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.

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.

Detection and identification of Giardia species using real-time PCR and sequencing.

We report a specific region of Giardia spp. 18S ribosomal RNA (18S rDNA) that serves as an ideal target for quantitative PCR (qPCR) detection and sequencing to identify Giardia species, including the clinically-relevant G. duodenalis, in clinical and environmental samples. The presence of multiple copies of the 18S rDNA gene and variations in the selected 18S genomic region enabled the development of a rapid, sensitive qPCR screening method for the detection of Giardia spp. The analytical sensitivity of the Giardia qPCR assay was determined to be a cyst equivalent of 0.4 G. duodenalis cysts per PCR reaction. Amplicon sequencing of the PCR product confirmed Giardia spp. detection and among the 35 sequences obtained, 31, 3 and 1 isolates were classified as belonging to G. duodenalis, G. microti and G. muris, respectively. The TaqMan assay reported here may be useful for the detection of low levels of Giardia in clinical and environmental samples, and further enables the effective use of direct sequencing of the PCR product for Giardia confirmation and to identify major species of Giardia, including G. duodenalis.

Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go.

Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy.

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.

Cryptosporidium and Giardia in feral water buffalo (Bubalus bubalis) in the South East Arnhem Land Indigenous Protected Area, Australia.

Global investigations have implicated water buffalo (Bubalus bubalis) as a potential source of zoonotic Cryptosporidium and Giardia parasites which may pose a threat to human health. In Australia, buffalo are a feral pest that have colonised the floodplains, wetlands and woodlands of Indigenous owned and managed Arnhem Land, in tropical monsoonal Northern Australia. Indigenous people from the remote community Ngukurr have raised concerns about the potential threat to their health from shared use of surface waters inhabited by buffalo, in the South East Arnhem Land Indigenous Protected Area (SEAL IPA), Northern Australia. Surface waters are valued by local Indigenous people for spiritual and customary reasons, bush foods, medicines and drinking water. Here, we used molecular methods to characterise Cryptosporidium spp. and Giardia duodenalis assemblages from feral water buffalo living in the SEAL IPA to determine potential zoonotic risks to health of Indigenous people through co-use of surface water billabongs. Buffalo faecal DNA was screened for Cryptosporidium and Giardia using the 18S rRNA gene. Giardia were also screened using Glutamate hydrogenase (gdh) and ßeta-giardin (ß-giardin) genes. DNA sequencing identified C. ryanae in 9.9% (31/313) and G. duodenalis assemblage E 1.9% (6/313) in buffalo. Cryptosporidium ryanae is not considered zoonotic and G. duodenalis assemblage E is a livestock assemblage that has been reported in humans. Carriage of G. duodenalis assemblage E in buffalo may present a disease risk for Indigenous people utilising billabongs, according to customary practice.

Microtubule organelles in Giardia.

Giardia lamblia is a widespread parasitic protist with a complex MT cytoskeleton that is critical for motility, attachment, mitosis and cell division, and transitions between its two life cycle stages-the infectious cyst and flagellated trophozoite. Giardia trophozoites have both highly dynamic and highly stable MT organelles, including the ventral disc, eight flagella, the median body and the funis. The ventral disc, an elaborate MT organelle, is essential for the parasite's attachment to the intestinal villi to avoid peristalsis. Giardia's four flagellar pairs enable swimming motility and may also promote attachment. They are maintained at different equilibrium lengths and are distinguished by their long cytoplasmic regions and novel extra-axonemal structures. The functions of the median body and funis, MT organelles unique to Giardia, remain less understood. In addition to conserved MT-associated proteins, the genome is enriched in ankyrins, NEKs, and novel hypothetical proteins that also associate with the MT cytoskeleton. High-resolution ultrastructural imaging and a current inventory of more than 300 proteins associated with Giardia's MT cytoskeleton lay the groundwork for future mechanistic analyses of parasite attachment to the host, motility, cell division, and encystation/excystation. Giardia's unique MT organelles exemplify the capacity of MT polymers to generate intricate structures that are diverse in both form and function. Thus, beyond its relevance to pathogenesis, the study of Giardia's MT cytoskeleton informs basic cytoskeletal biology and cellular evolution. With the availability of new molecular genetic tools to disrupt gene function, we anticipate a new era of cytoskeletal discovery in Giardia.

