The Seroprevalence of Toxoplasma gondii in Wild Boars from Three Voivodeships in Poland, MAT Analyses.

PURPOSE: The European wild boar (Sus scrofa) is a popular game animal species. Its meat, however, can represent a reservoir of dangerous foodborne diseases and can play an important role in the transmission of many pathogens, including Toxoplasma gondii, in humans and animals worldwide. The aim of the present study was to determine the presence of antibodies to T. gondii in the serum of hunted wild boars in Poland. METHODS: Using the commercial direct agglutination test, 398 serum samples collected during the hunting season 2009/2010 were tested for the presence of T. gondii antibodies, and the titre of 40 was considered indicative of T. gondii infection in analysed samples. RESULTS: It was found that nationwide, 37.7% were seropositive to T. gondii, although seroprevalence varied from 11.6 to 50% depending on the Voivodeship. Significant differences were observed between the Great Poland and Lubusz Voivodeships and between Great Poland and Warmian-Masurian. CONCLUSION: Serological test indicated widespread exposure to T. gondii by wild boar; therefore, consumption of raw or undercooked game meat of infected animals can carry a significant risk of T. gondii infection.

First record of Giardia assemblage D infection in farmed raccoon dogs (Nyctereutes procyonoides).

The presence of Giardia genotypes was investigated in 18 raccoon dogs (Nyctereutes procyonoides) and 80 red foxes (Vulpes vulpes) on one farm. To demonstrate Giardia cysts, fresh and trichrome stained smears were microscopically screened. Two molecular markers were used for Giardia genotyping: a fragment of the beta-giardin gene and a fragment of the glutamate dehydrogenase gene. All faecal samples obtained from red foxes were negative. Giardia cysts were identified only in 2 of the 18 raccoon dogs. The result of genotyping and phylogenetic analysis showed that the G. duodenalis from both raccoon dogs belonged to the D assemblage. This finding of a new animal reservoir of G. duodenalis canids-specific genotypes is important in order to eliminate the risk of infecting other animals bred for fur. Further molecular analyses of Giardia isolates in raccoon dogs are required. The present study represents the first contribution to knowledge of G. duodenalis genotypes in raccoon dogs.

Axenic in vitro culture and molecular characterization of Giardia duodenalis from red deer (Cervus elaphus) and Thomson's gazelle (Gazella thomsonii).

Giardia duodenalis is an ubiquitous flagellate that infects humans and many species of animals. This species exhibits great biotypic and genetic diversity. In the present study, we established short- and long-term in vitro cultures of G. duodenalis trophozoites originating from red deer and Thomson's gazelle (artiodactyls) and genetically characterised the isolates by their glutamate dehydrogenase and triose phosphate isomerase gene sequences. The G. duodenalis isolates from red deer and the gazelle represented assemblages A (AIII sub-assemblage) and B. In conclusion, G. duodenalis assemblages and sub-assemblages can be associated with differences in growth rate in vitro cultures.

The first report of Toxoplasma gondii antibodies in free-living European bison (Bison bonasus bonasus Linnaeus).

Toxoplasma gondii Nicolle et Manceaux, 1908 is an apicomplexan parasite with a worldwide distribution. It is of great medical and veterinary importance because it may cause abortion or congenital disease in its intermediate hosts, including man. The European bison, the largest herbivorous animal in Europe, is a species that has been saved from extinction. Twenty-four of 95 examined sera of the European bison (Bison bonasus bonasus) from the Bialowieza Forest, Poland collected from 2008 to 2011 were found to be positive for the presence of T. gondii-specific IgG antibodies using a direct agglutination test, with the antibody titre in positive animals ranging from 40 to 18000. Statistically significant differences were observed only between years of sample collection. This is the first report on T. gondii in lowland European bison living in the natural environment.

First detection of microsporidia in raised pigeons in Poland.

