Diagnosis of gastric cryptosporidiosis in birds using a duplex real-time PCR assay

Nakamura, Alex Akira; Homem, Camila Guariz; Silva, Adriana M. J. da; Meireles, Marcelo Vasconcelos

doi:10.1016/j.vetpar.2014.07.033

Cited by 27 publications

(37 citation statements)

References 29 publications

(54 reference statements)

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“…Duplex real-time PCR was performed to simultaneously detect C. galli and Cryptosporidium avian genotype III (NAKAMURA et al, 2014), amplifying 134-bp and 138-bp amplicons, respectively (Table 1), under the following reaction conditions: 25 μL of solution containing 12.5 μL of JumpStart  TaqReady Mix (Sigma-Aldrich, St. Louis, USA), 4.5 mM MgCl 2 , 250 nM of each probe, 600 nM of each primer, 0.6 μg/μL of non-acetylated bovine serum albumin (Sigma-Aldrich, St. Louis, USA), and 5 μL of target DNA. PCR cycles consisted of 2 minutes of denaturation at 94°C followed by 50 cycles of 30 seconds at 94°C and 1 minute at 61°C in the CFX96  Real-Time PCR Detection System (Bio-Rad, Hercules, USA).…”

Section: Duplex Real-time Pcrmentioning

confidence: 99%

“…In Brazil, C. avium, C. baileyi, C. meleagridis, C. parvum, the avian genotypes I, II and III and the duck genotype were reported to occur in fecal samples of domestic and wild birds (MEIRELES & FIGUEIREDO, 1992;SANTOS et al, 2005;MEIRELES et al, 2006;HUBER et al, 2007;NAKAMURA et al, 2009NAKAMURA et al, , 2014SEVÁ et al, 2011;NARDI, 2015;CUNHA et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…In canaries (Serinus canaria), C. galli, C. avium and C. avian genotypes I and III infections have been described (NG et al, 2006;ANTUNES et al, 2008;NAKAMURA et al, 2009NAKAMURA et al, , 2014NARDI, 2015). Most epidemiological studies on avian cryptosporidiosis were performed using convenience sampling with fecal samples from several bird species (NG et al, 2006;NAKAMURA et al, 2009, SEVÁ et al, 2011BAMAIYI et al, 2013;REBOREDO-FERNÁNDEZ et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Nested PCR is more expensive than microscopy, despite being highly sensitive and specific and identifying the species after amplicon sequencing (JEX et al, 2008). Another option for specific species/genotype diagnosis of gastric cryptosporidiosis in birds is through duplex real-time PCR (NAKAMURA et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Camargo

Santana

Ferrari

et al. 2018

Rev. Bras. Parasitol. Vet.

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This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.Keywords: Cryptosporidiosis, birds, diagnosis, epidemiology. ResumoEste trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.

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Section: Duplex Real-time Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Camargo

Santana

Ferrari

et al. 2018

Rev. Bras. Parasitol. Vet.

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“…ya ha sido reportada para Passeriformes silvestres (Yabsley 2008, Berto et al 2011, incluyendo a dos especies en el continente americano de la familia Tyrannidae: Casiornis rufus y Myiarchus ferox (Berto et al 2008(Berto et al , 2009 (Nakamura et al 2014).…”

Section: Resultados Y Discusiónunclassified

Prevalencia parasitaria en el gorrión altiplanero (Spizella wortheni): especie endémica de México y en peligro de extinción

Canales-del-Castillo

Pérez-Rangel

Ruvalcaba‐Ortega

et al. 2017

Huitzil Rev. Mex. Ornitol.

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ResumenEl gorrión altiplanero o de Worthen (Spizella wortheni) es endémico del altiplano mexicano y debido a su limitada distribución y tamaño poblacional es considerada una especie en peligro de extinción a nivel nacional e internacional. De manera general, las especies en peligro han estado expuestas, al menos históricamente, a una fuerte reducción en su tamaño poblacional y su diversidad genética, factores que en su conjunto aumentan el riesgo y susceptibilidad a infecciones. Por lo tanto, con el propósito de determinar dicha propensión en esta especie, analizamos la prevalencia de parásitos intestinales a partir de muestras fecales en 11 individuos, así como hemoparásitos, hematocrito y cuantificación diferencial de leucocitos a partir una muestra sanguínea. Los resultados mostraron que el 91% de los individuos presentaron por lo menos un taxón de parásitos intestinales, siendo el género Cryptosporidium (64%) el de mayor prevalencia, seguido por Eimeria (55%) y Ascaridia (9%). Sin embargo, los valores medios de oocistos/huevo por gramo de heces fecales indicaron una baja infección parasitaria. Además, no se observaron parásitos sanguíneos y el conteo de células blancas fue similar a lo previamente reportado para otras especies de la familia Emberizidae y Passerellidae.Palabras clave: Gorrión altiplanero, Spizella wortheni, peligro de extinción, parásitos fecales, hemoparásitos, conteo diferencial de leucocitos. AbstractThe Worthen's sparrow is an endemic bird of the Mexican Plateau that due to its limited distribution and population size is considered to be endangered, both nationally and globally. In general, species at risk have been, at least historically, under population size and genetic diversity reductions, which are factors that can act together to increase infections risk and susceptibility. Therefore, with the purpose to determine such propensity in this species, we analyzed the intestinal parasitic infection through fecal samples from 11 individuals, and hemoparasites, hematocrit and differential leukocyte quantification from one sample. Results indicated that 91% of the samples had one parasite taxon, with genus Cryptosporidium showing the highest prevalence (64%), followed by Eimeria (55%), and Ascaridia (9%). However, mean values of oocysts/eggs per gram indicated a low parasitic infection. We found no blood parasites, and the white blood cell counts were among reference values for other sparrow species.

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Infectious Diseases

Samanta

Bandyopadhyay

2017

Pet Bird Diseases and Care

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Diagnosis of gastric cryptosporidiosis in birds using a duplex real-time PCR assay

Cited by 27 publications

References 29 publications

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Prevalencia parasitaria en el gorrión altiplanero (Spizella wortheni): especie endémica de México y en peligro de extinción

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