A survey of the inter- and intraspecific RAPD markers of Eimeria spp. of the domestic fowl and the development of reliable diagnostic tools.
Coccidiosis of domestic fowl is a protozoan disease, caused by seven distinct species of the genus Eimeria, which is responsible for important economic losses in poultry production. In order to select RAPD primers for the discrimination of these seven Eimeria species, we carried out an initial screening using samples of E. acervulina, E. tenella and E. maxima. Out of 150 primers tested, 110 generated band profiles specific for each one of these species. A subset of 14 oligonucleotides were also tested for the simultaneous differentiation of the seven species, resulting in 11 discriminative primers. The intraspecific discrimination was assessed for five different species, using samples from different geographic regions including three continents. Numerous primers exhibited highly discriminative band profiles containing strain-specific markers, with a higher variability being observed among strains of E. acervulina than among E. tenella and E. maximastrains. However, no major differences were observed in the band patterns from strains collected in locations near to one another compared to strains originating from distantly located regions. Because RAPD is a technique performed under low stringency conditions, it suffers from poor reproducibility. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific RAPD fragments into SCAR markers. An initial conversion of 25 RAPD markers into SCARs, followed by validation of their specificity, resulted in 14 totally new Eimeria species-specific markers that can be used for the molecular diagnosis of the seven species that infect domestic fowl. This work represents a first step in the development of a set of species-specific SCARs that will be useful as tools for molecular diagnosis, genome mapping, and genetic diversity studies.
Fernandez
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S
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, USP, Av. Prof. Orlando Marques de Paiva 87, 05508-000, São Paulo SP, Brazil.
Costa
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AC
Katsuyama
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AM
Madeira
A M B N
AM
Gruber
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A
D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000469:parasitology*; D003001:Cloning, Molecular; D003048:Coccidiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D004539:Eimeria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005819:Genetic Markers; D011201:Poultry Diseases / Q000175:diagnosis / Q000469:parasitology*; D019105:Random Amplified Polymorphic DNA Technique; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D017931:DNA Primers; D016054:DNA, Protozoan; D005819:Genetic Markers