Surface waters as a potential source of Giardia and Cryptosporidium in Serbia.

Giardiasis and cryptosporidiosis are recognized by the WHO as important emerging diseases of the 21st century. Symptoms are similar and include diarrhoea and vomiting, which may be severe, even life-threatening, for the immunocompromised and children under five years of age. Between 2013 and 2017, the Institute for Public Health in Serbia recorded 10 waterborne epidemics that manifested as gastrointestinal disease. Routine testing for enteropathogenic bacteria and viruses did not identify the aetiological agents of these outbreaks. As water is not examined for the presence of protozoa in Serbia, we performed a pilot study to analyse samples from four major rivers and their tributaries using a newly implemented methodology for detection of Giardia and Cryptosporidium, based on the ISO 15553:2006 standard. Using immunofluorescence microscopy, Giardia was detected in 10 out of the 31 samples, Cryptosporidium in five, while two samples were positive for both. Presence of G. duodenalis gDNA was confirmed by amplification of the ß-giardin gene in eight samples, of which one and two, respectively, were identified by RFLP as potentially zoonotic assemblages A and B. The results suggest that surface water in Serbia may be a potential source of infection and call for more in-depth studies using sophisticated molecular tools.

Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go

Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020.

Prevalence and molecular characterization of Cryptosporidium and Giardia in pre-weaned native calves in the Republic of Korea.

Cryptosporidium spp. and Giardia duodenalis are protozoan parasites that cause diarrhea in humans and animals. Molecular data on Cryptosporidium spp. and G. duodenalis in calves in the Republic of Korea (ROK) is limited; thus, we investigated the prevalence of Cryptosporidium and Giardia in pre-weaned calves, analyzed the association between these parasites and diarrhea, and identified their zoonotic potential for human infection. Fecal samples were collected from 315 pre-weaned calves aged 1-60 days from 10 different regions in the ROK and screened for Cryptosporidium spp. and G. duodenalis using PCR. Overall prevalence of Cryptosporidium spp. and G. duodenalis was 4.4% (n = 14) and 12.7% (n = 40), respectively. Co-infection was not detected. All Cryptosporidium-positive samples were identified as C. parvum after sequence analysis of a small subunit rRNA fragment and further subtyped into zoonotic IIaA15G2R1 (n = 13) and IIaA18G3R1 (n = 1) by DNA sequencing of the 60-kDa glycoprotein gene. To our knowledge, this is the first report of C. parvum IIaA15G2R1 subtype in calves in the ROK. Based on ß-giardin (bg) gene, G. duodenalis-positive samples belonged to assemblages E (n = 36) and A (n = 4), with the latter belonging to subtype A1, the zoonotic genotype. Six subtypes of assemblage E were identified at the bg locus: E1 (n = 6), E2 (n = 3), E3 (n = 13), E5 (n = 1), E8 (n = 1), and E11 (n = 1). The occurrence of C. parvum and G. duodenalis was not associated with diarrhea in pre-weaned Korean native calves. The present results suggest that the prevalence of C. parvum is not related to calf age; in contrast, the prevalence of G. duodenalis was significantly higher in 41-50-day-old calves (odds ratio = 9.90, 95% confidence interval 2.37-41.34; P = 0.001) than in 1-10-day-old calves. Therefore, calves are a potential source of zoonotic transmission, which may have significant public health implications.

Prevalence and genetic characterization of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut, Canada.

