Cryptosporidium Genotypes Research Articles

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.

First report of Enterocytozoon bieneusi and Cryptosporidium spp. in peafowl (Pavo cristatus) in China.

Enterocytozoon bieneusi and Cryptosporidium spp. are important pathogens causing diarrhea in humans and animals. However, few studies have been conducted on the infection of E. bieneusi and Cryptosporidium spp. in peafowl up to now. The purpose of the present study was to determine the prevalence and the involved genotypes of Cryptosporidium spp. and E. bieneusi in peafowl in Beijing and Jiangxi Province, China. In total, 258 peafowl fecal samples were collected. Overall, both Cryptosporidium spp. and E. bieneusi had the same prevalence, i.e. 6.59% (17/258). Higher infection rates of E. bieneusi and Cryptosporidium spp. were found in the adolescent peafowl. The prevalence of E. bieneusi in Beijing and Jiangxi Province was 5.23% and 8.57% respectively. For Cryptosporidium spp., the prevalence was 4.58% and 9.52% in Beijing and Jiangxi Province, respectively. Three zoonotic genotypes of E. bieneusi were confirmed, including two known genotypes, genotype Peru 6 and D, and one novel genotype, JXP1. Two avian specific species/genotypes of Cryptosporidium, Avian genotype Ⅲ and Goose genotype Ⅰ, were identified. To our knowledge, this is the first report of E. bieneusi and Cryptosporidium spp. occurrence in peafowl in China. The findings suggest that peafowl could be reservoirs of E. bieneusi and Cryptosporidium spp. which could be potentially transmitted to humans and other animals, and the present survey have implications for controlling E. bieneusi and Cryptosporidium spp. infection in peafowl.

A retrospective molecular study of Cryptosporidium species and genotypes in HIV-infected patients from Thailand

BackgroundOpportunistic infections represent a serious health problem for HIV-infected people. Among enteric infections, cryptosporidiosis, a severe and life-threatening diarrheal disease, is of particular importance in low economic settings where access to anti-retroviral therapy is limited. Understanding transmission routes is crucial in establishing preventive measures, and requires the use of informative genotyping methods. In this study, we performed a retrospective analysis of Cryptosporidium species in 166 stool samples collected from 155 HIV-infected patients during 1999–2004 at the Siriraj Hospital in Bangkok, Thailand.ResultsMicroscopic examination of stools identified 104 of the 155 patients as positive for Cryptosporidium. Other common pathogens identified were microsporidia, Isospora, Giardia, Strongyloides and Opisthorchis. All samples were tested by amplification of a fragment of the 18S rDNA locus, and sequencing showed the presence of Cryptosporidium hominis (n = 42), C. meleagridis (n = 20), C. canis (n = 12), C. felis (n = 7), C. suis (n = 6) and C. parvum (n = 5). Genotyping at the glycoprotein 60 (gp60) locus revealed substantial variability in isolates of C. hominis and C. meleagridis. Among C. hominis isolates, subtype IeA11G3T3 was the most prevalent, but allelic family Id was the more diverse with four subtypes described, two of which were identified for the first time. Among C. meleagridis isolates, seven subtypes, two of which were new, were found in the allelic family IIIb, along with new subtypes in allelic families IIIe and IIIg. In the four C. parvum isolates, subtype IIoA16G1, a rare subtype previously reported in a Swedish patient who had traveled to Thailand, was identified.ConclusionsThis study confirms the high susceptibility of HIV-infected individuals to infection with different Cryptosporidium species and subtypes, and further stresses the importance of surveillance for opportunistic intestinal protozoans.

Statewide Cross-Sectional Survey of Cryptosporidium and Giardia in California Cow-Calf Herds

