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| Author(s): |
Alex Akira Nakamura
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2013-03-08 |
| Examining board members: |
Marcelo Vasconcelos Meireles;
Eliana Reiko Matushima;
Enio Mori;
Adriana Marques Joppert da Silva;
Rodrigo Martins Soares
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| Advisor: | Marcelo Vasconcelos Meireles |
| Abstract | |
Cryptosporidiosis has been described in several animal species, including many species of birds, and is considered a major protozoan infection of the orders Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes and Struthioniformes. Three species of Cryptosporidium are valid in birds: Cryptosporidium baileyi, Cryptosporidium meleagridis and Cryptosporidium galli; beside these species, there are several genotypes genetically distinct, as avian genotypes I, II, III, IV and V. Cryptosporidium galli and Cryptosporidium avian genotype III are related to chronic infections in proventriculus, similar to Cryptosporidium serpentis infection in snakes. Several methods are used for cryptosporidiosis diagnosis, but only those based on molecular biology are able to determine the Cryptosporidium species and genotypes. The aim of this project was the development of a duplex real-time PCR targeting 18S subunit of rRNA gene, by TaqMan assay, to detect DNA of C. galli and Cryptosporidium avian genotype III, and to evaluate the diagnostic attributes of the duplex real-time PCR compared to nested PCR, using 1027 fecal samples from birds of the orders Passeriformes and Psittaciformes. The duplex real time PCR showed positivity in 580/1027 (56.47%) for C. galli, whereas nested PCR was positive for Cryptosporidium spp. in 104/1027 (10.13%) samples. For Cryptosporidium avian genotype III, there was positivity in 21/1027 (2.04%) samples. The duplex real time PCR resulted in high analytical specificity when tested using DNA samples from other Cryptosporidium species and genotypes. The only exception was the amplification of DNA from C. serpentis with primers and probe for the detection of Cryptosporidium avian genotype III, although with lower efficiency. New avian hosts were identified for both gastric species, as well as it was possible to identify C. baileyi and, for the first time in Brazil, Cryptosporidium avian genotype V. It was concluded that the duplex real time PCR developed in this study is a fast and reliable tool for diagnosis of gastric cryptosporidiosis in birds. (AU) | |