Tpi Loci Research Articles

Prevalence and genotype analysis of Cryptosporidium and Giardia duodenalis from shelter dogs in South Korea

Cryptosporidium and Giardia duodenalis are common gastrointestinal protozoan parasites that cause diarrhea in humans and animals. Although dogs in animal shelters in South Korea can be reintroduced into human society as companions, no continuous investigations have been conducted on the prevalence and genetic characteristics of the protozoan parasites. In the present study, 345 fecal samples from dogs were obtained from animal shelters in six provinces between January and December 2022. The prevalence of Cryptosporidium and G. duodenalis were molecularly identified at the 18S rRNA gene. Cryptosporidium canis isolates were subtyped at the gp60 locus, and G. duodenalis assemblages were identified at the tpi, bg and gdh loci. Overall, 6.67% of the fecal samples tested positive for Cryptosporidium spp. and two species were identified: C. canis (5.51%) and Cryptosporidium parvum (1.16%). Significant regional differences in prevalence were identified for C. canis. Subtyping analysis of C. canis isolates revealed a predominance of the subtype families XXa and XXe over XXb, and XXc. G. duodenalis was detected in 17.68% of the samples, and significant regional differences were identified; its prevalence was also significantly highest in diarrhoeic animals. Molecular characterization of G. duodenalis reveal that most isolates belonged to the canine-specific assemblages C and D. The present results can contribute to further insights into the prevalence and genotypes of Cryptosporidium and G. duodenalis in shelter dogs in South Korea.

Prevalence and Genetic Characterization of Giardia duodenalis and Blastocystis spp. in Black Goats in Shanxi Province, North China: From a Public Health Perspective.

Blastocystis spp. and Giardia duodenalis are two prevalent zoonotic intestinal parasites that can cause severe diarrhea and intestinal diseases in humans and many animals. Black goat (Capra hircus) farming is increasingly important in China due to the remarkable adaptability, high reproductive performance, rapid growth rate, and significant economic value of black goats. A number of studies have indicated that black goats are the potential reservoir of multiple zoonotic protozoans in China; however, the prevalence and zoonotic status of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province is still unknown. Thus, a total of 1200 fecal samples of black goats were collected from several representative regions at different altitudes in Shanxi Province and were examined for the presence and genotypes of G. duodenallis and Blastocystis spp. by amplifying the beta-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci of G. duodenalis and SSU rRNA of Blastocystis spp. using PCR and sequence analysis methods, respectively. The overall prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province were 7.5% and 3.5%, respectively. Two assemblages (B and E) of G. duodenalis and four subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp. were identified, with assemblage E and ST10 as the prevalent genotype and subtype in black goats, respectively. One novel multilocus genotype (MLG) was identified in MLG-E and was designated as MLG-E12. For both G. duodenalis and Blastocystis spp., the prevalence was significantly related to the region and age groups (p < 0.05). This is the first report on the prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province. These results not only provide baseline data for the prevention and control of both parasites in black goats in Shanxi Province, but also enhance our understanding of the genetic composition and zoonotic potential of these two parasites.

Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene. This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran. From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy. Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA. A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes. G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively. Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively. MLG data for the two loci were available for only five samples. Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)]. In the present study, no assemblages suited for non-human animal hosts (e.g., CF) were detected. This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic. Further molecular-based analyses are necessary to confirm and expand upon these findings. These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

Detection and Molecular Diversity of Cryptosporidium spp. and Giardia duodenalis in the Endangered Iberian Lynx (Lynx pardinus), Spain.

