John McEvoy

Cryptosporidium avium n. sp. (Apicomplexa: Cryptosporidiidae) in birds

The morphological, biological, and molecular characteristics of Cryptosporidium avian genotype V are described, and the species name Cryptosporidium avium is proposed to reflect its specificity for birds under natural and experimental conditions. Oocysts of C. avium measured 5.30–6.90xa0μm (meanu2009=u20096.26xa0μm)u2009×u20094.30–5.50xa0μm (meanu2009=u20094.86xa0μm) with a length to width ratio of 1.29 (1.14–1.47). Oocysts of C. avium obtained from four naturally infected red-crowned parakeets (Cyanoramphus novaezealandiae) were infectious for 6-month-old budgerigars (Melopsittacus undulatus) and hens (Gallus gallus f. domestica). The prepatent periods in both susceptible bird species was 11xa0days postinfection (DPI). The infection intensity of C. avium in budgerigars and hens was low, with a maximum intensity of 5000 oocysts per gram of feces. Oocysts of C. avium were microscopically detected at only 12–16 DPI in hens and 12 DPI in budgerigars, while PCR analyses revealed the presence of specific DNA in fecal samples from 11 to 30 DPI (the conclusion of the experiment). Cryptosporidium avium was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus). Naturally or experimentally infected birds showed no clinical signs of cryptosporidiosis, and no pathology was detected. Developmental stages of C. avium were detected in the ileum and cecum using scanning electron microscopy. Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. avium is genetically distinct from previously described Cryptosporidium species.

Cryptosporidium galli and novel Cryptosporidium avian genotype VI in North American red-winged blackbirds (Agelaius phoeniceus)

Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoeniceus; order Passeriformes) were examined for the presence of Cryptosporidium by PCR amplification and sequence analysis of the 18S ribosomal RNA (18S rRNA), actin, and 70-kDa heat shock protein (HSP70) genes. Twelve birds (17.1xa0%) were positive for the Cryptosporidium 18S rRNA gene: six birds were positive at the proventriculus site only and six birds were positive at the proventriculus and intestinal sites. Sequence analysis of the 18S rRNA, actin and HSP70 genes showed the presence of the gastric species Cryptosporidium galli in a single proventriculus sample and a closely related genotype, which we have named Cryptosporidium avian genotype VI, in all other positive samples. These findings contribute to our understanding of Cryptosporidium diversification in passerines, the largest avian order.

Cryptosporidium apodemi sp. n. and Cryptosporidium ditrichi sp. n. (Apicomplexa: Cryptosporidiidae) in Apodemus spp.

Faecal samples from striped field mice (nu202f=u202f72) and yellow-necked mice (nu202f=u202f246) were screened for Cryptosporidium by microscopy and PCR/sequencing. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences revealed the presence of C. parvum, C. hominis, C. muris and two new species, C. apodemi and C. ditrichi. Oocysts of C. apodemi are smaller than C. ditrichi and both are experimentally infectious for yellow-necked mice but not for common voles. Additionally, infection by C. ditrichi was established in one of three BALB/c mice. The prepatent period was 7-9 and 5-6u202fdays post infection for C. apodemi and C. ditrichi, respectively. The patent period was greater than 30u202fdays for both species. Infection intensity of C. ditrichi ranged from 4000-50,000 oocyst per gram of faeces and developmental stages of C. ditrichi were detected in the jejunum and ileum. In contrast, neither oocysts nor endogenous developmental stages of C. apodemi were detected in faecal or tissue samples, although C. apodemi DNA was detected in contents from the small and large intestine. Morphological, genetic, and biological data support the establishment of C. apodemi and C. ditrichi as a separate species of the genus Cryptosporidium.

Cryptosporidium occultus sp. n. (Apicomplexa: Cryptosporidiidae) in rats

Cryptosporidium parvum VF383 has been reported in humans, domesticated ruminants, and wild rats worldwide and described under several names including Cryptosporidium suis-like, based on its close phylogenetic relationship to C. suis. Unlike C. suis, however, it has never been detected in pigs. In the present work, C. parvum VF383 originating from wild brown rats was not infectious for piglets or calves but was infectious for laboratory brown rats, BALB/c mice, and Mongolian gerbils. The prepatent period was 4-5u202fdays for all rodents. The patent period was longer for rats (>30u202fdays) than other rodents (<20u202fdays). None of the rodents developed clinical signs of infection. In all rodents, life cycle stages were detected in the colon by histology and electron microscopy. Oocysts were morphometrically similar to those of C. parvum and smaller than those of C. suis, measuring 5.20u202f×u202f4.94u202fμm. Phylogenetic analyses of 18S rRNA, actin, and HSP70 gene sequences revealed C. parvum VF383 to be genetically distinct from, C. suis, and other described species of Cryptosporidium. Morphological, genetic, and biological data support the establishment of C. parvum VF383 as a new species, and we propose the name Cryptosporidium occultus sp. n.

Cryptosporidium ubiquitum, C. muris and Cryptosporidium deer genotype in wild cervids and caprines in the Czech Republic.

A total of 269 faecal samples of various game animals, including 136 red deer (Cervus elaphus Linnaeus), 64 European fallow deer (Dama dama [Linnaeus]), 26 white-tailed deer (Odocoileus virginianus [Zimmermann]), and 43 mouflon sheep (Ovis orientalis musimon Pallas) were collected at 15 game preserves across the Czech Republic and examined for infection with species of Cryptosporidium Tyzzer, 1910 using microscopy (following aniline-carbol-methyl violet staining) and molecular tools. Oocysts of Cryptosporidium spp. were detected in one faecal sample originating from red deer. Ten positive cases of infection with cryptosporidia, including the case that was positive by microscopy, were detected using nested PCR. No associations between infection with cryptosporidia and diarrhoea were detected. Phylogenetic analyses based on the small subunit of the rRNA gene revealed the presence of three Cryptosporidium species/genotypes in ten positive samples: Cryptosporidium ubiquitum Fayer, Santin et Macarisin, 2010 was identified in five red deer, C. muris Tyzzer, 1907 in three samples (from a red deer, white-tailed deer and mouflon sheep), and Cryptosporidium deer genotype in two white-tailed deer. Subtyping of isolates of C. ubiquitum based on sequence analysis of the 60-kDa glycoprotein gene revealed that they belong to the XIId family. Finding C. muris and C. ubiquitum XIId for the first time in various wild cervids and caprines broadens their host range.

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.

First description of Cryptosporidium ubiquitum XIIa subtype family in farmed fur animals

This study investigated the prevalence of Cryptosporidium in farmed fur animals in the Czech Republic and Poland. A total of 480 faecal samples were collected from fur animals, including 300 American mink (Mustela vison), 60 silver foxes (Vulpes vulpes), 50 long-tailed chinchillas (Chinchilla lanigera), and 70 nutrias (Myocastor coypus), at 14 farms. Samples were examined for the presence of Cryptosporidium using microscopy (following aniline-carbol-methyl violet staining) and sequence analysis of PCR amplified products. Three mink and two chinchillas from two different farms tested positive for Cryptosporidium ubiquitum DNA. The presence of C. ubiquitum DNA was not associated with diarrhoea. Subtyping of C. ubiquitum isolates by sequence analysis of the 60-kDa glycoprotein gene showed that isolates belonged to the XIIa subtype family, which was previously restricted to humans and ruminants. This suggests that C. ubiquitum subtype XIIa has a broader host range than previously reported.

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.

Stray cats are more frequently infected with zoonotic protists than pet cats

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.