Cryptosporidium spp. and Giardia duodenalis are common protozoal parasites in livestock including beef cattle on rangeland and irrigated pasture. A statewide cross-sectional study was conducted to determine the prevalence, species or genotype, and risk factors for fecal shedding of Cryptosporidium and Giardia by cattle from California cow-calf operations. Species and genotypes of Cryptosporidium and Giardia were determined by molecular fingerprinting. Prevalence of Cryptosporidium (19.8%) and Giardia (41.7%) in fecal samples from calves were approximately twice as high as fecal samples from cows (9.2% and 23.1%, respectively). In addition to age, multivariable logistic regression showed that higher stocking density and a higher number of replacement heifers were positively associated with fecal shedding of Cryptosporidium while longer calving interval, a winter/spring calving season, and higher numbers of replacement heifers were positively associated with shedding of Giardia. The dominant species and genotypes of Cryptosporidium and Giardia in feces from these cow-calf herds were Cryptosporidium ryanae (75%) and assemblage E for Giardia duodenalis (90%), which have low impact on public health compared with other zoonotic species/genotypes of these two parasites. We identified host and potential management practices that can be used to protect cattle health and reduce the risk of surface water contamination with protozoal parasites from cow-calf operations. In addition, this work updated the scientific data regarding the predominance of low zoonotic genotypes of Cryptosporidium and Giardia shed in the feces of commercial cow-calf herds on California rangeland and irrigated pasture.

First detection of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus) in China.

Cryptosporidium spp. are opportunistic pathogens that cause diarrhea in a variety of animal hosts. Although they have been reported in many animals, no information has been published on the occurrence of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus). A total of 287 fecal specimens were collected from Sichuan province in China; the prevalence of Cryptosporidium spp., measured by nested-PCR amplification of the partial small-subunit (SSU) rRNA gene, was 1.4% (4/287). Three different Cryptosporidium species or genotypes were identified: Cryptosporidium parvum (n = 1), Cryptosporidium wrairi (n = 1), and Cryptosporidium rat genotype II (n = 2). The present study is the first report of Cryptosporidium infection in red-bellied tree squirrels in China. Although there is a relatively low occurrence of Cryptosporidium, the presence of C. parvum and C. wrairi, which were previously reported in humans, indicates that red-bellied tree squirrels may be a source of zoonotic cryptosporidiosis in China.

Host-adapted Cryptosporidium and Enterocytozoon bieneusi genotypes in straw-colored fruit bats in Nigeria.

Few data are available on the distribution and human infective potential of Cryptosporidium and Enterocytozoon bieneusi genotypes in bats. In this preliminary study, we collected 109 fecal specimens during April–July 2011 from a colony of straw-colored fruit bats (Eidolon helvum) in an urban park (Agodi Gardens) of Ibadan, Nigeria, and analyzed for Cryptosporidium spp., Giardia duodenalis and E. bieneusi using PCR targeting the small subunit rRNA gene, triosephosphate isomerase gene, and ribosomal internal transcribed spacer, respectively. Genotypes of these enteric parasites were determined by DNA sequencing of the PCR products. Altogether, 6 (5.5%), 0 and 16 (14.7%) specimens were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. DNA sequence analysis of the PCR products indicated the presence of two novel Cryptosporidium genotypes named as bat genotype XIV (in 5 specimens) and bat genotype XV (in 1 specimen) and one known E. bieneusi genotype (Type IV in 1 specimen) and two novel E. bieneusi genotypes (Bat1 in 13 specimens and Bat2 in 2 specimens). In phylogenetic analysis of DNA sequences, the two novel Cryptosporidium genotypes were genetically related to Bat genotype II previously identified in fruit bats in China and Philippines, whereas the two novel E. bieneusi genotypes were genetically related to Group 5, which contains several known genotypes from primates. With the exception of Type IV, none of the Cryptosporidium and E. bieneusi genotypes found in bats in this study are known human pathogens. Thus, straw-colored fruit bats in Nigeria are mainly infected with host-adapted Cryptosporidium and E. bieneusi genotypes.

A novel three-dimensional microTAS chip for ultra-selective single base mismatched Cryptosporidium DNA biosensor

The development of new rapid and reliable detection technology of Cryptosporidium, a deadly waterborne pathogen, using a three-dimensional micro total analysis systems (3D μTAS) is the main goal of this work. An approach to enhance DNA detection on a patterned configurable 3D μTAS electrochemical DNA biosensor to human genotype of Cryptosporidium was successfully developed in present study. A novel 3D μTAS, comprising three layers, was fabricated to achieve better result comparing to the commercial SPE. The first layer was comprised of a circular gold coated area (gold dot) in the center of a PI sheet and the third layer consisted of a common homemade screen printed electrode (SPE). The second layer was included PDMS channels and reservoir for sample delivery. The biosensor was fabricated on the gold dot of the first layer and electrochemical signal was recorded using the differential pulse voltammetry (DPV) and impedance methods on the third layer and the results were compared with biosensor fabricated on commercial SPE. The calibration curve of 3D μTAS DNA biosensor shows a detection limit of 1.8 ng/mL and a sensitivity of 12.844 μA/(μg mL−1) in a linear range of 2.5 ng/mL to 0.1 μg/mL, which are 6 times lower detection limit and 23 times higher sensitivity of commercial SPE in a similar linear range. The specificity of prepared DNA sensor was conducted by using non-complementary, single, three and five mismatches strands and the potential of the biosensor in discriminating single mismatch detection was experimentally appraised.