Cryptosporidium spp. and Giardia duodenalis are the main non-viral causes of diarrhoea in humans and domestic animals globally. Comparatively, much less information is currently available in free-ranging carnivore species in general and in the endangered Iberian lynx (Lynx pardinus) in particular. Cryptosporidium spp. and G. duodenalis were investigated with molecular (PCR and Sanger sequencing) methods in individual faecal DNA samples of free-ranging and captive Iberian lynxes from the main population nuclei in Spain. Overall, Cryptosporidium spp. and G. duodenalis were detected in 2.4% (6/251) and 27.9% (70/251) of the animals examined, respectively. Positive animals to at least one of them were detected in each of the analysed population nuclei. The analysis of partial ssu rRNA gene sequences revealed the presence of rodent-adapted C. alticolis (n = 1) and C. occultus (n = 1), leporid-adapted C. cuniculus (n = 2), and zoonotic C. parvum (n = 2) within Cryptosporidium, and zoonotic assemblages A (n = 5) and B (n = 3) within G. duodenalis. Subgenotyping analyses allowed for the identification of genotype VaA19 in C. cuniculus (gp60 locus) and sub-assemblages AI and BIII/BIV in G. duodenalis (gdh, bg, and tpi loci). This study represents the first molecular description of Cryptosporidium spp. and G. duodenalis in the Iberian lynx in Spain. The presence of rodent/leporid-adapted Cryptosporidium species in the surveyed animals suggests spurious infections associated to the Iberian lynx's diet. The Iberian lynx seems a suitable host for zoonotic genetic variants of Cryptosporidium (C. parvum) and G. duodenalis (assemblages A and B), although the potential risk of human transmission is regarded as limited due to light parasite burdens and suspected low excretion of infective (oo)cysts to the environment by infected animals. More research should be conducted to ascertain the true impact of these protozoan parasites in the health status of the endangered Iberian lynx.

Prevalence and assemblage identified of Giardia duodenalis in zoo and farmed Asiatic black bears (Ursus thibetanus) from the Heilongjiang and Fujian Provinces of China.

Captive and free-living wildlife serve as significant hosts for Giardia duodenalis. Asiatic black bears, valued for their economic and medicinal importance, are extensively farmed in China and also prevalent in zoos. However, studies on G. duodenalis in these animals in China are limited. Here, 218 feces samples of Asiatic black bears were collected: 36 from a zoo in Heilongjiang Province, and 182 from a farm in Fujian Province. Nested PCR of the SSU rRNA gene, followed by sequencing, was employed to determine the frequency and assemblage distribution of G. duodenalis. Positive samples underwent further analysis through multilocus genotyping (MLG) by amplifying the genes for glutamate dehydrogenase (gdh), β-giardin (bg), and triosephosphate isomerase (tpi). Of the 218 samples, G. duodenalis was detected in 22 cases at the SSU rRNA gene locus, including three from Heilongjiang and 19 from Fujian. Three assemblages were identified: A (n=1), B (n=16), and E (n=2) in Fujian; and B (n=3) in Heilongjiang. Out of the 22 positive samples, 20, 19, and 9 were effectively amplified and sequenced across the tpi, gdh, and bg loci, respectively. Seven samples were genotyped successfully at all three loci, identifying MLG-B1 (n=1), MLG-B2 (n=1), and MLG-B3 (n=1), MLG-B4 (n=1), MLG-B5 (n=2), and MLG-B6 (n=1) as the six assemblage B MLGs. This study marks the first documentation of G. duodenalis in Asiatic black bears in captivity in Fujian and Heilongjiang. The identification of zoonotic assemblages A and B, along with E, underscores potential public health concerns.

Comparison of the Efficiency of Diagnostic Tests Used to Prove Giardiasis in Terms of their Practicality and Use in the Veterinary Clinical Practice

Abstract Giardiasis is a protozoan disease that affects the health of animals, as well as other humans all over the world. Based on its host spectrum and genetic variability, Giardia duodenalis is classified into 8 assemblages (A–H). The present study was aimed at comparing the efficiency of the three most frequently used methods (the flotation method, the SNAP test and the ELISA assay) for the detection of giardiasis in carnivores in terms of the applicability thereof for the scientific purposes and the practicality of their application in the veterinary clinical practice. In the period from March 2020 to February 2022, a total of 173 faecal samples (141 samples collected from shelter dogs; 28 samples from pet dogs; and 6 samples from working dogs) were examined by applying the flotation method. The prevalence of Giardia duodenalis identified by the flotation method was 25 %. The SNAP test conducted with the fresh faecal samples revealed the high-level efficiency of 96 %, whereas the enzyme-linked immunosorbent assay (ELISA) achieved the efficiency of 65 %. By applying the nested PCR method, five samples were positively tested for assemblages C and D (G. canis) by the amplification of the bg and tpi loci. The dogs from shelters which were positive for G. duodenalis were also presented with a coinfection caused by other intestinal parasites, such as Trichuris vulpis (28.0 %) and parasites from the Ancylostomatidae family (8.0 %).

