Prevalence and multilocus genotyping of Giardia duodenalis in Tan sheep (Ovis aries) in northwestern China

Giardia duodenalis is a common intestinal protozoa, which can cause the occurrence of diarrhea, weight loss, and even death in animals or human, this threatens the husbandry industry and public health. It can infect virtually humans and all domestic animals including sheep. Tan sheep is one of the most important sheep breeds, which is short-tailed indigenous sheep breed used for production of high quality meat and pelts in China. However, there are no report regarding the occurrence and multilocus genotyping of G. duodenalis in Tan sheep in northwestern China. Thus, the objective of the present study was to investigate the prevalence and multilocus genotypes of G. duodenalis in Tan sheep. 1014 fecal samples were collected from Tan sheep from Ningxia Hui Autonomous Region, and three loci (β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes) were amplified by nested PCR. The prevalence of G. duodenalis in Tan sheep was 14.5% (147/1014), two assemblages (assemblage A, n = 43; and E, n = 90) were detected, including one novel assemblage A at bg locus, one novel assemblage A at tpi locus, and 10 and 11 novel subtypes of assemblage E were detected at the bg and gdh loci, respectively. One MLGs was formed based on sequence variation among the three loci. Moreover, 9 Tan sheep were infected with two assemblages (A and E) based on the three loci. These findings expand the host range of G. duodenalis and revealed genetic diversity of G. duodenalis assemblages in Tan sheep.     

Infection rate and genetic diversity of Giardia duodenalis in pet and stray dogs in Henan Province,China

Giardia duodenalis is an important protozoan parasite that is known to be zoonotic. To assess the potential zoonotic transmission of giardiasis from dogs and to identify genetic diversity of G. duodenalis in dog populations, we examined the infection rate and genotypes of G. duodenalis in both pet dogs (from pet dog farms, pet shops, pet hospitals, pet markets) and stray dogs of different ages in Henan Province, China. A total of 940 fresh fecal specimens were collected from 2007 to 2013 in Henan Province. The overall infection rate of G. duodenalis was 14.3% (134/940) as determined by microscopy, with the highest infection rate (17.3%) observed in dogs from shelters. Young dogs were more likely to be infected with G. duodenalis than adult dogs, and G. duodenalis cysts were found more frequently in diarrheic dogs. All G. duodenalis-positive isolates were characterized at the triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) loci, and 37, 51, and 48 sequences were obtained, respectively. The dog-specific assemblages C and D were identified using multi-locus sequence analysis. Six novel sequences of the tpi locus, one novel sequence of the gdh locus and two novel sequences of the bg locus were detected among the G. duodenalis assemblage C isolates, while two novel sequences of the gdh locus were found among the G. duodenalis assemblage D isolates. Our data indicate that G. duodenalis is a common parasite and cause of diarrheal disease in dogs in Henan Province. However, there was no evidence for zoonotic G. duodenalis assemblages in the study population.     

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in experimental rats in China

Cryptosporidium and Giardia are ubiquitous protozoan parasites that infect a broad range of hosts. The presence of Cryptosporidium spp. and G. duodenalis was detected in 355 fecal samples of laboratory experimental rats from four experimental rat rearing facilities in China by PCR amplification of the small subunit (SSU) rRNA gene. The G. duodenalis positive samples were further characterized in the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 0.6% (2/355) and 9.3% (33/355), respectively, with no co-infection. Among the four facilities, only the rats in Zhengzhou1 were found positive for the two pathogens. Undetermined Cryptosporidium genotype was observed in one sample and C. ubiquitum in another sample. Assemblage G was identified in all the 33 G. duodenalis positive isolates at SSU rRNA gene, out of which 19, 20, and 21 isolates were also subtyped as assemblage G at tpi, gdh and bg gens, respectively. To our knowledge, this is the first report of Cryptosporidium and G. duodenalis infections in laboratory experimental rats in China. The infections of these pathogens in laboratory animals should be monitored routinely since they may interfere the biological experiments in these animals.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

