A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents

Silva, Sheila O. S.; Richtzenhain, Leonardo José; Gomes, Alessandra M.M. C.; Silva, Aristeu Vieira da; Kozerski, Noemila Débora; Ceranto, Jaqueline B. de Araújo; Keid, Lara Borges; Soares, Rodrigo Martins

doi:10.1016/j.exppara.2013.09.003

Cited by 40 publications

(45 citation statements)

References 24 publications

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“…In addition, a total of six new host species were identified; Black Ghost Knife Fish (Apteronotus albifrons), Kribensis (Pelvicachromis), Orange clownfish (Amphiprion percula), Platyfish (Xiphophorus maculatus), Red-striped angelfish (Centropyge eibli) and Yellowheaded Jaw fish (Opistognathus aurifrons). Re-amplification and sequencing of 14 piscine-derived Cryptosporidium isolates from previous studies with the Silva et al (2013) primers confirmed the assignment to genotype in previous studies (Reid et al, 2010;Zanguee et al, 2010;Morine et al, 2012;Koinari et al, 2013) with the exception of one isolate (MM192). The five novel genotypes identified at the 18S locus (LC01, LC38, LC51, CA68 and KS02), exhibited substantial genetic distances (3.9-9%) from known genotypes and species.…”

Section: Discussionsupporting

confidence: 51%

“…A sound taxonomy of piscine-derived Cryptosporidium species is also important as previous research suggests that they may be the most primitive of Cryptosporidium species (Ryan et al, 2004(Ryan et al, , 2015Palenzuela et al, 2010;Reid et al, 2010;Zanguee et al, 2010;Morine et al, 2012;Koinari et al, 2013) and therefore, provide important information on the evolution of the genus. The purpose of the present study was to examine the extent of genetic diversity of Cryptosporidium species in fish hosts using a longer region (∼553 bp) of the 18S gene (Silva et al, 2013) and to construct a molecular phylogeny at the actin locus to better understand the phylogenetic relationships of piscine Cryptosporidium species and genotypes. Table 1 Fish species screened for Cryptosporidium.…”

Section: Q3mentioning

confidence: 99%

“…These included mullet-derived isolates M09 and M20 (piscine genotype 3), NZ107-Upsidedown catfish and NZ127-Wedgetailed blue tang (C. molnari-like), NZ106-Kupang damsel and NZ16-Golden algae eater (piscine genotype 4), oscarfish-derived isolates NZ13 and NZ113 (piscine genotype 2), NZ121-Golden algae eater, NZ129-butter bream and NZ95-angelfish (piscine genotype 5), neon tetra-derived isolates MM188 and MM192 (Piscine genotype 7) and silver-biddy isolate G018 (piscine genotype 8) (Reid et al, 2010;Zanguee et al, 2010;Morine et al, 2012;Koinari et al, 2013) (Table 2). All samples were screened at the 18S rRNA locus using previously described primers and conditions (Silva et al, 2013). Isolates positive at the 18S locus were also analyzed at the actin locus using PCR primers optimized for amplification of piscinederived Cryptosporidium species (which produce a 392 bp product), as previously described (Koinari et al, 2013).…”

Section: Genomic Dna Extraction and Pcr Amplificationmentioning

confidence: 99%

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Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections

Yang

Palermo

Chen

et al. 2015

Veterinary Parasitology

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a b s t r a c tCryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n = 14), Cryptosporidium huwi (n = 8), piscine genotype 2 (n = 4), piscine genotype 3-like (n = 1), piscine genotype 4 (n = 2), piscine genotype 5 (n = 13), piscine genotype 5-like (n = 1) and five novel genotypes (n = 5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.

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Section: Discussionsupporting

confidence: 51%

Section: Q3mentioning

confidence: 99%

Section: Genomic Dna Extraction and Pcr Amplificationmentioning

confidence: 99%

See 1 more Smart Citation

Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections

Yang

Palermo

Chen

et al. 2015

Veterinary Parasitology

View full text Add to dashboard Cite

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“…All samples were screened at the 18S rRNA locus using previously described primers and conditions (Silva et al, 2013). Isolates positive at the 18S locus were also analysed at the actin locus using a hemi-nested PCR optimized for amplification of piscine-derived Cryptosporidium species, as previously described (Koinari et al, 2013).…”

Section: Methodsmentioning

confidence: 99%

Molecular characterisation of a disseminated Cryptosporidium infection in a Koi carp (Cyprinus carpio)

Yang

Dorrestein

Ryan

2016

Veterinary Parasitology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…All samples were screened for the presence of Cryptosporidium at the 18S rRNA locus using a quantitative PCR (qPCR) previously described (King et al 2005;Yang et al 2014 Samples that were positive by qPCR were amplified at the 18S locus using primers which produced a 611-bp product as previously described (Silva et al 2013) with minor modifications; the annealing temperature used in the present study was 57 °C for 30 s, and the number of cycles was increased from 39 to 47 cycles for both primary and secondary reactions. PCR contamination controls were used including negative controls and separation of preparation and amplification areas.…”

Section: Pcr Amplification Of the 18s Rrna Genementioning

confidence: 99%

First report of Cryptosporidium species in farmed and wild buffalo from the Northern Territory, Australia

Zahedi

Phasey²,

Boland³

et al. 2016

Parasitol Res

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A molecular epidemiological survey of Cryptosporidium from water buffalo (Bubalus bubalis) in the Northern Territory in Australia was conducted. Fecal samples were collected from adult farmed (n = 50) and wild buffalo (n = 50) and screened using an 18S quantitative PCR (qPCR). Positives were typed by sequence analysis of 18S nested PCR products. The qPCR prevalence of Cryptosporidium species in farmed and wild buffalo was 30 and 12 %, respectively. Sequence analysis identified two species: C. parvum and C. bovis, with C. parvum accounting for ~80 % of positives typed from the farmed buffalo fecal samples compared to 50 % for wild buffalo. Subtyping at the 60 kDa glycoprotein (gp60) locus identified C. parvum subtypes IIdA19G1 (n = 4) and IIdA15G1 (n = 1) in the farmed buffalo and IIaA18G3R1 (n = 2) in the wild buffalo. The presence of C. parvum, which commonly infects humans, suggests that water buffaloes may contribute to contamination of rivers and waterways with human infectious Cryptosporidium oocysts, and further research on the epidemiology of Cryptosporidium in buffalo populations in Australia is required.

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A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents

Cited by 40 publications

References 24 publications

Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections

Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections

Molecular characterisation of a disseminated Cryptosporidium infection in a Koi carp (Cyprinus carpio)

First report of Cryptosporidium species in farmed and wild buffalo from the Northern Territory, Australia

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