Cryptosporidium sciurinum n. sp. (Apicomplexa: Cryptosporidiidae) in Eurasian Red Squirrels (Sciurus vulgaris).

Cryptosporidium spp. are common protozoan pathogens in mammals. The diversity and biology of Cryptosporidium in tree squirrels are not well studied. A total of 258 Eurasian red squirrels (Sciurus vulgaris) from 25 and 15 locations in the Czech Republic and Slovakia, respectively, were examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, TRAP-C1, COWP, and gp60 loci. Out of 26 positive animals, only juveniles (9/12) were microscopically positive (18,000 to 72,000 OPG), and molecular analyses revealed the presence of Cryptosporidium sp. ferret genotype in all specimens. Oocysts obtained from naturally-infected squirrels measured 5.54-5.22 µm and were not infectious for laboratory mice (BALB/c and SCID), Mongolian gerbils, Guinea pigs, Southern multimammate mice, chickens, or budgerigars. None of naturally infected squirrels showed clinical signs of disease. The frequency of occurrence of the ferret genotype in squirrels did not vary statistically based on host age, gender or country of capture. Phylogenetic analysis of sequences from six loci revealed that Cryptosporidium sp. ferret genotype is genetically distinct from the currently accepted Cryptosporidium species. Morphological and biological data from this and previous studies support the establishment of Cryptosporidium sp. ferret genotype as a new species, Cryptosporidium sciurinum n. sp.

Cryptosporidium myocastoris n. sp. (Apicomplexa: Cryptosporidiidae), the Species Adapted to the Nutria (Myocastor coypus).

Cryptosporidium spp., common parasites of vertebrates, remain poorly studied in wildlife. This study describes the novel Cryptosporidium species adapted to nutrias (Myocastor coypus). A total of 150 faecal samples of feral nutria were collected from locations in the Czech Republic and Slovakia and examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, and gp60 loci. Molecular analyses revealed the presence of C. parvum (n = 1), C. ubiquitum subtype family XIId (n = 5) and Cryptosporidium myocastoris n. sp. XXIIa (n = 2), and XXIIb (n = 3). Only nutrias positive for C. myocastoris shed microscopically detectable oocysts, which measured 4.8-5.2 × 4.7-5.0 µm, and oocysts were infectious for experimentally infected nutrias with a prepatent period of 5-6 days, although not for mice, gerbils, or chickens. The infection was localised in jejunum and ileum without observable macroscopic changes. The microvilli adjacent to attached stages responded by elongating. Clinical signs were not observed in naturally or experimentally infected nutrias. Phylogenetic analyses at SSU, actin, and HSP70 loci demonstrated that C. myocastoris n. sp. is distinct from other valid Cryptosporidium species.

Occurrence and genetic diversity of Cryptosporidium spp. in wild foxes, wolves, jackals, and bears in central Europe.

Parasites of the genus Cryptosporidium Tyzzer, 1910 are one of the most common protistan parasites of vertebrates. Faecal samples from 179 red foxes (Vulpes vulpes [Linnaeus]), 100 grey wolves (Canis lupus Linnaeus), 11 golden jackals (Canis aureus Linnaeus), and 63 brown bears (Ursus arctos Linnaeus) were collected in the Czech Republic, Poland and Slovakia. Samples were examined for the presence of Cryptosporidium spp. using microscopy and PCR/sequence analysis. Phylogenetic analysis based on the small subunit ribosomal RNA (SSU), actin and 60-kDa glycoprotein (gp60) genes using the maximum likelihood method revealed the presence of Cryptosporidium tyzzeri Ren, Zhao, Zhang, Ning, Jian et al., 2012 (n = 1) and C. andersoni Lindsay, Upton, Owens, Morgan, Mead et Blackburn, 2000 (n = 2) in red foxes, C. canis Fayer, Trout, Xiao, Morgan, Lai et Dubey, 2001 (n = 2) and C. ubiquitum Fayer, Santín et Macarisin, 2010 (n = 2) in grey wolves, and C. galli Pavlásek, 1999 in brown bears (n = 1) and red foxes (n = 1). Subtyping of isolates of C. ubiquitum and C. tyzzeri based on sequence analysis of gp60 showed that they belong to the XIId and IXa families, respectively. The presence of specific DNA of C. tyzzeri, C. andersoni and C. galli, which primarily infect the prey of carnivores, is probably the result of their passage through the gastrointestinal tract of the carnivores. Finding C. ubiquitum XIId in wolves may mean broadening the host spectrum of this subtype, but it remains possible this is the result of infected prey passing through the wolf - in this case deer, which is a common host of this parasite. The dog genotype of C. canis was reported for the first time in wolves.

