The objective of this study was to determine the occurrence of Cryptosporidium spp. in domestic chickens raised in different chicken production systems in Brazil using three nested PCR protocols. The purification and concentration of oocysts present in 190 fecal samples from chickens raised in extensive, semi-intensive and intensive production systems were accomplished by centrifugal flotation in Sheather's solution and were followed by the extraction of genomic DNA. The detection and molecular characterization of Cryptosporidium species and genotypes were performed using three nested polymerase chain reaction (nested PCR) protocols targeting the 18S rRNA gene followed by sequencing of the amplified fragments. Subgenotyping of C. meleagridis was performed using a nested PCR reaction targeting the gp60 gene. Sample identified as Cryptosporidium sp. genetically similar to Cryptosporidium xiaoi and Cryptosporidium bovis by 18S rRNA gene sequencing were further analyzed by nested PCR targeting the actin gene and subsequent sequencing of the amplified fragment. Positive amplification for Cryptosporidium spp. was observed in 12.6% (24/190) of the samples, including C. baileyi (9.8%; 18/190), C. meleagridis (0.5%, 1/190), C. parvum (2.1%; 4/190) and Cryptosporidium sp. (0.5%; 1/190). Subgenotyping of C. meleagridis revealed the presence of the zoonotic subtype IIIgA23G3R1. Sequencing of the 18S rRNA gene and the actin gene fragments revealed a Cryptosporidium genotype in an extensive poultry system genetically related to C. xiaoi and C. bovis. There was no significant difference in the frequency of positive results obtained by the three nested PCR protocols (p > 0.05); additionally, the agreement obtained by Kappa index ranged from substantial (0.70) to almost perfect (0.9).

The aim of this study was to evaluate the prevalence of and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. Oocysts were purified from fecal samples from 463 psittacines by centrifugal-flotation in Sheather's sugar solution. Cryptosporidium spp. were detected by malachite green negative staining and nested PCR targeting the 18S rRNA gene. Cryptosporidium species were identified by sequencing nested PCR amplicons. Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. determined by microscopy and nested PCR were 3.0% (14/463) and 5.0% (23/463), respectively. The nested PCR/sequencing identified avian genotype III (1.7%; 8/463), Cryptosporidium parvum (0.9%; 4/463) and Cryptosporidium canis (0.2%; 1/463). Duplex real-time PCR was positive for gastric Cryptosporidium in 9.5% (44/463) of the samples. Among them, 1.9% (9/463) were positive for C. galli, 5.8% (27/463) were positive for avian genotype III and 1.7% (8/463) showed mixed infections with C. galli and avian genotype III. With regards to the positive detection of Cryptosporidium spp., there was no statistically significant difference between nested PCR and microscopic analysis (p = .1237), and a fair agreement existed between them (Kappa = 0.242). A statistically significant difference (p < .0001) and fair agreement (Kappa = 0.317) were obtained between nested PCR/sequencing and duplex real-time PCR for the detection of gastric Cryptosporidium. We determined that nested PCR and duplex real-time PCR are the best options for the detection of Cryptosporidium spp. and gastric Cryptosporidium, respectively, and that avian genotype III is the most common Cryptosporidium genotype/species in psittacines.

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.