A modified method for molecular identification of Baylisascaris transfuga in European brown bears (Ursus arctos).

Baylisascaris transfuga is a roundworm that has been reported worldwide in most bear species. In mammals and possibly humans, the larvae of B. transfuga can migrate in the tissues of aberrant hosts with larva migrans syndrome. The current study was performed to identify B. transfuga in faecal samples from free-ranging brown bears in the Tatra Mountains National Park in southern Poland. A commercial kit was used to extract genomic DNA directly from faecal samples. Additionally, a Chelex resin-based technique was successfully implemented to prepare a PCR template from eggs retrieved by flotation. Based on the flotation results of 32 collected faecal samples, the prevalence of B. transfuga was 15.6%. The parasite was confirmed in samples found to be positive during the initial flotation by a molecular assay using DNA isolated directly from faeces. The retrieved eggs were confirmed as B. transfuga after their DNA was extracted using the Chelex protocol. Based on PCR amplification and sequencing of a 413-bp segment of cytochrome c oxidase 1 (COI), the obtained sequence was 100% identical to the COI segment of B. transfuga after a BLAST comparison to the GenBank™ database. The current study includes the first molecular confirmation of B. transfuga in brown bears in the western part of the Carpathians. We show that direct extraction of parasite DNA from bear faeces is efficient for molecular assays. As an alternative, we present the effectiveness of a Chelex-based technique for fast and convenient DNA isolation from the difficult-to-disrupt eggs of B. transfuga for PCR. Molecular tests of parasite DNA extracted directly from faecal material have limits of detection related to the amount of eggs in the samples. Thus, using classical flotation to obtain eggs for PCR may increase the credibility of the results, particularly in cases with a low number of excreted eggs. The Chelex resin protocol has potential for application in studies of intestinal parasites in wildlife for which conventional flotation is routinely used for microscopy.

Cystic echinococcosis in Poland: genetic variability and the first record of Echinococcus granulosus sensu stricto (G1 genotype) in the country.

Cystic echinococcosis is one of the most important zoonotic diseases affecting humans and livestock worldwide, and is endemic in Poland. A set of six isolates on larval stages of Echinococcus granulosus sensu lato tapeworms collected from three humans, two pigs and one sheep from Polish foci of CE was examined by DNA sequencing of two mitochondrial genes (cox1, rrnS). The results demonstrated the presence of E. canadensis and E. granulosus sensu stricto in the investigated hydatid cysts. The former species was found in all five isolates from pigs and humans derived from central Poland. In a sheep hydatid cyst originating from Lesser Poland Voivodeship in southern Poland, E. granulosus s. s. (G1 genotype) was identified. This is the first report of an unambiguously autochthonous infection with E. granulosus s. s. in Poland. The global distribution and host affiliations of the commonly occurring G1 microvariant with nucleotide change 56C/T in cox1, detected here in Polish sheep, are discussed. The finding that sheep harboured E. granulosus s. s. may have important consequences for developing effective hydatid control programmes in Poland due to its longer maturation rate in dogs compared with E. canadensis G7. This may lead to greater expenditures for purchasing anthelmintics to provide an appropriate dosing regime in sheep-raising areas of the country.

Alveolar echinococcosis in Europe and Poland. Threats to humans

The increasing number of detected cases of alveolar echinococcosis (AE) in recent years in Central-Eastern Europe strongly support the need of accurate diagnosis of the disease in patients and its differentiation from cystic echinococcosis. Accordingly to the EU notification requirements both parasitoses are reported jointly as echinococcosis, which makes it impossible to assess epidemiological situation of each disease. AE threat to humans is due to the environmental contamination with eggs of the tapeworm Echinococcus multilocularis excreted by infected foxes. Also contribution of dogs to the parasite transmission should be taken into account in endemic areas. The article presents current data on human cases of AE registered in humans in Europe and Poland, threats of this parasitosis to humans and results of studies on E. multilocularis infection in animals, the definitive hosts.

First cases of alveolar echinococcosis in dogs in Poland.