Cryptosporidium genotyping and land use mapping for hazard identification and source tracking in a small mixed rural-urban watershed in Southeastern Brazil.

Abstract Cryptosporidium, faecal indicator organisms and physical and chemical water quality variables were monitored in a small mixed rural–urban watershed in southeastern Brazil. Cryptosporidium was present in 43% of 117 water samples analysed by microscopy. Concentrations varied from non-detects to 14 oocysts L−1. All samples were further analysed by nested-PCR, and Cryptosporidium spp. were detected in 24% (28) of them. Sequencing at the 18S rRNA locus gave high quality sequences in eight samples, revealing the presence of Cryptosporidium parvum. Cryptosporidium was not correlated with faecal indicator organisms (total coliforms, Escherichia coli, Enterococcus and coliphages), nor with physical and chemical water quality variables (e.g. turbidity, electrical conductivity and chemical oxygen demand), but it was with farm animal density (number of animals per ha). Land use mapping reinforced the suggestions from Cryptosporidium genotyping that both animals (livestock) and humans are potential sources to environmental contamination with oocysts within the watershed.

Studies on occurrence, molecular detection and genotyping of Cryptosporidium parvum along with associated risk factors in cattle and human from Aizawl district, Mizoram, India

ABSTRACTAn epidemiological study was conducted along with molecular detection of Cryptosporidium in cattle and human from Aizawl district of Mizoram, India. A total of 200 samples (100 each of cattle faeces and human stool) were collected randomly for detection of Cryptosporidium ocysts by mZN staining after concentration of ocysts by Sheather’s sucrose floatation technique. Genomic DNA was extracted from the microscopically positive faecal/stool samples for PCR-based detection of 18S (SSU) rRNA gene of Cryptosporidium. All the microscopically positive cattle faecal and human stool samples were found to be positive for 18S (SSU) rRNA gene of Cryptosporidium, and subsequently the species and genotype of Cryptosporidium were detected by PCR- restriction fragment length polymorphism (RFLP). The overall prevalence rates of Cryptosporidium were 13% and 5% in cattle and human, respectively. Significantly (P < 0.01) higher prevalence rate of Cryptosporidium was recorded in 0–6 months than above 6 months of age group of cattle. The sex-wise and breed-wise prevalence rates of Cryptosporidium did not vary significantly in both cattle and human. Diarrhoeic cattle and human were found to be significantly (P < 0.01) more infected with Cryptosporidium than non-diarrhoeic. Significantly (p < 0.05) higher prevalence rates of Cryptosporidium were found in cattle during monsoon season than winter, and the seasonal variation was non-significant in human. The PCR-RFLP revealed C. parvum (genotype II) in nine numbers and C. andersoni in four numbers of cattle faecal samples and the two numbers of human stool samples revealed genotype I and genotype II, one each.

The first report of animal genotypes of Cryptosporidium parvum in immunosuppressed and immunocompetent humans in Slovakia.

The aim of our study was to determine species and genotypes of Cryptosporidium in patients suffering from immunosuppressive illnesses, but also in immunocompetent patients suffering from diarrhoea. A total of 80 samples of faeces were collected from both immunosuppressed and immunocompetent patients. The immunosuppressed patients (65 samples) - 35 adult patients (group A) and 30 children (group B) were hospitalized at the Clinic of Oncohemathology. Samples from immunocompetent humans (15 samples, group C) were taken from patients with clinical signs of acute diarrhoea. With the use of molecular methods targeting the 60kDa glycoprotein (GP60) gene region, we have identified multiple genotypes of Cryptosporidium.parvum and Cryptosporidium.hominis in immunocompromised, but also in immunocompetent individuals (C.hominis IbA10G2, IeA12G3T3; C.parvum IIaA10G1R1, IIaA11G2R1, IIaA12G2R1, IIaA13G1R1, IIaA14G1R1, IIaA14G2R1, IIaA17G1R1 and IIaA18G1R1). This is the first report of the occurrence of genotypes IIaA10G1R1, IIa12G2R1 and IIaA18G1R1 in human hosts.