There are few epidemiologic studies on the role of dogs in zoonotic parasitic transmission in the Circumpolar North. The objectives of this study were to: (a) estimate the faecal prevalence of Giardia spp. and Cryptosporidium spp. in dogs; (b) investigate potential associations between the type of dog population and the faecal presence of Giardia spp. and Cryptosporidium spp.; and (c) describe the molecular characteristics of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut. We conducted two cross-sectional studies in July and September 2016. In July, the team collected daily faecal samples for 3 days from each of 20 sled dogs. In September, the team collected three faecal samples from each of 59 sled dogs, 111 samples from shelter dogs and 104 from community dogs. We analysed faecal samples for the presence of Giardia spp. and Cryptosporidium spp. using rapid immunoassay and flotation techniques. Polymerase chain reaction (PCR) and sequencing of target genes were performed on positive faecal samples. Overall, the faecal prevalence of at least one of the target parasites, when one faecal sample was chosen at random for all dogs, was 8.16% (CI: 5.52-11.92), and for Giardia spp. and Cryptosporidium spp., prevalence was 4.42% (CI: 2.58-7.49) and 6.12% (CI: 3.88-9.53), respectively. The odds of faecal Giardia spp. in sled dogs were significantly higher than those in shelter and community dogs (OR 10.19 [CI: 1.16-89.35]). Sequence analysis revealed that 6 faecal samples were Giardia intestinalis, zoonotic assemblage B (n = 2) and species-specific assemblages D (n = 3) and E (n = 1), and five faecal samples were Cryptosporidium canis. Giardia intestinalis is zoonotic; however, Cryptosporidium canis is rare in humans and, when present, usually occurs in immunosuppressed individuals. Dogs may be a potential source of zoonotic Giardia intestinalis assemblage B infections in residents in Iqaluit, Nunavut, Canada; however, the direction of transmission is unclear.

Detection of Cryptosporidium and Giardia in the slaughterhouse, sewage and river waters of the Qinghai Tibetan plateau area (QTPA), China.

The occurrence of Cryptosporidium and Giardia species in slaughter, sewage and river waters of the Qinghai Tibetan Plateau Area (QTPA), China, was investigated. A total of 456 samples were collected from different locations in the QTPA to study the contamination rates of Cryptosporidium spp. and Giardia via PCR and subsequent sequence analysis. Ten samples were Cryptosporidium positive, and 97 were Giardia positive, as confirmed by PCR amplification of the SSU rRNA gene. The percentages of positive Cryptosporidium and Giardia detection were 2.2% (10/456) and 21.3% (97/456), respectively. Cryptosporidium was detected in only sewage and river waters. Six species of Cryptosporidium were identified: Cryptosporidium hominis (n = 5), C. andersoni (n = 1), C. environmental (n = 1), C. struthionis (n = 1), C. canis (n = 1), and C. parvum (n = 1). G. duodenalis assemblage A was identified in almost all positive samples (n = 96), and one sample harboured G. duodenalis assemblage E. The results suggest that Cryptosporidium and Giardia species circulate through the aqueous environment and different hosts. Therefore, we strongly recommend that the local government and health authorities in China undertake control measures to reduce the contamination of water sources by these protozoa to protect the health of humans and animals.

Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples.

Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.

Molecular genotyping, diversity studies and high-resolution molecular markers unveiled by microsatellites in Giardia duodenalis.

BACKGROUND: Giardia duodenalis (synonyms G. lamblia and G. intestinalis) is an enteric protozoan parasite of a wide range of mammalian hosts, including humans and various domestic and wild animals. There is considerable genetic variability in G. duodenalis and isolates of this parasite have been divided into eight genetic assemblages. Microsatellites markers can be used to discriminate isolates with a high level of sensitivity. This study was conducted to identify and characterize genomic microsatellites (simple sequence repeats-SSRs), sequences of one- to six-nucleotide motifs repeated in tandem, present in the available genomes of G. duodenalis and to develop new markers that can serve as a tool for detection and for characterizing the genetic diversity of this parasite. METHODOLOGY/ PRINCIPAL FINDINGS: For each genetic assemblage, polymorphism levels for the microsatellite markers were evaluated. After performing the analysis using the MISA and SciRoKo software, 1,853 simple sequence repeats (SSRs) were identified. In all the genomes, trinucleotide repeats were the most common class followed by tetranucleotide. Many of the SSR loci are assemblage-specific, and 36 SSR loci shared among all the genomes were identified. Together with hypothetical proteins, variant-specific surface proteins represented nearly half of the annotated SSR loci. The results regarding the most common repeat among the SSRs led us to infer that positive selection occurred to avoid frameshift mutations. Additionally, based on inter- and intra-genetic assemblages polymorphism analyses, we unveiled previously undetected genetic variation, indicating that the microsatellite markers we developed are useful molecular tools for epidemiological inferences based on population genetics patterns and processes. CONCLUSIONS: There is increasing demand for the development of new molecular markers and for the characterization of pathogens at a higher resolution level. In this study, we present 60 G. duodenalis microsatellites markers that exhibited high polymerase chain reaction (PCR) amplification efficiency among the different genetic assemblages. Twenty of these markers presented nucleotide sequence polymorphisms and may be used as a genotyping tool. The monomorphic markers can be used for the detection of the parasite at the species and genetic assemblage level. These polymorphic markers revealed a genetic diversity that was previously undetectable, thus they can be considered valuable molecular tools for high resolution markers in future studies investigating Giardia and may also be used for epidemiological inferences based on populations genetics patterns and processes.

