<i>Cryptosporidium</i>species and subtype analysis from dairy calves in Spain

Quílez, Joaquín; Torres, Eucaris; Chalmers, Rachel M.; Robinson, Guy; Cacho, Emilio del; Sánchez-Acedo, Caridad

doi:10.1017/s0031182008005088

Cited by 84 publications

(55 citation statements)

References 49 publications

(108 reference statements)

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“…Probably one of the most interesting findings of this study is that a majority of isolates of C. parvum infecting domestic ruminants in our geographical area are host associated, which supports the data previously reported by GP60 sequencing concluding that calves and lambs/goat kids are mostly infected by different allelic subtype families (IIa and IId, respectively) (23,41). Comparison with the previous multilocus study in calves using the same sixlocus subtyping scheme revealed the presence of host-associated alleles and even nucleotide polymorphisms in shared alleles between calves and lambs/goat kids (24).…”

Section: Discussionsupporting

confidence: 91%

Host Association of Cryptosporidium parvum Populations Infecting Domestic Ruminants in Spain

Quílez

Vergara-Castiblanco

Monteagudo

et al. 2013

Appl Environ Microbiol

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A stock of 148 Cryptosporidium parvum DNA extracts from lambs and goat kids selected from a previous study examining the occurrence of Cryptosporidium species and GP60 subtypes in diarrheic lambs and goat kids in northeastern Spain was further characterized by a multilocus fragment typing approach with six mini-and microsatellite loci. Various degrees of polymorphism were seen at all but the MS5 locus, although all markers exhibited two major alleles accounting for more than 75% of isolates. A total of 56 multilocus subtypes (MLTs) from lambs (48 MLTs) and goat kids (11 MLTs) were identified. Individual isolates with mixed MLTs were detected on more than 25% of the farms, but most MLTs (33) were distinctive for individual farms, revealing the endemicity of cryptosporidial infections on sheep and goat farms. Comparison with a previous study in calves in northern Spain using the same six-locus subtyping scheme showed the presence of host-associated alleles, differences in the identity of major alleles, and very little overlap in MLTs between C. parvum isolates from lambs and those from calves (1 MLT) or isolates from lambs and those from goat kids (3 MLTs). The Hunter-Gaston index of the multilocus technique was 0.976 (95% confidence interval [CI], 0.970 to 0.982), which supports its high discriminatory power for strain typing and epidemiological tracking. Population analyses revealed the presence of two host-associated subpopulations showing epidemic clonality among the C. parvum isolates infecting calves and lambs/goat kids, respectively, although evidence of genetic flow between the two subpopulations was also detected.

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Section: Discussionsupporting

confidence: 91%

Host Association of Cryptosporidium parvum Populations Infecting Domestic Ruminants in Spain

Quílez

Vergara-Castiblanco

Monteagudo

et al. 2013

Appl Environ Microbiol

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“…It is also worth mentioning that all alleles differed by multiples of the repeat unit, suggesting that polymorphisms were due to changes in the numbers of satellite units rather than to insertions or deletions of nucleotides within the fragment, although single-nucleotide mutations outside the repeat region were occasionally seen in some sequenced isolates. The previous characterization by sequence analysis of the GP60 gene revealed that all isolates belonged to GP60 subtypes previously described for human patients (28). This observation was confirmed by polymorphisms at some microsatellite loci and further supports the hypothesis that most isolates from diarrheic calves in northern Spain have zoonotic potential.…”

Section: Vol 77 2011 Multilocus Fragment Typing Of Cryptosporidium supporting

confidence: 82%

“…Genomic DNA samples of C. parvum isolates from 140 diarrheic preweaned calves selected from a previous study examining the Cryptosporidium species and GP60 subtypes responsible for outbreaks of neonatal diarrhea in dairy cattle farms in Spain were included in this analysis (28). These animals originated from 61 farms (mean, 2.29 Ϯ 1.81 calves/farm) in 14 provinces across northern Spain.…”

Section: Methodsmentioning

confidence: 99%

“…A range of host-adapted Cryptosporidium spp. have been described for cattle, but only preweaned calves are frequently infected by C. parvum and contribute significantly to zoonotic cryptosporidiosis (10,18,28). Nevertheless, human-specific, animal-specific, and zoonotic C. parvum subtypes have been identified, revealing that molecular subtyping tools are essential for unraveling the transmission dynamics of Cryptosporidium infections in humans and animals, although fundamental questions concerning population genetics, such as the extent of sexual recombination in populations of parasites in the field, are not yet fully resolved (37).…”

mentioning

confidence: 99%

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Multilocus Fragment Typing and Genetic Structure of Cryptosporidium parvum Isolates from Diarrheic Preweaned Calves in Spain

Quílez

Vergara-Castiblanco

Monteagudo

et al. 2011

Appl Environ Microbiol

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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.

