Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection.

Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host-parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Blastocystis sp. Prevalence and Subtypes Distribution amongst Syrian Refugee Communities Living in North Lebanon.

Molecular data concerning the prevalence and subtype (ST) distribution of the intestinal parasite Blastocystis sp. remain scarce in the Middle East. Accordingly, we performed the first molecular epidemiological survey ever conducted in the Syrian population. A total of 306 stool samples were collected from Syrian refugees living in 26 informal tented settlements (ITS) subjected or not to water, sanitation, and hygiene (WASH) interventions in North Lebanon, then screened for the presence of Blastocystis sp. by real-time polymerase chain reaction followed by subtyping. The overall prevalence of the parasite was shown to reach 63.7%. Blastocystis sp. colonization was not significantly associated with gender, age, symptomatic status, abdominal pain or diarrhea. In contrast, WASH intervention status of ITS was identified as a risk factor for infection. Among a total of 164 subtyped isolates, ST3 was predominant, followed by ST1, ST2, and ST10. No particular ST was reported to be associated with age, gender, symptomatic status, digestive disorders, or WASH intervention status of ITS. Intra-ST diversity of ST1 to ST3 was low suggesting large-scale anthroponotic transmission. Moreover, comparative analysis of ST1 to ST3 genotypes revealed that the circulation of the parasite between Syrian refugees and the host population was likely limited.

Molecular Characterization of Novel Cryptosporidium Fish Genotypes in Edible Marine Fish.

Current knowledge of Cryptosporidium species/genotypes in marine fish is limited. Following phylogenetic analysis at the 18S rDNA locus, a recent study identified six new genotypes of Cryptosporidium colonizing edible fish found in European seas. Of these, five grouped in a clade together (#Cryptofish 1-5) and one grouped separately (#Cryptofish 7). In the present study, after phylogenetic analyses of #Cryptofish1, #Cryptofish2, #Cryptofish4, #Cryptofish5 and #Cryptofish7 at the actin locus, the presence of two major clades was confirmed. In addition, when possible, longer 18S amplicons were generated. In conclusion, the small genetic distances between these genotypes designated as a novel marine genotype I (#Cryptofish 1-5) suggest that they may be genetic variants of the same species, while the designated novel marine genotype 2 (#Cryptofish 7) is clearly representative of a separate species.

Cryptosporidium and Colon Cancer: Cause or Consequence?

The number of cancers attributable to infectious agents represents over 20% of the global cancer burden. The apicomplexan intracellular parasite Cryptosporidium is currently considered one of the major causes of mild and severe diarrhea worldwide. However, less attention has been paid to its tumorigenic potential despite the high exposure of humans and animals to this ubiquitous parasite. Herein, we discuss the potential causal link between Cryptosporidium infection and digestive cancer, with particular emphasis on colon cancer, based on increasing clinical, epidemiological and experimental pieces of evidence supporting this association. In addition, we highlight the current knowledge about the potential mechanisms by which this parasite may contribute to cell transformation and parasite-induced cancer.

Prevalence and Subtype Distribution of Blastocystis sp. in Senegalese School Children.

Blastocystis sp. is an enteric protozoan that frequently colonizes humans and many animals. Despite impacting on human health, data on the prevalence and subtype (ST) distribution of Blastocystis sp. remain sparse in Africa. Accordingly, we performed the first multicenter and largest epidemiological survey ever conducted on Blastocystis sp. for this continent. A total of 731 stool samples collected from healthy school children living in 10 villages of the northwestern region of Senegal were tested for the presence of Blastocystis sp. by real-time polymerase chain reaction followed by subtyping of positive samples. Considerable variation in prevalence between villages (51.7 to 100%) was evident with the overall prevalence being 80.4%. Mixed infections were identified in 23% of positive individuals. Among 453 school children with a single infection, ST2 was predominant, followed by ST1, ST3, ST7, ST10, and ST14; this is the first report of ST10 and ST14 in humans. Genetic polymorphisms were evident at the intra-ST level with the identification of numerous ST1 to ST3 genotypes. ST1 showed the greatest intra-ST diversity followed by ST2 and ST3. The prevalence and distribution of STs and genotypes varied among target villages, pointing to several potential infection sources, including human-to-human, zoonotic, and waterborne transmission.