A shotgun metagenomic analysis of the fecal microbiome in humans infected with Giardia duodenalis

BackgroundThe mechanisms underlying the clinical outcome disparity during human infection with Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite’s genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis.MethodsThe genetic heterogeneity of G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal–Wallis non-parametric test with a post hoc Dunn test.ResultsWe found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite’s genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers.ConclusionsThese findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections.Graphical

A One-Year Retrospective Analysis of Viral and Parasitological Agents in Wildlife Animals Admitted to a First Aid Hospital.

This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020-2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance.

Molecular Detection and Multilocus Genotyping of Giardia duodenalis in Pigs in Fujian Province, Southeastern China.

Giardia duodenalis, an intestinal parasite, is widely distributed in humans and various animals, such as pigs, cattle and cats. The clinical symptoms of giardiasis are characterized as including abdominal pain, acute or chronic diarrhea, and bloating and weight loss in humans and animals, leading to public and veterinary health problems worldwide. However, the prevalence and genotypes of G. duodenalis in pigs in Fujian Province, southeastern China, have not been reported. In the present study, 725 fecal samples were collected from six cities (Fuqing, Putian, Nanping, Longyan, Sanming, Zhangzhou) in Fujian Province and analyzed for G. duodenalis prevalence and genotypes using nested PCR targeting the beta-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. The results shown that total occurrence rate of G. duodenalis was 26.9% (195/725) in pigs, with significant differences in the prevalence among different regions (χ2 = 86.508, p &lt; 0.05) and groups (χ2 = 12.748, p &lt; 0.05). 195, 11 and 6 samples were detected at the bg, tpi and gdh loci, respectively. Each one belonged to a subtype of assemblage E and was analyzed using sequences obtained in this study. Based on phylogenetic analyses of sequences from the three genetic loci, only one MLG E1 was found. The results indicated that pigs may present a potential zoonotic risk of spreading G. duodenalis infection from animals to humans in this area. The findings of the present study also provide basic data for the prevention and control of G. duodenalis infection in pigs and humans in China.

Multilocus sequence typing of Giardia duodenalis genotypes circulating in humans in a major metropolitan area.

Giardia duodenalis is an intestinal protozoan parasite of humans and animal hosts and comprises eight microscopically indistinguishable molecularly-diverse lineages designated as assemblages A–H. Assemblages A and B are the primary sources of infections in humans and a wide range of mammals. Here, we identified assemblages, and inter-/intra-assemblage genetic diversity of human G. duodenalis isolates based on the multilocus sequence typing of the triosephosphate isomerase (tpi), β -giardin (bg), and glutamate dehydrogenase (gdh) loci. Multilocus sequence analysis of 62 microscopically-positive G. duodenalis fecal samples identified 26 (41.9%), 27 (43.5%), and nine (14.5%) isolates belonging to assemblages A, B, and discordant assemblages, respectively. The tpi locus assemblage-specific primers identified dual infections with A and B assemblages (45.2%). The sequence analysis of multiple alignments and phylogenetic analysis showed low genetic polymorphism in assemblage A isolates, classified as sub-assemblage AII at three loci, subtype A2 at tpi and gdh loci, and subtype A2 or A3 at bg locus. High genetic variations were found in assemblage B isolates with 14, 15, and 23 nucleotide patterns at tpi, bg, and gdh loci, respectively. Further concatenated sequence analysis revealed four multilocus genotypes (MLG) in 24 assemblages A isolates, two previously-identified (AII-1 and AII-5), with one novel multilocus genotype. However, the high genetic variations observed in assemblage B isolates among and within the three genetic loci prevented the definitive designation of specific MLGs for these isolates. Multilocus sequence typing may provide new insight into the genetic diversity of G. duodenalis isolates in Tehran, suggesting that humans are likely a potential source of G. duodenalis infection. Further host-specific experimental transmission studies are warranted to elucidate the modes of transmission within multiple host populations.

