Skunk Genotype Research Articles

Molecular detection and genetic variability of Cryptosporidium spp. in wild Asian house shrews (Suncus murinus) from southern Zhejiang province, China

Shrews play a crucial role as repositories for diverse pathogens linked to zoonotic infectious diseases. However, the genetic information regarding Cryptosporidium in Chinese shrews remains unexplored. The objectives of this study were twofold: to determine the occurrence rate of Cryptosporidium spp. in wild shrews residing in the southern part of Zhejiang Province, China, and to investigate their genetic characteristics. A total of 282 wild shrews were captured between April and October of 2023. The detection of Cryptosporidium in fecal samples, collected from each animal's rectum, was performed using PCR and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. The 60-kDa glycoprotein (gp60) gene was utilized to further subtype the positive samples of C. viatorum and C. parvum. All animals were identified as Suncus murinus, and a positive result for Cryptosporidium was obtained in 14.2 % (40/282) of the samples. The following species and genotypes were identified: C. ratti (n = 19), C. parvum (n = 2), C. viatorum (n = 1), Cryptosporidium rat genotype IV (n = 13), and Cryptosporidium skunk genotype (n = 5). Furthermore, the subtypes IIdA15G1 and XVdA3 were detected within C. parvum and C. viatorum, respectively. Molecular evidence indicates that S. murinus is concurrently infected with rodent-adapted and zoonotic species/genotypes, actively contributing to the dissemination of cryptosporidiosis.

The Occurrence of Cryptosporidium spp. in Wild-Living Carnivores in Poland—A Question Concerning Its Host Specificity

Cryptosporidium is an apicomplexan protozoan parasite that primarily infects the gastrointestinal epithelium in humans and domestic and wild animals. The majority of studies have been focused on human, livestock, and pet infections. Hence, Cryptosporidium spp. in wildlife, including wild carnivores, remained neglected. There are several studies reporting the occurrence of Cryptosporidium spp. in wild foxes, but these are only a few molecular surveys; no data is available concerning the occurrence of this parasite in raccoon dogs and martens in Europe, and to the best of our knowledge to date, only one study has reported Cryptosporidium from badgers in Spain. Therefore, we used molecular analyses to identify and genotype Cryptosporidium spp. in wild-living mesocarnivores in Poland. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine, and beech martens were collected and then analyzed for the presence of Cryptosporidium spp. using the nested PCR method. The appearance of PCR products in the reaction with Cryptosporidium-specific primers against the 18S rRNA and actin genes demonstrated that Cryptosporidium spp. occurred in 23.0% of all examined species of animals. Performed sequence analyses showed the presence of the Cryptosporidium skunk genotype, Cryptosporidium vole genotype II, Cryptosporidium canis dog and fox genotypes, as well as Cryptosporidium erinacei, Cryptosporidium ditrichi, Cryptosporidium suis, and Cryptosporidium alticolis, in these hosts. Molecular data presented here indicate that examined mesocarnivores may be a significant reservoir of specific and non-specific Cryptosporidium species, including those with zoonotic potential. Most studies of carnivores have described the presence of non-specific Cryptosporidium spp. in carnivore hosts, and this is probably the result of the transfer of these parasites from prey species through the digestive tract or the transfer of the parasite from a contaminated environment.

Molecular characterization of Cryptosporidium skunk genotype in raccoons (Procyon lotor) in Iran: concern for zoonotic transmission.

Cryptosporidium spp. are significant zoonotic parasites in humans and animals worldwide. This study aimed to investigate the prevalence of Cryptosporidium infection among raccoon (Procyon lotor) in north of Iran. The fecal samples (n = 30) were collected from raccoons. After DNA extraction, all samples were examined by nested PCR amplification of the 18S ribosomal RNA (rRNA) gene. From 30 raccoon samples, 4 (13.3%) were positive, and the isolates were identified as Cryptosporidium skunk genotype based on sequence analysis. The large distribution of raccoons in northern provinces of Iran and their potency for carrying some human-infecting parasites like Cryptosporidium spp. propose this mammalian as asource for zoonotic parasites.

Pathology of cryptosporidiosis in raccoons: case series and retrospective analysis, 1990-2019.

