Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate

This study was undertaken in order to characterize a Cryptosporidium muris-like parasite isolated from cattle in Hungary and to compare this strain with other Cryptosporidium species. To date, the large-type oocysts isolated from cattle were considered as C. muris described from several mammals. The size, form, and structure of the oocysts of the Hungarian strain were identical with those described by others from cattle. An apparent difference between the morphometric data of C. muris-like parasites isolated from cattle or other mammals was noted, which is similar in magnitude to the differences between Cryptosporidium meleagridis and Cryptosporidium felis or between Cryptosporidium serpentis and Cryptosporidium baileyi. The cross-transmission experiments confirmed the findings of others, as C. muris-like oocysts isolated from cattle fail to infect other mammals. The sequence of the variable region of small subunit (SSU) rRNA gene of the strain was 100% identical with that of the U.S. Cryptosporidium andersoni and C. andersoni-like isolates from cattle. The difference between the SSU rRNA sequence of bovine strains and C. muris is similar in magnitude to the differences between C. meleagridis and Cryptosporidium parvum anthroponotic genotype or between Cryptosporidium wrairi and C. parvum zoonotic genotype. Our findings confirm that the Cryptosporidium species responsible for abomasal cryptosporidiosis and economic losses in the cattle industry should be considered a distinct species, C. andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000.     

Genotyping Cryptosporidium andersoni in Cattle in Shaanxi Province,Northwestern China

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.     

Multilocus fragment typing and genetic structure of Cryptosporidium parvum Isolates from diarrheic preweaned calves in Spain

A collection of 140 Cryptosporidium parvum isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the C. parvum isolates from cattle farms in this geographical area.     

Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus

To further validate previous observations in the taxonomy of Cryptosporidium parasites, the phylogenetic relationship was analyzed among various Cryptosporidium parasites at the actin locus. Nucleotide sequences of the actin gene were obtained from 9 putative Cryptosporidium species (C. parvum, C. andersoni, C. baileyi, C. felis, C. meleagridis, C. muris, C. saurophilum, C. serpentis, and C. wrairi) and various C. parvum genotypes. After multiple alignment of the obtained actin sequences, genetic distances were measured, and phylogenetic trees were constructed. Results of the analysis confirmed the presence of genetically distinct species within Cryptosporidium and various distinct genotypes within C. parvum. The phylogenetic tree constructed on the basis of the actin sequences was largely in agreement with previous results based on small subunit rRNA, 70-kDa heat shock protein, and Cryptosporidium oocyst wall protein genes. The Cryptosporidium species formed 2 major clades; isolates of C. andersoni, C. muris, and C. serpentis formed the first major group, whereas isolates of all other species, as well as various C. parvum genotypes, formed the second major group. Intragenotype variations were low or absent at this locus.     

Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.     

Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world.     

Occurrence and Genetic Characteristics of Cryptosporidium hominis and Cryptosporidium andersoni in Horses from Southwestern China

A total of 333 fecal specimens from horses in southwestern China were genotyped based on analysis of the small subunit rRNA (SSU rRNA) gene. Cryptosporidium hominis and Cryptosporidium andersoni were identified in 2 and 4 stool specimens, respectively. The identification of C. hominis was confirmed by sequence analysis of the 70‐kDa heat shock protein (HSP70) and oocyst wall protein (COWP) genes. Subtyping analysis of the 60‐kDa glycoprotein (GP60) gene sequence of C. hominis revealed a new rare subtype Id, named IdA15; only three Id isolates have been reported in humans to date. Multilocus sequence typing (MLST) analysis indicated that the C. andersoni subtype was A6, A5, A2, and A1 at the four minisatellite loci (MS1, MS2, MS3, and MS16, respectively). This is the first report to identify the presence of C. andersoni and C. hominis in horses in southwestern China and the first to identify a rare zoonotic subtype Id of C. hominis in horses. These findings suggest that infected horses may act as potential reservoirs of Cryptosporidium to transmit infections to humans.     

Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.     

Molecular characterization of Cryptosporidium sp. isolated from northern Alaskan caribou (Rangifer tarandus)

Cryptosporidium sp. was found in 3 out of 49 caribou (Rangifer tarandus) from northern Alaska. Segments of both the 18S ribosomal RNA and the heat shock protein genes were amplified from the caribou isolate and compared with that obtained from an isolate from a wild white-tailed deer (Odocoileus virginianus) in Virginia as well as other species and isolates available from GenBank. Analyses showed the white-tailed deer isolate to be identical with the C. parvum cattle genotype; however, the caribou isolate represents a new genotype closely related to C. serpentis, C. muris, and C. andersoni. Giardia sp. was not detected in any of the caribou samples nor was Cryptosporidium sp. or Giardia sp. detected in any of the 42 moose (Alces alces) samples examined.     

Characteristics of a novel type of bovine Cryptosporidium andersoni

We isolated oocysts that resemble Cryptosporidium andersoni from cattle grazing on a farm in Japan. The partial sequences of genes from the isolate were coincident with published sequences of genes of C. andersoni. Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of C. andersoni.     