Genotypic Characterization of Cryptosporidium Species in Humans and Peri-Domestic Animals in Ekiti and Oyo States, Nigeria.

Cryptosporidiosis is one of the leading causes of diarrhea in humans and several other vertebrate species. Because surveys of Cryptosporidium genotypes from animals and humans living in the same region are rare, our understanding of the importance of zoonotic transmission in the epidemiology of cryptosporidiosis remains superficial. PCR was used to amplify a portion of the Cryptosporidium 18S small subunit ribosomal RNA gene from fecal DNA from humans and livestock living in Ekiti and Oyo states, Nigeria. PCR-positive samples were further analyzed using PCR targeting the heat-shock protein HSP-70, the actin, and the sporozoite glycoprotein gene gp60. A questionnaire was used to collect demographic information. Sixteen of 187 samples collected were Cryptosporidium 18S PCR positive. Of these, 5 samples originating from HIV-positive patients, 5 from otherwise healthy children, 2 from chickens, 3 from goats, and 1 from a dog were positive for at least 1 marker. Sequencing of the 18S rRNA amplicons revealed the presence of Cryptosporidium parvum in 2 HIV positive patients and in a child; the actin sequence confirmed the presence of this species. Two samples of HIV-positive patients amplified Cryptosporidium hominis 18S rRNA, one of them confirmed by the HSP-70, actin, and gp60 sequences. Cryptosporidium meleagridis was found in another HIV patient, while C. hominis was detected in 3 children (of which 2 were confirmed by gp60). Cryptosporidium muris was found in 1 child. In birds, we found C. meleagridis and, significantly, C. parvum, whereas we detected C. parvum and C. muris in 1 goat each. The only dog sampled was positive for Cryptosporidium canis. We conclude that, in the environment we surveyed, humans and animals are a potential part of the same transmission cycle. Measures to prevent zoonotic transmission should therefore be considered to reduce the prevalence of cryptosporidiosis.

INTESTINAL PARASITOSIS AND PEDIATRIC HEPATIC DISEASE: COPROSCOPY AND IMMNO MOLECULAR ASSAYS

Parasitic infections in children with liver diseases could be a serious health problem. This study documented a true prevalence of intestinal parasitosis in children with pediatric liver diseases and estimate risk factors for parasitic infection. Faecal samples were collected from 95 children suffering liver diseases. Samples were examined microscopically for parasitic stages and coproscopy of acid fast (AF) stained fecal smear for Cryptosporidium oocysts, by ELISA for E. histolytica and Giardia and by PCR genotyping for E. histolytica and Cryptosporidium spp. copro-DNA. G. intestinalis was the prevailing protozoa (14.7%) followed by Hymenolepis nana (8.4%) and C. hominis (4.2%). The pathogenic E. histolytica was not detected by genetic differentiation or ELISA. Only age group (P value .004) was a risk factor for intestinal parasitosis among these children. Giardiasis, hymenolepiasis nana and cryptosporidiosis are the most prevailing parasites in children with liver disease that physicians must consider. Accurate diagnosis using molecular technique is a must with E.histolytica and helpful with Cryptosporidium genotyping for epidemiological purposes.

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination.

Identification of a novel piscine Cryptosporidium genotype and Cryptosporidium parvum in cultured rainbow trout (Oncorhynchus mykiss).

This study reports for the first time the presence and molecular characterization of Cryptosporidium in farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792). A total of 360 fish, with no apparent clinical signs of disease, were collected and classified into groups according to their size. Cryptosporidium oocysts were detected by immunofluorescence microscopy in 33 specimens (9.2%), which were located in pyloric caeca samples (42.4%), intestinal scrapings (39.4%), or at both locations (18.2%). In the smallest (youngest) fish group, a higher percentage of positive samples were detected in the pyloric caeca relative to the intestinal location (58.8 vs. 17.6%; P = 0.01), including a cluster with more than 10 oocysts observed in the pyloric caeca of one specimen. PCR amplification and sequencing of fragments of SSU-rDNA and hsp70 genes identified a novel Cryptosporidium piscine genotype (genotype 9) in two specimens and Cryptosporidium parvum in seven fish, including the specimen in which the oocyst cluster was observed. Moreover, Cryptosporidium oocysts were detected in farm water samples (41.7 and 16.7% from influent and effluent, respectively). Although Giardia was not found in gastrointestinal samples, Giardia cysts were observed in 50.0 and 33.3% of the influent and effluent water samples, respectively. The results support the existence of natural infections by C. parvum in freshwater cultured fish, suggesting that the rainbow trout could shed infectious oocysts in aquatic environments and it may be a potential source of human infection when this edible fish is handled.