Cryptosporidiosis is an intestinal protozoal disease of public health importance caused by Cryptosporidium spp. Despite the high synanthropism of raccoons, studies describing the pathology of Cryptosporidium spp. infections in this species are lacking. Therefore, we characterized the pathology of cryptosporidiosis in 2 juvenile raccoons. In addition, we conducted a retrospective search of the database of the California Animal Health and Food Safety laboratory for 1990-2019 and found 6 additional cases of cryptosporidiosis in raccoons. Sequencing of cryptosporidia was performed in one autopsied raccoon, and PCR on formalin-fixed, paraffin-embedded tissues in archived cases. The Cryptosporidium skunk genotype (CSkG), a strain of zoonotic relevance, was detected in 6 of 8 cases (75%). Frequently, cryptosporidiosis was associated with enteritis, eosinophilic infiltrates, villus atrophy or blunting and/or fusion, and crypt abscesses or necrosis. In 7 of the 8 cases, there was confirmed concurrent coinfection with canine distemper virus; 1 case was coinfected with canine parvovirus. Although crypt necrosis is considered a classic lesion of canine parvoviral infection in mesocarnivores and not a hallmark of cryptosporidiosis, results suggest that canine distemper virus is capable of mimicking such lesions in combination with cryptosporidia and immunosuppression.

Cryptosporidium sp. skunk genotype in wild raccoons (Procyon lotor) naturally infected with Baylisascaris procyonis from Central Germany.

Cryptosporidium spp. are apicomplexan parasites of public health concern. They are one of the main causes of intestinal diseases in humans and animals. Contaminated water is among the main sources of infection for humans and mammals. Raccoons are an introduced species in Germany. They are anthropogenic adapters with a natural affinity for water bodies. We collected samples from wild raccoons in the Federal States of Saxony and Thuringia, Central Germany. Through molecular genotyping, we found Cryptosporidium sp. skunk genotype in one raccoon from Saxony (1/24) and in one animal from Thuringia (1/27). Both raccoons were also infected with the zoonotic nematode Baylisascaris procyonis. This is the first report of co-infection with these two parasites in raccoons from Germany. Our study highlights the potential of these animals as carriers of zoonotic pathogens. Since raccoons can thrive in human settlements, this study provides data that can be used as a baseline for preventive programs.

Prevalence and Genotypes of Cryptosporidium in Wildlife Populations Co-Located in a Protected Watershed in the Pacific Northwest, 2013 to 2016.

Between October 2013 and May 2016, 506 scat samples were collected from 22 species of wildlife located in a protected watershed of a major municipal water supply in the Pacific Northwest, USA. Overall prevalence of Cryptosporidium in the wildlife scat was 13.8% (70/506), with 15 species of wildlife found positive for Cryptosporidium. Prevalence of Cryptosporidium varied among species of wildlife, with higher prevalences observed in cougars (50.0%), mountain beavers (40.0%), and bobcats (33.3%), but none of these species are riparian-dependent. Genotyping of Cryptosporidium by sequencing PCR amplicons from the 18S rRNA gene were successful for seven species of wildlife, including bobcat, unknown predator, black-tailed deer, deer mouse, snowshoe hare, mountain beaver, and western spotted skunk. BLAST and phylogenetic analyses indicated that multiple species and genotypes of Cryptosporidium were present, with some isolates possibly co-circulating within and between wildlife populations in this protected watershed. Evidence of oocyst exchange between infected prey and their predators was also found. During the study period, several zoonotic Cryptosporidium species and genotypes that are uncommon in humans were detected in bobcat (99.58% identical to Cryptosporidium felis), unknown predator (100% identical to Cryptosporidium canis), snowshoe hare (100% identical to Cryptosporidium sp. skunk genotype), and mountain beaver (100% identical to Cryptosporidium ubiquitum). Novel sequences were also found in mountain beaver. To our knowledge, this is the first published report of a unique genotype or species of Cryptosporidium in mountain beaver (Aplodontia rufa).

First surveillance and molecular identification of the Cryptosporidium skunk genotype and Cryptosporidium parvum in wild raccoons (Procyon lotor) in Osaka, Japan.

Recent research suggests that raccoons (Procyon lotor) can transmit several important pathogens affecting humans, including protozoans. In Japan, the number of wild raccoons has increased since they were first introduced more than 50years ago. Here, we report the first survey of Cryptosporidium infection using fecal swabs of raccoons captured in Osaka, Japan. Of 116 raccoons examined by PCR targeting of the Cryptosporidium 18S rRNA gene, 7 (6.03%; 2 adults and 5 young animals) were positive, and the isolates were identified as Cryptosporidium skunk genotype (subtype XVIa) and C. parvum based on sequence and phylogenetic analyses. Both species and the genotype are zoonotic; thus, our results suggest that raccoons could transmit Cryptosporidium infections to humans in Japan.

