Detección de Cryptosporidium spp. en terneras de lecherías de la Región Metropolitana mediante Ziehl Neelsen y confirmada por inmunocromatografía y ensayo molecular

Muñoz, Pamela; Fredes, Fernando; Díaz-Lee, A; Mercado, Rubén; Ozaki, LS

doi:10.4067/s0301-732x2011000200003

Cited by 4 publications

(11 citation statements)

References 26 publications

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“…Thirty-six (36) diarrheic calves, less than 30 days old, from two dairy farms located in Melipilla and El Monte counties in the Metropolitan Region (MR) 33°27′S 70°40′W, were selected for fecal sample collection. Another similar set of 36 calves were studied from dairy farms located in Mariquina, Rio All DNA samples were tested by PCR with SSU-rDNA Cryptosporidium specific primers [25] and COX1 bovine specific primers [26] to rule out PCR inhibitory activity. The DNA samples positive in both tests were then submitted to PCR for amplification of the GP60 gene, using 2.5 μl of extracted DNA and the primers gp15-ATG (5' ATG AGA TTG TCG CTC ATT ATC 3') and gp15-STOP (5' TTA CAA CAC GAA TAA GGC TGC 3') [15], resulting in an expected amplicon of about 1,000 bp.…”

Section: Methodsmentioning

confidence: 99%

Cryptosporidium parvum GP60 subtypes present in diarrheic dairy calves of two biogeographical regions of Chile

Peña¹,

Muñoz²,

Raffo³

et al. 2019

Preprint

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Background Cryptosporidium is an apicomplexan zoonotic pathogen primary causing diarrhea in vertebrate hosts notably bovines and humans. Here, we characterized Cryptosporidium isolates by using the GP60 gene fragment of C. parvum to observe the dynamics of cryptosporidiosis transmission in dairy calves from two distant biogeographical regions of Chile (Metropolitan and Los Rios Regions). We collected 72 fecal samples from diarrheic calves screening the parasite carried out microscopy of an acid-fast staining smear and molecular characterization employing PCR to directly detect the Sanger GP60 C. parvum subtype and simultaneously in one selected sample the NGS profile of the GP60 same gene fragment to determine same and/or others Cryptosporidium subtypes Results The IIaA15G2R1 subtype was present in the 100% of the bovine fecal samples studied from Los Rios Region. Along with this same subtype, another two were observed in the Metropolitan Region, IIaA17G2R1 and IIaA17G4R1. The NGS analysis of a single selected GP60 PCR amplicon of one selected sample of our study showed similarly the Sanger sequencing determined subtype, the IIaA17G4R1 in 90% of readable sequences observed. By using this approach another multiple low frequency IIa subtypes of C. parvum were observed confirming that in an infected host multiple subtypes of the parasite can be present. Conclusions Cryptosporidiosis in these dairy farms calves in Chile is produced by C. parvum limited number of subtypes, being IIaA15G2R1 the most frequent. The IIa subtype family is considered prevalent in calves in South America. Subtypes IIaA17G2R1 and IIaA17G4R1 had been worldwide distribution. As all C. parvum subtypes observed in calves in Chile were isolated from diarrheic animals, so, it can be possible to relate its presence with the pathogenic role in the bovine host and with a potential digestive disease risk for humans.

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Section: Methodsmentioning

confidence: 99%

Cryptosporidium parvum GP60 subtypes present in diarrheic dairy calves of two biogeographical regions of Chile

Peña¹,

Muñoz²,

Raffo³

et al. 2019

Preprint

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“…With respect to the prevalence of the disease, the first reports in Chile of the discovery of Cryptosporidium in cattle, are produced in the year 1986, a study where the presence of this protozoan was determined in 12 calves from a total of 51 diarrheic animals. Then in 1989, a study conducted in the Metropolitan region found lower prevalence levels, 2.2% in healthy animals and 23% in diarrheic animals (5,6). Then in 1997, a study conducted on diarrheic calves, reported a prevalence of 30.6% in the south of Chile (6).…”

Section: Introductionmentioning

confidence: 99%

“…Then in 1989, a study conducted in the Metropolitan region found lower prevalence levels, 2.2% in healthy animals and 23% in diarrheic animals (5,6). Then in 1997, a study conducted on diarrheic calves, reported a prevalence of 30.6% in the south of Chile (6). The latest study in the country is that conducted by Diaz-Lee et al (5) in the Metropolitan region, where 205 samples of diarrheic valves belonging to 2 dairy farms were analyzed, determining an infection rate of 57%.…”

Section: Introductionmentioning

confidence: 99%

“…Con respecto a la prevalencia de la enfermedad, los primeros reportes en Chile de hallazgo de Cryptosporidium en bovinos se producen en el año 1986, estudio en el que se determinó la presencia de este protozoo en 12 terneros de un total de 51 animales diarreicos. Luego en 1989, un estudio realizado en la región Metropolitana determinó niveles de prevalencia menores, el 2.2% en animales sanos y el 23% en animales diarreicos (5,6). Después en el año 1997, un estudio realizado en terneros diarreicos, reportó una prevalencia de 30.6% en el sur de Chile (6).…”

