Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Genetic characterization of Cryptosporidium spp. in the North African hedgehog (Atelerix algirus) in the Canary Islands, Spain.

The North African hedgehog (Atelerix algirus) is an introduced species from Northwest Africa and is currently distributed in the Canary Islands. This species of hedgehog has been studied as a reservoir of enteropathogens, including Cryptosporidium spp. However, there are no data at species level. Therefore, the aim of the present study was to identify the Cryptosporidium species present in a population of hedgehogs (n = 36) in the Canary Islands. Molecular screening was performed using conventional polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene of Cryptosporidium spp. Seven of the 36 fecal samples (19.45%) were positive and confirmed by nested PCR targeting the 18S rRNA gene and Sanger sequencing. Cryptosporidium parvum and Cryptosporidium muris were identified in 11.1% (4/36) and 5.6% (2/36) of the samples, respectively, while one sample could only be identified at the genus level. The zoonotic subtypes IIdA15G1 (n = 1), IIdA16G1b (n = 1), and IIdA22G1 (n = 1) of C. parvum were identified by nested PCR followed by analysis of the 60 kDa glycoprotein (gp60) gene sequence. This study is the first genetic characterization of Cryptosporidium spp. in A. algirus, identifying zoonotic species and subtypes of the parasite.

Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Complete sequencing of the Cryptosporidium suis gp60 gene reveals a novel type of tandem repeats-Implications for surveillance.

Cryptosporidiosis is an infectious enteric disease caused by species (some of them zoonotic) of the genus Cryptosporidium that in many countries are under surveillance. Typing assays critical to the surveillance of cryptosporidiosis typically involve characterization of Cryptosporidium glycoprotein 60 genes (gp60). Here, we characterized the gp60 of Cryptosporidium suis from two samples-a human and a porcine faecal sample-based on which a preliminary typing scheme was developed. A conspicuous feature of the C. suis gp60 was a novel type of tandem repeats located in the 5' end of the gene and that took up 777/1635 bp (48%) of the gene. The C. suis gp60 lacked the classical poly-serine repeats (TCA/TCG/TCT), which is usually subject to major genetic variation, and the length of the tandem repeat made a typing assay incorporating this region based on Sanger sequencing practically unfeasible. We therefore designed a typing assay based on the post-repeat region only and applied it to C. suis-positive samples from suid hosts from Norway, Denmark, and Spain. We were able to distinguish three different subtypes; XXVa-1, XXVa-2, and XXVa-3. Subtype XXVa-1 had a wider geographic distribution than the other subtypes and was also observed in the human sample. We think that the present data will inform future strategies to develop a C. suis typing assay that could be even more informative by including a greater part of the gene, including the tandem repeat region, e.g., by the use of long-read next-generation sequencing.

Diversity of gastrointestinal parasites and molecular characterization of Giardia duodenalis in free-living and captive howler monkeys (Alouatta guariba clamitans) in southern Brazil.

Non-human primates (NHPs) are the group that most share infectious agents with humans due to their close taxonomic relationship. The southern brown howler monkeys (Alouatta guariba clamitans) are endemic primates from Brazil and Argentina's Atlantic Forest. This study aimed to investigate the presence of intestinal parasites in free-living (FL) and captive (CA) southern brown howler monkeys. Thirty-nine stool samples were collected in two areas in southern Brazil, 15 FL and 24 CA. Stool sediments obtained by centrifugal sedimentation technique were used for microscopic analysis and direct immunofluorescence assay and evaluated by molecular analysis through amplification and sequencing of TPI fragments. Intestinal parasites Giardia duodenalis, Cryptosporidium spp., and Trypanoxyuris minutus were detected at coproparasitological analysis. This is the first report of the presence of Cryptosporidium spp. in free-living howlers. The molecular characterization of G. duodenalis isolates indicated assemblage B for the first time found in free-living A. guariba clamitans. The high prevalence of G. duodenalis transmission in CA howler monkeys can be explained by direct contact with humans and frequent soil contact. The presence of a potentially zoonotic assemblage in these animals indicates that the process of fragmentation and cohabitation with humans and livestock affects the wildlife, thus indicating a need for eco-health measures.