Multilocus genotyping of Giardia duodenalis isolates from household cats and pet shop kittens.

Giardia duodenalis is a pathogenic protozoan that causes diarrhea in mammalian hosts including humans and companion animals, and the host specificity differs depending on the genetic assemblages. The purpose of the present study is to perform multilocus genotyping at four loci of G. duodenalis isolates from household cats and pet shop kittens in Japan and evaluate the zoonotic potential. Fifty-seven fecal samples from cats (household cats: 13, pet shop kittens: 44), which were positive for Giardia-specific antigen as determined by an ELISA kit, were the subjects of our analysis. Nested or semi-nested PCRs targeting 18S ribosomal RNA (18S rRNA), glutamate dehydrogenase (GDH), beta-giardin (BG), and triosephosphate isomerase (TPI) were performed on all samples. All DNA amplicons were sequenced for genotyping. Forty-four isolates (household cats: 11, pet shop kittens: 33) were positive for at least one of the PCRs. Assemblage F was the most frequently detected (75%; 33/44), followed by assemblage A (13.6%; 6/44) and assemblage B (2.3%; 1/44). Four isolates (9.1%) showed the results as mixed assemblages F and A. Sub-genotyping of assemblage A isolates based on three loci (GDH, BG, and TPI) identified all of them as assemblage AI. DNA sequences of zoonotic G. duodenalis assemblages were detected from 25% of the genotyped specimens, and these were found at several shops and hospitals in Japan, which suggests that zoonotic G. duodenalis is prevalent among domestic cats in Japan, and that the considerable risk of zoonotic transmission of G. duodenalis from household cats or pet shop kittens to humans exists.

Occurrence and distribution of Giardia species in wild rodents in Germany.

BACKGROUND: Giardiasis is an important gastrointestinal parasitic disease in humans and other mammals caused by the protozoan Giardia duodenalis. This species complex is represented by genetically distinct groups (assemblages A-H) with varying zoonotic potential and host preferences. Wild rodents can harbor potentially zoonotic assemblages A and B, and the rodent-specific assemblage G. Other Giardia spp. found in these animals are Giardia muris and Giardia microti. For the latter, only limited information on genetic typing is available. It has been speculated that wild rodents might represent an important reservoir for parasites causing human giardiasis. The aim of this study was to investigate the occurrence and distribution of Giardia spp. and assemblage types in wild rodents from different study sites in Germany. RESULTS: Screening of 577 wild rodents of the genera Apodemus, Microtus and Myodes, sampled at eleven study sites in Germany, revealed a high overall Giardia prevalence. Giardia species determination at the SSU rDNA gene locus revealed that Apodemus mice, depending on species, were predominantly infected with one of two distinct G. muris sequence types. Giardia microti was the predominant parasite species found in voles of the genera Microtus and Myodes. Only a few animals were positive for potentially zoonotic G. duodenalis. Subtyping at the beta-giardin (bg) and glutamine dehydrogenase (gdh) genes strongly supported the existence of different phylogenetic subgroups of G. microti that are preferentially harbored by distinct host species. CONCLUSIONS: The present study highlights the preference of G. muris for Apodemus, and G. microti for Microtus and Myodes hosts and argues for a very low prevalence of zoonotic G. duodenalis assemblages in wild rodents in Germany. It also provides evidence that G. muris and G. microti subdivide into several phylogenetically distinguishable subgroups, each of which appears to be preferentially harbored by species of a particular rodent host genus. Finally, the study expands the database of sequences relevant for sequence typing of G. muris and G. microti isolates which will greatly help future analyses of these parasites' population structure.