High prevalence Giardia duodenalis assemblage B and potentially zoonotic subtypes in sporadic human cases in Western Australia

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Fecal samples from sporadic human clinical cases of giardiasis in Western Australia were analysed at two loci; 18S rRNA and glutamate dehydrogenase (gdh), and G. duodenalis assemblage B isolates were identified in 75% of isolates. Sequence analyses of 124 isolates at the 18S rRNA locus identified 93 isolates as assemblage B and 31 as assemblage A. Analyses of 109 isolates at the gdh locus identified 44 as B3, 38 as B4 and 27 were A2. Infection with Giardia was highest amongst children <5 years of age, with >56% of infections in this age group. The majority of the isolates were from rural areas (91/124) compared with urban areas (33/124). The assemblage A isolates were completely homogenous genetically at the gdh locus, while assemblage B isolates showed variability at the nucleotide but not at the amino acid level at this locus. Some of the assemblage B3 and B4 subtypes identified in humans were previously identified in marsupials in Australia and in a fox, indicating potential zoonotic transmission.     

Limitations of tpi and bg genes sub-genotyping for characterization of human Giardia duodenalis isolates

The intestinal protozoan Giardia duodenalis includes 2 genetically distinct assemblages, A and B, which are responsible for human infections. Little is known so far on the genotypes of G. duodenalis human isolates in France. The present characterization of 19 French clinical isolates was aimed at determining their genotype patterns and associations with clinical symptoms, and in vivo metronidazole resistance, respectively. Based on both triose-phosphate isomerase (tpi) and β-giardin (bg) gene sequences, twelve isolates were identified as assemblage A, and 7 as assemblage B for the 2 gene loci. Sub-genotyping heterogeneities were observed in 15/19 isolates attributed to either A or B assemblage. They include frequent mismatches and intra-assemblage discordances and mixed positions, which were found more frequently in tpi than in bg sequences, and in assemblage B than in assemblage A sequences. No association was found between sub-genotypes, clinical symptoms and metronidazole sensitivity. Present data underline the need for improvements in the standardization of G. duodenalis multilocus genotyping approach for further molecular epidemiologic studies of giardiasis.     

Genetic Characterizations of Giardia duodenalis in Sheep and Goats in Heilongjiang Province,China and Possibility of Zoonotic Transmission

Background

Giardia duodenalis is a widespread intestinal protozoan of both humans and mammals. To date, few epidemiological studies have assessed the potential and importance of zoonotic transmission; and the human giardiasis burden attributable to G. duodenalis of animal origin is unclear. No information about occurrence and genotyping data of sheep and goat giardiasis is available in China. The aim of the present study was to determine prevalence and distribution of G. duodenalis in sheep and goats in Heilongjiang Province, China, and to characterize G. duodenalis isolates and assess the possibility of zoonotic transmission.

Methodology/Principal Findings

A total of 678 fecal specimens were collected from sheep and goats on six farms ranging in age from one month to four years in Heilongjiang Province, China. The average prevalence of G. duodenalis infection was 5.0% (34/678) by microscopy after Lugol''s iodine staining, with 5.6% (30/539) for the sheep versus 2.9% (4/139) for the goats. Molecular analysis was conducted on 34 G. duodenalis isolates based on the triosephosphate isomerase (tpi) gene. 29 tpi gene sequences were successfully obtained and identified as assemblages A (n = 4), B (n = 2) and E (n = 23). High heterogeneity was observed within assemblage E at the tpi locus, with five novel subtypes found out of seven subtypes. Two subtypes of assemblage A were detected, including subtype AI (n = 3) and a novel subtype (designated as subtype AIV) (n = 1). Two assemblage B isolates were identical to each other in the tpi gene sequences.

Conclusions/Significance

This is the first report of G. duodenalis infections in sheep and goats in China. The present data revealed the unique endemicity on prevalence, distribution and genetic characterization of G. duodenalis in sheep and goats in Heilongjiang Province. The findings of assemblages A and B in sheep and goats implied the potential of zoonotic transmission.     

