Disease syndromes caused by T. parva infections

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Background

Cattle theileriosis caused by Theileria parva infections is associated with high mortality, primarily in exotic and crossbred cattle, but also in indigenous calves and adult cattle in endemically unstable areas (Perry and Young, 1995). This results in major constraints on cattle production and the expansion of the dairy industry. In 1989 the cost of cattle theileriosis, East Coast fever (ECF), was estimated at $186 million (Mukhebi et al., 1992) in 11 affected countries in the African region. Following the introduction of ECF to South Africa in 1902, an estimated 5.5 million deaths of cattle were attributed to ECF.
The control and the ultimate eradication of the disease cost the country R100 million (Anonymous, 1981). Theileria parva infections in cattle manifest in three different disease syndromes, namely, ECF, Corridor disease and January disease. East Coast fever was introduced into southern Africa at the turn of the 19th century and was eventually eradicated after a costly campaign involving quarantine of infected farms and compensated slaughter of infected cattle herds (Anonymous, 1981). After the eradication of ECF, Corridor disease became the most important form of theileriosis in South Africa. Corridor disease is still a serious threat in areas where there are common grazing grounds between cattle and infected buffalo and where the tick vectors, Rhipicephalus appendiculatus and R.
zambeziensis occur (Uilenberg, 1999). Since the South African cattle population is completely naïve to T. parva, it should be protected against exposure. Therefore, in South Africa today, cattle theileriosis is a controlled disease and authorities try to keep infected buffalo separated from livestock to prevent the spread of the disease. Theileria parva has existed in Cape buffalo (Syncerus caffer) for thousands of years (Uilenberg, 1981; Young, 1981) and the parasite still circulates in the buffalo population in South Africa.
Consequently, in South Africa, infections by T. parva parasites do not only impact on the cattle industry, but also affect the game farming industry. Buffalo are natural reservoirs of the parasite, and when infected by T. parva their value significantly decreases to ten times less than their “clean” counterparts. The financial implication of this extends to the loss of revenue if the game properties lose their attraction for tourists or hunters due to the absence of buffalo. Corridor disease is still endemic in buffalo populations in some parts of South Africa. As a result, buffalo are required to be tested at least five consecutive times before they can be relocated to a disease-free area, as a means of controlling of the spread of the parasite to Corridor disease-free areas.

Thesis rationale

Corridor disease is a controlled disease in South Africa. The Agricultural Research Council- Onderstepoort Veterinary Institute (ARC-OVI) is the only institution in the country with a mandate from the South African government to test for T. parva infections in cattle and buffalo. Previously, a package of tests including microscopic examination of blood smears, the indirect fluorescent antibody test (IFAT) and a convetional PCR/probing test was used for this purpose.
These tests, particularly the microscopic examination of blood smears and IFAT, lacked the desired sensitivity and specificity to detect T. parva infections which often occur in the presence of other Theileria species, as mixed infections. The application of the PCR/probing test improved the sensitivity and specificity that conventional diagnostic tests suffered over the years. However, because this assay is both time- and labour-intensive, it was no longer an ideal test for processing large numbers of samples resulting from the increasing demand for disease-free buffalo in South Africa. The ARC-OVI needed a more sensitive, more specific and less time-consuming diagnostic technique to detect T. parva-positive animals. Therefore, the use of real-time PCR technology to improve molecular diagnostics of T. parva infection was evaluated in this study.

TABLE OF CONTENTS :