Prevalence and genetic diversity of Giardia duodenalis in pet dogs from Zhengzhou, central China and the association between gut microbiota and fecal characteristics during infection

As a common zoonotic intestinal parasite, Giardia duodenalis could infect humans and various mammals worldwide, including pet dogs, leading to giardiasis. This study detected the infection of G. duodenalis in asymptomatic pet dogs in Zhengzhou, and evaluated the possibility of zoonosis and the relationship between gut microbiota and fecal characteristics. We randomly collected 448 fresh fecal samples from Zhengzhou, and G. duodenalis was screened based on the beta-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes. The difference of gut microbiota between five G. duodenalis-positive and five G. duodenalis-negative samples was investigated by 16S rRNA gene sequencing. The overall prevalence of G. duodenalis was 7.1% (32/448) based on bg, gdh, and tpi locus, two G. duodenalis assemblages (C = 13, D = 14) and five (15.6%) mixed infection (C + D) were identified. Moreover, compared with the G. duodenalis-negative group, the diversity of gut microbiota increased in G. duodenalis-positive group. The decrease of Lactobacillus spp. and considerable increase of Prevotella spp. were associated with the fecal characteristics. These results show that the transmission of zoonotic giardiasis between humans and pet dogs is rare in Zhengzhou, central China, and support the use of Lactobacillus spp. as a potential probiotic agent to improve intestinal health in dogs, or even humans, by treating G. duodenalis. Therefore, the public health significance of G. duodenalis to humans, companion animals, and the environment should be further evaluated from One Health perspective.

Prevalence and molecular characterization of Giardia duodenalis in small ruminants of Shiraz, southwestern Iran: A zoonotic concern

Livestock are commonly affected by gastrointestinal protozoan parasites, including Giardia duodenalis. In small ruminants, G. duodenalis infection may decrease carcass weight and dressing percentage. Current study was done to determine the prevalence, assemblage distribution, and zoonotic significance of Giardia infection in sheep and goats in Shiraz, southwestern Iran. In total, 200 fecal samples were randomly collected from 100 sheep and 100 goats in 10 farms in Shiraz, southwest of Iran (June 2021-February 2022) and directly examined for G. duodenalis cyst by saline/iodine wet mount examination using a light microscope (400 ×). Positive samples were further genotyped using a nested-PCR and sequencing methods. A mean prevalence of 5.5% (11/200) was estimated for G. duodenalis infection among small ruminants, with 7% and 4% for sheep and goats, respectively. Next, 9 out of 11 positive samples were amplified and only 5 were successfully sequenced at the tpi locus. Our results showed that 80% (4/5) isolates belonged to assemblage E, while only 20% (1/5) were associated with the assemblage A (subtype AI). Of note, 2 E assemblages were isolated from goats and 2 E assemblages plus 1 assemblage A were isolated from sheep. The major finding in the present study was the isolation of assemblage A from sheep in Shiraz, highlighting the zoonotic transmission of Giardia infection in the study area. In general, the information mentioned in the present study is very limited and more extensive studies in this field are needed to achieve more conclusive results.

Population genetics reveal multiple independent invasions of Spodoptera frugiperda (Lepidoptera: Noctuidae) in China.

