Molecular Characterization Of Cryptosporidium Research Articles

Prevalence and molecular characterization of Cryptosporidium spp. in pre-weaned diarrheic dairy calves and their bedding materials in northern China.

Cryptosporidiosis, primarily caused by Cryptosporidium parvum, is a significant cause of diarrhea in pre-weaned dairy calves. To investigate the prevalence of Cryptosporidium among pre-weaned diarrheic dairy calves and identify potential sources of infection in northern China, 234 fecal samples from 18 farms in six regions were analyzed for Cryptosporidium. Furthermore, 217 bedding samples from both occupied and unoccupied calf hutches, heating lamp pens, and individual calving pens in eight farms in Beijing were also examined for the presence of the parasite. All samples were screened for Cryptosporidium spp. using nested PCR targeting the SSU rRNA gene fragment, and C. parvum was subtyped with nested PCR targeting the 60kDa glycoprotein gene. The prevalence of Cryptosporidium was 33.3%, with C. parvum and C. bovis constituting 29.9% and 3.4% of cases, respectively. The positive rate of Cryptosporidium in 1- to 4-week-old calves ranged from 9.6 to 63.6%. Analysis of the gp60 fragment of C. parvum revealed four subtypes: IIdA15G1, IIdA17G1, IIdA19G1, and IIdA20G1. Besides the bedding samples in heating lamp pens, both C. parvum and C. bovis were detected in bedding samples throughout the other regions. A significant positive correlation between the detection rate of Cryptosporidium in fecal samples and that in the bedding materials of occupied calf hutches (R = 0.93, P = 0.002). These findings suggest that C. parvum is the predominant species among pre-weaned diarrheic dairy calves in northern China. Contaminated bedding materials may act as sources of infection for newborn calves.

The Prevalence of Cryptosporidium spp. and Giardia duodenalis in Marmota himalayana (Rodentia: Sciuridae) in the Qinghai Tibetan Plateau area, China.

Cryptosporidium and Giardia are well-known important intestinal zoonotic pathogens that can infect various hosts and cause diarrhoeal diseases. We aimed to determine the epidemiological prevalence and molecular characterization of Cryptosporidium and Giardia species in Himalayan marmot (Marmota himalayana, class Marmota) in the Qinghai Tibetan Plateau Area of Qinghai Province, Northwest China. Overall, 243 Himalayan marmot fecal samples were collected in 2017 and in 2019 and a two-step nested PCR technique was performed to amplify the fragments of the SSU rRNA gene of Cryptosporidium and 18S ribosomal RNA gene of Giardia. Molecular characterization of Cryptosporidium was performed with the primary primers NDIAGF2 and N-DIAGR2, the secondary primers CPB-DIAGF and CPB-DIAGR. Similarly, molecular characterization of Giardia was used the first primers Gia2029 and Gia2150c, the secondary primers RH11 and RH4. The positive PCR products were sequenced and the sequences were processed by Clustal Omega and BLAST. Phylogenetic analysis was achieved by NJ method in MEGA. The infection rate of Cryptosporidium spp. and G. duodenalis was 4.9% (12/243) and 0.8% (2/243) in M. himalayana, respectively. Cryptosporidium spp. are characterized as novel genotypes Cryptosporidium marmot genotype I (n=3) and Cryptosporidium marmot genotype II (n=9); G. duodenalis assemblage A (n=2) was found in M. himalayana. This is the first report of Cryptosporidium spp. and G. duodenalis infections in M. himalayana in Qinghai of Northwest China. The results indicate the existence of Cryptosporidium species and G. duodenalis infections that may have a potential public health significance.

Molecular characterization of Cryptosporidium spp. in Bactrian camels (Camelus bactrianus) from Yili Kazak Autonomous Prefecture of Xinjiang, China.

