Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia

Abstract

Background and Aim: Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia. Materials and Methods: Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp. Results: Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two. Conclusion: Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.

Molecular identification of Eimeria species in cattle from Veracruz, Mexico

“…In our study, 31/57 oocyst concentrations were negative in agarose gel. There are some factors, such as the quantity and quality of DNA, the presence of mixed Eimeria oocysts, contamination with PCR inhibitors, resistance of the oocyst wall to rupture or restricted access to template DNA, which can interfere with the molecular diagnosis of Eimeria in cattle (Oliveira et al, 2011;Lee et al, 2018;Ekawasti et al, 2022;Melo et al, 2022). Regarding the quantity and quality of the DNA, failures in the molecular identi cation of Eimeria spp.…”

“…Several techniques have been proposed for oocyst rupture, either mechanically or chemically, but a reproducible method for disrupting the oocyst wall and recovering high yields of DNA has not yet been developed (Oliveira et al, 2011). In order to create PCR tests, emphasis should be placed on the purity of the sample, the number of oocysts and the characteristics of the primers to validate the geographical origins of the strains and verify that they can be used in the region studied (Ekawasti et al, 2022).…”

“…Although 21 species of Eimeria have been reported to infect cattle worldwide, only E. bovis, E. zuernii, E. alabamensis, E. auburnensis, E. cylindrical and E. ellipsoidalis are reported as pathogenic (Lee et al, 2018;Bangoura & Bardsley, 2020). The rest of the species are considered non-pathogenic or are rarely associated with the presence of clinical signs (Bangoura & Bardsley, 2020); however, the high prevalence of some non-pathogenic species or their unknown involvement, either alone or in mixed infections, warrants future epidemiological and clinical studies throughout the year (Lee et al, 2018).Therefore, a proper Eimeria diagnose approach is essential to effectively control coccidiosis (Ekawasti et al, 2022).…”

“…Also, there is the possibility of morphological changes due to the application of some antiparasitic treatment before sampling or due to the manipulation of the samples in the sporulation process (Li et al, 2010). Molecular identi cation of Eimeria species is described as a more sensitive, species-speci c, reliable and timee cient technique for the diagnosis and treatment implementation in infected calves (Ekawasti et al, 2022). Kawahara et al (2010) proposed the use of the transcribed spacer region − 1 (ITS-1) as an attractive DNA purpose for phylogenetic analysis and for the design of speci c oligonucleotides for Eimeria spp.…”

“…Kawahara et al (2010) proposed the use of the transcribed spacer region − 1 (ITS-1) as an attractive DNA purpose for phylogenetic analysis and for the design of speci c oligonucleotides for Eimeria spp. PCR reports of Eimeria species in cattle are limited worldwide (Lee et al, 2018;Pyziel et al, 2020;Ekawasti et al, 2022;Hastutiek et al, 2022). So far, no molecular studies have been conducted in Mexico to determine the genetics of Eimeria spp.…”