Alveolar echinococcosis caused by Echinococcus multilocularis is common parasitic disease among humans and animals in the northern hemisphere. Dogs, foxes, and other wild canids are definitive hosts, whereas small rodents play the role of intermediate hosts. In rare cases, after incidental ingestion of tapeworm eggs, dogs can become an intermediate host. The study describes briefly two cases of alveolar echinococcosis in dogs in Poland, including clinical management, diagnostic, treatment and molecular confirmation. Diagnostic procedures included laparotomy, cytology, histopathology and molecular analysis. Obtained sequences data were 100% homologous to E. multilocularis dehydrogenase subunit 1 gene sequences in GenBank®. To the authors' knowledge, alveolar echinococcosis has not been reported previously in a dog in Poland.

[Potential risk factors for alveolar echinococcosis in humans in Poland]. / Potencjalne czynniki ryzyka zachorowania na alweolarna echinokokoze dla ludzi w Polsce.

The high prevalence of Echinococcus multilocularis recorded in foxes in Poland creates the risk of alveolar echinococcosis in humans. Sensational press releases on threats which had appeared during last few years do not favour the rational opinion on the risk of this dangerous disease. Based on accessible epidemiological data the potential risk factors of alveococcosis are addressed in this article.

Occurrence of Echinococcus spp. in red foxes and wolves in the protected area of the Tatra National Park in southern Poland - a threat to human health.

INTRODUCTION: Echinococcus multilocularis has been endemic in red foxes in eastern and central parts of Europe, and E. granulosus s. l. identified in wolves in some countries. In recent years, wolves hale emerged as potentially important definitive hosts of E. multilocularis. OBJECTIVE: This aim of the survey was to record indirectly using nested-PCR test with faecal samples the presence of Echinococcus multilocularis and E. granulosus s. l. in the two species of wild canids in the protected area of the Tatra National Park (TNP) in Western Carpathian, southern mountainous part of Poland. MATERIAL AND METHODS: From February to June 2019, experienced staff of TNP randomly collected fox and wolf faeces on and off hiking trails at altitudes from 850 m to 2,000 m above sea level. In total, 91 faecal samples from red foxes and 19 from wolves were collected. Genomic DNA was obtained by direct extraction from faecal samples using a commercial kit, and from taeniid eggs retrieved from the same samples after flotation. RESULTS: A nested PCR screening of 91 red fox faeces indicated the prevalence of E. multilocularis of 4.4%. Positive samples were confirmed by sequencing parts of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Neither genomic DNA of E. multilocularis nor of E. granulosus s.l. was obtained from 19 wolves faeces, nor from taeniid eggs retrieved from these samples by initial flotation. CONCLUSIONS: The current results show that humans might be exposed to a risk of fox tapeworm infection in nature, even at high altitude inan alpine zone, in an environment contaminated by roaming red foxes encouraged by food leftovers on mountain trails.

[Occurrence of Toxocara spp. eggs in household environment of children with diagnosed toxocariasis in Lódz voivodeship]. / Czestosc wystepowania jaj inwazyjnych Toxocara spp. w srodowisku przydomowym dzieci ze zdiagnozowana toksokaroza w woj. lódzkim.

Toxocariasis is a zoonosis due to infection of humans by dog or cat roundworm (Toxocara canis, T. cati). Humans become infected by ingestion of infective eggs either from soil, dirty hands, raw fruits and vegetables or larvae from undercooked meat of paratenic hosts. The aim of the study was to evaluate the level of contamination of soil samples from households of children with diagnosed toxocariasis in rural and urban areas of Lódz voivodeship. In the years 2004-2007 toxocariasis was confirmed in 178 patients of the Polish Memorial Hospital in Lódz. The soil samples were collected from 53 courtyards of patients' domiciles. Toxocara spp. eggs were isolated from the samples using flotation technique (Dada 1979). The examinations revealed the high prevalence of ground contamination with Toxocara eggs in both, rural (30.4%) and urban areas (23.3%). The presence of Toxocara eggs in households enlarges the risk of re-infection for children with diagnosed toxocariasis, especially in rural areas where the high level of contamination was detected.

Morphometric identification of equid cyathostome (Nematoda: Cyathostominae) infective larvae.