Cryptosporidium spp. is a significant zoonotic parasite. The prevalence and infection characteristics of Cryptosporidium spp. in Bactrian camels in Yili Kazak Autonomous Prefecture have yet to be fully understood. Thus, the molecular epidemiology of cryptosporidiosis in camels was investigated in this region. A total of 1,455 fecal samples were collected from 6 counties in three regions (Altay, Tacheng, and Yili) in Yili Prefecture. Nested PCR targeting the small subunit ribosomal RNA (ssu rRNA) gene was used to identify the species or genotypes of Cryptosporidium infection in camels. For C. parvum positive samples, the subtypes were identified using the 60-kDa glycoprotein (gp60) gene. The overall infection rate was 8.7% (126/1,455), ranging from 5.6% to 11.7% in different regions, and 4.2% to 15.8% in different counties. A significant difference was observed amongst the counties (p < 0.001). Three species were detected, namely C. andersoni (65.1%, 82/126), C. parvum (34.1%, 43/126), and C. occultus (0.8%, 1/126). Three C. parvum subtypes, If-like-A15G2 (n = 29), IIdA15G1 (n = 4), and IIdA19G1(n = 1) were detected, with If-like-A15G2 being the most prevalent subtype. Camels aged 3-12 months exhibited the highest infection rate (11.4%, 44/387), with no significant difference among age groups (p > 0.05). C. parvum was predominant in camels under 3 months, while C. andersoni prevailed in camels over 3 months. There was an extremely significant difference observed among seasons (p < 0.001), summer had the highest infection rates (16.9%, 61/360). This study collected nearly 1,500 samples and, for the first time, investigated Cryptosporidium spp. infection in camels based on different age groups and seasons. All three Cryptosporidiumspecies identified were zoonotic, posing a potential threat to human health and requiring close attention.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Cattle in Heilongjiang Province, Northeast China.

Crytosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are important diarrheal pathogens with a global distribution that threatens the health of humans and animals. Despite cattle being potential transmission hosts of these protozoans, the associated risks to public health have been neglected. In the present study, a total of 1155 cattle fecal samples were collected from 13 administrative regions of Heilongjiang Province. The prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi were 5.5% (64/1155; 95% CI: 4.2-6.9), 3.8% (44/1155; 95% CI: 2.7-4.9), and 6.5% (75/1155; 95% CI: 5.1-7.9), respectively. Among these positive fecal samples, five Cryptosporidium species (C. andersoni, C. bovis, C. ryanae, C. parvum, and C. occultus), two G. duodenalis assemblages (E and A), and eight E. bieneusi genotypes (BEB4, BEB6, BEB8, J, I, CHS7, CHS8, and COS-I) were identified. Phylogenetic analysis showed that all eight genotypes of E. bieneusi identified in the present study belonged to group 2. It is worth noting that some species/genotypes of these intestinal protozoans are zoonotic, suggesting a risk of zoonotic disease transmission in endemic areas. The findings expanded our understanding of the genetic composition and zoonotic potential of Cryptosporidium spp., G. duodenalis, and E. bieneusi in cattle in Heilongjiang Province.

Molecular characterization of Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi in eleven wild rodent species in China: Common distribution, extensive genetic diversity and high zoonotic potential

Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi are common zoonotic pathogens in humans and animals. Although rodents are important parts of the ecosystem and common hosts for these pathogens, little is known of the distribution, genetic diversity and zoonotic potential of these pathogens in wild rodents. A total of 442 fecal samples were collected from eleven wild rodent species in three provinces of China, and analyzed for these pathogens by PCR and DNA sequencing. The infection rates of Cryptosporidium spp., Giardia spp. and E. bieneusi were 19.9% (88/442), 19.8% (75/378) and 12.2% (54/442), respectively. Altogether, 23 known Cryptosporidium species/genotypes were identified and their distribution varied among different sampling locations or rodent species. Subtyping of the zoonotic Cryptosporidium species identified two novel subtype families XVe and XVf in C. viatorum, the subtype family XIIh and a novel subtype family XIIj in C. ubiquitum, and the subtype family IId in C. parvum. Three Giardia species were identified, including G. microti (n = 57), G. muris (n = 15) and G. duodenalis (n = 3), with G. duodenalis assemblages A and G identified in brown rats in urban areas of Guangdong. In addition, 13 E. bieneusi genotypes including eight known and five novel ones were identified, belonging to Groups 1, 2, 10, 14 and 15. Within nine genotypes in the zoonotic Group 1, common human-pathogenic genotypes D, Type IV, PigEbITS7 and Peru8 were detected only in brown rats and Lesser rice-field rats in urban areas of Guangdong. Apparent host adaptation and geographical differences were observed among Cryptosporidium spp., Giardia spp. and E. bieneusi genotypes in wild rodents in the present study. Furthermore, the zoonotic Cryptosporidium species and E. bieneusi genotypes commonly found here suggest a high zoonotic potential of these pathogens in wild rodents, especially in brown rats in urban areas. Hygiene and One Health measures should be implemented in urban streets and food stores to reduce the possible direct and indirect transmission of these rodent-related pathogens.

