Molecular Identification and Subtype Analysis of Blastocystis sp. Isolates from Wild Mussels (Mytilus edulis) in Northern France.

Blastocystis sp. is the most common single-celled eukaryote colonizing the human gastrointestinal tract worldwide. Because of the proven zoonotic potential of this protozoan, sustained research is therefore focused on identifying various reservoirs of transmission to humans, and in particular animal sources. Numerous groups of animals are considered to be such reservoirs due to their handling or consumption. However, some of them, including mollusks, remain underexplored. Therefore, a molecular epidemiological survey conducted in wild mussels was carried out in Northern France (Hauts-de-France region) to evaluate the frequency and subtypes (STs) distribution of Blastocystis sp. in these bivalve mollusks. For this purpose, 100 mussels (Mytilus edulis) were randomly collected in two sampling sites (Wimereux and Dannes) located in the vicinity of Boulogne-sur-Mer. The gills and gastrointestinal tract of each mussel were screened for the presence of Blastocystis sp. by real-time polymerase chain reaction (qPCR) assay followed by direct sequencing of positive PCR products and subtyping through phylogenetic analysis. In parallel, sequences of potential representative Blastocystis sp. isolates that were previously obtained from temporal surveys of seawater samples at marine stations offshore of Wimereux were integrated in the present analysis. By taking into account the qPCR results from all mussels, the overall prevalence of the parasite was shown to reach 62.0%. In total, more than 55% of the positive samples presented mixed infections. In the remaining mussel samples with a single sequence, various STs including ST3, ST7, ST14, ST23, ST26 and ST44 were reported with varying frequencies. Such distribution of STs coupled with the absence of a predominant ST specific to these bivalves strongly suggested that mussels might not be natural hosts of Blastocystis sp. and might rather be carriers of parasite isolates from both human and animal (bovid and birds) waste. These data from mussels together with the molecular identification of isolates from marine stations were subsequently discussed along with the local geographical context in order to clarify the circulation of this protozoan in this area. The identification of human and animal STs of Blastocystis sp. in mussels emphasized the active circulation of this protozoan in mollusks and suggested a significant environmental contamination of fecal origin. This study has provided new insights into the host/carrier range and transmission of Blastocystis sp. and emphasized its potential as an effective sentinel species for water quality and environmental contamination.

Frequency and Molecular Identification of Cryptosporidium in Adult Prim'Holstein Dairy Cattle Farms in the North of France.

Cryptosporidium apicomplexan protozoa are ubiquitous intracellular agents affecting humans and animals. In particular, bovine cryptosporidiosis is recognized as endemic worldwide. However, epidemiological investigations remain limited in France regarding the burden of these parasites in cattle. To improve our understanding of the epidemiology of cryptosporidiosis, the main aim of this study was to determine the frequency and the genetic diversity of Cryptosporidium in adult Prim'Holstein dairy cattle farms in the north of France. Fecal specimens were collected from 1454 non-diarrheic and non-pregnant animals (nulli-, primi-, or multiparous) throughout 20 farms in an area of 110 km around Lille. For Cryptosporidium species identification, nested PCR followed by sequence and phylogenetic analyses were used. The overall frequency of Cryptosporidium spp. in-fection was 30.00% (C.I. 95%: 12.83-54.33) in farms and 0.89% (C.I. 95%: 0.498-1.57) at the individual level. In primi- or multiparous cows, only C. andersoni was found. C. ryanae, C. bovis/xiaoi and C. andersoni were detected in heifers. The phylogenetic tree confirmed that analyzed sequences were grouped with known reference sequences reported in dairy cattle. Further studies on the cumulative prevalence, risks factors and pathogenicity are needed to give a more accurate assessment of the impact of Cryptosporidium infection in dairy cattle in France.

Molecular Epidemiology and Genetic Diversity of the Enteric Protozoan Parasite Blastocystis sp. in the Northern Egypt Population.

