Lada Hofmannová

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.

Cryptosporidium erinacei n. sp. (Apicomplexa: Cryptosporidiidae) in hedgehogs

The morphological, biological, and molecular characteristics of Cryptosporidium hedgehog genotype are described, and the species name Cryptosporidium erinacei n. sp. is proposed to reflect its specificity for hedgehogs under natural and experimental conditions. Oocysts of C. erinacei are morphologically indistinguishable from Cryptosporidium parvum, measuring 4.5-5.8 μm (mean=4.9 μm) × 4.0-4.8 μm (mean=4.4 μm) with a length to width ratio of 1.13 (1.02-1.35) (n=100). Oocysts of C. erinacei obtained from a naturally infected European hedgehog (Erinaceus europaeus) were infectious for naïve 8-week-old four-toed hedgehogs (Atelerix albiventris); the prepatent period was 4-5 days post infection (DPI) and the patent period was longer than 20 days. C. erinacei was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus), Mongolian gerbils (Meriones unguiculatus), or golden hamsters (Mesocricetus auratus). Phylogenetic analyses based on small subunit rRNA, 60 kDa glycoprotein, actin, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, and heat shock protein 70 gene sequences revealed that C. erinacei is genetically distinct from previously described Cryptosporidium species.

Lethal Encephalitozoon cuniculi genotype III infection in Steppe lemmings (Lagurus lagurus).

Microsporidia are ubiquitous, spore-forming, intracellular parasites infecting invertebrates and vertebrates. Some of them are important opportunistic pathogens in humans, including three species of genus Encephalitozoon. Intraspecies genetic variation with a different range of hosts is known in Encephalitozoon cuniculi distinguishing four genotypes. Recently, E. cuniculi is often observed in pet animals, mainly E. cuniculi genotype I in pet rabbits. This study described a fatal encephalitozoonosis in a group of pet rodents Steppe lemmings (Lagurus lagurus). The animals were presented with progressive weight loss, aggression, cannibalism, purulent conjunctivitis and hind limb paresis. Death occurred within 48 h after the onset of clinical signs. The group comprised of 15 animals was affected and died within a period of three months. Post-mortal examination did not show any macroscopic changes. Microsporidial vacuoles with typical spores were found in brain and kidney tissues and E. cuniculi DNA in all tested organs. The internal transcribed spacer region (ITS) of rRNA gene showed 100% homology with E. cuniculi genotype III previously identified in dogs, tamarin colonies from zoos, swine, birds and humans. Pet lemmings could represent a new potential source of the infection for their breeders.

Variability in susceptibility of voles (Arvicolinae) to experimental infection with Cryptosporidium muris and Cryptosporidium andersoni

The infectivity of Cryptosporidium muris and Cryptosporidium andersoni in various species of voles was studied using experimental infections. None of the experimental voles inoculated with 1 × 105 oocysts of Cryptosporidium spp. shed any oocysts during 40 DPI, except Brandts vole (Lasiopodomys brandtii), which was susceptible to C. muris infection. Experiments confirmed the resistance of voles of the genus Microtus sensu stricto to infection with mammalian gastric cryptosporidia, which provides a new study model with prospects to more fully understand the processes involved in the phenomenon of host specificity of this group of protists.

Native and introduced squirrels in Italy host different Cryptosporidium spp.

The present study was undertaken to describe Cryptosporidium spp. infection in tree squirrels from 17 locations in Northern Italy. A total of 357 squirrels were examined, including species native to Europe (Sciurus vulgaris; n=123), and species introduced from North America (Sciurus carolinensis; n=162) and Southeast Asia (Callosciurus erythraeus; n=72). Faecal samples of all squirrels were examined for the presence of Cryptosporidium infection by microscopy (flotation method) and PCR/sequence analysis of the Cryptosporidium 18S rRNA, actin, and gp60 genes. Despite the overlapping ranges of native and introduced tree squirrel species in the study area, they host different Cryptosporidium spp. Sciurus vulgaris were exclusively infected with Cryptosporidium ferret genotype (n=13) belonging to three novel gp60 subtypes, VIIIb-VIIId. Sciurus carolinensis hosted C. ubiquitum subtype XIIb (n=2), Cryptosporidium skunk genotype subtype XVIa (n=3), and chipmunk genotype I subtype XIVa (n=1). Cryptosporidium chipmunk genotype I subtype XIVa was also found in two C. erythraeus. Comparing data from this and previous studies, we propose that Cryptosporidium skunk genotype and possibly C. ubiquitum subtype XIIb were introduced to Europe with eastern grey squirrels. Cryptosporidium chipmunk genotype I and ferret genotype were associated with high intensity infections, but there was no association with diarrhoea.