Prevalence and molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common intestinal pathogens that infect humans and animals. To date, research regarding these three protozoa in the Ningxia Hui Autonomous Region (Ningxia) has mostly been limited to a single pathogen, and comprehensive data on mixed infections are unavailable. This study aimed to evaluate the zoonotic potential of these three protozoa. In this study, small subunit ribosomal RNA (SSU rRNA) and 60 kDa glycoprotein (gp60) genes of Cryptosporidium; internal transcribed spacer (ITS) gene of E. bieneusi; and SSU rRNA, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis were examined. DNA extraction, polymerase chain reaction, and sequence analysis were performed on fecal samples collected from 320 dairy cattle at three intensive dairy farms in Ningxia in 2021 to determine the prevalence and genetic characteristics of these three protozoa. The findings revealed that 61.56% (197/320) of the samples were infected with at least one protozoan. The overall prevalence of Cryptosporidium was 19.38% (62/320), E. bieneusi was 41.56% (133/320), and G. duodenalis was 29.38% (94/320). This study identified four Cryptosporidium species (C. bovis, C. andersoni, C. ryanae, and C. parvum) and the presence of mixed infections with two or three Cryptosporidium species. C. bovis was the dominant species in this study, while the dominant C. parvum subtypes were IIdA15G1 and IIdA20G1. The genotypes of E. bieneusis were J, BEB4, and I alongside the novel genotypes NX1-NX8, all belonging to group 2, with genotype J being dominant. G. duodenalis assemblages were identified as assemblages E, A, and B, and a mixed infection involving assemblages A + E was identified, with assemblage E being the dominant one. Concurrently, 11 isolates formed 10 different assemblage E multilocus genotypes (MLGs) and 1 assemblage A MLG and assemblage E MLGs formed 5 subgroups. To the best of our knowledge, this is the first report on mixed infection with two or three Cryptosporidium species in cattle in Ningxia and on the presence of the C. parvum subtype IIdA20G1 in this part of China. This study also discovered nine genotypes of E. bieneusis and novel features of G. duodenalis assemblages in Ningxia. This study indicates that dairy cattle in this region may play a significant role in the zoonotic transmission of Cryptosporidium spp., E. bieneusi, and G. duodenalis.

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

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

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

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

Occurrence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in Lambs and Calves in Southeastern Anatolia, Turkey

Occurrence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in Lambs and Calves in Southeastern Anatolia, Turkey

Molecular characterization of Cryptosporidium in wild rodents from the Inner Mongolian Autonomous Region and Liaoning Province, China: assessing host specificity and the potential for zoonotic transmission.