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

First Report on the Prevalence and Subtype Distribution of Blastocystis sp. in Edible Marine Fish and Marine Mammals: A Large Scale-Study Conducted in Atlantic Northeast and on the Coasts of Northern France.

Blastocystis is frequently identified in humans and animal hosts and exhibits a large genetic diversity with the identification of 17 subtypes (STs). Despite its zoonotic potential, its prevalence and ST distribution in edible marine fish and marine mammals remain unknown. A large-scale survey was thus conducted by screening 345 fish caught in Atlantic Northeast and 29 marine mammals stranded on the coasts of northern France for the presence of the parasite using real-time Polymerase Chain Reaction PCR. The prevalence of the parasite was about 3.5% in marine fish. These animals were mostly colonized by poikilotherm-derived isolates not identified in humans and corresponding to potential new STs, indicating that fish are natural hosts of Blastocystis. Marine fishes are also carriers of human STs and represent a likely limited source of zoonotic transmission. 13.8% of the marine mammals tested were colonized and 6 different STs were identified including 3 potential new STs. The risk of zoonotic transmission through marine mammals is insignificant due to the lack of repeated contact with humans. The present survey represents the first data regarding the prevalence and ST distribution of Blastocystis in marine fish and marine mammals and provides new insights into its genetic diversity, host range and transmission.

Prevalence, Molecular Identification, and Risk Factors for Cryptosporidium Infection in Edible Marine Fish: A Survey Across Sea Areas Surrounding France.

Cryptosporidium, a zoonotic pathogen, is able to infect a wide range of hosts including wild and domestic animals, and humans. Although it is well known that some parasites are both fish pathogens and recognized agents of zoonosis with a public health impact, little information is available concerning the prevalence of Cryptosporidium in wild aquatic environments. To evaluate the prevalence of Cryptosporidium spp. in commercially important edible marine fish in different European seas (English channel, North sea, Bay of Biscay, Celtic sea and Mediterranean sea), 1,853 specimens were collected as part of two surveys. Nested PCR followed by sequence analysis at the 18S rRNA gene locus was used to identify Cryptosporidium spp. The overall prevalence of Cryptosporidium spp. in sampled fish reached 2.3% (35 out of 1,508) in a first campaign and 3.2% (11 out of 345) in a second campaign. Sequence and phylogenetic analysis of positive samples identified Cryptosporidium parvum (n = 10) and seven genotypes which exhibited between 7.3 and 10.1% genetic distance from C. molnari, with the exception of one genotype which exhibited only 0.5-0.7% genetic distance from C. molnari. Among 31 analyzed fish species, 11 (35.5%) were identified as potential hosts for Cryptosporidium. A higher prevalence of Cryptosporidium spp. was observed in larger fish, in fish collected during the spring-summer period, and in those caught in the North East Atlantic. Pollachius virens (saithe) was the most frequently Cryptosporidium positive species. In fish infected by other parasites, the risk of being Cryptosporidium positive increased 10-fold (OR: 9.95, CI: 2.32-40.01.04, P = 0.0002). Four gp60 subtypes were detected among the C. parvum positive samples: IIaA13G1R1, IIaA15G2R1, IIaA17G2R1, and IIaA18G3R1. These C. parvum subtypes have been previously detected in terrestrial mammals and may constitute an additional source of infection for other animals and in particular for humans. Microscopical examination of histological sections confirmed the presence of round bodies suggestive of the development of C. parvum within digestive glands. We report herein the first epidemiological and molecular data concerning the detection of Cryptosporidium in edible marine fish in European seas surrounding France broadening its host range and uncovering potential novel infection routes.

First report on the prevalence and subtype distribution of Blastocystis sp. in dairy cattle in Lebanon and assessment of zoonotic transmission.