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.     

Multi-locus analysis of Giardia duodenalis intra-Assemblage B substitution patterns in cloned culture isolates suggests sub-Assemblage B analyses will require multi-locus genotyping with conserved and variable genes

Recent research concerning Giardia duodenalis has focused on resolving possible sub-assemblages within Assemblages A and B to better understand host-specific and zoonotic relationships. In the present study nine cloned, cultured, Assemblage B isolates were used to investigate the intra-Assemblage B substitution patterns of conserved (ssrDNA, ef, h2b, h4) and variable (tpi, gdh, bg) genes to assess their suitability for further application to sub-assemblage analyses. The resolution of each gene was found to be proportional to its substitution rate and for the genetically narrow sample set examined, the variable genes best represented the consensus phylogeny while the conserved genes only established fractions. However it was demonstrated that the spectra of conserved and variable genes were required to ensure accuracy of inferred phylogeny and it was therefore concluded that further research into sub-Assemblage B groups would require a mixture of conserved and variable genes for the multi-locus analyses of this genetically broad assemblage.     

LNA probes in a real‐time TaqMan PCR assay for genotyping of Giardia duodenalis in wastewaters

Aims: This study describes an approach for genotyping Giardia cysts obtained from wastewater treatment plants (WTPs) in Spain using real‐time PCR (qPCR) in combination with immunomagnetic beads. Methods and Results: A 50‐cycle amplification of a 74‐bp fragment of the Giardia beta‐giardin gene was adopted from a previous qPCR method. Additionally, two locked nucleic acid (LNA) probes were designed (LNA P434 P1 for assemblage A and LNA P434 H3 for assemblage B). All 16 wastewater samples analysed were positive with the immunofluorescence assay (IFA). Assemblage A was detected in all WTP samples using primer–LNA probe P434 P1 set. Giardia duodenalis identification was confirmed by PCR–RFLP analysis and sequencing of the β‐giardin gene in the water samples found positive by IFA and qPCR. Among the 16 assemblage A isolates that were sequenced, two subtypes were identified; 11 corresponded to the A2 subgenotype, whereas three corresponded to the subgenotype A3. A mixture of subgenotypes was found in the remaining two isolates. Conclusions: The newly developed qPCR assays were able to discern G. duodenalis assemblages A and B in wastewater. Significance and Impact of the Study: The real‐time PCR assays provided a rapid method for detection and one‐step genotyping of G. duodenalis from wastewater samples, and its application would contribute to understanding the distribution and abundance of G. duodenalis assemblages A and B in wastewater.     

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.     

Multilocus genotyping of Giardia duodenalis from pigs in Korea

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is an important zoonotic parasite infecting livestock (including pigs) through ingesting cysts in contaminated food or water. This parasite has been classified into eight different genetic assemblages, A to H. Here, we examined the individual-level prevalence of G. duodenalis in domestic pig farms and confirmed host specificity by genotype comparisons. Samples were collected from southern and central Korea, between May 2017 and January 2019. DNA directly extracted from 745 pig fecal specimens were tested by PCR for G. duodenalis small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), and β-giardin gene sequences. Based on ssu rRNA PCR, 110 (14.8%) were positive for G. duodenalis. Infection risk was the highest in the fattener group (31/139, 22.3%) and during the autumn season (52/245, 21.2%: p < .001). No statistically significant differences in risk for infection were observed between fecal types (normal versus diarrheal). Fifty ssu rRNA samples, three gdh samples, and five β-giardin samples were successfully sequenced and genotyped. Ssu rRNA assemblage sequence analysis identified E (40.0%, 20/50), D (34.0%, 17/50), C (24.0%, 12/50), and A (2.0%, 1/50). The gdh locus identified three samples as assemblage E, and the β-giardin locus identified four samples as assemblage E and one as assemblage C. Assemblage A sequences obtained (ssu rRNA; MK430919) had 100% identity with Giardia sequences isolated from a Korean individual (AJ293301), indicating the potential of zoonotic transmission. Continuous management and monitoring for prevention of transmission and protection of animal and human health are essential.     