Cryptosporidium proventriculi sp. n. (Apicomplexa: Cryptosporidiidae) in Psittaciformes birds.

Cryptosporidiosis is a common parasitic infection in birds that is caused by more than 25 Cryptosporidium species and genotypes. Many of the genotypes that cause avian cryptosporidiosis are poorly characterized. The genetic and biological characteristics of avian genotype III are described here and these data support the establishment of a new species, Cryptosporidium proventriculi. Faecal samples from the orders Passeriformes and Psittaciformes were screened for the presence of Cryptosporidium by microscopy and sequencing, and infections were detected in 10 of 98 Passeriformes and in 27 of 402 Psittaciformes. Cryptosporidium baileyi was detected in both orders. Cryptosporidium galli and avian genotype I were found in Passeriformes, and C. avium and C. proventriculi were found in Psittaciformes. Cryptosporidium proventriculi was infectious for cockatiels under experimental conditions, with a prepatent period of six days post-infection (DPI), but not for budgerigars, chickens or SCID mice. Experimentally infected cockatiels shed oocysts more than 30 DPI, with an infection intensity ranging from 4,000 to 60,000 oocysts per gram (OPG). Naturally infected cockatiels shed oocysts with an infection intensity ranging from 2,000 to 30,000 OPG. Cryptosporidium proventriculi infects the proventriculus and ventriculus, and oocysts measure 7.4 × 5.8 µm. None of the birds infected C. proventriculi developed clinical signs.

The opportunistic pathogen Encephalitozoon cuniculi in wild living Murinae and Arvicolinae in Central Europe.

Encephalitozoon spp. is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. This study was conducted to determine the prevalence of Encephalitozoon spp. in wild living rodents from Poland, the Czech Republic and Slovakia. Faecal and spleen samples were collected from individuals of Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, and Myodes glareolus (n = 465) and used for DNA extraction. PCR, targeting the ITS region of the rRNA gene was performed. The overall prevalence of microsporidia was 15.1%. The occurrence of Encephalitozoon cuniculi in the abovementioned host species of rodents has been presented for the first time, with the highest infection rate recorded for A. flavicollis. Sequence analysis showed that the most frequent species was E. cuniculi genotype II (92.5%). E. cuniculi genotypes I (1.5%) and III (6.0%) were also identified.

Host specificity and age-dependent resistance to Cryptosporidium avium infection in chickens, ducks and pheasants.

Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chickens (Gallus gallus), domestic ducks (Anas platyrhynchos) and ring-necked pheasants (Phasianus colchicus) under experimental conditions. Cryptosporidium avium was not infectious for ring-necked pheasants, but it was infectious for ducks and chickens at all age categories. The course of infection in ducks did not differ among age categories, but 365-day-old chickens had less severe infections than 1- and 21-day-old chickens. The patent period in chickens and ducks was >30 DPI, but ducks started to shed oocysts of C. avium earlier (5-6 DPI) and at a lower intensity (accumulated value of infection intensity of 58,000-65,000 OPG) than chickens (9-11 DPI and accumulated value of infection intensity of 100,000-105,000 OPG). Experimentally infected birds showed no clinical signs of cryptosporidiosis.

Effect of Fascioloides magna (Digenea) on fecundity, shell height, and survival rate of Pseudosuccinea columella (Lymnaeidae).

Infection with Fascioloides magna (Digenea) causes serious damage to liver tissue in definitive hosts represented by ruminants, especially cervids. The distribution of F. magna includes the indigenous areas in North America, and the areas to which F. magna was introduced-Central Europe, Southeast Europe, and Italy. The North American intermediate host of F. magna, the freshwater snail Pseudosuccinea columella (Lymnaeidae), is an invasive species recorded in South America, the Caribbean, Africa, Australia, and west and Southeast Europe. In Europe, Galba truncatula is the snail serving for transmission, but P. columella has potential to become here a new intermediate host of F. magna. Little is known about interactions between F. magna and P. columella. In this study, the susceptibility of P. columella (Oregon, USA) to the infection by a single miracidium of the Czech strain of F. magna and the influence of F. magna on snail fecundity, shell height, and survival were evaluated. The data show that the Oregon strain of P. columella is a highly suitable host for the Czech strain of F. magna, with the infection rate of 74 %. In addition, a negative effect on survival rate of infected snails was recorded only in the late phase of infection. The infection was accompanied by a major reduction in egg mass production and by a decrease in the number of eggs per egg mass. The shell height of infected snails did not significantly differ from that in unexposed controls.

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.

A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae).

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 µm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 µm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 µm long in F. hepatica while subtelocentric and 4.7 µm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica.

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.