Blastocystis sp. is frequently identified in a wide range of animal hosts, including bovids. Because of its burden and zoonotic potential, this parasite has been sought in domestic cattle from various countries, since this livestock may also represent a possible reservoir of human infection. However, epidemiological data regarding the prevalence and ST distribution of Blastocystis sp. in this animal group is lacking in Lebanon. Therefore, faecal samples were collected from a total of 254 dairy cattle raised on 55 farms located in the North Lebanon region and screened for the presence of the parasite by quantitative real-time PCR. The overall prevalence of Blastocystis sp. was shown to reach 63.4% in cattle livestock. Sequence analysis of positive samples indicated the presence of seven STs, with predominance of ST10 (44.0%) and ST14 (36.8%) and lower proportions of ST2 (8.0%), ST1 (7.2%), ST5 (2.4%), ST3 and ST7 (0.8% each). This survey was the first conducted worldwide reporting ST2 and ST7 in domestic cattle and confirmed that ST10 and ST14 represent cattle-adapted STs in view of their high prevalence. Faecal samples from in-contact dairy farmers and patients hospitalised in the same Lebanese governorate who reported no contact with cattle livestock were also analysed for the presence of Blastocystis sp. The same three STs were identified in both human cohorts, with predominance of ST3, followed either by ST1 or ST2 depending of the group. No other STs, including ST10 or ST14, have been reported. Moreover, even though ST1, ST2 and ST3 were found to be common to dairy cattle and farmers cohorts, only one ST3 isolate showed 100% sequence identity between both hosts. Consequently, the presence and low prevalence of ST1, ST2, ST3, ST5 and ST7 identified herein in domestic cattle, most of which exhibit low host specificity, could be derived from occasional direct exposure to faecal material from human and non-human hosts or by ingestion of contaminated drinking water or food in the enclosure of the farms. Together with the absence of ST10 and ST14 in the human population, these data suggest that cattle play a negligible role as zoonotic reservoirs of Blastocystis sp.

Prevalence and subtype distribution of Blastocystis sp. isolates from poultry in Lebanon and evidence of zoonotic potential.

BACKGROUND: Blastocystis sp. is a common protozoan parasite frequently identified in the digestive tract of humans and a large variety of animal hosts worldwide, including birds. It exhibits a large genetic diversity with the identification of 17 subtypes (STs), most of them with low host specificity. ST6 and ST7 were identified in birds and suggested to represent avian STs only in the context of scarce small-scale epidemiological surveys. Moreover, these two STs also account for a significant proportion of human infections whose zoonotic origin has never been clearly confirmed. Therefore, molecular screening of Blastocystis sp. was conducted by quantitative real-time PCR for fecal samples from poultry farms and their in-contact humans from slaughterhouses in Lebanon. In parallel, a control group consisting of patients hospitalized in the same geographical area and reporting no contact with poultry was also screened for the presence of the parasite. RESULTS: The overall prevalence of Blastocystis sp. was shown to reach around 32% in chicken samples and 65% in the farms screened. All the avian isolates were subtyped and belonged to either ST6 or ST7, with a large predominance of ST6. Fifty-four percent of slaughterhouse staff members were positive for Blastocystis sp. compared with a similar prevalence of 56% in hospitalized patients. ST3 was predominant in both human cohorts followed by either ST1 then ST2 among slaughterhouse staff or by ST2 then ST1 among hospitalized patients. ST6 was also identified in two slaughterhouse workers and not in the group of hospitalized patients. Gene sequence identity was observed between chicken and human ST6 isolates from the same slaughterhouse. CONCLUSIONS: Our data revealed a high prevalence of Blastocystis sp. in chicken samples and confirmed that ST6 and ST7 represented avian-adapted STs. Among both human cohorts, Blastocystis sp. infection was shown to exceed 50% with a predominance of ST3. The identification of ST6 in slaughterhouse staff members confirmed the zoonotic transmission of this ST through repeated and direct contact between chickens and their handlers.

Prevalence, transmission, and host specificity of Cryptosporidium spp. in various animal groups from two French zoos.