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.

Subtype analysis of zoonotic pathogen Cryptosporidium skunk genotype

Cryptosporidium skunk genotype is a zoonotic pathogen commonly identified in surface water. Thus far, no subtyping tool exists for characterizing its transmission in humans and animals and transport in environment. In this study, a subtyping tool based on the 60kDa glycoprotein (gp60) gene previously developed for Cryptosporidium chipmunk genotype I was used in the characterization of Cryptosporidium skunk genotype in animal and storm runoff samples from a watershed in New York. Altogether, 17 positive samples from this watershed and 5 human and animal specimens from other areas were analyzed. We identified 14 subtypes of Cryptosporidium skunk genotype, 11 of which were seen in the watershed. In phylogenetic analysis, these subtypes belonged to 4 subtype families (XVIa, XVIb, XVIc, and XVId). No host-adapted subtypes were identified and the two subtypes in humans were genetically similar to some in raccoons, otters, and storm runoff samples from the watershed. The characteristics of gp60 protein sequences of the Cryptosporidium skunk genotype are similar to those of other Cryptosporidium species, but only its XVIb subtype family has a putative furin cleavage site. This subtyping tool might be useful in characterizing Cryptosporidium skunk genotype in clinical and environmental samples.

Cryptosporidium spp. and Enterocytozoon bieneusi in introduced raccoons (Procyon lotor)-first evidence from Poland and Germany.

The raccoon (Procyon lotor) carnivore native to North America is a fast spreading, invasive species in the Europe now. At the moment, the highest population occupies areas near the German-Polish border. The data on the occurrence of Cryptosporidium spp. and microsporidia in raccoons is limited to North America’s territory and is totally lacking in the case of their introduction to Europe. Therefore, the objective of this study was to investigate the occurrence of microparasites, i.e., Cryptosporidium spp. and microsporidia in the introduced raccoons obtained from localities in Poland and Germany. A PCR-based approach that permitted genetic characterization via sequence analysis was applied to raccoon fecal samples (n = 49), collected during 2012–2014. All fecal samples were simultaneously tested with the use of genetic markers, and DNA of microsporidia and Cryptosporidium spp. was detected among the examined raccoons. The results of our research confirmed the presence of Cryptosporidium skunk genotype and Enterocytozoon bieneusi NCF2 genotype. The results suggest a possible role of raccoons in the contamination of the environment, including urban areas, with pathogens of zoonotic significance as well as their role in the transmission and introduction of new genotypes of microparasites in the areas where P. lotor has not been observed yet. To our knowledge, there has been no literature data on the above genotypes detected previously in humans or animals from the examined study sites so far.

North American tree squirrels and ground squirrels with overlapping ranges host different Cryptosporidium species and genotypes

Wildlife-associated Cryptosporidium are an emerging cause of cryptosporidiosis in humans. The present study was undertaken to determine the extent to which North American tree squirrels and ground squirrels host zoonotic Cryptosporidium species and genotypes. Fragments of the Cryptosporidium 18S rRNA and actin genes were amplified and sequenced from fecal samples obtained from three tree squirrel and three ground squirrel species. In tree squirrels, Cryptosporidium was identified in 40.5% (17/42) of American red squirrels (Tamiasciurus hudsonicus), 40.4% (55/136) of eastern gray squirrels (Sciurus carolinensis), and 28.6% (2/7) of fox squirrels (Sciurus niger). Human-pathogenic Cryptosporidium ubiquitum and Cryptosporidium skunk genotype were the most prevalent species/genotypes in tree squirrels. Because tree squirrels live in close proximity to humans and are frequently infected with potentially zoonotic Cryptosporidium species/genotypes, they may be a significant reservoir of infection in humans. In ground squirrels, Cryptosporidium was detected in 70.2% (33/47) of 13-lined ground squirrels (Ictidomys tridecemlineatus), 35.1% (27/77) of black-tailed prairie dogs (Cynomys ludovicianus), and the only golden-mantled ground squirrel (Callospermophilus lateralis) that was sampled. Cryptosporidium rubeyi and ground squirrel genotypes I, II, and III were identified in isolates from these ground squirrel species. In contrast to the Cryptosporidium infecting tree squirrels, these species/genotypes appear to be specific for ground squirrels and are not associated with human disease.