Wild rodents are key hosts for Cryptosporidium transmission, yet there is a dearth of information regarding their infection status in the Inner Mongolian Autonomous Region and Liaoning Province of China. Therefore, the present study was conducted to determine the prevalence and genetic characteristics of Cryptosporidium among wild rodents residing in these two provinces. A total of 486 rodents were captured, and fresh feces were collected from each rodent's intestine for DNA extraction. Species identification of rodents was performed through PCR amplification of the vertebrate cytochrome b (cytb) gene. To detect the presence of Cryptosporidium in all fecal samples, PCR analysis and sequencing of the partial small subunit of the ribosomal RNA (rRNA) gene were performed. Four species of rodents were identified: Rattus norvegicus, Mus musculus, Apodemus agrarius, and Cricetulus barabensis. Positive results for Cryptosporidium were obtained for 9.2% (18/195), 6.6% (7/106), 5.6% (5/89), and 6.3% (6/96) of these rodents, respectively, with an average infection rate of 7.4% (36/486). The identification revealed the presence of five Cryptosporidium species, C. ubiquitum (n = 8), C. occultus (n = 5), C. muris (n = 2), C. viatorum (n = 1), and C. ratti (n = 1), along with two Cryptosporidium genotypes: Rat genotype III (n = 10) and Rat genotype IV (n = 9). Based on the molecular evidence presented, the wild rodents investigated were concurrently infected with zoonotic (C. muris, C. occultus, C. ubiquitum and C. viatorum) as well as rodent-adapted (C. ratti and Rat genotype III and IV) species/genotypes, actively participating in the transmission of cryptosporidiosis.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy and beef cattle in Shanxi, China.

Cryptosporidium spp. are key gastrointestinal protists in humans and animals worldwide. Infected cattle are considered the main source of cryptosporidiosis outbreaks in humans. However, little is known about the genetic makeup of Cryptosporidium populations in Shanxi province, China. We analyzed 858 fecal samples collected from farms in Shanxi. The presence of Cryptosporidium spp. was determined via polymerase chain reaction and subsequent sequence analysis of the small subunit rRNA gene as well as restriction fragment length polymorphism analysis. Cryptosporidium parvum was subtyped following sequence analysis of the 60 kDa glycoprotein gene (gp60). The overall prevalence of Cryptosporidium in cattle was 11.19%, with a prevalence of 13.30% and 8.67% in Lingqiu and Yingxian, respectively. The overall prevalence of Cryptosporidium in dairy and beef cattle was 10.78% and 11.50%, respectively. Cryptosporidium infection was detected across all analyzed age groups. The overall prevalence of Cryptosporidium in diarrhea and nondiarrhea samples was 18.24% and 9.72%, respectively, whereas that in intensively farmed and free-range cattle was 17.40% and 3.41%, respectively. We identified five Cryptosporidium species, with C. andersoni being the dominant species. Further, two cases of mixed infections of Cryptosporidium species were detected. All identified C. parvum isolates belonged to the subtype IIdA17G1.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli in Dairy Goat Kids with Diarrhea in Partial Regions of Shaanxi Province, China.

Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are important diarrheal pathogens threatening the health of humans and various animals. Goats, especially pre-weaned goat kids, that carry these pathogens are important reservoirs related to human infection. In the present study, PCR-based sequencing techniques were applied to characterize Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in 202 fecal samples of diarrheal kids for Guanzhong dairy goats from five locations in Shaanxi Province. The positive rates of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli were 37.6% (76/202), 16.3% (33/202), 55.4% (112/202) and 78.7% (159/202) in these goat kids, respectively. Co-infection of two to four pathogens was found in 114 of 202 fecal samples. Significant differences (p < 0.001) in the positive rates of Cryptosporidium spp. and G. duodenalis were found among locations and age groups. Furthermore, two Cryptosporidium species (C. parvum and C. xiaoi), two G. duodenalis assemblages (E and A), nine E. bieneusi genotypes (CHG3, CHG1, BEB6, CHG5, CHG2, SX1, CHG28, COS-II and CD6) and two E. coli pathotypes (EPEC and EHEC) were identified. As for Cryptosporidium, two (IIdA19G1 and IIdA19G2) and two (XXIIIa and XXIIIg) subtypes were recognized in samples positive for C. parvum and C. xiaoi, respectively. A phylogenetic analysis based on the ITS locus of E. bieneusi indicated that all nine genotypes of E. bieneusi identified in this study belonged to the group 2. Four virulence factors (ehxA, eae, stx2 and stx1) of EPEC and EHEC were found in E. coli strains. Collectively, this study explored the colonization frequency of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats in Shaanxi Province and expanded our understanding of the genetic composition and zoonotic potential of these pathogens in goats.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China

BackgroundCryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia.MethodsWe focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium.ResultsSSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1.ConclusionsTo the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Diarrheic and Non-Diarrheic Calves from Ningxia, Northwestern China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are significant parasitic gastrointestinal pathogens with global distribution in humans and domestic animals, including calves. The main symptoms of calf infection are severe diarrhea, dehydration, growth retardation, and sometimes even death. To date, there has been limited information on the prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi infections in calves in Ningxia, China, especially between diarrheic and non-diarrheic calves. A total of 438 fecal samples were collected from diarrheic (201) and non-diarrheic (237) calves in Ningxia. PCR and DNA sequencing were used to find the prevalence of Cryptosporidium spp. at 46.8% (205/438), G. duodenalis at 16.9% (74/438), and E. bieneusi at 10.0% (44/438). The prevalence of Cryptosporidium spp. infection in diarrheic and non-diarrheic calves was 54.0% (128/237) and 38.3% (77/201), respectively, and statistical analysis showed a positive correlation between the prevalence of Cryptosporidium spp. infection and calf diarrhea (p < 0.01). However, in this study, there was no statistical correlation between the prevalence of G. duodenalis infection as well as E. bieneusi infection and calf diarrhea (p > 0.05). Furthermore, four known Cryptosporidium species were successfully identified by comparing them with SSU rRNA gene sequences, including C. parvum, C. bovis, C. ryanae, and C. andersoni. In addition, all 74 G. duodenalis-positive samples were identified as assemblage E by comparative analysis of bg gene sequences. Among the 44 E. bieneusi-positive samples sequenced in the present study, 4 distinct E. bieneusi genotypes were successfully identified by comparative analysis of ITS sequences, including 3 known genotypes (J, BEB4, and N) and 1 novel genotype, the latter of which was identified and designated as NX1. These findings indicated that the high genetic diversity and complex population structures of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia diarrhea calves and non-diarrhea calves, which provide new data for understanding the epidemiological status of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia calves.

Molecular characterization of Cryptosporidium and Giardia in environmental samples and faecal samples from biogas users in Bac Giang, Vietnam

Little is known about Cryptosporidium and Giardia in biogas waste and humans in Vietnam. There is a potential risk of infections during or after using the biogas system. The detected protozoan genotypes are zoonotic pathogens, and contamination of vegetables may relay through runoff to the surface waters and soil. The objective of this study was to understand the role of the environment in the epidemiology of human infections in Bac Giang province, Vietnam, with a focus on investigating the presence of Cryptosporidium spp. genotypes and Giardia assemblages among 239 environmental samples and 94 faecal samples of biogas users. PCR and sequencing analysis were used to identify the occurrence and genotypes of Cryptosporidium and Giardia in these samples. Results showed that 13/333 (3.9 %) and 9/333 (2.7 %) samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively. Characterization revealed the presence of Cryptosporidium scrofarum, C. suis, C. meleagridis, C. bailey and Giardia intestinalis assemblage A and E. C. scrofarum and Giardia assemblage E were identified for the first time in humans in Bac Giang. The current information from the above investigations will be valuable for protozoan source tracking and control interventions against Cryptosporidium and Giardia infection associated with biogas wastes in Vietnam.

Prevalence and molecular characterization of Cryptosporidium in captive-bred Mustela putorius furo in Jiangsu Province

To investigate the prevalence and molecular features of Cryptosporidium in captive-bred Mustela putorius furo in Jiangsu Province. A total of 290 fresh stool samples were collected from a ferret farm in Jiangsu Province on May 2017, and the small subunit rRNA (SSU rRNA) gene of Cryptosporidium was amplified in stool samples using nested PCR assay. The actin, cowp and gp60 genes were amplified in positive samples and sequenced to characterize Cryptosporidium species/genotypes. A total of 18 stool samples were tested positive for Cryptosporidium SSU rRNA gene, with a detection rate of 6.2%. Sequence and phylogenetic analyses of SSU rRNA, actin and cowp genes characterized Cryptosporidium isolated from captive-bred ferrets as Cryptosporidium sp. ferret genotype. In addition, gp60 gene was amplified in 10 out of 18 stool samples tested positive for Cryptosporidium. Cryptosporidium is widely prevalent in captive-bred ferrets in Jiangsu Province, and Cryptosporidium sp. ferret genotype is the only Cryptosporidium genotype in ferrets.

