Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission.
ABSTRACT: BACKGROUND: Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China. METHODOLOGY/PRINCIPAL FINDINGS: 378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather's sugar flotation technique and Lugol's iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n?=?3) and a new subtype VbA21 (n?=?6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples. CONCLUSIONS/SIGNIFICANCE: The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.
Project description:BACKGROUND: Giardia duodenalis is a common intestinal parasite that infects humans and many other mammals, mainly distributing in some areas with poor sanitation. The proportion of the human giardiasis burden attributable to G. duodenalis of animal origin differs in different geographical areas. In Mainland China, genetic data of the gdh and bg genes of G. duodenalis from animals are only limited in dogs and cats. The aim of the study was to provide information on the genetic characterizations of animal-derived G. duodenalis isolates (from rabbits, sheep and cattle) at both loci in Heilongjiang Province, Northeastern China, and to assess the potential for zoonotic transmission. METHODOLOGY/PRINCIPAL FINDINGS: 61 G. duodenalis isolates from animal feces (dairy and beef cattle, sheep and rabbits) in Heilongjiang Province were characterized at the gdh and bg loci in the present study. The gdh and bg gene sequences of sheep-derived G. duodenalis assemblage AI, and the gdh sequences of rabbit-derived G. duodenalis assemblage B had 100% similarity with those from humans, respectively. Novel subtypes of G. duodenalis were identified, with one and seven subtypes for assemblages A and E at the gdh locus, and two and three subtypes for assemblages B and E at the bg locus, respectively. Three pairs of the same bg sequences of assemblage E were observed in sheep and cattle. CONCLUSIONS/SIGNIFICANCE: This is the first description of genetic characterizations of the gdh and bg genes of G. duodenalis from rabbits, sheep and cattle in Mainland China. Homology analysis of assemblages AI and B implied the possibility of zoonotic transmission. The novel subtypes of assemblages of G. duodenalis may represent the endemic genetic characteristics of G. duodenalis in Heilongjiang Province, China.
Project description:Cryptosporidium spp. and Enterocytozoon bieneusi are two prevalent opportunistic pathogens in humans and animals. Currently, few data are available on genetic characterization of both pathogens in rabbits in China. The aim of the present study was to understand prevalence and genetic characterization of Cryptosporidium spp. and E. bieneusi in rabbits. We collected 215 fecal samples from 150 Rex rabbits and 65 New Zealand White rabbits on two different farms in Heilongjiang Province, China. Cryptosporidium spp. and E. bieneusi were tested by polymerase chain reaction (PCR) and sequencing the partial small subunit of ribosomal DNA (SSU rDNA) and the internal transcribed spacer (ITS) region of rDNA, respectively. Cryptosporidium was detected in 3.3% (5/150) of Rex rabbits and 29.2% (19/65) of New Zealand White rabbits. All the 24 Cryptosporidium isolates were identified as C. cuniculus. Enterocytozoon bieneusi was only found in 14.7% (22/150) of Rex rabbits. Five known genotypes: CHN-RD1 (n = 12), D (n = 3), Type IV (n = 2), Peru6 (n = 1), and I (n = 1), and three novel ones CHN-RR1 to CHN-RR3 (one each) were detected. By analyzing the 60-kDa glycoprotein (gp60) gene sequences of C. cuniculus isolates, three subtypes were obtained: VbA28 (n = 2), VbA29 (n = 16), and VbA32 (n = 3). All these three C. cuniculus subtypes were reported previously in humans. Four known E. bieneusi genotypes have been found to be present in humans. The three novel ones fell into zoonotic group 1. The results suggest zoonotic potential of C. cuniculus and E. bieneusi isolates in rabbits.
Project description: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.