Baylisascaris procyonis in raccoons (Procyon lotor) from eastern Colorado, an area of undefined prevalence

Baylisascaris procyonis is a zoonotic parasite that has been documented in raccoons throughout much of the United States; however, no published information on its occurrence is available for the transition zone from the Great Plains to the Rocky Mountains. Because this parasite can cause neural larva migrans and diffuse unilateral subacute neuroretinitis in humans (as well as other hosts), a more complete understanding of the distribution of this parasite seems warranted for public health reasons. The purpose of this study was to begin to fill in the gaps in our knowledge of the distribution of B. procyonis in an area of the US where there is, currently, no published information available. Fifty-three raccoons were collected throughout eastern Colorado during 2007–2010. Forty-six were examined by necropsy and seven by fecal flotation. Age (11 juveniles, 25 adults) and sex (16 males, 19 females) of the raccoons were recorded when intact carcasses were available. When available, feces were further processed for the detection of Giardia and Cryptosporidium using a direct fluorescent antibody detection method. B. procyonis was found in 31 of 53 raccoons (58.5%, 95% CI=44.1%, 71.9%). Mean intensity was 11.7 with a range of 1–49 worms per infected individual. There was no significant difference between age or sex, and the presence of ascarids or the number of ascarids. Cryptosporidium spp. oocysts and Giardia spp. cysts were detected in 11/44 (25%; 95% CI: 13.2, 40.3) and 3/44 (6.9%; 95% CI: 1.4, 18.7) raccoons, respectively. The genotype of the Giardia present could not be determined. The genotype of five of six cryptosporidial isolates was 100% homologous to the skunk genotype while the sixth was 100% homologous to Cryptosporidium parvum. Based on these results, both B. procyonis and Cryptosporidium spp. appear to be prevalent in raccoons of eastern Colorado.

The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000–2008

Routine typing of 14 469 isolates from human cryptosporidiosis cases between 2000 and 2008 revealed that 7439 (51·4%) were Cryptosporidium (C.) hominis, 6372 (44·0%) C. parvum, 51 (0·4%) both C. hominis and C. parvum, 443 (3·1%) were not typable and 164 (1·1%) were other Cryptosporidium species or genotypes. Of the latter, 109 were C. meleagridis, 38 C. felis, 11 C. ubiquitum, one C. canis, two horse, two novel and one skunk genotype. C. hominis monkey genotype and C. cuniculus were identified in a separate study. Patients with unusual infections were older than those with C. hominis (P<0·01) or C. parvum (P<0·01) and were more likely to be immunocompromised (Fisher's exact P<0·01). Forty-one percent of unusual cases had travelled abroad, mainly to the Indian subcontinent. Significant risk factors in those with unusual species were travel abroad (C. meleagridis, P<0·01), being immunocompromised (C. felis, Fisher's exact P=0·02), and contact with cats (C. felis, Fisher's exact P=0·02).

