Genetic diversity of piroplasms in plains zebra and Cape mountain zebra

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Quantitative real-time PCR assays

The recent development of quantitative real-time PCR (qPCR) has greatly improved the molecular detection and diagnosis of many organisms of veterinary and medical importance (Jeong et al., 2003; Lindh et al., 2007; O’Grady et al., 2008; Wengi et al., 2008). Quantitative PCR assays provide several advantages over the use of conventional PCR and probe-based assays, which are relatively sensitive but involve complex procedures that are time consuming and labour intensive (Allsopp et al., 1993; Bashiruddin et al., 1999; Nicolaiewsky et al., 2001; Rampersad et al., 2003; Alhassan et al., 2005). Detection and quantification of a qPCR product takes place in a single tube during the cycling process, thus eliminating the need for post-PCR manipulation and reducing the risk of contamination. Quantitative PCR tests have recently been developed for a number of haemoparasitic disease agents including Theileria sergenti (Jeong et al., 2003), Babesia bovis and Babesia bigemina (Buling et al., 2007), Anaplasma marginale (Carelli et al., 2007) and Theileria parva (Sibeko et al., 2008). These qPCR assays have significantly improved the sensitivity and specificity of parasite detection. A qPCR assay, based on the 18S rRNA gene, was recently developed for the detection of T. equi infections in horses (Kim et al., 2008). This assay proved to be highly sensitive and specific for T. equi, and it allowed for the simultaneous detection and quantification of experimental samples. Several parasite outer membrane protein gene sequences have also been targeted in the development of molecular diagnostic assays for equine piroplasmosis (Nicolaiewsky et al., 2001; Ueti et al., 2003; Alhassan et al., 2005; Alhassan et al., 2007; Heim et al., 2007). A qPCR assay targeting the gene (ema-1) encoding the equi merozoite antigen-1 (EMA-1) was developed to determine the number of T. equi parasites in the midgut of Rhipicephalus (Boophilus) microplus ticks (Ueti et al., 2003). A multiplex assay, using this test and a qPCR based on the rhoptryassociated protein (BC 48) gene of B. caballi, was subsequently developed and used to determine the prevalence of both T. equi and B. caballi parasites in horses in Brazil (Heim et al., 2007).

Objectives and overview of this study

Improved diagnosis and detection of piroplasmosis carrier animals would be of great benefit to the horse industry both locally and internationally. The main objective of this study was therefore to develop a quantitative real-time PCR assay to complement and improve on the current diagnostic tests. Many molecular diagnostic assays developed for the detection of Theileria and Babesia species have been based on the amplification of the 18S rRNA gene. A preliminary study in our laboratory, however, provided evidence of sequence heterogeneity in the V4 hypervariable region of the 18S rRNA gene within Theileria and Babesia parasite species infecting horses in South Africa. In Chapter 2 of this thesis, the extent of genetic heterogeneity within the 18S rRNA genes of T. equi and B. caballi parasites is explored and conclusions regarding the usefulness of current diagnostic assays employing the V4 hypervariable region in detecting these two piroplasm species in South Africa are made. The identification of extensive sequence variation in the 18S rRNA gene of T. equi and B. caballi parasites in South Africa explained the failure of previous molecular assays in detecting these parasites. In Chapter 3, the recently reported T. equi-specific TaqMan qPCR assay targeting the 18S rRNA gene (Kim et al., 2008) is evaluated for its ability to detect all T. equi 18S rRNA variants that have been shown to occur in South Africa. The development of a TaqMan minor groove binder (MGB™) qPCR assay for the detection of B. caballi in equine field blood samples is also described.
Despite the knowledge that piroplasm parasites occur in our zebra populations, the molecular epidemiology and the possible influence through genetic recombination that their existence may have on horse piroplasms, has largely been overlooked. In Chapter 4 zebra samples are screened to identify piroplasm parasites, and 18S rRNA genes of T. equi-like piroplasms of zebra are sequenced to further elucidate genetic variation in T. equi parasites in South Africa.
Although a qPCR assay based on the 18S rRNA gene has been developed (Kim et al., 2008) and was evaluated in this study, the identification of additional T. equi 18S variants in zebras suggests that we cannot rule out the possible existence of as yet undetected 18S gene sequence variants. In chapter 5, a second qPCR assay recently developed for the detection of T. equi targeting the equi merozoite antigen-1 gene (ema-1) (Ueti et al., 2003) is evaluated. Following its poor performance, the ema-1 gene from South African T. equi samples is characterized and a more sensitive TaqMan MGB™ qPCR assay that targets a conserved region of the ema-1 gene is developed.
Finally in Chapter 6 the characterization of rap-1 gene homologues from South African B. caballi isolates is described in an attempt to provide reasons for the failure of the commercial cELISA to detect B. caballi antibody in blood samples that tested positive using the IFAT.

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Field samples and tissue-culture stabilates

A total of 488 samples were investigated (Table 2.1). Blood samples, collected from 148 yearlings on six different stud farms in different locations in South Africa (Table 2.2), were obtained from the blood bank of the Equine Research Centre, Faculty of Veterinary Science, University of Pretoria. Also, 39 blood samples were collected from South African horses housed in the premises of this Faculty. Serum samples were collected at the South African National Two-year Sale (2005) and the National Yearling Sale (2006) and sent to the Agricultural Research Council-Onderstepoort Veterinary Institute where they were tested using IFAT. Based on these results, 211 horses, which tested positive serologically for T. equi and/or B. caballi, were identified and whole-blood samples were collected from each of them. In addition, 90 tissue-culture samples, which included 17 Cape mountain zebra (Equus zebra zebra) samples from the Bontebok National Park, Western Cape province, South Africa (Zweygarth et al., 1997; Zweygarth et al., 2002) were investigated.

CHAPTER 1 Literature Review  
1.1 Introduction
1.2 Etiology
1.3 Epidemiology
1.4 Transmission
1.5 Pathogenesis
1.6 Clinical signs
1.7 Economical impact of equine piroplasmosis – Globally and in South Africa
1.8 Control and treatment
1.9 Diagnosis
1.10 Objectives and overview of this study
1.11 Reference List
CHAPTER 2 Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa  
2.1 Abstract
2.2 Introduction
2.3 Materials and methods
2.4 Results
2.5 Discussion
2.6 Reference List
CHAPTER 3 Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa  
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.4 Results
3.5 Discussion
3.6 Reference List
CHAPTER 4 Genetic diversity of piroplasms in plains zebra (Equus quagga burchellii) and Cape mountain zebra (Equus zebra zebra) in South Africa  
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.4 Results
4.5 Discussion
4.6 Reference List
CHAPTER 5 Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB™ assay for the detection of T. equi 
5.1 Abstract
5.2 Introduction
5.3 Materials and Methods
5.4 Results
5.5 Discussion
5.6 Reference List
CHAPTER 6 Sequence heterogeneity in the gene encoding the rhoptry associated protein-1 (RAP-1) of Babesia caballi isolates from South Africa  
6.1 Abstract
6.2 Introduction
6.3 Materials and Methods
6.4 Results
6.5 Discussion
6.6 Reference List
CHAPTER 7 General Discussion  
7.1 Reference List
SCIENTIFIC PUBLICATIONS  
APPENDICES

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