Project description:Cattle are the main reservoir host of C. andersoni, which shows a predominance in yearlings and adults of cattle. To understand the subtypes of C. andersoni and the population genetic structure in Heilongjiang Province, fecal specimens were collected from 420 dairy cattle and 405 beef cattle at the age of 12-14 months in eight cattle farms in five areas within this province and were screened for the presence of Cryptosporidium oocysts by microscopy after Sheather's sugar flotation technique. The average prevalence of Cryptosporidium spp. was 19.15% (158/825) and all the Cryptosporidium isolates were identified as C. andersoni by the SSU rRNA gene nested PCR-RFLP using SspI, VspI and MboII restriction enzymes. A total of 50 C. andersoni isolates were randomly selected and sequenced to confirm the RFLP results before they were subtyped by multilocus sequence typing (MLST) at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16). Four, one, two and one haplotypes were obtained at the four loci, respectively. The MLST subtype A4,A4,A4,A1 showed an absolute predominance and a wide distribution among the six MLST subtypes obtained in the investigated areas. Linkage disequilibrium analysis showed the presence of a clonal population genetic structure of C. andersoni in cattle, suggesting the absence of recombination among lineages. The finding of a clonal population genetic structure indicated that the prevalence of C. andersoni in cattle in Heilongjiang Province is not attributed to the introduction of cattle. Thus, prevention and control strategies should be focused on making stricter measures to avoid the occurrence of cross-transmission and re-infection between cattle individuals. These molecular data will also be helpful to explore the source attribution of infection/contamination of C. andersoni and to elucidate its transmission dynamics in Heilongjiang Province, even in China.
Project description:Cryptosporidium spp. and Giardia duodenalis are important gastrointestinal protists in humans and animals worldwide. In China, bovine cryptosporidiosis and giardiasis are of increasing concern because cattle are important reservoirs of these parasites, which have become potential threats to public health and to large numbers of cattle in recent years.A total of 1366 fecal samples from the Ningxia Autonomous Region were examined. The overall infection rates for Cryptosporidium spp. and G. duodenalis were 1.61% and 2.12%, respectively. Cryptosporidium was only detected in preweaned calves and adults older than 2 years, whereas G. duodenalis was only detected in calves aged less than 11 months. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit rRNA gene. Three Cryptosporidium species were identified: C. parvum (n = 15) and C. bovis (n = 4) in preweaned calves, and C. andersoni (n = 4) in adults aged over 2 years. A DNA sequence analysis of the gp60 gene suggested that the 15 C. parvum isolates all belonged to subtype IIdA15G1. Twenty-nine G. duodenalis isolates were analyzed by DNA sequencing of the triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Two G. duodenalis assemblages were identified, assemblages E (n = 15) and B (n = 4, one subtype B1 and three subtype B2) in preweaned calves, and assemblage E (n = 10) in 3-11-month-old calves.The predominance of C. parvum detected in preweaned calves and the first identified subtype IIdA15G1 in dairy cattle, and the dominant G. duodenalis assemblage E in this study differed considerably from those found in Henan, Heilongjiang, and Shannxi Provinces. Our findings further confirm the dominance of C. parvum IId subtypes in China.
Project description:To assess the prevalence and public health significance of rabbit cryptosporidiosis, a total of 1,081 fecal specimens were collected between October 2007 and April 2008 from rabbits on eight farms in five different areas in Henan Province, China, and were examined by microscopy after Sheather's sucrose flotation and modified acid-fast staining. The average infection rate of Cryptosporidium was 3.4% (37/1,081 samples). There was a significant association between the prevalence of Cryptosporidium and the age of animals (chi(2) = 57.13; P < 0.01); the prevalence of cryptosporidiosis in 1- to 3-month-old rabbits was the highest (10.9%). The Cryptosporidium species in microscopy-positive specimens were genotyped by sequence analyses of the 18S rRNA, 70-kDa heat shock protein (HSP70), oocyst wall protein (COWP), and actin genes and were subtyped by sequence analysis of the 60-kDa glycoprotein (gp60) gene. Only the Cryptosporidium rabbit genotype was identified, with 100% sequence identity to published sequences of the 18S rRNA, HSP70, COWP, and actin genes, and the strains belonged to three gp60 subtypes (VbA36, VbA35, and VbA29). In view of the recent finding of the Cryptosporidium rabbit genotype in human outbreak and sporadic cases, the role of rabbits in the transmission of human cryptosporidiosis should be reassessed.
Project description:<h4>Background</h4>In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays.<h4>Methodology/principal findings</h4>Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1-10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67-55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79-11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16-7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58-0.95).<h4>Conclusions/significance</h4>This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites.
