Development of nested duplex PCR assays for detection of pathogen Eimeria species in cattle in Papua, Indonesia

Abstract Eimeria bovis and Eimeria zuernii are highly pathogenic Eimeria species in cattle that are the most prevalent causes of a severe clinical illness characterized by hemorrhagic diarrhea in calves and young cattle with potentially fatal effects over the world. The oocysts of a handful of the known bovine eimeriosis species are difficult to distinguish morphologically. For the specific differentiating evidence of Eimeria species, symptomatic research institutions are increasingly relying on DNA-based technologies. This research offers a duplex polymerase chain reaction (PCR) test based on the internal transcribed spacer-1 (ITS-1) gene that may be used to diagnose E. bovis and E. zuernii in cattle from various locations at the same time. The oocysts were concentrated and purified using a fecal harvesting method. The genomic DNA is extracted according to the instructions included with the kit. Primer pairs specific to each species, as well as a standard optimum annealing temperature of 55°C for these species, were discovered. The samples were amplified in a homogenous way, resulting in a homogeneous band ladder, revealing that the test could distinguish between two highly pathogenic Eimeria species in one tube reaction. This duplex PCR assay can detect a high pathogenic bovine Eimeria simultaneously in a rapid and low cost.