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.     

Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia

Multilocus sequence typing (MLST), a sequence-based method to characterize bacterial genomes, was used to examine the genetic structure in a large collection of Medicago-nodulating rhizobial strains. This is the first study where MLST has been applied in conjunction with eBURST analysis to determine the population genetic structure of nonpathogenic bacteria recovered from the soil environment. Sequence variation was determined in 10 chromosomal loci of 231 strains that predominantly originated from southwest Asia. Genetic diversity for each locus ranged from 0.351 to 0.819, and the strains examined were allocated to 91 different allelic profiles or sequence types (STs). The genus Medicago is nodulated by at least two groups of rhizobia with divergent chromosomes that have been classified as Sinorhizobium meliloti and Sinorhizobium medicae. Evidence was obtained that the degree of genetic exchange among the chromosomes across these groups is limited. The symbiosis with Medicago polymorpha of nine strains placed in one of these groups, previously identified as S. medicae, ranged from ineffective to fully effective, indicating that there was no strong relationship between symbiotic phenotype and chromosomal genotype.     

Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium

The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.     

Identification of novel Cryptosporidium genotypes from avian hosts

A total of 430 avian-derived fecal specimens were randomly collected from selected Western Australian commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo and screened for the presence of Cryptosporidium by PCR. Of these, 27 Cryptosporidium-positive isolates were detected, characterized, and compared with 11 avian-derived isolates from the Czech Republic at the 18S rRNA and actin gene loci. Sequence and phylogenetic analysis identified four genetically distinct genotypes, avian genotypes I to IV, from various avian hosts. In addition, the host range for Cryptosporidium galli was extended. Cryptosporidium muris and Cryptosporidium andersoni were also identified in a tawny frogmouth and a quail-crested wood partridge, respectively.     

Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCCmec) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCCmec was acquired at least 56 times by S. epidermidis. Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 (P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCCmec.     

Biallelic polymorphism in the intron region of beta-tubulin gene of Cryptosporidium parasites

Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.     

Population genetic characterisation of dominant Cryptosporidium parvum subtype IIaA15G2R1

The subtype IIaA15G2R1 at the 60 kDa glycoprotein (gp60) gene locus is the most dominant Cryptosporidium parvum infecting dairy cattle and humans in industrialised nations. The reasons for its high transmissibility are not clear, and it remains to be determined whether this subtype represents a homogeneous parasite population. In this study, we sequence-characterised 26 IIaA15G2R subtype specimens and 26 non-IIaA15G2R subtype specimens from the United States, Canada, United Kingdom and Spain at seven other known polymorphic loci, including CP47, CP56, DZ-HRGP, MSC6-5, MSC6-7, RPGR and ZPT. Extensive heterogeneity within IIaA15G2R1 and discordance in typing results between gp60 and other genetic markers were observed. Results of inter-locus and intra-ZPT linkage disequilibrium and recombination analyses indicated that the heterogeneity within IIaA15G2R1 and discordance in typing results among genetic loci were largely due to the occurrence of genetic recombination, mostly within the gp60 subtype IIaA15G2R1. Although there was no clear population diversion between IIaA15G2R and non-IIaA15G2R subtypes, results of STRUCTURE and F_ST analyses suggested the presence of at least two subpopulations; subpopulation 1 had an epidemic population structure and was widely distributed, whereas subpopulation 2 had a clonal population structure and consisted of geographically segregated multilocus subtypes. Genetic recombination between epidemic and geographically segregated C. parvum populations appeared to be a driving force in the emergence of a hyper-transmissible IIaA15G2R1 subtype. Genetic recombination was observed even between the zoonotic IIa subtype family and anthroponotic subtype family IIc at CP56, MSC6-7 and ZPT. Thus, the IIaA15G2R1 subtype at gp60 is likely a fitness marker for C. parvum and the wide spread of IIaA15G2R1 subtype around the world is probably independent of the sequence characteristics at other genetic loci.     

Temporal variation and host association in the Campylobacter population in a longitudinal ruminant farm study

Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g(-1)) and sheep (820 CFU g(-1)), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.     

中国地方黄牛的遗传多样性*

中国黄牛起源复杂,我国地方黄牛群体不同品种在毛色、形态外貌、细胞遗传学、血液蛋白座位分析均表现出多样性。计算我国黄牛群体6个毛色座位平均杂合度和6个血液蛋白座位平均杂合度分别为0.3144和0.4873,表明我国地方黄牛群体的遗传多样性非常丰富。计算我国黄牛群体的6个毛色座位和6个血液蛋白座位的基因分化系数分别为0.3404和0.095,表明我国黄牛群体毛色差异中有34.04％是由品种间的差异造成的。血液蛋白的多态性有9.5％是由品种间的差异造成的。我国黄牛群体的遗传多样性主要来自品种内的遗传多样性。保存我国黄牛品种资源多样性不仅要从整个中国黄牛群体上考虑,而且要针对每个品种（或类群）进行保种。