Project description:BACKGROUND:The transmission of Cryptosporidium spp. and Giardia duodenalis into humans varies according to species/genotypes of the pathogens. Although infections with both parasites are recorded in Egypt, few data are available on the distribution of Cryptosporidium species and G. duodenalis genotypes. The present study assessed the occurrence and genetic diversity of Cryptosporidium spp. and G. duodenalis in Egyptian children. METHODS:In the present study, 585 fecal specimens were collected from children eight years old and younger in three provinces (El-Dakahlia, El-Gharbia and Damietta) during March 2015 to April 2016. PCR-RFLP analysis of the small subunit rRNA gene and sequence analysis of the 60 kDa glycoprotein gene were used to detect and subtype Cryptosporidium spp., respectively, whereas PCR and sequence analyses of the triose phosphate isomerase, glutamate dehydrogenase and ?-giardin genes were used to detect and genotype Giardia duodenalis. RESULTS:The overall infection rates of Cryptosporidium spp. and G. duodenalis were 1.4% and 11.3%, respectively. The Cryptosporidium species identified included C. hominis and C. parvum, each with three subtype families. The C. hominis subtypes were IbA6G3 (n = 2), IdA17 (n = 1), IdA24 (n = 1) and IfA14G1R5 (n = 1), while C. parvum subtypes were IIdA20G1 (n = 1), IIaA15G2R1 (n = 1), and IIcA5G3a (n = 1). The G. duodenalis identified included both assemblages A (n = 31) and B (n = 34). All G. duodenalis assemblage A belonged to the anthroponotic sub-assemblage AII, while a high genetic heterogeneity was seen within assemblage B. CONCLUSIONS:Data from this study are useful in our understanding of the genetic diversity of Cryptosporidium spp. and G. duodenalis in Egypt and the potential importance of anthroponotic transmission in the epidemiology of both pathogens.
Project description:Giardia duodenalis and Cryptosporidium spp. are common human and animal pathogens. They have increasingly been reported in dairy calves in recent years; however, multilocus genotyping information for G. duodenalis and Cryptosporidium infecting pre-weaned dairy calves in southwestern China is limited. In the present study, the prevalence of G. duodenalis and Cryptosporidium spp. in pre-weaned dairy calves in central Sichuan province was determined and the pathogens were analyzed molecularly. Of 278 fecal samples from pre-weaned dairy calves, 26 (9.4%) were positive for G. duodenalis and 40 (14.4%) were positive for Cryptosporidium spp. Cryptosporidium bovis (n = 28), Cryptosporidium ryanae (n = 5) and Cryptosporidium parvum (n = 7) were detected. All seven C. parvum isolates were successfully subtyped based on the gp60 gene sequence, and only IIdA15G1 was detected. Multilocus sequence typing of G. duodenalis based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes revealed 19 different assemblage E multilocus genotypes (two known and 17 unpublished genotypes). Based on eBURST analysis, a high degree of genetic diversity within assemblage E was observed in pre-weaned dairy calves in Sichuan province. To the best of our knowledge, this is the first study using multilocus sequence typing and eBURST analysis to characterize G. duodenalis in pre-weaned dairy calves in southwestern China.
Project description:Little is known about the prevalence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in deer in China. In this study, 662 fecal samples were collected from 11 farms in Henan and Jilin Provinces between July 2013 and August 2014, and were screened for the presence of Cryptosporidium and G. duodenalis with genotyping and subtyping methods.Cryptosporidium spp. and G. duodenalis were detected in 6.80% (45/662) and 1.21% (5/662) of samples, respectively. Six Cryptosporidium species/genotypes were identified based on the small subunit ribosomal ribonucleic acid (SSU rRNA) gene: C. parvum (n = 11); C. andersoni (n = 5); C. ubiquitum (n = 3); C. muris (n = 1); C. suis-like (n = 1); and Cryptosporidium deer genotype (n = 24). When five of the 11 C. parvum isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene, zoonotic subtypes IIaA15G2R2 (n = 4) and IIdA19G1 (n = 1) were found. According to a subtype analysis, three C. ubiquitum isolates belonged to XIIa subtype 2. In contrast, only assemblage E was detected in the five Giardia-positive samples with small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequencing.To our knowledge, this is the first study to report C. andersoni, as well as C. parvum zoonotic subtypes IIaA15G2R2 and IIdA19G1 in cervids. These data, though limited, suggest that cervids may be a source of zoonotic Cryptosporidium and Giardia. Cervids in the present study are likely to be of low zoonotic potential to humans, and more molecular epidemiological studies are required to clarify the prevalence and public health significance of Cryptosporidium and G. duodenalis in cervids throughout China.