Microsporidia are single-celled intracellular parasites which occur in a number of animals, both vertebrates and invertebrates. Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. However, the sources of human infection and the routes of transmission of microsporidia have not yet been fully determined, although more and more researchers are of the opinion that microsporidia in humans is zoonotic. The aim of the presented study was to identify the most common microsporidial species in the droppings of raised and wild pigeons in Poland. A total of 139 collective samples of droppings (33 samples of droppings from feral pigeons and 106 samples from raised pigeons) were examined using conventional staining and molecular techniques. Using chromotrope staining and multiplex FISH techniques, the microsporidial spores were found in 12 (8.6%) of the 139 samples of droppings. The spores of Encephalitozoon hellem were detected in five samples of pigeon droppings (3.6%), spores of E. intestinalis in four samples of droppings (2.9%), while spores of E. cuniculi and E. bieneusi were only detected in two samples (1.4%). Furthermore, a mixed infection of E. bieneusi and E. cuniculi was found in a single sample of droppings (0.7%). The presence of microsporidial spores in droppings collected from raised pigeons indicates a risk of infection to humans, mainly pigeon fanciers.

Multilocus genotyping of Giardia duodenalis isolates from red deer (Cervus elaphus) and roe deer (Capreolus capreolus) from Poland.

A total of 181 faecal samples were collected from wild cervids in two regions of Poland. Giardia cysts were detected in one faecal specimen from red deer and in two samples from roe deer. Fragments of the beta-giardin (bg) triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes were successfully amplified from the Giardia isolate obtained from red deer, whereas only amplicons of bg and gdh were obtained from Giardia isolates derived from two roe deer. The result of genotyping and phylogenetic analysis showed that the G. duodenalis isolate from red deer belonged to sub-assemblage AIII, which has never been identified in humans, whereas isolates from roe deer clustered within zoonotic sub-assemblage AI. Further studies are necessary to explain which Giardia assemblages and/or sub-assemblages occur in wild cervids in various regions of the world. Moreover, the impact of Giardia infection on the health of wild cervids should also be elucidated.

Occurrence of Encephalitozoon intestinalis in the Red ruffed lemur (Varecia rubra) and the Ring-tailed lemur (Lemur catta) housed in the Poznan Zoological Garden, Poland.

Encephalitozoon intestinalis is one of the most common microsporidial species found in humans worldwide but it has rarely been identified in animals. The presence of this pathogen has been detected in a few species of domestic, captive and wild mammals as well as in three species of birds. The aim of the present study was to examine fecal samples obtained from mammals housed in the Poznan Zoological Garden, Poland, for the presence of potentially human-infectious microsporidia. A total of 339 fresh fecal samples collected from 75 species of mammals belonging to 27 families and 8 orders were examined for the presence of microsporidian spores. Microsporidian spores were identified in 3 out of 339 (0.9%) examined fecal samples. All samples identified as positive by chromotrope 2R and calcofluor white M2R were also positive by the FISH assay. Using multiplex FISH in all 3 fecal samples, only spores of E. intestinalis were identified in 2 out of 14 Ring-tailed lemurs (Lemur catta) and in one out of 17 Red ruffed lemurs (Varecia variegata rubra). To our knowledge this is the first diagnosis of E. intestinalis in Ring-tailed and Red ruffed lemurs. It should be mentioned that both lemur species are listed by the IUCN Red List of Threatened Species. Although the lemurs were asymptomatically infected, the possibility of widespread infection or death of these animals remains in the event of an elevated stress or a decrease in their immunological functions.

Giardia prevalence in wild cervids in Poland.

A total of 181 faecal samples were collected from wild cervids in two regions of Poland. Specimens were taken from 65 fallow deer (Dama dama), 61 red deer (Cervus elaphus), 50 roe deer (Capreolus capreolus), and five moose (Alces alces). Giardia cysts were detected in one faecal specimen from the red deer and in two samples from the roe deer. Although this study has demonstrated that Giardia infection is remarkably rare in wild cervids, it should be emphasized that there are large populations of these animals in Poland.

Prevalence and multilocus genotyping of Giardia from animals at the zoo of Poznan, Poland.