The prevalence of infection with cyathosthomes of horses is high worldwide. Identification of species on infective larvae based on available morphological keys is not fully accurate and the aim of the present study was to provide a reliable identification key of cyathostomes infective larvae. At the abattoir, horse large intestines were examined and the cyathostomes females extracted and identified. The eggs from uteruses of identified cyathostomes females were used for breeding the infective larvae. Morphological parameters of Cyathostominae infective larvae, i.e. the length of the larvae with sheath, length of oesophagus, intestine, end of the intestine to the end of the larvae body, and finally the body breadth measured and the cells arrangement of the intestine cells (A-D) noted. These morphological parameters of the third infective larvae were determined of the following cyathostomes species: Cylicocyclus nassatus, Cylicostephanus goldi, Cyathostomum catinatum, Cylicostephanus longibursatus, Cyathostomum pateratum, Cylicocyclus ashworti, Cylicocyclus insigne, Cylicostephanus calicatus and Coronocyclus coronatus. The qualitative assignment into A, C and D intestinal type and then the combination of morphological measures allows specific identification for most species.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

Environmental and personal risk factors for toxocariasis in children with diagnosed disease in urban and rural areas of central Poland.

To investigate the epidemiology of human toxocariasis a field survey was carried out at homes of 194 children (80 of rural and 114 of urban origin) with diagnosed disease from central Poland. A questionnaire referring to the possible risk factors was directed to their parents. Overall contamination rate of soil by Toxocara eggs was 27.5% in rural and 21.1% in urban environment in the households examined, with difference not significant (chi2=1.08, p=0.2986). In rural settlements 29.3% of yards surrounding houses were found contaminated, whereas in urban 25.0% of family gardens, 26.4% of private yards and 10.7% of public sandpits were positive. Frequency of positive samples differs only for rural yards and urban sandpits (chi2=3.85, p=0.0499). The study showed a high risk of reinfection for the ill children in sites of their residence. Despite diagnosed toxocariasis kids were not adequately supervised by their parents with no measures undertaken to avoid further infection. These data present strong need for educational programs which should be implemented for prevention of Toxocara infections in children.

[Toxocariasis in children--difficult clinical problem]. / Toksokaroza u dzieci--trudny problem kliniczny.

Toxocariasis in humans is a zoonosis due to the migration of Toxocara canis or T. cati larvae in human body. This review provides basic information on pathology of infection and clinical signs and symptoms of toxocariasis in children. The cases diagnosed in the recent years in central Poland are presented. The disease was recognised accidentally based on the results of laboratory analysis performed for other reasons, when abnormalities suggesting an active helminthiasis were found (eosinophilia, elevated IgE level and/or anemia). The high rate of soil contamination in households of the patients demonstrated elevated risk of infection and reinfection in both, rural and urban areas. Presented data show the need for educational programs which should be implemented for prevention of Toxocara infection in children.

[An attempt to determine intermediate hosts of the tapeworm Echinococcus multilocularis in Poland]. / Próba wykrycia zywicieli posrednich tasiemca Echinococcus multilocularis na terenie Polski.

Echinococcus multilocularis, a tiny tapeworm (2-3 mm in length) occurs in the small intestine of red foxes and raccoon dogs in Poland. Small rodents, intermediate hosts of the tapeworm can ingest parasite eggs excreted in faeces by final host. Humans can become infected by accidental ingestion of the eggs. The resulting disease, alveolar echinococcosis, potentially lethal zoonosis, typically presents as an infiltrative tumor-like growth in the liver, which may invade neighbouring organs. The objective of the study was to determine intermediate host species for E. multilocularis in Poland. During the years 2004-2006, a total of 1425 animals, i.e. 1170 rodents (14 species), 238 insectivores (4 species) and 17 Mustelids (3 species) were trapped and autopsied for the presence of E. multilocularis protoscolices (larvae). Animals were captured in northern, north-eastern, central and southern Poland, areas where the high prevalence of the tapeworm was recorded in foxes. In 44 (3.1%) of examined animals were found cysts in the liver, however no typical lesions were affirmed. Infection was not confirmed using PCR technique with primers specific for E. multilocularis (EmCA90, EmTriple83).

[Estimation of eosinophilia, immunoglobulin E and eosinophilic cationic protein concentration during the treatment of toxocariasis]. / Ocena eozynofilii, stezenia immunoglobuliny E i eozynofilowego bialka kationowego podczas leczenia toksokarozy.