Cryptosporidium erinacei and C. parvum in a group of overwintering hedgehogs.

This study describes cryptosporidiosis in an overwintering group of 15 European hedgehogs (Erinaceus europaeus), comprising 3 adults and 12 juveniles. Four juvenile hedgehogs were hospitalised with anorexia, malodorous diarrhoea and dehydration. Immediate parasitological examinations revealed the presence of Cryptosporidium sp. in these animals and also in 5 other juveniles. All hedgehogs were coproscopically monitored for 4 months over the winter season. Shedding of Cryptosporidium oocysts persisted from 6 to 70 days. Repeated shedding of Cryptosporidium oocysts occurred in 3 animals after 4 months subsequent to the first outbreak. Clinical signs were observed only at the beginning of the outbreak (apathy, anorexia, general weakness, mild dehydration, and malodorous faeces with changed consistence - soft/diarrhoea) in the 4 hospitalised juveniles. Overall 11 hedgehogs were Cryptosporidium-positive, both microscopically and by PCR methods. Sequence analyses of SSU rRNA and gp60 genes revealed the presence of C. parvum IIdA18G1 subtype in all positive hedgehogs. Moreover, 3 hedgehogs had a mixed infection of the zoonotic C. parvum and C. erinacei XIIIaA19R13 subtype. Cryptosporidium infections can be rapidly spread among debilitated animals and the positive hedgehogs released back into the wild can be a source of the infection for individuals weakened after hibernation.

Response of cell lines to actual and simulated inoculation with Cryptosporidium proliferans

The need for an effective treatment against cryptosporidiosis has triggered studies in the search for a working in vitro model. The peculiar niche of cryptosporidia at the brush border of host epithelial cells has been the subject of extensive debates. Despite extensive research on the invasion process, it remains enigmatic whether cryptosporidian host-parasite interactions result from an active invasion process or through encapsulation. We used HCT-8 and HT-29 cell lines for in vitro cultivation of the gastric parasite Cryptosporidium proliferans strain TS03. Using electron and confocal laser scanning microscopy, observations were carried out 24, 48 and 72 h after inoculation with a mixture of C. proliferans oocysts and sporozoites. Free sporozoites and putative merozoites were observed apparently searching for an appropriate infection site. Advanced stages, corresponding to trophozoites and meronts/gamonts enveloped by parasitophorous sac, and emptied sacs were detected. As our observations showed that even unexcysted oocysts became enveloped by cultured cell projections, using polystyrene microspheres, we evaluated the response of cell lines to simulated inoculation with cryptosporidian oocysts to verify innate and parasite-induced behaviour. We found that cultured cell encapsulation of oocysts is induced by parasite antigens, independent of any active invasion/motility.

Two new species of Eimeria (Apicomplexa: Eimeriidae) in Philippine tarsier (Tarsius syrichta)

Philippine tarsier (Tarsius syrichta) is a small nocturnal primate from the Philippines. Little is known about tarsier parasites, including coccidia (Apicomplexa: Eimeriidae), a highly prevalent parasitic protist group in all vertebrate classes. Only 7 valid species of the genus Eimeria, seven species of Isospora and 5 species of Cyclospora have been described in Primates. This study extends the number of coccidia known in primates by two new species obtained from faeces of Philippine tarsiers from Bohol Island. The newly described Eimeria syrichta n. sp. and Eimeria boholensis n. sp. differ morphologically from each other as well as from other coccidia reported from primates. Partial DNA sequences of three genes were obtained from oocysts of E. syrichta n. sp. and E. boholensis n. sp., and formed clusters according to their host specificity; however, there are no other sequentional data of coccidia from primates, except for the genus Cyclospora, which clusters inside the chicken eimerians, and Cystoisospora belli, which is phylogenetically related to Sarcocystidae. More molecular data on coccidia infecting primates are needed for further discussion.