CryptosporidiumPig Genotype II in Immunocompetent Man

To the Editor: Protozoan parasites from the genus Cryptosporidium have been described as a cause of diarrheal disease in immunodeficient and immunocompetent humans worldwide. Although C. hominis and C. parvum (cattle genotype) cause most infections, humans can be infected by several other Cryptosporidium species or genotypes: C. meleagridis; C. felis; C. canis; C. suis; C. muris; C. andersoni; C. hominis monkey genotype; C. parvum (mouse genotype); and Cryptosporidium rabbit genotype, deer genotype, skunk genotype, horse genotype, and chipmunk genotype I (1–4). Wild and domestic animals are sources of infection for humans (and other animals) and important contributors to contamination of food and drinking water; many nonhuman Cryptosporidium species or genotypes are detected in untreated water (5). We examined the diversity of Cryptosporidium spp. in immunocompetent persons in South Bohemia in the Czech Republic. Diarrheal fecal samples (n = 457) from 203 anonymous immunocompetent patients <69 years of age with suspected cryptosporidiosis (at least 2 samples/patient/3-day period) were obtained from local health departments and public hospitals in South Bohemia during 2005–2007. Samples were examined for Cryptosporidium oocysts by using aniline-carbol-methyl violet staining and light microscopy at × 1,000 magnification (6). The microscopically positive samples were confirmed by DNA sequencing of the small subunit (SSU) rRNA gene. Total DNA was extracted from 200–300 mg stool by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions, after previous homogenization and disruption of oocysts with the Mini-BeadBeater (Biospec Products, Bartlesville, OK, USA). An ≈830-bp fragment of the SSU rRNA gene was amplified by nested PCR according to Jiang et al. (7). Purified PCR products were sequenced in both directions on an ABI3130 sequencer analyzer (Applied Biosystems, Foster City, CA, USA) by using the secondary PCR primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were assembled by using Chromas Pro (www.technelysium.com.au/chromas.html) and aligned with reference sequences using ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX). The BLAST server (www.ncbi.nlm.nih.gov/BLAST) was used for DNA database searches. The SSU rRNA gene partial sequences of the 7 patient isolates have been submitted to GenBank (Table). Table Cryptosporidium genotypes identified by using sequencing of partial sequences of the small subunit rRNA gene in the stool samples of immunocompetent humans, Czech Republic Of the 203 patients, 7 (3.4%) (6 children and 1 adult) had positive results for Cryptosporidium spp. Moreover, all samples obtained from these persons during the 3-day period were Cryptosporidium spp. positive; partial sequences of the Cryptosporidium SSU rRNA gene were obtained from all positive samples identifying 3 different species or genotypes of Cryptosporidium. Five were C. parvum (bovine genotype), 1 was C. hominis, and 1 contained the Cryptosporidium pig genotype II (Table). Cryptosporidium pig genotype II was found in stool samples from a 29-year-old man who also was infected with Giardia intestinalis (assemblage A) (data not shown). Only C. parvum (bovine genotype), C. hominis, and Cryptosporidium rabbit genotype have been implicated in waterborne outbreaks of cryptosporidiosis in humans. Further studies are needed to determine the potential of other cryptosporidia of animal origin. Recent genetic and biologic characterization studies have identified 2 distinct host-adapted cryptosporidia in pigs, C. suis and Cryptosporidium pig genotype II. Furthermore, both above-mentioned cryptosporidia have been identified in untreated water (8). Pigs could be sources of Cryptosporidium water and food pollution and a consequent risk to public health. Although human infection with C. suis has been previously described (9), human infection with Cryptosporidium pig genotype II has been never reported. This genotype was found in diarrheal stool of 1 adult patient in this study. However, onset of diarrhea could have been caused by co-infection with G. intestinalis (assemblage A), which recently also has been described in pigs (10). Contact with infected animals and ingestion of contaminated food or water could be the source of both Cryptosporidium and Giardia infection in the Cryptosporidium pig genotype II–positive patient. The passage of oocysts can be excluded because of the number of oocysts detected in repeat samples (Table). Moreover, identification of the infection in an immunocompetent patient underlines the zoonotic potential of this pig genotype and possible presence of risk factors in rural areas with poor water treatment or inadequate biosecurity in pig units. Further evidence of the zoonotic potential of this Cryptosporidium genotype is needed to show its pathogenic potential in immunocompetent patients as a cause of gastroenteritis (in the absence of Giardia spp. and other established enteropathogens) and to demonstrate invasive tissue stages. The use of molecular techniques to identify Cryptosporidium spp. probably will show more zoonotic species or genotypes in humans.

Source Tracking Identifies Deer and Geese as Vectors of Human-Infectious Cryptosporidium Genotypes in an Urban/Suburban Watershed

This study identified Cryptosporidium genotypes in the Wissahickon watershed from May 2005 to April 2008. We analyzed 129 samples from Wissahickon Creek, 83 effluent samples from wastewater treatment plants (WWTPs), and 240 fecal droppings. Genotyping was based on the hypervariable region of the 18S rRNA gene. Oocysts were detected year-round, independent of wet weather events, in 22% of Wissahickon Creek samples, 5% of WWTP effluents, and 7% of fecal samples. Of the genotypes detected, 67% were human-infectious: 30% C. hominis or C. hominis-like, 12% C. parvum, 14% cervine genotype, 9% skunk genotype, and 1% chipmunk I genotype. Similar genotype profiles were detected in Wissahickon Creek each year, and human-infectious genotypes were detected year-round. Unusual genotypes detected in a deer (a C. hominis-like genotype) and geese (C. hominis-like genotypes, C. parvum, and muskrat genotype I) show that these animals are vectors of human-infectious genotypes in this watershed. Results suggest that deer, geese, and WWTPs are appropriate targets for source water protection in the Wissahickon watershed.