The fall armyworm (Spodoptera frugiperda), a destructive pest that originated in South and North America, spread to China in early 2019. Controlling this invasive pest requires an understanding of its population structure and migration patterns, yet the invasion genetics of Chinese S. frugiperda is not clear. Here, using the mitochondrial cytochrome oxidase subunit I (COI) gene, triose phosphate isomerase (Tpi) gene and eight microsatellite loci, we investigated genetic structure and genetic diversity of 16 S. frugiperda populations in China. The Tpi locus identified most S. frugiperda populations as the corn-strains, and a few were heterozygous strains. The microsatellite loci revealed that the genetic diversity of this pest in China was lower than that in South America. Furthermore, we found moderate differentiation among the populations, distinct genetic structures between adjacent populations and abundant genetic resources in the S. frugiperda populations from China sampled across 2 years. The survival rate of S. frugiperda was significantly higher when it was fed on corn leaves than on rice leaves, and the larval stage mortality rate was the highest under both treatments. Our results showed that S. frugiperda probably invaded China via multiple independent introductions and careful pesticide control, continuous monitoring and further studies will be needed to minimize its potential future outbreak.

Molecular characterizations of Giardia duodenalis based on multilocus genotyping in sheep, goats, and beef cattle in Southwest Inner Mongolia, China.

Giardia duodenalis is an important zoonotic parasite that causes economic losses to animal husbandry and threatens public health. In the present study, a total of 1466 fresh fecal samples were collected from sheep (n = 797), goats (n = 561) and beef cattle (n = 108) in Southwest Inner Mongolia, China. Giardia duodenalis was initially screened via nested polymerase chain reaction (PCR) targeting the β-giardin (bg) gene, and bg-positive samples were subjected to PCR amplification targeting the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. A total of 4.0% of samples (58/1466) were positive for G. duodenalis, with a prevalence of 3.4% in sheep, 3.7% in goats and 5.2% in beef cattle. Three G. duodenalis assemblages (A, B, and E) were identified, with E as the prevalent assemblage. Four and one novel assemblage E sequences were obtained for the gdh and tpi loci, respectively and four assemblage E multilocus genotypes (MLG) were obtained. This study demonstrates high genetic variations in G. duodenalis assemblage E, and provides baseline data for preventing and controlling G. duodenalis infection in livestock in Inner Mongolia.

Investigation of giardiasis in captive animals in zoological gardens with strain typing of assemblages in China.

Giardia duodenalis is a common zoonotic intestinal pathogen. It has been increasingly reported in humans and animals; however, genotyping information for G. duodenalis in captive animals is still limited. This study was conducted to assess the prevalence and multilocus genotyping of G. duodenalis in captive animals in zoological gardens in Shanghai, China. A total of 678 fresh fecal samples were randomly collected from captive animals including non-human primates (NHPs) (n = 190), herbivores (n = 190), carnivores (n = 151), birds (n = 138) and reptiles (n = 9) in a zoo and were examined for the presence of G. duodenalis using nested polymerase chain reaction (nested PCR). All G. duodenalis positive samples were assayed with PCR followed by sequencing at β-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. In this study, 42 specimens (6.2%) were tested G. duodenalis-positive of the 678 fecal samples examined based on a single locus. A total of 30 (4.4%), 30 (4.4%) and 22 (3.2%) specimens were successfully amplified and sequenced at gdh, tpi and bg loci, respectively. Assemblages A and B were identified with assemblage B dominating in NHPs. Sequence analysis demonstrated that one, two and five new isolates were identified at bg, gdh and tpi loci. DNA sequences and new assemblage-subtypes of zoonotic G. duodenalis assemblages A and B were identified in the current study. Our data indicate the occurrence and molecular diversity of G. duodenalis and the potential zoonotic transmission in captive animals in China.

Seasonal Prevalence and Novel Multilocus Genotypes of Giardia duodenalis in Yaks (Bos grunniens) in Qinghai Province, Western China.