Molecular detection of Cryptosporidium in Alpine musk deer (Moschus chrysogaster) in Gansu Province, Northwest China.

Cryptosporidium spp. are protozoa commonly found in domestic and wild animals. Limited information is available on Cryptosporidium in deer worldwide. In this study, 201 fecal samples were collected from Alpine musk deer on three farms in Gansu Province, China. Detection and subtyping of Cryptosporidium were performed by PCR and sequence analysis of the SSU rRNA and gp60 genes. The prevalence of Cryptosporidium infection in Alpine musk deer was 3.9% (8/201), with infection rates of 1.0% (1/100), 2.8% (1/36), and 9.2% (6/65) in three different farms. All positive samples for Cryptosporidium were from adult deer. Two Cryptosporidium species were identified, including C. parvum (n = 2) and C. xiaoi (n = 6). The C. parvum isolates were subtyped as IIdA15G1, while the C. xiaoi isolates were subtyped as XXIIIa (n = 2) and XXIIIg (n = 4). The IIdA15G1 subtype of C. parvum was found for the first time in deer. These results provide important insights into the identity and human infectious potential of Cryptosporidium in farmed Alpine musk deer.

Cryptosporidium occultus in disguise.

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Cryptosporidium spp. in large-scale sheep farms in China: prevalence and genetic diversity.

Cryptosporidium spp. are significant zoonotic intestinal parasites that induce diarrhea and even death across most vertebrates, including humans. Previous studies showed that sheep are important hosts for Cryptosporidium and that its distribution in sheep is influenced by geography, feeding patterns, age, and season. Environmental factors also influence the transmission of Cryptosporidium. Molecular studies of Cryptosporidium in sheep have been conducted in only a few regions of China, and studies into the effect of sheep-housing environments on Cryptosporidium transmission are even rarer. To detect the prevalence of Cryptosporidium in large-scale sheep-housing farms, a total of 1241 fecal samples were collected from sheep, 727 environmental samples were taken from sheep housing, and 30 water samples were collected in six regions of China. To ascertain the existence of the parasite and identify the species of Cryptosporidium spp., we conducted nested PCR amplification of DNA extracted from all samples using the small-subunit (SSU) rRNA gene as a target. For a more in-depth analysis of Cryptosporidium spp. subtypes, C. xiaoi-and C. ubiquitum-positive samples underwent separate nested PCR amplification targeting the 60 kDa glycoprotein (gp60) gene. The amplification of the Cryptosporidium spp. SSU rRNA gene locus from the whole genomic DNA of all samples yielded a positive rate of 1.2% (20/1241) in fecal samples, 0.1% (1/727) in environmental samples, and no positive samples were found in water samples. The prevalence of Cryptosporidium spp. infection in large-scale housed sheep was 1.7%, which was higher than that in free-ranging sheep (0.0%). The highest prevalence of infection was found in weaning lambs (6.8%). Among the different seasons, the peaks were found in the fall and winter. The most prevalent species were C. xiaoi and C. ubiquitum, with the former accounting for the majority of infections. The distribution of C. xiaoi subtypes was diverse, with XXIIIc (n = 1), XXIIId (n = 2), XXIIIe (n = 2), and XXIIIl (n = 4) identified. In contrast, only one subtype, XIIa (n = 9), was found in C. ubiquitum. In this study, C. xiaoi and C. ubiquitum were found to be the predominant species, and Cryptosporidium was found to be present in the environment. These findings provide an important foundation for the comprehensive prevention and management of Cryptosporidium in intensively reared sheep. Furthermore, by elucidating the prevalence of Cryptosporidium in sheep and its potential role in environmental transmission, this study deepens our understanding of the intricate interactions between animal health, environmental contamination, and public health dynamics.

Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi.

Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi, which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis.

Enterocytozoon bieneusi and Cryptosporidium bat genotype XXI and bat genotype XXII in fruit bats (Rousettus leschenaultii) inhabiting a tropical park in Hainan Province, China.

