Prevalence and diversity of Encephalitozoon spp. and Enterocytozoon bieneusi in wild boars (Sus scrofa) in Central Europe.

From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1-6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.

Cryptosporidium suis and Cryptosporidium scrofarum in Eurasian wild boars (Sus scrofa) in Central Europe.

From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa) 29 randomly selected localities (both forest areas and enclosures) across the Central European countries of Austria, the Czech Republic, Poland, and the Slovak Republic were investigated. Cryptosporidium oocysts were microscopicaly detected in 11 out of 460 faecal samples examined using aniline-carbol-methyl violet staining. Sixty-one Cryptosporidium infections, including the 11 infections that were detected by microscopy, were detected using genus- or species-specific nested PCR amplification of SSU rDNA. This represents a 5.5 fold greater sensitivity for PCR relative to microscopy. Combining genus- and species-specific PCR tools significantly changes the perspective on the occurrence of Cryptosporidium spp. in wild boars. While RFLP and direct sequencing of genus specific PCR-amplified products revealed 56 C. suis (20) and C. scrofarum (36) monoinfections and only 5 mixed infections of these species, species-specific molecular tools showed 44 monoinfections and 17 mixed infections with these species. PCR analysis of the gp60 gene did not reveal any other Cryptosporidium infections. Similar to domestic pigs, C. scrofarum was detected as a dominant species infecting adult Eurasian wild boars (Sus scrofa). Cryptosporidium infected wild boars did not show signs of clinical disease. This report is perhaps the most comprehensive survey of cryptosporidial infection in wild boars.

Molecular identification and genotyping of Microsporidia in selected hosts.

The work is described by microscopic analysis, the serological analysis (IFAT) and the molecular analysis of isolates from clinical samples (blood, faeces and urine) from ten domestic rabbits (Oryctolagus cuniculus), breed Malický, four New Zealand domestic rabbits, 11 sows of breed Slo0076akian Improved White and 15 clinically healthy laboratory BALB/c mice. The aim of the study was to validate the suitability of species-unspecific primer pairs 530F and 580R for genotype determination of the Microsporidia strain and species-specific primer pairs ECUNF and ECUNR, SINTF and SINTR and EBIER1 and EBIEF1 for the determination of E ncephalitozoon cuniculi, Encephalitozoon intestinalis and Enterocytozoon bieneusi species for diagnostic purposes. Sequences of animals were compared with those from the GenBank database. In rabbits, two murine genotypes II and four canine genotypes III were identified. Genotype II was identified in mice. The Encephalitozoon intestinalis identified in the sample from swine showed no genetic heterogeneity.

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.

First detection and genotyping of Encephalitozoon cuniculi in a new host species, gyrfalcon (Falco rusticolus).

Recently, the pathogenic species of microsporidia of the genus Encephalitozoon have been detected increasingly, also in representatives of the Aves class. Our study presents laboratory proof of Encephalitozoon cuniculi (E. cuniculi) genotype II in a new host, gyrfalcon (Falco rusticolus), with suspect microsporidiosis. E. cuniculi is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. Characterization of the internal transcribed spacer of the rRNA gene has identified three genotypes of E. cuniculi based on the number of 5'-GTTT-3' repeats present: a genotype I from rabbits and mice, containing three repeats; a genotype II from mice and dogs, containing two repeats; and a genotype III from dogs and fox, containing four repeats. Samples of faeces from 30 gyrfalcons were examined for the presence of microsporidia spores, using microscopical, molecular methods and sequencing. Microscopic analysis showed presence of brightly fluorescing oval shapes of size 1.5 × 3 µm, characteristic of the strain Microsporidia in five samples. The PCR method, using species non-specific (530F/580R) and species-specific (ECUNR/ECUNF) primers, proved the presence of E. cuniculi spores in two samples. After sequencing were confirmed, E. cuniculi genotype II which implies new host species for this parasite.

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals.

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p<0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p<0.0075).