Identical Assemblage of Giardia duodenalis in Humans,Animals and Vegetables in an Urban Area in Southern Brazil Indicates a Relationship among Them

Background

Giardia duodenalis infects humans and other mammals by ingestion of cysts in contaminated water or food, or directly in environments with poor hygiene. Eight assemblages, designated A–H, are described for this species.

Methodology/Principal Findings

We investigated by microscopy or by direct immunofluorescence technique the occurrence of G. duodenalis in 380 humans, 34 animals, 44 samples of water and 11 of vegetables. G. duodenalis cysts present in samples were genotyped through PCR-RFLP of β giardin and glutamate dehydrogenase (gdh) genes and sequencing of gdh. The gdh gene was amplified in 76.5% (26/34) of the human faeces samples with positive microscopy and in 2.9% (1/34) of negative samples. In 70.4% (19/27) of the positive samples were found BIV assemblage. In two samples from dogs with positive microscopy and one negative sample, assemblages BIV, C, and D were found. Cysts of Giardia were not detected in water samples, but three samples used for vegetable irrigation showed total coliforms above the allowed limit, and Escherichia coli was observed in one sample. G. duodenalis BIV was detected in two samples of Lactuca sativa irrigated with this sample of water. BIV was a common genotype, with 100% similarity, between different sources or hosts (humans, animals and vegetables), and the one most often found in humans.

Conclusions/Significance

This is the first study in Brazil that reports the connection among humans, dogs and vegetables in the transmission dynamics of G. duodenalis in the same geographic area finding identical assemblage. BIV assemblage was the most frequently observed among these different links in the epidemiological chain.     

Occurrence of Giardia duodenalis assemblages in alpacas in the Andean region

In this study, 352 fecal samples were analyzed for G. duodenalis from alpaca mothers and crias from three different areas of highland in Peru. The triosephosphate isomerase (TPI) gene of Giardia was amplified using a nested PCR protocol. Forty-six G. duodenalis-PCR positive samples were sequenced. G. duodenalis assemblage A was the most frequent followed by assemblage E. The former was seen in 37 animals whereas the latter was seen in nine. Most of the assemblage A infections were caused by the A1 subtype of sub-assemblage AI, except for three, which were caused by the A2 subtype of sub-assemblage AI. Assemblage A was found in all three geographic regions, while assemblage E was detected in crias from two regions. Among the four alpaca mothers positive for Giardia, three had assemblage AI and one had assemblage AII. Results of this study indicate that possible zoonotic transmission human to alpacas.     

Prevalence,Risk Factors and Multilocus Genotyping of Giardia intestinalis in Dairy Cattle,Northwest China

Giardia intestinalis is a cosmopolitan protozoan parasite that can infect a range of animals, including dairy cattle. As information regarding the prevalence and genotyping of G. intestinalis infection in dairy cattle in northwestern China is limited, 2,945 feces samples from 1,224 dairy cattle in Gansu Province and from 1,614 in Ningxia Hui Autonomous Region (NXHAR) were examined between December 2012 and March 2014. The overall prevalence of G. intestinalis was 3.63% (107/2,945), with 2.63% and 4.38% in Gansu and NXHAR, respectively. Logistic regression analysis showed region, age and season to be significant risk factors for G. intestinalis infection. Assemblage analysis identified 106 assemblage E and one assemblage A at the triose phosphate isomerase (tpi) locus in this study. Intravariations were also detected at tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci within assemblage E, showing seven, three, and five new subtypes, respectively. Moreover, 13 new multilocus genotypes (E20‐E32) were observed in assemblage E. Effective strategies and measures should be taken to prevent and control giardiasis in Gansu and NXHAR.     