Toxocara canis and Toxocara cati do not transform in human organism into a mature form and they circulate reaching various organs and tissues causing characteristic symptoms. Activated eosinophils, which play a significant role in parasitic invasion, contain in their granules eosinophilic cationic protein (ECP) of strong pro-inflammatory activity. The aim of the study was to estimate the concentration of immunoglobulin E, peripheral blood eosinophilia and serum concentration of eosinophilic cationic protein in children treated for toxocariasis and the analysis of their value as the markers of active invasion and the therapy efficacy. The study included 45 children, aged from 3 to 18 years with Toxocara canis infection diagnosed for the first time. The children were diagnosed and treated at the Department of Gastroenterology, in the Outpatient Gastrointestinal and Allergologic Clinic, Institute Polish Mother Health Centre. T. canis larva infection was diagnosed based on serological investigation using immunoenzymatic ELISA test (Bordier Affinity Products, Switzerland). The tests (percentage of eosinophilia in peripheral blood, ECP, IgE) were performed after diagnosis and every 3 months since the beginning of the therapy. In children with toxocariasis its covert form was diagnosed and mebendazole was administered. Among the children with toxocariasis eosinophilia was found in 14 (31.1%) before therapy. Mean percentage of peripheral blood eosinophils was 5.58% in children with toxocariasis. The concentration of IgE was elevated in these children and was significantly higher than in the control group (p=0.002). Mean IgE concentration after 3-and 6-month therapy decreased IgE(I) vs IgE(III) (p=0.01), but it was still higher than normal value. In children with toxocariasis the ECP concentration was 30.19 microg/l before the therapy and was significantly higher than in the control group (p<0.05); after 6 months of the therapy it decreased significantly (p<0.05). Eosinophilic cationic protein and eosinophilia can be the markers of Toxocara canis infection activity. The determination of immunoglobulin E and eosinophilic cationic protein concentration may be useful for toxocariasis therapy monitoring.

The benefits of analysing complete mitochondrial genomes: Deep insights into the phylogeny and population structure of Echinococcus granulosus sensu lato genotypes G6 and G7.

Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6.

[The communities of cyathostomes (Cyathostominae) in year-old and two-year-old Pure Blood Arabian mares]. / Zgrupowania slupkowców malych (Cyathostominae) u rocz- nych i dwuletnich klaczek czystej krwi arabskiej.

BACKGROUND: The objective of this study was to evaluate the strongyles species in 34 Pure Blood Arabian Horses: one-year-old mares (n = 12) and two-year old mares (n = 22) from the horse stud in 2005, kept under housed-pastured system and dewormed systematically, twice a year using the ivermectin compounds. MATERIAL AND METHODS: The practical method of in vivo determination of strongyles species in horses is collection of these nematodes from the feaces after treatment. Nematodes were collected 24 hours after deworming from 1000 g sample of feaces, then were classified to appropriate species and relative abundance of strongyles community was determined. RESULTS: In the feaces of horses after treatment 10 species of cyathostomes was determined: Cyathostomum catinatum (CAT), C. pateratum (PAT), Cylicocyclus nassatus (NAS), C. ashworti (ASH), C. leptostomus (LEP), C. insigne (INS), Cylicostephanus longibursatus (LNG), C. goldi (GLD), C. calicatus (CAL) and Coronocyclus coronatus (COR). No large strongyles (Strongylinae) was found. The relative abundance was the highest for following cyathostomes species: NAS (22%), CAT (18.1%), INS (17.7%), PAT (13.8%), LNG (9.1%) and LEP (8.2%). The relative abundance of cyathostomes species was similar in both group of mares. On the other hand CAL (15.8%) was common for the one-year-old mares and INS (25%) for the two-year-old mares. In vivo diagnositic of the infection is important from the perspective of individual small strongyle infections dynamics in horses and because of possible different pathogenicity of individual species of these nematodes.

Quantifying the risk of zoonotic geohelminth infections for rural household inhabitants in Central Poland.

The objective of this study was to evaluate the risk of soil transmitted zoonotic helminth infections for families with young children, inhabitants of villages in the Mazowieckie Province of Central Poland. Epidemiological survey was conducted at 33 randomly selected households with 2-3 children present. Examination of soil samples from yards surrounding the houses for the presence of geohelminth eggs was conducted, the households were inspected, and family members interviewed using a designed questionnaire. Among 55 localities examined, i.e. 33 backyards, 10 vegetable gardens and 12 sandpits, contamination was found in 2 backyards (6.1%) and 1 sandpit (8.3%) at 3 households (9.1%). Of the total 550 examined soil samples, 4 (0.7%) were found to contain Trichuris and Toxocara eggs, with an average density of 1.5 and 2.0 eggs per sample. The study showed a low level of soil contamination, which was the result of inhabitants care about the sanitation of their domiciles. However, the results of the questionnaire survey demonstrated the need to warn rural residents about the risk factors for zoonotic helmints infections. In particular, parents should be advised how to minimize the threat of parasitic diseases for children in the rural environment. The presented study showed that promotional campaigns implemented in recent years on the prevention of parasitic zoonoses have had little effect to increase the awareness of the rural community. The present results confirmed that Toxocara is the most common zoonotic agent among geohelminths in the rural environment.