Bats stand as one of the most diverse groups in the animal kingdom and are key players in the global transmission of emerging pathogens. However, their role in transmitting Enterocytozoon bieneusi and Cryptosporidium spp. remains unclear. This study aimed to evaluate the occurrence and genetic diversity of the two pathogens in fruit bats (Rousettus leschenaultii) in Hainan, China. Ten fresh fecal specimens of fruit bats were collected from Wanlvyuan Gardens, Haikou, China. The fecal samples were tested for E. bieneusi and Cryptosporidium spp. using Polymerase Chain Reaction (PCR) analysis and sequencing the internal transcribed spacer (ITS) region and partial small subunit of ribosomal RNA (SSU rRNA) gene, respectively. Genetic heterogeneity across Cryptosporidium spp. isolates was assessed by sequencing 4 microsatellite/minisatellite loci (MS1, MS2, MS3, and MS16). The findings showed that out of the ten specimens analyzed, 2 (20 %) and seven (70.0 %) were tested positive for E. bieneusi and Cryptosporidium spp., respectively. DNA sequence analysis revealed the presence of two novel Cryptosporidium genotypes with 94.4 to 98.6 % sequence similarity to C. andersoni, named as Cryptosporidium bat-genotype-XXI and bat-genotype-XXII. Three novel sequences of MS1, MS2 and MS16 loci identified here had 95.4 to 96.9 % similarity to the known sequences, which were deposited in the GenBank. Two genotypes of E. bieneusi were identified, including a novel genotype named HNB-I and a zoonotic genotype PigEbITS7. The discovery of these novel sequences provides meaningful data for epidemiological studies of the both pathogens. Meanwhile our results are also presented that the fruit bats infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Epidemiological investigation of Cryptosporidium in children with diarrhea in middle Inner Mongolia, China.

Cryptosporidia (Cryptosporidium) is a protozoan that is widely parasitic in the intestinal cells of humans and animals, and it is also an important zoonotic parasite. However, there is no epidemiological investigation on Cryptosporidium spp. infection in infants with diarrhea of Inner Mongolia, the largest livestock region in China. To investigate the prevalence of Cryptosporidium, 2435 fresh fecal samples were collected from children with diarrhea in Inner Mongolia Maternal and Child Health Care Hospital. Molecular characterization of Cryptosporidium was carried out based on its 18S rRNA and gp60 gene sequences. The overall prevalence was 12.85% (313/2435), and in Hohhot (12.15%), it was lower than that in the surrounding city (14.87%) (P < 0.05). Moreover, Cryptosporidium was detected in different seasons and sexes. Concerning the age of children with diarrhea, the prevalence of those age groups between 0 and 1 was obviously lower than others, and there were significant differences in the prevalence at different ages (P < 0.001). Analysis of the 18S rRNA gene sequence revealed that all the positive samples were Cryptosporidium parvum, and there were 5 subtypes (IIdA23G3, IIdA24G3, IIdA24G4, IIdA25G3, and IIdA25G4). To the best of our knowledge, the above subtypes have not been reported. Our results provide a relevant basis for control and education on food safety and foodborne illness prevention.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in pet cats in Henan Province, central China.