Blastocystis sp. is currently reported as the most frequent single-celled eukaryote inhabiting the intestinal tract of humans and a wide range of animal groups. Its prevalence is especially higher in developing countries linked with fecal peril. Despite a growing interest in this enteric protozoan, certain geographical regions potentially at high risk of infection, such as North Africa, remain under-investigated. Therefore, a large-scale molecular epidemiological survey, including 825 participants presenting digestive disorders or not, was conducted in five governorates located in Northern Egypt. A real-time polymerase chain reaction (qPCR) assay was performed to identify the parasite in stool samples, followed by direct sequencing of the positive PCR products for subtyping and genotyping of the corresponding isolates. The overall prevalence was shown to reach 72.4% in the Egyptian cohort, coupled with a variable frequency depending on the governorate (41.3 to 100%). Among the 597 positive participants, a large proportion of them (39.4%) presented mixed infections, as determined by sequencing. The remaining individuals with single infection were predominantly colonized by subtype 3 (ST3) (48.3%) followed by ST1 (39.5%), ST2 (10.8%), ST14 (1.1%), and ST10 (0.3%). This was the first report of ST10 and ST14 in North Africa. Age, sex, digestive symptoms, and health status of the participants or contact with animals were not identified as significant risk factors for Blastocystis sp. occurrence or affecting the ST distribution. In contrast, substantial variations in the prevalence and ST distribution of the parasite were reported according to the governorate. Genotyping of isolates revealed the lower intra-ST diversity for ST3, followed by ST1 and then ST2. By combining subtyping and genotyping data, a widespread inter-human transmission was strongly suggested for ST3 within the Egyptian cohort. Regarding ST1 and ST2, additional animal or environmental sources of infection by these STs have been proposed, whereas the few cases of colonization by ST10 and ST14 were likely the result of zoonotic transmission from bovid. These investigations clearly emphasized the active circulation of Blastocystis sp. in Northern Egypt and the necessity for health authorities to implement prevention campaigns towards the population and quality control of drinking water, with the aim of reducing the burden of this enteric protozoan in this endemic country.

Bact-to-Batch: A Microbiota-Based Tool to Determine Optimal Animal Allocation in Experimental Designs.

The basis of any animal experimentation begins with the housing of animals that should take into account the need for splitting animals into similar groups. Even if it is generally recommended to use the minimum number of animals necessary to obtain reliable and statistically significant results (3Rs rule), the allocation of animals is currently mostly based on randomness. Since variability in gut microbiota is an important confounding factor in animal experiments, the main objective of this study was to develop a new approach based on 16S rRNA gene sequencing analysis of the gut microbiota of animals participating in an experiment, in order to correctly assign the animals across batches. For this purpose, a pilot study was performed on 20 mouse faecal samples with the aim of establishing two groups of 10 mice as similar as possible in terms of their faecal microbiota fingerprinting assuming that this approach limits future analytical bias and ensures reproducibility. The suggested approach was challenged with previously published data from a third-party study. This new method allows to embrace the unavoidable microbiota variability between animals in order to limit artefacts and to provide an additional assurance for the reproducibility of animal experiments.

First Epidemiological Survey on the Prevalence and Subtypes Distribution of the Enteric Parasite Blastocystis sp. in Vietnam.

Although Blastocystis sp. is the most common enteric protozoan in human stools worldwide, various geographical areas remain to be investigated regarding the frequency and circulation of this parasite. Such is the case of some developing countries in Southeast Asia that exhibit a higher risk for parasitic infections due to unsanitary conditions. While several epidemiological surveys have been conducted, for instance, in Thailand, little or no data are available from neighboring countries, such as Vietnam. Therefore, in order to determine the prevalence and subtype (ST) distribution of Blastocystis sp. and to clarify the transmission of the parasite, the first molecular epidemiological survey ever conducted in this country was performed. For this purpose, a total of 310 stool specimens were collected from patients enrolled at the Family Hospital of Da Nang and then tested for the presence of Blastocystis sp. by real-time Polymerase Chain Reaction (qPCR), followed by subtyping of the isolates. The overall prevalence of the parasite reached 34.5% in this Vietnamese cohort. No significant association was found between parasite infection and gender, age, symptomatic status, contact with animals or source of drinking water. Out of the 107 positive patients, nearly half presented mixed infections. Therefore, some of the corresponding samples were reanalyzed by end-point PCR, followed by PCR products cloning and sequencing. Of the 88 total subtyped isolates, ST3 was predominant, followed by ST10, ST14, ST7, ST1, ST4, ST6 and ST8. Our study was, thus, the first to report ST8, ST10 and ST14 in the Southeast Asian population. The predominance of ST3 within this Vietnamese cohort, coupled with its low intra-ST genetic variability, reflected a large inter-human transmission, while ST1 transmission was suggested to be not only anthroponotic, but also likely correlated to animal or environmental sources. Strikingly, isolates considered of animal origin (ST6-ST8, ST10 and ST14) accounted for more than 50% of the subtyped isolates. These findings improved our knowledge of the epidemiology and circulation of Blastocystis sp. in Southeast Asia, and in particular, in Vietnam, and highlighted both a major burden of the parasite in this country and a high risk of zoonotic transmission, mainly from poultry and livestock.

