Multilocus Sequence Typing of an Emerging Cryptosporidium hominis Subtype in the United States

Feng, Yaoyu; Tiao, N.; Li, Na; Hlavsa, Michele C.; Xiao, Lihua

doi:10.1128/jcm.02973-13

Cited by 50 publications

(37 citation statements)

References 22 publications

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“…This is also a common C . hominis subtype identified in humans worldwide [28, 79, 80, 81, 74]. The source and human health significance of the novel C .…”

Section: Discussionmentioning

confidence: 99%

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments

et al. 2016

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Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney’s drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5’ half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3’ end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may have important implications for the management of drinking water catchments to minimize risk to public health.

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“…This is also a common C . hominis subtype identified in humans worldwide [28, 79, 80, 81, 74]. The source and human health significance of the novel C .…”

Section: Discussionmentioning

confidence: 99%

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments

et al. 2016

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“…Some of these studies have shown a panmictic population structure with frequent recombination in C. parvum (Herges et al 2012;De Waele et al 2013), whereas others have demonstrated the existence of a flexible reproductive strategy (co-occurrence of panmictic, clonal or epidemic structure) in this species (Tanriverdi et al 2008;Drumo et al 2012). Similarly, some genetic studies conducted on C. hominis identified largely a clonal population structure (Mallon et al 2003;Gatei et al 2007;Li et al 2013;Feng et al 2014), whereas others showed the common occurrence of genetic recombination in C. hominis in developing countries (Widmer and Sullivan, 2012). In areas with an overall clonal population structure of C. hominis, genetic recombination has been shown to be a driving force for the emergence of virulent subtypes such as IbA10G2 and IaA28R4 Feng et al 2014).…”

Section: Multilocus Typing and Population Geneticsmentioning

confidence: 99%

Cryptosporidiumspecies in humans and animals: current understanding and research needs

2014

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S U M M A R YCryptosporidium is increasingly recognized as one of the major causes of moderate to severe diarrhoea in developing countries. With treatment options limited, control relies on knowledge of the biology and transmission of the members of the genus responsible for disease. Currently, 26 species are recognized as valid on the basis of morphological, biological and molecular data. Of the nearly 20 Cryptosporidium species and genotypes that have been reported in humans, Cryptosporidium hominis and Cryptosporidium parvum are responsible for the majority of infections. Livestock, particularly cattle, are one of the most important reservoirs of zoonotic infections. Domesticated and wild animals can each be infected with several Cryptosporidium species or genotypes that have only a narrow host range and therefore have no major public health significance. Recent advances in next-generation sequencing techniques will significantly improve our understanding of the taxonomy and transmission of Cryptosporidium species, and the investigation of outbreaks and monitoring of emerging and virulent subtypes. Important research gaps remain including a lack of subtyping tools for many Cryptosporidium species of public and veterinary health importance, and poor understanding of the genetic determinants of host specificity of Cryptosporidium species and impact of climate change on the transmission of Cryptosporidium.

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“…The data have also facilitated the development of high-resolution molecular typing tools (4,9). Population genetic characterizations of C. parvum and C. hominis using these advanced molecular detection tools have started to improve our understanding of the genetic determinants for host specificity and virulence (10)(11)(12).…”

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confidence: 99%

Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

Guo

Lysen

et al. 2015

J Clin Microbiol

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c Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (C T ) values yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, <25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly <14.0) C T values were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing. C ryptosporidium spp. are an important cause of moderate to severe diarrhea in humans and various animals (1, 2). Over the past decade, great efforts have been made to understand the interaction between Cryptosporidium spp. and their hosts. Thus far, at least 26 species and more than 60 genotypes have been described (3), most with some host specificity. In contrast to the expanding knowledge of the taxonomic complexity of the genus Cryptosporidium, little progress has been made in understanding the molecular basis of phenotypic traits such as the host specificity. This is mainly due to the lack of whole-genome characterization of most Cryptosporidium species and genotypes.Thus far, the genomes of only four Cryptosporidium isolates have been sequenced. Sequence data of the whole genomes of C. parvum and C. hominis, the two species responsible for most human infections (4), were first published in 2004 (5, 6). Several years later, the genome of C. muris was sequenced, and data from the project are available on CryptoDB (http://cryptodb.org). More recently, the genome of anthroponotic subtype family IIc of C. parvum has been sequenced using the next-generation sequencing (NGS) tools (7). The availability of the whole-genome sequenc...

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Multilocus Sequence Typing of an Emerging Cryptosporidium hominis Subtype in the United States

Cited by 50 publications

References 22 publications

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments

Cryptosporidiumspecies in humans and animals: current understanding and research needs

Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

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