Occurrence of human-pathogenic Enterocytozoon bieneusi,Giardia duodenalis and Cryptosporidium genotypes in laboratory macaques in Guangxi,China

Captive nonhuman primates have been identified as common hosts of Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium hominis, and Cyclospora spp., thus are potential reservoirs of some enteric parasites in humans. However, few studies have examined the source and human-infective potential of enteric parasites in laboratory nonhuman primates. In the present work, 205 fecal specimens were collected from three groups of captive Macaca fascicularis kept in different densities in a laboratory animal facility in Guangxi, China, and examined by PCR for E. bieneusi, G. duodenalis, Cryptosporidium spp., and Cyclospora spp. The infection rates of E. bieneusi and G. duodenalis were 11.3% and 1.2% in Group 1 (young animals kept individually; n = 168), 72.2% and 11.1% in Group 2 (young animals kept in groups; n = 18), and 31.6% and 5.3% in Group 3 (adults kept in groups; n = 19), respectively. Sequence analysis of PCR products showed the presence of five E. bieneusi genotypes, with genotype D (in 16/36 genotyped specimens) and a new genotype (in 15/36 genotyped specimens) as the dominant genotypes. All five E. bieneusi genotypes belonged to the zoonotic group (Group 1). The G. duodenalis genotypes (assemblages AII and B) in five specimens and C. hominis subtype (IdA14) in one specimen were also known human-pathogens, although the Cyclospora seen in one animal appeared to be unique to macaque monkeys. The higher infection rate in younger animals reared in groups and common occurrence of zoonotic genotypes indicated that human-pathogenic E. bieneusi could be transmitted efficiently in captive nonhuman primates, and group-housing was a risk factor for transmission of zoonotic pathogens in young nonhuman primates in research facilities.     

Molecular characterization of Giardia duodenalis isolates from police and farm dogs in China

To assess the potential zoonotic transmission of giardiasis from dogs in China, a total of 205 fecal specimens from dogs were screened for Giardia duodenalis using PCR and sequence analysis of the triosephosphate isomerase gene. The prevalence of G. duodenalis in dogs was 13.2% (27/205). The potentially zoonotic assemblage A and the dog-specific assemblage C was identified in 25 (12.2%) and two (1.0%) dogs, respectively. All assemblage A isolates belonged to sub-assemblage AI, genotype AI-1. Likewise, one subtype was found in assemblage C. The high occurrence of potentially zoonotic G. duodenalis subtype AI-1 in dogs that are in close contact with humans is of public health concern.     

Age patterns of Cryptosporidium species and Giardia duodenalis in dairy calves in Egypt

Little is known of the occurrence and age patterns of species/genotypes and subtypes of Cryptosporidium spp. and Giardia duodenalis in calves in Egypt. In this study, 248 fecal specimens were collected from dairy calves aged 1?day to 6?months on eight farms in three provinces during March 2015 to April 2016. Cryptosporidium spp. were detected and genotyped by using PCR-RFLP analysis of the small subunit rRNA (SSU rRNA) gene, while G. duodenalis was detected and genotyped by using PCR and sequence analyses of the triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh) and β-giardin (bg) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 9.7 and 13.3%, respectively. The highest Cryptosporidium infection rate (26.7%) was in calves of age?≤?1?month while the highest G. duodenalis infection rate (44.4%) was in calves of 2?months. Three Cryptosporidium spp. were identified, including C. parvum (n?=?16), C. bovis (n?=?5) and C. ryanae (n?=?3), with the former being almost exclusively found in calves of ≤3?months of age and the latter two being only found in calves of over 3?months. Subtyping of C. parvum by PCR-sequence analysis of the 60?kDa glycoprotein gene identified subtypes IIaA15G1R1 (n?=?15) and IIaA15G2R1 (n?=?1). The G. duodenalis identified included both assemblages E (n?=?32) and A (n?=?1), with the latter belonging to the anthroponotic subtype A2. These data provide new insights into the genetic diversity and age patterns of Cryptosporidium spp. and G. duodenalis in calves in Egypt.