Prevalence, Subtype Distribution and Zoonotic Significance of Blastocystis sp. Isolates from Poultry, Cattle and Pets in Northern Egypt.

Blastocystis sp. is a widespread enteric protozoan that frequently infects human and animal groups. Despite its burden and zoonotic potential worldwide, epidemiological investigations remain limited in animal groups that come in contact with humans. Therefore, the largest survey ever conducted in North Africa was performed in Egypt with the aim to investigate the prevalence and subtype (ST) distribution of Blastocystis sp. in animals. For this purpose, a total of 889 fecal specimens were collected from chickens (217), cattle (373), dogs (144) and cats (155) from six governorates of northern Egypt. These specimens were then screened for the presence of Blastocystis sp. using a quantitative real-time PCR, followed by subtyping the isolates. The overall prevalence of Blastocystis sp. reached 9.2% (82/889), with the highest infection rates reported in chickens (17.0%) and domestic cattle (11.0%), highlighting an active circulation of the parasite in both animal groups. In contrast, the low prevalence in cats (2.6%) and the absence of the parasite in dogs suggested that pets are not natural hosts of Blastocystis sp. ST10 and ST14 were largely predominant in cattle, confirming that both STs represented cattle-adapted STs. The report of one ST3 and one ST4 isolate in this animal group could be explained by an accidental zoonosis from humans to animals. All but one of the subtyped isolates in poultry belonged to ST7, which was considered as an avian ST. The presence of a remaining isolate of ST14 likely reflected a transient infection from contact between birds and cattle feces. The same environmental contamination was also likely the source of the ST14 infection in three of the four positive cats, with the remaining animals infected by ST3 as the result of human-to-animal transmission. These occurrences and subtyping data, combined with those previously collected in the Egyptian population, implies that poultry could play a significant role as reservoir for zoonotic transmission, which would not be the case for cattle and pets.

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.

Detection, Molecular Identification and Transmission of the Intestinal Protozoa Blastocystis sp. in Guinea from a Large-Scale Epidemiological Study Conducted in the Conakry Area.

Blastocystis sp. is a single-celled parasite estimated to colonize the digestive tract of 1 to 2 billion people worldwide. Although it represents the most frequent intestinal protozoa in human stools, it remains still under-investigated in countries with a high risk of infection due to poor sanitary and hygiene conditions, such as in Africa. Therefore, the present study was carried out to determine the prevalence and subtype (ST) distribution of Blastocystis sp. in the Guinean population. For this purpose, fecal samples were collected from 500 individuals presenting or not digestive disorders in two hospitals of Conakry. Search for the parasite in stools was performed by real-time PCR targeting the small subunit rDNA gene followed by sequencing of the PCR products for subtyping of the isolates. A total of 390 participants (78.0%) was positive for Blastocystis sp. Five STs were identified in the Guinean cohort (ST1, ST2, ST3, ST4 and ST14) with varying frequency, ST3 being predominant. Among them, ST4 was found in only two patients confirming its global rarity in Africa whereas infections by ST14 were likely the result of zoonotic transmission from bovid. No significant association was detected between Blastocystis sp. colonization or ST distribution and the symptomatic status of Guinean subjects or the presence of digestive symptoms. In contrast, drilling water consumption represented a significant risk factor for infection by Blastocystis sp. Predominance of ST3 coupled with its low intra-ST diversity strongly suggested large-scale human-to-human transmission of this ST within this cohort. In parallel, the highest intra-ST diversity of ST1 and ST2 was likely correlated with various potential sources of infection in addition to anthroponotic transmission. These findings highlighted the active circulation of the parasite in Guinea as reported in some low-income African countries and the necessity to implement prevention and control measures in order to limit the circulation of this parasite in this endemic geographical area.