Cryptosporidium represents a major cause of gastrointestinal illness in humans and animals including domestic, wild, and in captivity animals, and more than 30 validated species of Cryptosporidium are recognized as infectious to different hosts such as mammals, birds, reptiles, amphibians, and fish. Therefore, numerous investigations have been conducted worldwide in order to shed light on the epidemiology of this parasite and to explore its potential reservoirs. Few surveys, targeting humans and animals have been carried out regarding the epidemiology of Cryptosporidium spp. in France and no data are available about the circulation of this parasite in French zoological gardens. Herein, we determined the prevalence of Cryptosporidium in animals housed in two French zoos. A total of 307 fecal samples belonging to 161 species were screened by nested PCR. Overall, Cryptosporidium DNA was detected in 1.9% of the 161 species and 1% of the total number of fecal samples tested. Additionally, three Cryptosporidium species were identified: C. galli, C. andersoni, and C. tyzzeri. To our knowledge, this is the first molecular study focused on Cryptosporidium infection in captivity animals in France. This study is of interest considering the exposure of a large number of humans and animals to this waterborne protozoan, found ubiquitously in the environment.

Molecular Epidemiology of Blastocystis sp. in Various Animal Groups from Two French Zoos and Evaluation of Potential Zoonotic Risk.

Blastocystis sp. is a common intestinal parasite infecting humans and a wide range of animals worldwide. It exhibits an extensive genetic diversity and 17 subtypes (STs) have thus far been identified in mammalian and avian hosts. Since several STs are common to humans and animals, it was proposed that a proportion of human infections may result from zoonotic transmission. However, the contribution of each animal source to human infection remains to be clarified. Therefore, the aim of this study was to expand our knowledge of the epidemiology and host specificity of this parasite by performing the largest epidemiological survey ever conducted in animal groups in terms of numbers of species screened. A total of 307 stool samples from 161 mammalian and non-mammalian species in two French zoos were screened by real-time PCR for the presence of Blastocystis sp. Overall, 32.2% of the animal samples and 37.9% of the species tested were shown to be infected with the parasite. A total of 111 animal Blastocystis sp. isolates were subtyped, and 11 of the 17 mammalian and avian STs as well as additional STs previously identified in reptiles and insects were found with a varying prevalence according to animal groups. These data were combined with those obtained from previous surveys to evaluate the potential risk of zoonotic transmission of Blastocystis sp. through the comparison of ST distribution between human and animal hosts. This suggests that non-human primates, artiodactyls and birds may serve as reservoirs for human infection, especially in animal handlers. In contrast, other mammals such as carnivores, and non-mammalian groups including reptiles and insects, do not seem to represent significant sources of Blastocystis sp. infection in humans. In further studies, more intensive sampling and screening of potential new animal hosts will reinforce these statements and expand our understanding of the circulation of Blastocystis sp. in animal and human populations.

Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk.

Several parasites including the protozoa Blastocystis sp. and Cryptosporidium spp. may be causative agents of gastrointestinal symptoms in domestic dogs, and there may be a potential risk of transmission to owners. While France is one of the largest European countries in terms of its canine population, little data is available about the molecular epidemiology of these two parasites. The purpose of this study was to determine the prevalence of intestinal parasites in household dogs in France, and to evaluate the zoonotic risk of Blastocystis sp. and Cryptosporidium spp. by genotyping the corresponding isolates. To this end, 116 faecal samples were collected from household dogs regardless of breed, age or gender, living in the Lyons area, France. Various intestinal protozoa and helminths were identified by light microscopy. Screening for Blastocystis sp. and Cryptosporidium spp. were subsequently performed by PCR targeting the small subunit (SSU) rDNA coding region, followed by direct sequencing of the PCR products and analysis of the sequences obtained for genotyping. The overall prevalence of dogs infected with at least one gastrointestinal parasite was 42.2% (49/116). After light microscopy examination of faecal samples, the most common parasites found were the protozoa Giardia sp. (25.0%) and Cystoisospora sp. (19.8%). Using molecular methods, four dogs (3.4%) were shown to be infected by Blastocystis sp. and carried either subtype (ST) 2, commonly identified in various animal groups, or ST10, frequently found in bovids. Three dogs (2.6%) were positive for C. canis, infecting humans episodically. The low prevalence of both parasites, combined with the identification of C. canis and Blastocystis sp. ST2 and ST10 in the canine population, strongly suggests that dogs play a negligible role as zoonotic reservoirs for both parasites and do not seem to be natural hosts of Blastocystis sp.