Section: Introductionunclassified

“…Luego en 1989, un estudio realizado en la región Metropolitana determinó niveles de prevalencia menores, el 2.2% en animales sanos y el 23% en animales diarreicos (5,6). Después en el año 1997, un estudio realizado en terneros diarreicos, reportó una prevalencia de 30.6% en el sur de Chile (6). El estudio más reciente en el país, corresponde al realizado por Díaz-Lee et al (5) en la región Metropolitana donde se analizaron 205 muestras de terneros diarreicos, pertenecientes a 2 predios lecheros, determinándose una frecuencia de infección de un 57%.…”

Section: Introductionunclassified

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Cryptosporidium spp., comparative diagnosis and geospatial distribution in diarrheic calves from dairy farms, Valdivia, Chile

Muñoz

Mercado

et al. 2014

Rev MVZ Córdoba

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Objective. To determine the Cryptosporidium spp. infection frequency by using Ziehl-Neelsen and Auramine stains on samples obtained from diarrheic calves from milking farms of the Valdivia province. To compare both diagnostic tests and to determine the geospatial distribution of the infections caused by this protozoan. Materials and methods. 221 fecal samples of diarrheic calves of 24 milking farms of the Valdivia province were studied. The processing and analysis of the samples was done by Ziehl-Neelsen (ZN) and Auramine (AU) staining techniques, and the results were compared by McNemar statistical test and the concordance level was determined by kappa index. A map was also generated to determine the geospatial distribution of Cryptosporidium infections. Results. 57.9% of all the animals tested were classified as positive with the ZN stain test, while 55.6% of all the animals turned out positive for the AU stain test. The McNemar test showed no significant difference between both diagnostic techniques (p>0.05), while the kappa index showed proper concordance between tests (κ=0.73). 100% of the farms studied showed protozoan presence demonstrating the broad distribution of the parasite, however, and considering the previous factor, it was not possible to determine geospatial associations for the parasite distribution. Conclusions. The infection frequency of Cryptosporidium is higher than 50% in the milking farms studied from the Valdivia province. No difference between the Ziehl-Neelsen and Auramine staining techniques was demonstrated showing very consistent results. It was possible to detect that the number of farms infected correspond to 100% of the farms analyzed.

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Detección y caracterización molecular de Cryptosporidium spp. en terneros de lechería de la provincia de Valdivia, Chile

Muñoz¹

2021

Rev MVZ Córdoba

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Objetivo: Detectar ooquistes de Cryptosporidium spp. en terneros mediante tinción Ziehl Neelsen (ZN) y aplicar herramientas moleculares con los genes SSU18S rARN (SSU18S) y glicoproteína de 60 kDa (gp60), para caracterizar la especie y subtipos presentes en la provincia de Valdivia. Materiales y métodos: Se obtuvieron 275 muestras de heces de terneros menores de 2 meses de 15 predios lecheros de la provincia de Valdivia, las que fueron analizadas mediante tinción de ZN. Los resultados positivos se sometieron a PCR con genes target SSU18S y gp60, con posterior secuenciación. Resultados: 30 muestras fueron positivas a la tinción de ZN, el 46,6% (14/30) logró ser confirmado mediante PCR convencional para SSU18S y de éstas, sólo 42,8% (6/14) fueron positivas a la PCR convencional para gp60. La secuenciación arrojó la presencia de C. parvum en el 100% de las muestras y los subtipos IIaA15G2R1 (3/6), IIaA14G1R1 (2/6) y IIaA17G1R1 (1/6). Conclusiones: El subtipo IIaA15G2R1 fue el predominante, encontrándose en dos comunas de la provincia. El subtipo IIaA14G1R1 fue encontrado en dos predios pertenecientes a una misma comuna y su hallazgo es el primero en bovinos en Chile y el continente. Por ultimo, el subtipo IIaA17G2R1 ha sido encontrado esporádicamente en terneros al igual que el presente estudio. Las tres variantes han sido reportadas en humanos, sin embargo, no hay registros de estudios que caractericen los subtipos de C. parvum en sistemas de crianza de terneros y personal trabajador simultáneamente en Chile, por lo tanto, se requiere indagar mas en este tema.

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Detección de Cryptosporidium spp. en terneras de lecherías de la Región Metropolitana mediante Ziehl Neelsen y confirmada por inmunocromatografía y ensayo molecular

Cited by 4 publications

References 26 publications

Cryptosporidium parvum GP60 subtypes present in diarrheic dairy calves of two biogeographical regions of Chile

Cryptosporidium parvum GP60 subtypes present in diarrheic dairy calves of two biogeographical regions of Chile

Cryptosporidium spp., comparative diagnosis and geospatial distribution in diarrheic calves from dairy farms, Valdivia, Chile

Detección y caracterización molecular de Cryptosporidium spp. en terneros de lechería de la provincia de Valdivia, Chile

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