Background:Giardia duodenalis is an important opportunistic zoonotic intestinal protozoon, which could parasitize yaks. However, a few studies have been conducted on the seasonal infection of G. duodenalis in yaks in China.Methods:Overall, 1,027 fecal samples were collected from yaks of two age groups in seven cities of Qinghai Province, China at four seasons between May 2016 and Sep 2017. The prevalence and assemblages were analyzed by nested PCR and multilocus sequence typing (MLST).Results:The overall prevalence of G. duodenalis was 2.04% (21/1027) based on triose phosphate isomease (tpi) locus. No significant differences in prevalence of the organism in yaks were found among different sampling areas. Additionally, same result was also presented in different seasons. However, there was statistically significant difference between young yaks within 6 months (8.33%, 4/48) and adult yaks over 6 months (1.73%, 17/979). The assemblage A recognized as a zoonotic assemblage (n=3) was found in yaks (>6 months) from Xining, while assemblage E (n=18) was detected from yaks in six cities. There were 5, 2 and 3 G. duodenalis subtypes detected positive at the tpi, the β-giardin (bg), and the glutamate dehydrogenase (gdh) loci, with 2, 2 and 3 novel subtypes, respectively. Three samples were successfully sequenced at all three loci, forming 1 assemblages A multilocus genotype (MLG) and 2 assemblages E MLGs, not reported.Conclusion:This study indicated a zoonotic potential of G. duodenalis in yaks from Qinghai Province and provides basic information about the epidemiology of G. duodenalis.

Prevalence and multi-locus genotyping of Giardia duodenalis in rabbits from Shaanxi province in northwestern China.

Giardia duodenalis is an important parasite with veterinary and public health significance worldwide. The presence and zoonotic assemblages of G. duodenalis have previously been reported in rabbits. In this study, to understand the infection status of G. duodenalis in rabbits from Shaanxi province, a total of 537 fecal samples were collected from two breeds of rabbits in four age groups (<30 days, 31–90 days, 91–200 days and >200 days) from four geographical origins (Fengxiang, Yangling, Tongchuan, and Shanyang). The presence of G. duodenalis in these samples was assessed using molecular assays based on beta-giardin (bg). The glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci were then amplified in the bg-positive samples for multi-locus genotype (MLG) analysis. The total prevalence of G. duodenalis in these rabbits was 3.54% (19/537). Giardia duodenalis infection was found in both breeds of rabbits, and in all farms and age groups, but with no statistically significant differences related to these factors (p > 0.05). Two assemblages, including B and E, were identified, with the former the predominant assemblage detected in both breeds, and in all age groups and farms. Sequence analysis revealed 2 (named as rbg1-2), 1 (named as rtpi1), and 2 (named as rgdh1-2) haplotypes at the gene loci of bg, tpi, and gdh, respectively, forming a multilocus genotype (MLG) of assemblage B (rbg1, rtpi1, and rgdh1). These findings reveal the significant zoonotic potential and genetic diversity of G. duodenalis in rabbits in Shaanxi Province, PR China.

Identification of Uncommon Cryptosporidiumviatorum (a Novel Subtype XVcA2G1c) and Cryptosporidium andersoni as Well as Common Giardia duodenalis Assemblages A and B in Humans in Myanmar.

Cryptosporidium and Giardia are two important zoonotic intestinal protozoa responsible for diarrheal diseases in humans and animals worldwide. Feces from infected hosts, water and food contaminated by Cryptosporidium oocysts and Giardia cysts as well as predictors such as poverty have been involved in their transmission. Myanmar is one of the world’s most impoverished countries. To date, there are few epidemiological studies of Cryptosporidium and Giardia in humans. To understand the prevalence and genetic characterization of Cryptosporidium spp. and Giardia duodenalis in humans in Myanmar, a molecular epidemiological investigation of the two protozoa was conducted in four villages of Shan State. 172 fecal specimens were collected from Wa people (one each) and identified for the presence of Cryptosporidium spp. and G. duodenalis by sequence analysis of their respective small subunit ribosomal RNA genes. 1.74% of investigated people were infected with Cryptosporidium spp.—C. andersoni (n = 2) and C. viatorum (n = 1) while 11.05% infected with G. duodenalis—assemblages A (n = 6) and B (n = 13). By sequence analysis of 60-kDa glycoprotein gene, the C. viatorum isolate belonged to a novel subtype XVcA2G1c. DNA preparations positive for G. duodenalis were further subtyped. Five of them were amplified and sequenced successfully: different assemblage B sequences (n = 2) at the triosephosphate isomerase (tpi) locus; sub-assemblage AII sequence (n = 1) and identical assemblage B sequences (n = 2) at the β-giardin (bg) locus. This is the first molecular epidemiological study of Cryptosporidium spp. and G. duodenalis in humans in Myanmar at both genotype and subtype levels. Due to unclear transmission patterns and dynamics of Cryptosporidium spp. and G. duodenalis, future research effort should focus on molecular epidemiological investigations of the two parasites in humans and animals living in close contact in the investigated areas, even in whole Myanmar. These data will aid in making efficient control strategies to intervene with and prevent occurrence of both diseases.