Cryptosporidium spp. and G. duodenalis often infect humans, cats, and other mammals, causing diarrhea and being responsible for numerous outbreaks of waterborne and foodborne infections worldwide. The rapid increase in the number of pet cats poses a substantial public health risk. However, there were few reports about the infection of Cryptosporidium spp. and G. duodenalis infections in pet cats in Henan Province, central China. Thus, to understand the prevalence and genetic distribution of Cryptosporidium spp. and G. duodenalis in pet cats, and to evaluate the zoonotic potential, possible transmission routes and public health implications of isolates, fecal samples (n = 898) were randomly collected from pet cats in 11 cities in Henan Province, central China. Nested PCR based on the SSU rRNA gene and bg gene was used to the prevalence of Cryptosporidium spp. and G. duodenalis, respectively. The prevalence was 0.8 % (7/898) and 2.0 % (18/898) for Cryptosporidium spp. and G. duodenalis respectively. Additionally, the Cryptosporidium spp. positive isolates were identified as C. parvum subtype IIdA19G1 by gp60 gene. In the present study, the IIdA19G1 subtype was discovered in pet cats for the first time in China, enriching the information on the host type and geographical distribution of Cryptosporidium spp. in China. For G. duodenalis, a total of 18 G. duodenalis positive samples were identified, belonging to four assemblages: a zoonotic assemblage A1 (4/898), three host-specific assemblages C (8/898), D (5/898), and F (1/898). Interestingly, we found that pet cats infected with Cryptosporidium spp. and G. duodenalis are more likely to experience emaciation symptoms compared to the negative group. More importantly, the prevalence of Cryptosporidium spp. and G. duodenalis detected in the present study were low, but the subtype IIdA19G1 of Cryptosporidium spp. and the assemblages A1, C, D, and F of G. duodenalis have the potential for zoonotic transmission. Thus, we should focus on preventing and controlling the risk of cross-species transmission that may occur in pet cats in Henan Province.

Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Cryptosporidium diagnosis in different groups of children and characterization of parasite species.

BACKGROUND: Microscopy and enzyme-linked immunosorbent assay (ELISA) are routinely used for Cryptosporidium diagnosis, without differentiating the parasite species. METHODS: Children's feces were analyzed by modified Ziehl-Neelsen (mZN) and ELISA for Cryptosporidium diagnosis and by polymerase chain reaction-restriction fragment length polymorphism for species identification. RESULTS: Cryptosporidium frequency was 2.6%. The sensitivity and specificity of ELISA were 85.7% and 99.7%, respectively, with excellent concordance with mZN (kappa=0.854). Parasite species were characterized as Cryptosporidium hominis (78.3%), Cryptosporidium felis (17.4%), and Cryptosporidium parvum (4.3%). CONCLUSIONS: Coproantigen ELISA is as efficient as mZN for Cryptosporidium diagnosis. Cryptosporidium genotyping suggests anthroponotic and zoonotic transmission to children.

Prevalence of intestinal protozoan parasites among school children in africa: A systematic review and meta-analysis.

INTRODUCTION: Parasitic infections, especially intestinal protozoan parasites (IPPs) remain a significant public health issue in Africa, where many conditions favour the transmission and children are the primary victims. This systematic review and meta-analysis was carried out with the objective of assessing the prevalence of IPPs among school children in Africa. METHODS: Relevant studies published between January 2000 and December 2020 were identified by systematic online search on PubMed, Web of Science, Embase and Scopus databases without language restriction. Pooled prevalence was estimated using a random-effects model. Heterogeneity of studies were assessed using Cochrane Q test and I2 test, while publication bias was evaluated using Egger's test. RESULTS: Of the 1,645 articles identified through our searches, 46 cross-sectional studies matched our inclusion criteria, reported data from 29,968 school children of Africa. The pooled prevalence of intestinal protozoan parasites amongst African school children was 25.8% (95% CI: 21.2%-30.3%) with E. histolytica/ dispar (13.3%; 95% CI: 10.9%-15.9%) and Giardia spp. (12%; 95% CI: 9.8%-14.3%) were the most predominant pathogenic parasites amongst the study participants. While E. coli was the most common non-pathogenic protozoa (17.1%; 95% CI: 10.9%-23.2%). CONCLUSIONS: This study revealed a relatively high prevalence of IPPs in school children, especially in northern and western Africa. Thus, poverty reduction, improvement of sanitation and hygiene and attention to preventive control measures will be the key to reducing protozoan parasite transmission.

Cryptosporidium felis differs from other Cryptosporidium spp. in codon usage.

