Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis
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ABSTRACT:
SUBMITTER: O’ Leary J
PROVIDER: S-EPMC7724200 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundNon-human primates are often infected with human-pathogenic Cryptosporidium hominis subtypes, but rarely with Cryptosporidium parvum. In this study, 1452 fecal specimens were collected from farmed crab-eating macaques (Macaca fascicularis) in Hainan, China during the period April 2016 to January 2018. These specimens were analyzed for Cryptosporidium species and subtypes by using PCR and sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively.ResultsAltogether, Cryptosporidium was detected using 18S rRNA-based PCR in 132 (9.1%) sampled animals, with significantly higher prevalence in females (12.5% or 75/599 versus 6.1% or 43/706), younger animals (10.7% or 118/1102 in monkeys 1-3-years-old versus 4.0% or 14/350 in those over 3-years-old) and animals with diarrhea (12.6% or 46/365 versus 7.9% or 86/1087). Four Cryptosporidium species were identified, namely C. hominis, C. parvum, Cryptosporidium muris and Cryptosporidium ubiquitum in 86, 30, 15 and 1 animal, respectively. The identified C. parvum, C. hominis and C. ubiquitum were further subtyped by using gp60 PCR. Among them, C. parvum belonged to subtypes in two known subtype families, namely IIoA14G1 (in 18 animals) and IIdA19G1 (in 2 animals). In contrast, C. hominis mostly belonged to two new subtype families Im and In, which are genetically related to Ia and Id, respectively. The C. hominis subtypes identified included ImA18 (in 38 animals), InA14 (in six animals), InA26 (in six animals), InA17 (in one animal) and IiA17 (in three animals). The C. ubiquitum isolates belonged to subtype family XIId. By subtype, ImA18 and IIoA14G1 were detected in animals with diarrhea whereas the remaining ones were mostly found in asymptomatic animals. Compared with C. parvum and C. muris, higher oocyst shedding intensity was observed in animals infected with C. hominis, especially those infected with the Im subtype family.ConclusionsData from the study suggest that crab-eating macaques are infected with diverse C. parvum and C. hominis subtypes. The C. parvum IIo subtype family previously seen in rodents in China has apparently expanded its host range.
| S-EPMC6631616 | biostudies-literature
Project description:Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ2 = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocyst-positive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.
| S-EPMC7087732 | biostudies-literature
Project description:The 2 main species of Cryptosporidium that infect humans are Cryptosporidium hominis and C. parvum. Here, multilocus fragment analysis of 3 microsatellite loci (ML1, ML2, and gp60) was used to subtype strains from sporadic cases of cryptosporidiosis in Wales and northwest England. Of 72 strains of C. parvum, 63 were typeable at all 3 loci, forming 31 subtypes. These strains formed 3 broad clusters, representing 74.6%, 20.6%, and 4.8% of typeable strains. Of 118 C. hominis strains, 106 were typeable at all 3 loci, forming 9 subtypes; however, 90% belonged to the same subtype. Analysis with epidemiologic data found an association between strains from case-patients who reported contact with farm animals and individual C. parvum microsatellite alleles. The strongest association was with ML1; all strains from case-patients that reported farm animal contact had the same allele (ML1-242). Microsatellite typing of C. parvum provides valuable additional information on the epidemiology of this pathogen.
| S-EPMC2725800 | biostudies-literature
Project description:BACKGROUND:Bamboo rats are widely farmed in southern China for meat, but their potential in transmitting pathogens to humans and other farm animals remains unclear. METHODS:To understand the transmission of Cryptosporidium spp. in these animals, 709 fecal samples were collected in this study from Chinese bamboo rats (Rhizomys sinensis) on nine farms in Jiangxi, Guangxi and Hainan provinces, China. They were analyzed for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. Cryptosporidium parvum, C. parvum-like and C. ubiquitum-like genotypes identified were subtyped by sequence analysis of the 60 kDa glycoprotein (gp60) gene. RESULTS:Altogether, Cryptosporidium spp. were detected in 209 (29.5%) samples. The detection rate in samples from animals under two months of age (70.0%,105/150) was significantly higher than in samples from animals above 2 months (18.6%, 104/559; ?2?=?150.27, df?=?1, P?<?0.0001). Four Cryptosporidium species/genotypes were identified: C. parvum (n?=?78); C. occultus (n?=?1); a new genotype that is genetically related to C. ubiquitum (n?=?85); and another new genotype that is genetically related to C. parvum (n?=?44). Among them, C. parvum (27,610?±?71,911 oocysts/gram of feces) and the C. parvum-like genotype (38,679?±?82,811 oocysts/gram of feces) had higher oocyst shedding intensity than the C. ubiquitum-like genotype (2470?±?7017 oocysts/gram of feces) and the C. occultus (1012 oocysts/gram of feces). The C. parvum identified belonged to three subtypes in two rare subtype families, including IIpA9 (n?=?43), IIpA6 (n?=?6) and IIoA15G1 (n?=?9), while the C. parvum-like and C. ubiquitum-like genotypes generated very divergent gp60 sequences. CONCLUSIONS:Results of the present study suggest that bamboo rats on the study farms were infected with diverse Cryptosporidium species and divergent C. parvum subtypes, which probably had originated from their native habitats. As similar C. parvum subtypes have been recently detected in humans and farmed macaques, attentions should be paid to the potential role of these new farm animals in the transmission of zoonotic pathogens.