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants’ opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe.

Molecular Screening and Genotyping of Cryptosporidium Species in Household Dogs and In-Contact Children in Egypt: Risk Factor Analysis and Zoonotic Importance.

Cryptosporidiosis is a parasitic zoonosis implicated in severe diarrhoea in pets and humans. This study aimed to determine the prevalence and genotypes of Cryptosporidium spp. in household dogs and in-contact children, and the risk factors associated with infection in children in Sharkia Province, Egypt. Fecal samples of 100 children (2-12 years old) and 50 dogs (3 months-1 year old) were randomly collected from both rural (children: n = 85, dogs: n = 40) and urban (children: n = 15, dogs: n = 10) households. Initial parasite detection was done by light microscopy, while, genotyping was based on molecular diagnostic assays. The overall prevalence of Cryptosporidium spp. infection in children was 35% using microscopy and 14% using nested polymerase chain reaction (PCR). In dogs, it was 34% using microscopy and 24% using nested PCR. Cryptosporidium spp. from children were identified as distinct genotypes, with the predominance of human genotype I (Cryptosporidium hominis) over the zoonotic genotype II (Cryptosporidium parvum). Moreover, only zoonotic genotype II (C. parvum) was identified in dog samples. The significant risk factors associated with the prevalence of Cryptosporidium infection in children were the presence of diarrheal episodes during time of survey, improper disposal of garbage, and dog feces and contact with other livestock (p ≤ 0.05). This study concluded that the existence of C. parvum in children and dogs residing the same households confirm the zoonotic transmission and its public health significance. Also, the study recommended the necessity of hygienic disposal of dog feces and preventing direct contact of dogs with other livestock.

A new avian Cryptosporidium genotype in a 1-month-old caged brown wood owl (Strix leptogrammica) with severe dehydration and diarrhea

A 1-month-old brown wood owlet (Strix leptogrammica) purchased from a wholesaler and housed as a companion bird by an individual owner in Japan showed severe dehydration and anorexia following a week of vomiting and severe diarrhea. A great number of approximately 5 × 4-μm-sized Cryptosporidium oocysts were found in the feces by microscopy. The owlet was administered subcutaneous fluid and intragastric tube feeding for 2weeks, resulting in improvement of the condition with a decreased number of oocysts in the feces. At days 51 and 119, no oocysts were found in the feces by microscope and PCR detection. These results suggested that this parasite was a possible agent of severe diarrhea in the affected bird. Molecular analysis of DNA extracted from oocysts based on the 18SrRNA loci identified C. avium; however, analysis of actin and hsp (heat shock protein) genes identified a novel genotype indicating a mixed infection with C. avium and a novel genotype.

Molecular characterizations of Cryptosporidium spp. and Enterocytozoon bieneusi in brown rats (Rattus norvegicus) from Heilongjiang Province, China

