of the European brown hare (Lepus europaeus Pallas, 1778) in south -western Slovakia

SummaryA total of 74 European brown hares (Lepus europaeus Pallas, 1778), hunted during the winter seasons of 2006 and 2007, were examined by dissection for the presence of helminths and coprologically for protozoa. The animals came from five districts with a high population density of this species. Our results revealed 54.5 % of specimens being infected with one or more helminth species and a high prevalence of eimeriid coccidia (91.89 %). The most prevalent helminth species was Trichuris leporis (55.41 %). Lower prevalence was found for Passalurus ambiguus (12.16 %) and Trichostrongylus retortaeformis (6.76 %). The intensity of infection was low for all parasite species. As for coccidia, Eimeria semisculpta (74.35 %) and E. leporis (61.54%) were recorded in all districts. Other coccidia showed lower prevalence rates: E. robertsoni (15.38 %), E. europaea (12.82 %), E. babatica (12.82 %), E. hungarica (5.13 %) and E. towsendi (2.56 %), occurring only in some districts. The highest infection rate was observed in E. semisculpta, 7657.8 oocysts per gram of faeces (OPG). The potential effect of protozoan infection on hare mortality is discussed.

Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions

The study of the genetic polymorphism of pathogens is important for phylogenetic and biogeographic studies and, in the case of foodborne pathogens, to trace the origin of food infection. Since its discovery in 1972, the nonencapsulated species Trichinella pseudospiralis has been detected in mammals and birds, and human infection has occurred, in some cases resulting in death. We studied DNA polymorphism among ten T. pseudospiralis isolates from the Palearctic, Nearctic, and Australian regions, screening the sequences of nine genes [18sRNA, a random amplified polymorphism DNA derived sequence, mitochondrial cytochrome oxidase subunit I (COI), cytochrome P450, cynate lyase, epithelial fusion failure-1, and three unknown genes of Tp3, Tp8, and Tp26]. A high identity of sequence for the nine gene loci was obtained among the seven isolates from the Palearctic region and between the two isolates from the Nearctic region. Genetic identity analysis indicated the distinct polymorphism among the three geographical origins. To easily identify T. pseudospiralis genotypes, a polymerase chain reaction-restriction fragment length polymorphism analysis of COI gene was performed, and the results confirmed the DNA polymorphism within T. pseudospiralis, corresponding to the three regions of origin. We have named the three genotypes as “T. pseudospiralis Palearctic genotype” (code T4P), “Nearctic genotype” (code T4N), and “Australian genotype” (code T4A). To further investigate polymorphism among the nonencapsulated Trichinella species, the sequences of four gene loci (COI, P450, cynate lyase, and SB147D) of T. pseudospiralis, T. papuae, and T. zimbabwensis were analyzed, and the results showed high polymorphism among the three species, strongly supporting their classification as separate species.

Occurrence of Cryptosporidium spp. in red foxes and brown bear in the Slovak Republic

Wild animals can be involved in epidemiology of many important diseases and often act as reservoirs of pathogens which cause disease in domestic animals and humans. This paper aims the role of red fox (Vulpes vulpes) and brown bear (Ursus arctos) in the circulation of coccidian parasites from the genus Cryptosporidium. Cryptosporidiosis is known as an important enteric pathogen, clinical symptoms in particular in immune-compromised individuals range from mild to severe diarrhoea and dehydration, which could be fatal. Fecal samples from 62 red foxes shot during September 2010 to February 2011 and 63 brown bears collected during June 2010 to March 2011 in central and eastern Slovakia were examined for the qualitative determination of Cryptosporidium spp. antigens in faeces by sandwich ELISA kit. Overall, 38.7% (24/62) of faecal samples of red foxes and 55.6% (35/63) of faecal samples of brown bear were positive. Our preliminary results emphasize prevalence of Cryptosporidium spp. amongst brown bears and red foxes in Slovakia and highlight the potential risk for transmission of cryptosporidiosis to humans using the countryside for professional or recreational purposes.

Kinetics of specific humoral immune response of mice infected with low doses of Trichinella spiralis, T. britovi, and T. pseudospiralis larvae

SummaryThe specific humoral immune response of the host (mouse) to the infection with low doses of larvae of encapsulating (Trichinella spiralis, T. britovi) and non-encapsulating (T. pseudospiralis) species was studied. Mice were experimentally infected with 10 muscle larvae of the parasite to simulate natural conditions of the infection in rodents, important reservoirs of trichinellosis. The low infective dose of T. spiralis and T. britovi did not evoke an increased specific IgM response, which is typical for the acute infection. Only T. pseudospiralis induced a higher specific IgM level in the intestinal phase of the infection, till day 30 p.i. The low infective dose of T. spiralis larvae stimulated a specific IgG1 production from day 20 p.i. with a strong increase on day 45 p.i., but T. britovi infection on day 60 p.i. Specific IgG1 antibodies were not detected in T. pseudospiralis infection. The production of IgG2a and IgG2b antibodies was again earlier and more expressive in T. spiralis infection from day 45 p.i., in contrast to T. britovi, where these antibodies were increased on day 60 p.i. Only IgG2b isotype was detected in T. pseudospiralis infection on days 45 and 60 p.i., however in very low values in comparison with encapsulating species.Results conclude the low infective dose of T. spiralis, T. britovi, and T. pseudospiralis induced a late seroconversion in infected mice. T. spiralis caused earlier and more intensive specific antibody response, from day 45 p.i, when antigens from newborn and muscle larvae were accumulated, on the contrary to T. britovi and T. pseudospiralis, which induced specific antibody response from day 60 p.i.

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae.

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20 dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30 dpi. The IgG antibodies appeared at 40 dpi in inbred mice, and at 50 dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30 dpi. Antibody levels increased until the end of the experiment (80 dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.

Toxoplasma gondii in protected wildlife in the Tatra National Park (TANAP), Slovakia

Toxoplasma gondii is an obligatory intracellular protozoan parasite that infects a broad spectrum of warm-blooded vertebrate species. As a part of the food chain, farm animals play a significant role in transmission of T. gondii to humans, while rats and mice serve as a main source of infection for free-living animals. The spread of toxoplasmosis in the human population is due to the interchange of the domestic and sylvatic cycles. During 2009-2011, a survey on toxoplasmosis distribution was conducted in wildlife of the Tatra National Park (TANAP) in Slovakia. A total of 60 animals were examined. The presence of T. gondii was detected by means of molecular methods based on TGR1E gene analyses. The highest prevalence was recorded in birds (40.0%), followed by carnivores (30.8%) and rodents (18.2%). RFLP analyses of SAG2 locus confirmed in birds the genotype II and III, belonging to the avirulent strain; rodents exclusively had genotype I, characterised as a virulent train, and in carnivores all three genotypes were detected. These results present the first survey on the parasites occurrence in several species of free-living animals in the TANAP area. An epidemiological study confirmed the prevalence of 30.0%, implicitly referring to the level of environmental contamination with T. gondii oocysts.