• DEDICATION
• DECLARATION
• ACKNOWLEDGEMENTS
• TABLE OF CONTENTS
• LIST OF FIGURES
• LIST OF TABLES
• THESIS SUMMARY
• CHAPTER 1 General Introduction
  • 1.1 Background
  • 1.2 Thesis rationale
  • 1.3 Thesis objectives
  • 1.4 References
• CHAPTER 2 Literature Review
  • 2.1 Introduction
  • 2.2 The parasite: Theileria parva
  • 2.2.1 The life cycle of T. parva
    • 2.2.1.1 In the mammalian host
    • 2.2.1.2 In the vector tick
  • 2.3 Disease syndromes caused by T. parva infections
  • 2.3.1 East Coast fever (ECF)
  • 2.3.2 Corridor disease
  • 2.3.3 January disease (Zimbabwean theileriosis)
  • 2.4 Epidemiology of theileriosis in southern Africa
  • 2.4.1 Introduction and eradication of cattle theileriosis, East Coast fever, in southern Africa
  • 2.4.2 Emergence of other theilerial disease syndromes
  • 2.4.3 Transformation of buffalo-derived T. parva into cattle-derived T. parva
  • 2.4.4 Carrier state of T. parva
  • 2.5 Treatment and control of theileriosis
  • 2.5.1 Tick control
  • 2.5.2 Chemotherapy
  • 2.5.3 Immunization
  • 2.6 Detection of T. parva infections
  • 2.6.1 Conventional methods
  • 2.6.2 Serological methods
    • 2.6.2.1 Indirect immunofluorescent antibody test
    • 2.6.2.2 Enzyme-linked immunosorbent assay (ELISA)
  • 2.6.3 Molecular techniques
    • 2.6.3.1 Conventional PCR assays
    • 2.6.3.2 PCR-based hybridization assays
    • 2.6.3.3 PCR-based RFLP assays
    • 2.6.3.4 Real-time PCR assays
  • 2.7 Characterization of T. parva stocks
  • 2.7.1 Monoclonal antibody screening assays
  • 2.7.2 Molecular characterization
  • 2.8 Aim
  • 2.9 Thesis overview
  • 2.10 References
• CHAPTER 3 Development and evaluation of a real-time PCR test for detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle
  • 3.1 Abstract
  • 3.2 Introduction
  • 3.3 Materials and methods
  • 3.3.1 Sample collection
  • 3.3.2 DNA extraction
  • 3.3.3 Design of primers and hybridization probes
  • 3.3.4 Optimized real-time PCR conditions
  • 3.3.5 Specificity of the real-time PCR assay
  • 3.3.6 Sensitivity of the real-time PCR assay
  • 3.3.7 Comparison of the real-time PCR assay with other molecular tests
  • 3.3.8 Proficiency testing
  • 3.4 Results
  • 3.4.1 Specific detection of T. parva using the real-time PCR assay with hybridization probes
  • 3.4.2 Analytical sensitivity
  • 3.4.3 Comparison of molecular tests
  • 3.4.4 Proficiency testing
  • 3.5 Discussion
  • 3.6 Summary
  • 3.7 References
• CHAPTER 4 Four p67 alleles identified in South African Theileria parva field samples
  • 4.1 Abstract
  • 4.2 Introduction
  • 4.3 Materials and methods
  • 4.3.1 Sample collection
  • 4.3.2 DNA isolation
  • 4.3.3 PCR amplification of the p67 gene from T. parva
  • 4.3.4 Cloning and sequencing of p67 amplicons
  • 4.3.5 Sequence analysis
    • 4.3.5.1 Sequence editing
    • 4.3.5.2 Sequence alignment
  • 4.3.6 Phylogenetic analysis
  • 4.4 Results
  • 4.4.1 Amplicon analysis by agarose gel electrophoresis
  • 4.4.2 Sequence analysis
  • 4.4.3 Phylogenetic analysis
  • 4.5 Discussion
  • 4.6 Conclusion
  • 4.7 References
• CHAPTER 5 Characterization of Theileria parva parasites occurring in buffalo (Syncerus caffer) in South Africa: In search of cattle-type p104 alleles
  • 5.1 Abstract
  • 5.2 Introduction
  • 5.3 Materials and methods
  • 5.3.1 Sample collection
  • 5.3.2 DNA isolation
  • 5.3.3 Analysis of the p104 gene from T. parva samples using PCR-RFLP
  • 5.3.4 PCR-RFLP profile analysis
  • 5.3.5 Cloning and sequencing of p104 PCR products
  • 5.3.6 Sequence analysis
  • 5.4 Results
  • 5.4.1 p104 PCR-RFLP profile analysis
  • 5.4.2 p104 gene sequence analysis
  • 5.5 Discussion
  • 5.6 Summary
  • 5.7 References
• CHAPTER 6 Analysis of the gene encoding the Theileria parva polymorphic immunodominant molecule (PIM) reveals evidence of the presence of cattle-type alleles in South Africa
  • 6.1 Abstract
  • 6.2 Introduction
  • 6.3 Materials and methods
  • 6.3.1 Sample collection
  • 6.3.2 DNA isolation and selection of T. parva-positive samples
  • 6.3.3 Amplification of the PIM gene from T. parva samples
  • 6.3.4 Analysis of the PIM gene from T. parva samples using PCR-RFLP
  • 6.3.5 Cloning and sequencing of PIM PCR products
  • 6.3.6 Sequence analysis
  • 6.4 Results
  • 6.4.1 PIM PCR-RFLP profile analysis
  • 6.4.2 PIM gene sequence analysis
  • 6.5 Discussion
  • 6.6 Summary
  • 6.7 References
• CHAPTER 7 General Discussion and Conclusion
  • 7.1 Improvement of molecular diagnosis of T. parva infections
  • 7.2 Molecular characterization of South African T. parva parasites
  • 7.2.1 Evidence of cattle-type p67, p104 and PIM alleles in T. parva parasite populations in South Africa
    • 7.2.1.1 Identification of cattle-type alleles from cattle T. parva samples
    • 7.2.1.2 Identification of cattle-type alleles from buffalo T. parva samples
    • 7.2.2 Extensive genetic diversity among South African T. parva parasites
  • 7.3 Conclusion
  • 7.4 References
  • LIST OF PUBLICATIONS

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