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.

Revisiting the Pneumocystis host specificity paradigm and transmission ecology in wild Southeast Asian rodents.

Pneumocystis fungi are opportunistic parasites of mammalian lungs whose evolution, ecology and host specificity in natural host populations remain poorly understood and controversial. Using an extensive collection of 731 lung samples from 27 rodent species sampled in five Southeast Asian countries, and nested PCR amplification of mitochondrial and nuclear genes, we investigated the host specificity and genetic structure of Pneumocystis lineages infecting wild rodents. We also identified the rodent species playing a central role in the transmission of these parasites using network analysis and centrality measurement and we characterized the environmental conditions allowing Pneumocystis infection in Southeast Asia using generalized linear mixed models. Building upon an unprecedented Pneumocystis sampling from numerous rodent species belonging to closely related genera, our findings provide compelling evidence that the host specificity of Pneumocystis lineages infecting rodents is not restricted to a single host species or genus as often presented in the literature but it encompasses much higher taxonomic levels and more distantly related rodent host species. The phylogenetic species status at both mitochondrial and nuclear genetic markers of at least three new Pneumocystis lineages, highly divergent from Pneumocystis species currently described, is also suggested by our data. Our models show that the probability of Pneumocystis infection in rodent hosts is positively correlated to environmental variables reflecting habitat fragmentation and landscape patchiness. Synanthropic and habitat-generalist rodents belonging to the Rattus, Sundamys and Bandicota genera played a role of bridge host species for Pneumocystis spreading in these heterogeneous habitats, where they can reach high population densities. These are critical findings improving our understanding of the ecology of these enigmatic parasites and the role played by cospeciation and host switches in their evolution. Our results also confirmed the role of land-use change and habitat fragmentation in parasite amplification and spillover in rodents.

Changes in the Human Gut Microbiota Associated With Colonization by Blastocystis sp. and Entamoeba spp. in Non-Industrialized Populations.

Human gut microbial communities are mainly composed of bacteria, but also include fungi, viruses, archaea, and protozoa, whose role in the gut ecosystem has only recently begun to be recognized. For example, humans colonized by Blastocystis (a gut protozoan with controversial pathogenicity) host a more diverse bacterial microbiota than individuals not carrying it, suggesting that its presence may be beneficial for the host. In parallel, the presence of non-pathogenic Entamoeba spp. has been associated with an increased diversity and compositional shifts in the bacterial microbiota of healthy rural individuals in Cameroon. However, Entamoeba and Blastocystis, the two most prevalent human gut protozoa, have never been studied in the same individuals, preventing the study of their interaction. As Blastocystis is one of the few gut protozoa commonly found in industrialized populations, which are otherwise mostly devoid of gut eukaryotes, we need to focus on rural "traditional" populations, who harbor a higher diversity of gut eukaryotes (whether pathogenic or commensal) in order to study protozoa interactions in the gut ecosystem. To this end, we profiled the gut bacterial microbiota of 134 healthy Cameroonian adults using 16S rRNA gene amplicon sequencing data. Entamoeba and Blastocystis presence and co-occurrence pattern in the same individuals were determined using metagenomic shotgun data. We found that, when taking into account both protozoa jointly, Blastocystis was associated with both a higher richness and a higher evenness of the gut bacterial microbiota, while Entamoeba was associated only with a higher richness. We demonstrated a cumulative influence of these protozoa on bacterial microbiome diversity. Furthermore, while the abundance of several common taxa (for example, Ruminococcaceae, Coprococcus and Butyrivibrio) varied according to Blastocystis colonization, only a single Bacteroides amplicon sequence variant was found to be differentially abundant between Entamoeba-negative and Entamoeba-positive samples. Given the specific signature of each protozoan on the gut microbiota and the seemingly stronger association for Blastocystis, our results suggest that Blastocystis and Entamoeba interact with gut bacteria each in its own way, but experimental studies are needed to explore the precise mechanisms of these interactions.

Genomic insights into the host specific adaptation of the Pneumocystis genus.

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

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.

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.

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species.

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.IMPORTANCEPneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

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.