In this study total of 266 fecal samples from 242 animals belonging to 113 species kept in the Poznan Zoological Garden were examined for Giardia. The cysts of Giardia were found only in five samples of feces collected from a giant toad (Bufo marinus), tamandua (Tamandua tetradactyla) and three individuals of cactus mouse (Peromyscus eremicus). Fragments of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes were successfully amplified only from the Giardia isolate obtained from the tamandua. Sequence and phylogenetic analyses showed that the Giardia isolate from the tamandua belonged to the B assemblage and showed homologies of 99% to 100% at bg, gdh and tpi loci of the same markers of parasites isolated from humans and animals in various parts of the world. This is the first molecular characterization of G. duodenalis from tamandua.

[Genotype analysis of Giardia duodenalis isolates obtained from humans in west-central Poland]. / Genotypowanie izolatów Giardia duodenalis uzyskanych od ludzi w zachodnio-centralnej Polsce.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a cosmopolitan flagellate organism belonging to the most common intestinal protozoan parasites of humans and animals. Great genetic heterogeneity has been found within G. duodenalis, where only genotypes representing assemblages A and B have zoonotic potential. Fecal samples (447 specimens) obtained from 232 humans in West-central region of Poland were examined by microscopy and PCR. The total prevalence of Giardia in humans was 1.3%. DNA was extracted from three positive fecal samples and PCR products were obtained after amplification using the beta-giardin primers G7 and G759. Sequence and phylogenetic analyses showed that G. duodenalis isolates from humans belonged to A and B genotypes. Moreover, three subgenotypes, including a cosmopolitan subgenotype A2 and two new subgenotypes A and B were detected.

A survey of the prevalence and genotypes of Giardia duodenalis infecting household and sheltered dogs.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is an intestinal protozoan parasite that infects humans and a wide range of mammals that includes dogs. Highly significant genetic heterogeneity has been found within this species, while only genotypes from assemblages A and B have zoonotic potential. Although Giardia infection in dogs has been reported worldwide, there is increasing molecular evidence that dogs may be infected with host-specific genotypes (C and D) as well as zoonotic ones (A and B). Therefore, dogs play a role as a potential source of Giardia infection impacting humans and other Canidae. Fecal samples from privately owned dogs and from dogs kept in two shelters in the west-central region of Poland were examined using a microscope and polymerase chain reaction. The total prevalence of Giardia in specimens was 1.9%. Deoxyribonucleic acid was extracted only from two out of the three Giardia-positive fecal samples. After amplification using the G7 and G759 beta-giardin primers, 753 bp amplicons were obtained, and both amplification products were sequenced. Sequence and phylogenetic analyses showed that both G. duodenalis isolates were dog-specific genotypes (C and D).

The role of free-ranging, captive, and domestic birds of Western Poland in environmental contamination with Cryptosporidium parvum oocysts and Giardia lamblia cysts.

As Cryptosporidium parvum and Giardia lamblia can be disseminated in the environment by avian hosts, a total of 499 fecal dropping from 308 free-ranging, 90 captive, and 101 domestic birds were tested by conventional, immunological, and molecular techniques for these human enteropathogens. Twenty-six (5.2%) tested positive for G. lamblia cysts and 19 (3.8%) for C. parvum oocysts. A bird total of 23 (7.5%) free-ranging, two (2.2%) captive, and one (0.1%) domestic tested positive for cysts, whereas 18 (5.8%) free-ranging, one (1.1%) captive, and zero livestock birds tested positive for oocysts. G. lamblia cysts and C. parvum oocysts were found significantly more frequently in fecal droppings of free-ranging aquatic birds than in birds not normally associated with water. No specimen tested positive for both pathogens simultaneously. Aquatic birds represent an important epidemiologic link in water-associated transmission cycles of Cryptosporidium and Giardia and play a significant role in environmental contamination of aquatic habitats with these anthropozoonotic pathogens.

The role of birds in dissemination of human waterborne enteropathogens.

Cryptosporidiosis, giardiasis and microsporidiosis are serious human diseases of waterborne origin; their etiologic agents and a substantial fecal coliform load can enter surface, drinking and recreational water resources from aquatic birds. The aim of this article is to present interactions between waterfowl and these waters that imply a negative public health impact, reinforcing the need for either better water-quality indicators or for water monitoring specifically for Cryptosporidium, Giardia and microsporidia. Where justifiable, the presence of waterfowl should be supported; however, management of drinking and recreational water resources needs to be improved by incorporating effective protection measures for pathogens linked to these birds.