| S-EPMC7092434 | biostudies-literature
Project description:Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.
| S-EPMC10464362 | biostudies-literature
Project description:BackgroundFeline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70.MethodsWe have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker.ResultsSequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed.ConclusionsWe have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations.
| S-EPMC6979280 | biostudies-literature
Project description:Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.
| S-EPMC130769 | biostudies-literature
Project description:Bamboo rats (Rhizomys sinensis) are widely farmed in Guangdong, China, but the distribution and public health potential of Cryptosporidium spp. in them are unclear. In this study, 724 fecal specimens were collected from bamboo rats in Guangdong Province and analyzed for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. The overall detection rate of Cryptosporidium spp. was 12.2% (88/724). By age, the detection rate in animals under 2 months (23.2% or 13/56) was significantly higher than in animals over 2 months (11.2% or 75/668; ?2 = 6.95, df = 1, p = 0.0084). By reproduction status, the detection rate of Cryptosporidium spp. in nursing animals (23.1% or 27/117) was significantly higher than in other reproduction statuses (6.8% or 4/59; ?2 = 7.18, df = 1, p = 0.0074). Five Cryptosporidium species and genotypes were detected, including Cryptosporidium bamboo rat genotype I (n = 49), C. parvum (n = 31), Cryptosporidium bamboo rat genotype III (n = 5), C. occultus (n = 2), and C. muris (n = 1). The average numbers of oocysts per gram of feces for these Cryptosporidium spp. were 14,074, 494,636, 9239, 394, and 323, respectively. The genetic uniqueness of bamboo rat genotypes I and III was confirmed by sequence analyses of the 70 kDa heat shock protein and actin genes. Subtyping C. parvum by sequence analysis of the 60 kDa glycoprotein gene identified the presence of IIoA15G1 (n = 20) and IIpA6 (n = 2) subtypes. The results of this study indicated that Cryptosporidium spp. are common in bamboo rats in Guangdong, and some of the Cryptosporidium spp. in these animals are known human pathogens.
| S-EPMC7761605 | biostudies-literature
Project description:BackgroundCryptosporidium parvum is a zoonotic pathogen worldwide. Extensive genetic diversity and complex population structures exist in C. parvum in different geographical regions and hosts. Unlike the IIa subtype family, which is responsible for most zoonotic C. parvum infections in industrialized countries, IId is identified as the dominant subtype family in farm animals, rodents and humans in China. Thus far, the population genetic characteristics of IId subtypes in calves in China are not clear.MethodsIn the present study, 46 C. parvum isolates from dairy and beef cattle in six provinces and regions in China were characterized using sequence analysis of eight genetic loci, including msc6-7, rpgr, msc6-5, dz-hrgp, chom3t, hsp70, mucin1 and gp60. They belonged to three IId subtypes in the gp60 gene, including IIdA20G1 (n = 17), IIdA19G1 (n = 24) and IIdA15G1 (n = 5). The data generated were analyzed for population genetic structures of C. parvum using DnaSP and LIAN and subpopulation structures using STRUCTURE, RAxML, Arlequin, GENALEX and Network.ResultsSeventeen multilocus genotypes were identified. The results of linkage disequilibrium analysis indicated the presence of an epidemic genetic structure in the C. parvum IId population. When isolates of various geographical areas were treated as individual subpopulations, maximum likelihood inference of phylogeny, pairwise genetic distance analysis, substructure analysis, principal components analysis and network analysis all provided evidence for geographical segregation of subpopulations in Heilongjiang, Hebei and Xinjiang. In contrast, isolates from Guangdong, Shanghai and Jiangsu were genetically similar to each other.ConclusionsData from the multilocus analysis have revealed a much higher genetic diversity of C. parvum than gp60 sequence analysis. Despite an epidemic population structure, there is an apparent geographical segregation in C. parvum subpopulations within China.
| S-EPMC7437029 | biostudies-literature
Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.
| S-EPMC3227725 | biostudies-literature