BackgroundCryptosporidium spp. and Enterocytozoon bieneusi are prevalent zoonotic pathogens responsible for the high burden of diarrheal diseases worldwide. Rodents are globally overpopulated and are known as reservoirs or carriers of a variety of zoonotic pathogens including Cryptosporidium spp. and E. bieneusi. However, few data are available on genetic characterizations of both pathogens in rodents in China. The aim of the present work was to determine the prevalence and genetic characterizations of Cryptosporidium spp. and E. bieneusi in brown rats (Rattus norvegicus) from Heilongjiang, China.MethodsA total of 242 wild brown rats were captured in Heilongjiang Province of China. A fresh fecal specimen was collected directly from the intestinal and rectal content of each brown rat. All the fecal specimens were examined for the presence of Cryptosporidium spp. and E. bieneusi by PCR and sequencing of the partial small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) region of the rRNA gene of the two pathogens, respectively.ResultsThe infection rate was 9.1% (22/242) for Cryptosporidium spp. and 7.9% (19/242) for E. bieneusi. Sequence analysis confirmed the presence of C. ubiquitum (1/22, 4.5%) and three genotypes of Cryptosporidium, including Cryptosporidium rat genotype I (14/22, 63.6%), Cryptosporidium rat genotype IV (6/22, 27.3%) and Cryptosporidium suis-like genotype (1/22, 4.5%). Meanwhile, two E. bieneusi genotypes were identified, including D (17/19, 89.5%) and Peru6 (2/19, 10.5%).ConclusionsTo the best of our knowledge, Enterocytozoon bieneusi genotype Peru6 was identified in rodents for the first time globally and Cryptosporidium rat genotype I and Cryptosporidium rat genotype IV were found in rats in China for the first time. The finding of zoonotic C. ubiquitum and C. suis-like genotype, as well as E. bieneusi genotypes, suggests that brown rats pose a threat to human health. It is necessary to control brown rat population in the investigated areas and improve local people’s awareness of the transmission risk of the two pathogens from brown rats to humans.

Occurrence, genotyping, and health risk of Cryptosporidium and Giardia in recreational lakes in Tianjin, China

Cryptosporidium and Giardia are critical parasites in the etiology of diarrhea worldwide, and often cause waterborne outbreaks. The presence of Cryptosporidium and Giardia in recreational lakes was investigated with molecular characterization, and a comprehensive quantitative microbial risk assessment (QMRA) of protozoan infections was performed, considering multiple exposure pathways, differences in age, sex, and disease severity, and the genotypes of the protozoa. Forty-three (82.7%) and 51 (98.1%) water samples were positive for Cryptosporidium oocysts and Giardia cysts, respectively, with average counts of 3.65 oocysts/10 L and 12.58 cysts/10 L, respectively. Six Cryptosporidium species and three Giardia lamblia assemblages were confirmed with molecular analyses. The protozoan concentration was significantly associated with water turbidity, but not with the total coliform numbers. Swimming in the lakes entailed the highest incidence risk of 5.72 × 10−4 per person per year (pppy) (95% confidence interval (CI): 0.03–43.33 × 10−4) for Cryptosporidium and 4.04 × 10−4 pppy (95% CI: 0.01–32.66 × 10−4) for Giardia, whereas wading entailed the lowest risk (2.20 × 10−4 and 1.70 × 10−4 pppy, respectively). The annual burdens attributable to recreational-water-associated cryptosporidiosis and giardiasis were 3.44 (95% CI: 0.04–23.51) and 1.81 (95% CI: 0.01–12.96) disability-adjusted life years per 1,000,000 individuals per year, respectively. Children were more likely to have an individual disease burden than adults, and males were more likely than females. Sensitivity analysis highlighted the great importance of controlling the proportion of exposed individuals and reducing the frequency of exposure. The methodology and results of this study will allow us to better evaluate and reduce the burden of Cryptosporidium and/or Giardia infections associated with recreational water use in China and other countries.

Understanding human infectious Cryptosporidium risk in drinking water supply catchments

Treating drinking water appropriately depends, in part, on the robustness of source water quality risk assessments, however quantifying the proportion of infectious, human pathogenic Cryptosporidium oocysts remains a significant challenge. We analysed 962 source water samples across nine locations to profile the occurrence, rate and timing of infectious, human pathogenic Cryptosporidium in surface waters entering drinking water reservoirs during rainfall-runoff conditions. At the catchment level, average infectivity over the four-year study period reached 18%; however, most locations averaged <5%. The maximum recorded infectivity fraction within a single rainfall runoff event was 65.4%, and was dominated by C. parvum. Twenty-two Cryptosporidium species and genotypes were identified using PCR-based molecular techniques; the most common being C. parvum, detected in 23% of water samples. Associations between landuse and livestock stocking characteristics with Cryptosporidium were determined using a linear mixed-effects model. The concentration of pathogens in water were significantly influenced by flow and dominance of land-use by commercial grazing properties (as opposed to lifestyle properties) in the catchment (p < 0.01). Inclusion of measured infectivity and human pathogenicity data into a quantitative microbial risk assessment (QMRA) could reduce the source water treatment requirements by up to 2.67 log removal values, depending on the catchment, and demonstrated the potential benefit of collating such data for QMRAs.