Quantitative assessment of contamination of fresh food produce of various retail types by human-virulent microsporidian spores.

This study demonstrated that fresh food produce, such as berries, sprouts, and green-leafed vegetables, sold at the retail level can contain potentially viable microsporidian spores of human-virulent species, such as Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon cuniculi, at quantities representing a threat of food-borne infection.

Asymptomatic Enterocytozoon bieneusi microsporidiosis in captive mammals.

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic transmission of microsporidian spores. A survey utilizing chromotrope 2R stain and fluorescent in situ hybridization techniques for testing feces from 193 captive mammals demonstrated that 3 animals (1.6%) shed Encephalitozoon bieneusi spores. These include two critically endangered species (i.e., black lemurs, Eulemur macaco flavifrons; and Visayan warty pig, Sus cebifrons negrinus) and a threatened species (mongoose lemur, Eulemur mongoz). The concentration of spores varied from 2.7 x 10(5) to 5.7 x 10(5)/g of feces, and all infections were asymptomatic. The study demonstrates that E. bieneusi spores can originate from captive animals, which is of particular epidemiologic importance because the close containment of zoological gardens can facilitate pathogen spread to other animals and also to people such as zoo personnel and visitors.

Microsporidian species known to infect humans are present in aquatic birds: implications for transmission via water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 x 10(5) spores/g) than nonaquatic bird droppings contained (mean, 4.4 x 10(4) spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 x 10(8) microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.

Equine Cryptosporidium parvum infections in western Poland.

A total of 564 fecal specimens from 318 horses used for recreational riding, child hippotherapy, and racing at ten commercial and government-run stables in western Poland were tested for Cryptosporidium parvum oocysts by microscopic examination of Ziehl-Neelsen stained smears, enzyme immunoassay, and combined direct immunofluorescent antibody and fluorescent in situ hybridization. Also, seven stool specimens from five personnel who had repeated contact with these horses were tested for C. parvum oocysts. Eleven horses that shed C. parvum oocysts were found in five of ten stables (50%). The prevalence of infection varied from 0% to 11.5%. The overall prevalence of equine C. parvum-associated cryptosporidiosis in the Wielkopolska region of western Poland was 3.5%. C. parvum oocysts were found only in fecal samples from mature horses, the number of oocysts was low, and infections were not associated with clinical signs. Oocysts were not found in human fecal specimens.

[Evaluation of usefulness of different methods for detection of Cryptosporidium in human and animal stool samples]. / Ocena uzytecznosci metod wykrywania Cryptosporidium w kale ludzi i zwierzat.

There are many methods for detection of Cryptosporidium oocysts. Most of them (more than 20) enable the microscopic detection of Cryptosporidium oocysts in faecal smears. Such a great variability of diagnostic methods may lead to confusion as far as the choice of an appropriate technique by a given laboratory is concerned. This study evaluated the diagnostic usefulness of Cryptosporidium oocysts and coproantigen detection methods in the diagnosis of cryptosporidiosis in human (266 stool specimen) and animals (205 from cattle, 160 from sheep, 30 from horses, 80 from cats, 227 from dogs and 11 from wild animals). The total number of human and animal stool specimens processed was 266 and 713, respectively. In this study the usefulness of several diagnostic methods was compared. The following techniques were taken into account: wet mounts, hematoxylin staining, four different specific methods (modified Zeihl-Neelsen, Kinyoun's, safranin-methylene blue, as well as carbol-methyl violet and tartrazyne) and commercially available kit based on enzyme-linked immunoassay (ProspecT(r) Cryptosporidium Microplate Assay). The final number of positive specimens was 123. Out of them 77 were positive in all specific methods. The oocysts found in stool specimens were measured. Humans were infected with C. parvum and animals with C. parvum, C. andersoni or C. felis. The statistical analysis has shown that EIA test was a better than microscopy method for identification of Cryptosporidium in faecal samples in human and wild animal. Sensitivity and specificity are important factors for the choice of a proper diagnostic method for Cryptosporidium detection, however other factors such as cost, simplicity and ease of interpretation of results are also important considerations.