Phylogenetic analysis of the hypervariable region of the 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes

To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.     

Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.     

Host-Adapted Cryptosporidium spp. in Canada Geese (Branta canadensis)

The prevalence and distribution of Cryptosporidium spp. in the fecal droppings of the free-living waterfowl Canada geese were examined at 13 sites in Ohio and Illinois. On the basis of the analysis of the small-subunit rRNA gene by PCR, followed by restriction fragment length polymorphism analysis and DNA sequencing, 49 (23.4%) of 209 fecal specimens collected from 10 sites (76.9%) were positive for Cryptosporidium spp. The following five Cryptosporidium species and genotypes were identified: Cryptosporidium goose genotype I (in 36 specimens), Cryptosporidium goose genotype II (in 9 specimens), Cryptosporidium duck genotype (in 1 specimen), Cryptosporidium parvum (in 4 specimens), and C. hominis (in 2 specimens). Cryptosporidium goose genotype I was the most prevalent parasite and was found at all five Cryptosporidium-positive sites in Ohio and at four of five positive sites in Illinois, followed by Cryptosporidium goose genotype II, which was found at two of five positive sites in Ohio and at four of five positive sites in Illinois. Cryptosporidium goose genotype II was detected for the first time, and it is phylogenetically related to goose genotype I and the duck genotype. All three genotypes have not so far been reported in humans, and their pathogenicity in geese has not been determined. Only 10.2% of the Cryptosporidium-positive specimens had C. parvum and C. hominis. The results of this study indicate that Canada geese might only serve as accidental carriers of cryptosporidia infectious to humans and probably play a minor role in the animal-to-human transmission cycle of the pathogen.     

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.     

Phylogenetic Relationships of Cryptosporidium Parasites Based on the 70-Kilodalton Heat Shock Protein (HSP70) Gene

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.     

Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.     

Sequence Differences in the Diagnostic Target Region of the Oocyst Wall Protein Gene of Cryptosporidium Parasites

Nucleotide sequences of the Cryptosporidium oocyst wall protein (COWP) gene were obtained from various Cryptosporidium spp. (C. wrairi, C. felis, C. meleagridis, C. baileyi, C. andersoni, C. muris, and C. serpentis) and C. parvum genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of Cryptosporidium spp. and genotypes.     

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.     

Tracking Host Sources of Cryptosporidium spp. in Raw Water for Improved Health Risk Assessment

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.     

Cryptosporidium spp. in Domestic Dogs: the “Dog” Genotype

Genetic and phylogenetic characterization of Cryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the “dog” genotype is, in fact, a valid species.     

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.     

Analysis of Multiple Tsetse Fly Populations in Uganda Reveals Limited Diversity and Species-Specific Gut Microbiota

The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse''s commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10⁴ to 10⁶ normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse''s gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.     

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60 × 3.99 μm (n = 50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.     

Natural Communities of Achromatium oxaliferum Comprise Genetically, Morphologically, and Ecologically Distinct Subpopulations

The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A. oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A. oxaliferum communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A. oxaliferum cells corresponded to genetically distinct Achromatium subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit.     

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.     

Morphologic, Host Specificity, and Molecular Characterization of a Hungarian Cryptosporidium meleagridis Isolate

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.     

Ecological Significance of Microdiversity: Identical 16S rRNA Gene Sequences Can Be Found in Bacteria with Highly Divergent Genomes and Ecophysiologies

A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the B. alba strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.     

Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Genotypic variations in field isolates of Theileria species infecting giraffes (Giraffa camelopardalis tippelskirchi and Giraffa camelopardalis reticulata) in Kenya

Recently, mortalities among giraffes, attributed to infection with unique species of piroplasms were reported in South Africa. Although haemoparasites are known to occur in giraffes of Kenya, the prevalence, genetic diversity and pathogenicity of these parasites have not been investigated.In this study, blood samples from 13 giraffes in Kenya were investigated microscopically and genomic DNA extracted. PCR amplicons of the hyper-variable region 4 (V4) of Theileria spp. small subunit ribosomal RNA (18S rRNA) gene were hybridized to a panel of genus- and species-specific oligonucleotide probes by reverse line blot (RLB). Two newly designed oligonucleotide probes specific for previously identified Theileria spp. of giraffes found single infections in eight of the specimens and mixed infections in the remaining five samples.Partial 18S rRNA genes were successfully amplified from 9 samples and the PCR amplicons were cloned. A total of 28 plasmid clones representing the Kenyan isolates were analyzed in the present study and compared with those of closely-related organisms retrieved from GenBank. In agreement with RLB results, the nucleotide sequence alignment indicated the presence of mixed infections in the giraffes. In addition, sequence alignment with the obtained 18S rRNA gene sequences revealed extensive microheterogeneities within and between isolates, characterized by indels in the V4 regions and point mutations outside this region. Phylogeny with 18S rRNA gene sequences from the detected parasites and those of related organisms places Theileria of giraffes into two major groups, within which are numerous clades that include the isolates reported in South Africa. Collectively, these data suggest the existence of at least two distinct Theileria species among giraffes, and extensive genetic diversity within the two parasite groups.     

Novel Cryptosporidium Genotype in Wild Australian Mice (Mus domesticus)

A total of 250 mouse fecal specimens collected from crop farms in Queensland, Australia, were screened for the presence of Cryptosporidium spp. using PCR. Of these, 19 positives were detected and characterized at a number of loci, including the 18S rRNA gene, the acetyl coenzyme A gene, and the actin gene. Sequence and phylogenetic analyses identified two genotypes: mouse genotype I and a novel genotype (mouse genotype II), which is likely to be a valid species. Cryptosporidium parvum, which is zoonotic, was not detected. The results of the study indicate that wild Australian mice that are not in close contact with livestock are probably not an important reservoir of Cryptosporidium infection for humans and other animals.