Cryptosporidium spp. are important enteric pathogens in a wide range of vertebrates including humans. Previous comparative analysis revealed conservation in genome composition, gene content, and gene organization among Cryptosporidium spp., with a progressive reductive evolution in metabolic pathways and invasion-related proteins. In this study, we sequenced the genome of zoonotic pathogen Cryptosporidium felis and conducted a comparative genomic analysis. While most intestinal Cryptosporidium species have similar genomic characteristics and almost complete genome synteny, fewer protein-coding genes and some sequence inversions and translocations were found in the C. felis genome. The C. felis genome exhibits much higher GC content (39.6 %) than other Cryptosporidium species (24.3-32.9 %), especially at the third codon position (GC3) of protein-coding genes. Thus, C. felis has a different codon usage, which increases the use of less energy costly amino acids (Gly and Ala) encoded by GC-rich codons. While the tRNA usage is conserved among Cryptosporidium species, consistent with its higher GC content, C. felis uses a unique tRNA for GTG for valine instead of GTA in other Cryptosporidium species. Both mutational pressures and natural selection are associated with the evolution of the codon usage in Cryptosporidium spp., while natural selection seems to drive the codon usage in C. felis. Other unique features of the C. felis genome include the loss of the entire traditional and alternative electron transport systems and several invasion-related proteins. Thus, the preference for the use of some less energy costly amino acids in C. felis may lead to a more harmonious parasite-host interaction, and the strengthened host-adaptation is reflected by the further reductive evolution of metabolism and host invasion-related proteins.

Tandem mass tag (TMT)-based proteomic analysis of Cryptosporidium andersoni oocysts before and after excystation.

BACKGROUND: Cryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process. METHODS: Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation. RESULTS: Proteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data. CONCLUSIONS: This study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium.

Prevalence and Molecular Characterization of the Zoonotic Enteric Protozoans Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis from Pallas's Squirrels (Callosciurus erythraeus) in Kanagawa Prefecture, Japan.

Pallas's squirrel (Callosciurus erythraeus) was introduced in Japan in the 1930s and has since established itself in several areas across the country. Although wild Sciuridae populations have been demonstrated to be potential reservoirs for zoonotic enteric protozoa, epidemiological studies of such pathogens in Japan are scarce. Here, we examined 423 fecal samples from Pallas's squirrels captured in Kanagawa Prefecture, Japan, using PCR and DNA sequencing to determine the occurrence of Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis. The overall prevalence of Cryptosporidium spp., E. bieneusi, and Blastocystis was 4.3% (18/423 samples), 13.0% (55/423 samples), and 44.0% (186/423 samples), respectively. The prevalence of Blastocystis and E. bieneusi was significantly higher in spring (60.1% and 17.4%, respectively) than in winter (27.6% and 8.6%, respectively [P < 0.01]). Sequence analysis of Cryptosporidium spp., targeting the partial small subunit ribosomal RNA gene (SSU rDNA), showed 100% identity (541/541 bp) to Cryptosporidium ubiquitum, and analysis of the gp60 gene showed 99.76% (833/835 bp) identity to C. ubiquitum subtype XIIh. The sequences of the ribosomal internal transcribed spacer region of E. bieneusi and the partial SSU rDNA of Blastocystis were identified as E. bieneusi genotype SCC-2 and Blastocystis subtype 4, respectively. This study confirmed the presence of C. ubiquitum, E. bieneusi, and Blastocystis in Pallas's squirrels in Kanagawa Prefecture. Because Pallas's squirrels inhabit urban areas, living close to humans, the species may serve as a potential source of infection in human populations. IMPORTANCE Pallas's squirrel is designated a "regulated organism" under the Invasive Alien Species Act in Japan, and municipal authorities are introducing control measures to reduce its populations. It has been suggested that wild mammals may play a role in contaminating the environment with zoonotic pathogens. The present study detected the enteric pathogens Cryptosporidium ubiquitum, Enterocytozoon bieneusi, and Blastocystis in the feces of Pallas's squirrels inhabiting Kanagawa Prefecture, Japan. These pathogens persist in the environment and contaminate soils and water, which may potentially infect humans. Because Pallas's squirrels in Kanagawa Prefecture are found in urban areas, where they are in close contact with human populations, continued monitoring of zoonotic diseases among squirrel populations will be important for evaluating the significance of wildlife in pathogen transmission.

Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea.

Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1-60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11-20 days (26.1%, 95% CI 17.1-35.1), whereas the lowest rate was observed in calves aged 21-30 days (7.7%, 95% CI 0.0-16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.