Prevalence and molecular characterization of Cryptosporidium spp. in calves in the Siirt Province, Türkiye

Cryptosporidiosis, one of the main protozoan infections of the last century, is especially dangerous for calves and causes significant economic losses. This research was carried out to determine the prevalence of Cryptosporidium spp. by microscopic and molecular methods and to determine subtypes in 100 calves up to 6 months old and with diarrhoea in the Siirt Province, Türkiye. As a result of the microscopic examination (Kinyoun’s acid-fast), Cryptosporidium spp. oocysts were found in 8 (8%) of 100 samples. As a result of nested PCR, 826-864 bp specific bands for Cryptosporidium spp. were obtained in 13 (13%) of 100 samples. When the DNA sequences of the SSU rRNA gene were compared with the NCBI Basic Local Alignment Search Tool database, it was determined that eight samples sequence analyses showed 100% similarity with the C. parvum, C. ryanae, and C. bovis samples. The detection of C. parvum, which has zoonotic importance in this study, suggests that calves with diarrhoea may be a source of contamination for other animals and humans. Therefore, animal owners and people in close contact with animals should be informed about the public health of cryptosporidiosis.

Molecular characterization of Cryptosporidium in ruminants and observation of natural infection by Cryptosporidium andersoni in sheep from Paraná, Brazil.

The aim of this study was to identify Cryptosporidium species found in cattle and sheep in Paraná, southern region of Brazil. Individual fecal samples from 458 bovines and 101 sheep were submitted for molecular analysis by PCR and nested PCR using specific primers for sequences of the 18S ribosomal unit (rRNA). Positive samples were analyzed using restriction fragment length polymorphism (RFLP), followed by genetic sequencing for species confirmation. The occurrence of Cryptosporidium was 11.27% (63/559). The highest occurrence was detected in lambs (12/59, 20.33%). From the 63 positive samples, it was possible to identify the species in 58 of them by RFLP and genetic sequencing. Five species of Cryptosporidium were identified: Cryptosporidium andersoni, Cryptosporidium bovis, Cryptosporidium ryanae, Cryptosporidium xiaoi, and Cryptosporidium parvum. The most prevalent species was C. andersoni (41.38%) and the least predominant was C. parvum (10.34%). The most abundant species of Cryptosporidium in dairy calves were C. andersoni (11/25) and C. ryanae (6/25). Of the 17 positive sheep, nine (52.94%) were infected with C. andersoni. This finding is the first report on the occurrence of C. andersoni in naturally infected sheep in Brazil and the first observation of a high absolute occurrence of this Cryptosporidium species in sheep.

First detection of Cryptosporidium proventriculi from wild birds in Cyprus.

Cryptosporidium is an important intestinal zoonotic pathogen that can infect various hosts and cause diarrheal disease. There are no reports on the prevalence and molecular characterization of Cryptosporidium in wild birds in Cyprus. Therefore, the present study aimed to determine the prevalence of Cryptosporidium spp. and genotypes in wild birds found at Phassouri Reedbeds (Akrotiri Wetlands), Cyprus. Fecal samples of 75 wild birds (Eurasian coot Fulica atra, N = 48; Eurasian teal Anas crecca, N = 20; duck - Anas spp., Ν = 7) were screened for Cryptosporidium by PCR amplification and sequencing. Only one sample (1.3%) belonging to a Eurasian coot was PCR-positive for Cryptosporidium. Based on sequencing of the 18S rRNA locus, this species was identified as Cryptosporidium proventriculi. This is the first report on the molecular identification of this Cryptosporidium species in a Eurasian coot.