Cryptosporidium and Giardia in dam water on sheep farms – An important source of transmission?

Cryptosporidium and Giardia infections can negatively impact livestock health and reduce productivity, and some species and genotypes infecting livestock have zoonotic potential. Infection occurs via the faecal-oral route. Waterborne infections are a recognised source of infection for humans, but the role of livestock drinking water as a source of infection in livestock has not been described. This study aimed to determine whether contaminated drinking water supplies, such as farm dams, are a likely transmission source for Cryptosporidium and Giardia infections for extensively managed sheep. Dam water samples (n = 47) were collected during autumn, winter and spring from 12 farm dams located on six different farms in south west Western Australia, and faecal samples (n = 349) were collected from sheep with access to these dams. All samples were initially screened for Cryptosporidium spp. at the 18S locus and Giardia spp. at the gdh gene using qPCR, and oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR. Cryptosporidium-positive sheep faecal samples were typed and subtyped by sequence analysis of 18S and gp60 loci, respectively. Giardia-specific PCR and Sanger sequencing targeting tpi and gdh loci were performed on Giardia- positive sheep faecal samples to characterise Giardia duodenalis assemblages. To identify Cryptosporidium and Giardia spp. in dam water samples, next-generation sequencing analysis of 18S and gdh amplicons were performed, respectively. Two species of Cryptosporidium (Cryptosporidium xiaoi and Cryptospordium ubiquitum (subtype family XIIa)) were detected in 38/345 sheep faecal samples, and in water from 9/12 farm dams during the study period, with C. xiaoi the species most frequently detected in both faeces and dam water overall. Giardia duodenalis assemblages AI, AII and E were detected in 36/348 faecal samples and water from 10/12 farm dams. For dam water samples where oo/cysts were detected by qPCR, Cryptosporidium oocyst concentration ranged from 518–2429 oocysts/L (n = 14), and Giardia cyst concentration ranged from 102 to 1077 cysts/L (n = 17). Cryptosporidium and Giardia with zoonotic potential were detected in farm dam water, including C. ubiquitum, C. hominis, C. parvum, C. cuniculus, C. xiaoi, and G. duodenalis assemblages A, B and E. The findings suggest that dam water can be contaminated with Cryptosporidium species and G. duodenalis assemblages that may infect sheep and with zoonotic potential, and farm dam water may represent one source of transmission for infections.

Molecular prevalence and genotyping of Giardia duodenalis in cattle in Central Anatolia Region of Turkey.

The molecular prevalence and genotypes of Giardia duodenalis in cattle were investigated. A total of 450 fecal samples were collected from cattle in three provinces of Central Anatolia from August 2017 to July 2019. Genomic DNA was extracted from the fecal samples and used in molecular analysis carried out by nested PCR analyses of the β-giardin (bg) gene of G. duodenalis. Positive samples were further analyzed by nested PCR at two gene loci (triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh)) for genotyping of G. duodenalis isolates. PCR analyses of the bg gene indicated that the overall prevalence of G. duodenalis was 30.2%. However, lower rates were determined with PCR analyses for gdh and tpi loci. The sequence analyses of the bg, gdh, and tpi genes revealed the presence of zoonotic assemblage A and livestock-specific assemblage E. Combined-sequence analyses revealed that assemblage E was the most common in the study area. Our study provides the first data on the wide prevalence of livestock-specific assemblages E in cattle in Turkey. The prevalence of assemblage A in cattle also reveals the importance of cattle for zoonotic transmission of giardiasis in Turkey.