Risk of soil-transmitted helminth infections on agritourism farms in central and eastern Poland.

Agritourism provides ecological tourist services for urban dwellers in rural areas. Agritourism farms offer space and attractive scenery for people seeking to rest in quiet place and wanting healthy, outdoor recreational activities. The high epidemiological standard of agritourism farms is beneficial for the health of the farm owners and the guests. Upgraded level of the farm sanitation, also from parasitological point of view is of great importance, especially that among agritourism farms guests predominate families with small children. A field survey was carried out in 57 farms in central-eastern Poland to evaluate the environmental risk factors for geohelminth infections on agritourism farms offering tourist services for urban dwellers. Samples of soil were collected from 76 sites, i.e. yards surrounding houses, vegetable, fruit and flower gardens, playgrounds and sandpits. In addition, samples were taken from 27 public places of recreation (playgrounds at forest clearing) visited by agritourism farm guests. During visits the farms were inspected and the owners were questioned about their awareness of the threat of parasitic infections. Soil contamination with geohelminth eggs was found in 4 examined farms (7.0%), in one locality on each farm. The eggs of Toxocara spp. and Ascaris spp. were detected in single samples from 3 backyards (6.4%) and one sandpit (10.0%). In the soil samples from places of recreation outside the farms eggs of human or animal helminths were not identified. The results of this study showed that the risk of helminth infections on agritourism farms is low, since geohelminth eggs (1-3 per sample) were detected only in four samples (0.5%) among 760 collected from farms households. The farm owners must be aware of the importance of preventive measures to eliminate the environmental contamination with eggs of zoonotic soiltransmitted helminths. Special attention should be paid to the risk of intestinal parasites of cats of semi domestic behaviour migrating from neighbouring yards as well as of red foxes frequently observed in the vicinity of agritourism farms.

[Risk of alveococcosis for humans in Poland]. / Zagrozenie bablowica wielojamowa (alweokokoza) dla ludzi w Polsce.

The paper presents data on occurrence, diagnosis and treatment of alveolar echinococcosis in humans as well as possible methods of its prevention. Studies done in 2001-2003 in Poland have shown the high prevalence of the tapeworm in red foxes in the north-east (34.5%) and south-east (39.3%) of Poland with foci of infection in some counties (up to 70% foxes infected). It makes the high potential risk for human infection in these areas.

The contamination of the environment with Toxocara eggs in Mazowieckie voivodship as a risk of toxocarosis in children.

The home surroundings (109 places) of 120 children with diagnosed toxocarosis were examined to assess if the presence of Toxocara spp. eggs in the soil corresponds with the occurrence of clinical cases. 31 places (28.4%) were found to be contaminated. The highest level of soil contamination was revealed in rural and suburban regions (15.6% and 11.9%, respectively), while in urban areas it was 0.9%. These results confirmed that soil contamination might be recognized as a very important factor in the epidemiology of human toxocarosis.

Risk of human toxocarosis in Poland due to Toxocara infection of dogs and cats.

The purpose of this paper was to present the current data on the risk of toxocarosis in humans in Poland and to give an overview of the clinical and diagnostic aspects of the disease. The number of reported clinical cases of Toxocara infection in children in Poland in medical literature has increased recently. The results of field surveys aimed to evaluate the soil contamination with geohelminth eggs conducted during the last few years showed that Toxocara is the most common zoonotic agent in urban public sites and in rural settlements. The questionnaire revealed rural inhabitants' low awareness of zoonotic parasite threats to humans. In particular parents should be advised as to what proper preventive measures to undertake to eliminate the toxocarosis risk factors for children in rural environment. Prevention of initial environment contamination with Toxocara canis and T. cati eggs, which includes proper treatment regimes to eliminate patent infections in dogs and cats and preventing pets from defecating in public areas and private households is vital. To provide the public with suitably presented information as well as pet owners with uniform recommendations, a close collaboration between veterinary and public health professionals is crucial.