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“…The sample that tested positive for the C. parvum genotype by SSU-rRNA PCR was subgenotyped by sequence analysis of the GP60 gene. The GP60 nucleotide sequences of the C. parvum isolate (BRAcalf72) revealed 100% identity with C. parvum subtype IIaA15G2R1, GenBank accession number EU549719 (QUILEZ et al, 2008).…”

Section: Sequencing Analysis Of Dna Sequencesmentioning

confidence: 99%

Identification of Cryptosporidiumspecies and genotypes in dairy cattle in Brazil

Silva

Lopes

Araújo-Junior

2013

Rev. Bras. Parasitol. Vet.

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In this study, we identified Cryptosporidium species and genotypes present in dairy cattle in the central region of São Paulo state, Brazil. Fecal specimens were collected from 200 animals (100 calves and 100 cows) in ten dairy farms. Fecal samples were examined using microscopic examination (ME), enzyme immunoassay (EIA) and polymerase chain reaction (PCR). Cryptosporidium species and genotypes were determined by restriction fragment length polymorphism (RFLP) or DNA sequencing analysis of the SSU-rRNA and GP60 genes. The occurrence of Cryptosporidium spp. infection was 14% (28/200). The occurrence in calves (26%) was significantly higher than in cows (2%). Of the 27 Cryptosporidium-positive specimens submitted to genotyping, C. andersoni was identified in 23 (85.1%), C. bovis in three (11.1%), and the zoonotic C. parvum subtype IIaA15G2R1 in one (3.7%). The study demonstrates that Cryptosporidium spp. infection was common and widespread in dairy cattle in this region and that calves have a high prevalence of C. andersoni. Furthermore, the presence of C. parvum subtype IIaA15G2R1 indicates that dairy calves from this region should be considered a potential source of zoonotic Cryptosporidium oocysts.Keywords: Cryptosporidium spp., cattle, PCR-RFLP, sequencing, genotyping. ResumoNo presente estudo foram identificadas espécies e genótipos de Cryptosporidium originadas de bovinos leiteiros na região central do estado de São Paulo, Brasil. Amostras fecais foram coletadas de 200 animais (100 bezerros e 100 vacas) em 10 propriedades leiteiras. As amostras foram examinadas utilizando os métodos de microscopia óptica (MO), ensaio imunoenzimático (EI) e reação em cadeia da polimerase (PCR). As espécies e genótipos de Cryptosporidium foram determinados pelo método de polimorfismo no tamanho dos fragmentos de restrição (RFLP) ou sequenciamento dos genes SSU-rRNA e GP60. A infecção por Cryptosporidium spp. teve ocorrência de 14% (28/200). A ocorrência em bezerros (26%) foi significativamente maior do que em vacas (2%). Do total de 27 amostras positivas submetidas à caracterização genética, C. andersoni foi identificado em 23 (85.1%), C. bovis em três (11.1%) e C. parvum subtipo IIaA15G2R1 em uma (3.7%). O presente estudo demonstrou que a infecção por Cyptosporidium é comum e difundida em bovinos leiteiros nessa região e que bezerros possuem uma alta prevalência de C. andersoni. A presença de C. parvum subtipo IIaA15G2R1 indica que bezerros leiteiros dessa região devem ser considerados uma fonte de oocistos de Cryptosporidium com potencial zoonótico.

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Cryptosporidiumspecies and subtype analysis from dairy calves in Spain

Cited by 84 publications

References 49 publications

Host Association of Cryptosporidium parvum Populations Infecting Domestic Ruminants in Spain

Host Association of Cryptosporidium parvum Populations Infecting Domestic Ruminants in Spain

Multilocus Fragment Typing and Genetic Structure of Cryptosporidium parvum Isolates from Diarrheic Preweaned Calves in Spain

Identification of Cryptosporidiumspecies and genotypes in dairy cattle in Brazil

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