Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites

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Abstract

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

Introduction

Bovine coccidiosis is the disease caused by Eimeria infection in cattle and the most economically important disease of cattle throughout the world (Fitzgerald, 1980, Fox, 1985, Cornelissen et al., 1995, Marshall et al., 1998). Calves age 3 weeks to 6 months are particularly susceptible to clinical coccidiosis (Taylor and Catchpole, 1994), reflecting pre-exposed naive states to Eimeria infections (Conlogue et al., 1984, Svensson et al., 1996). More than 20 species of bovine coccidia have been reported (Levine and Ivens, 1967). There are 11 species identified in Germany and Japan, and 13 species in the United States (Levine and Ivens, 1967, Ernst and Todd, 1977, Oda and Nishida, 1990, Faber et al., 2002). Of these species, Eimeria (E.) alabamensis, E. auburnensis, E. bovis, E. ellipsoidalis and E. zuernii are recognized pathogenic (Daugschies and Najdrowski, 2005). E. bovis and E. zuernii are highly pathogenic that they usually cause bloody stool (Stockdale et al., 1981, Chibunda et al., 1997, Mundt et al., 2003, Bangoura et al., 2007). Moderate infections caused by the abovementioned species or other species may exhibit subclinical signs to transient non-hemorrhagic diarrhea (Daugschies and Najdrowski, 2005).

Currently, morphological observation of oocysts is the only practical method to identify species within bovine coccidia (Daugschies and Najdrowski, 2005). The detailed features described by Levine and Ivens, 1967, Levine and Ivens, 1970 for bovine coccidial oocysts have been cited in many reports to determine the species (Oda and Nishida, 1990, Faber et al., 2002, Sánchez et al., 2008). However, the morphological method is not fully reliable since several species have confusing features alongside the presence of intra-species variation (Long and Joyner, 1984). Furthermore, morphological observations combined with fecal examination are very labor-intensive and require skillful technique. It is essential to develop a more rapid, convenient and cost effective method.

An inter-species variation of 18S rRNA gene sequences in bovine coccidia has been known to be rare (Li et al., 2007), and that is not efficient for the identification of species based on PCR assays that have been reported. Therefore, the development of sensitive and reliable technique to detect and identify correct species is the primary requirement.

Knowledge of apicomplexa at the genomic level has been progressing continuously, and certain molecular methods for the species identification have been presented using PCR technique (Burg et al., 1989, Stucki et al., 1993, Müller et al., 1996, Tsuji et al., 1997). In these methods, one of attractive genomic DNA target is the internal transcribed spacer 1 (ITS-1) region derived from the ribosomal RNA (rRNA) genes (Cai et al., 1992, Payne and Ellis, 1996, Schnitzler et al., 1998, Schnitzler et al., 1999). This region is separated between the 3′ end of 18S rRNA gene and the 5′ end of the 5.8S rRNA gene in each transcription unit. Due to heterogeneity of both sequence compositions and lengths among different species, the ITS-1 region is a promising target to design the specific primers. Furthermore, the ITS-1 region belonging to a multiple copy gene family provides large number of targets for PCR assays. In a recent report (Lew et al., 2003), inter-species-specific diversity were shown within the ITS-1 regions from 7 chicken Eimeria with high heterogeneity with each other, and the species-specific DNA sequences were applied for the diagnostic purpose. The conventional or the real-time PCR assays have been developed using species-specific sequences of ITS-1 region to differentiate species of chicken Eimeria (Lew et al., 2003, Su et al., 2003, Jenkins et al., 2006, Kawahara et al., 2008).

To date, a phylogenetic analysis based on PCR assay has not been reported in Eimeria species of cattle. In this study, we report the results of the analysis conducted for 21 ITS-1 sequences to define the phylogenetic relationship, inter- and intra-species variation existing among 18 Eimeria isolates, and to describe the reliability and applicability of PCR assays depending on the specific ITS-1 region for identification of bovine Eimeria.

Section snippets

Parasites

More than 100 samples of cattle feces were collected from commercial farms throughout Japan. Feces were examined for the presence of oocysts using a microscope. Species of Eimeria (E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis, E. zuernii and others) were determined morphologically with 50 oocysts in each sample according to the standard presented by Levine and Ivens, 1967, Levine and Ivens, 1970. Oocysts were separated from the fecal debris and concentrated by

Morphometric classification on field isolates

Fecal samples were collected from commercial cattle farms and isolated bovine coccidia oocysts. Based on the morphological characteristics of oocysts in these samples, field isolates were selected on the condition that they predominantly contain single Eimeria species.

Three representative samples of each species were obtained from different farms, resulted in a total of 18 isolates of the 6 species (Table 1). Among them, 6 isolates (one isolate of E. alabamensis, E. bovis, E. cylindrica and E.

Discussion

In the present investigation, inter-species-specific DNA sequences situating in the ITS-1 region of the rRNA gene of six cattle Eimeria were examined to study their diversity. The ITS-1 regions are flexible corresponding with species variation, as compared with whole rRNA genes, showing a pattern of low intra-specific and high inter-specific variations in the DNA sequence. These features make easy to design primers accurately, thereby minimizes the risk of cross-reactions with different species.

Conclusions

Present results showed the phylogenetic relationship between bovine Eimeria and the diversity of species based on the ITS-1 sequences. These findings suggest that the sequence variation in species could be correlated with the morphological characteristics of oocysts. We developed the assays to differentiate these species by PCR targeting the species-specific ITS-1 region. Results demonstrated that the PCR assay targeting the ITS-1 region of Eimeria species in cattle can be used for detection

Conflict of interest statement

None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias this paper.

Acknowledgements

We would like to thank all farmers and veterinarians concerning this study for their assistance when sampling. We also thank Eri Tomeno for technical assistance in the laboratory.

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