GENETIC CHARACTERIZATION OF CRYPTOSPORIDIUM SPP. IN DIARRHOEIC CHILDREN FROM FOUR PROVINCES IN SOUTH AFRICA

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Cryptosporidium is a coccidian protozoan parasite of the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidiida, suborder Eimeriina and family Cryptosporidiidae (Carmena, 2010). To date, 26 Cryptosporidium species have been recognized (Chalmers and Katzer, 2013) and nearly 50 genotypes have been described (Xiao and Feng, 2008a), while new genotypes are continually being discovered (Feng et al., 2007a). Cryptosporidium has the ability to infect a large variety of animals, including humans, worldwide. Humans can acquire Cryptosporidium infections through direct contact with infected persons (anthroponotic transmission) or animals (zoonotic transmission) via ingestion of contaminated food (foodborne transmission) or water (waterborne transmission) (Xiao, 2010). Young individuals appear to be more susceptible to infection and disease, while infections in adults are often asymptomatic. Therefore cryptosporidiosis occurs most commonly in young animals and children in association with yellow watery diarrhoea which causes dehydration, weight loss, fever, and inappetence (O’Donoghue, 1995; Ramirez et al., 2004). In immunocompetent hosts these symptoms are usually self- limiting, while in immunosuppressed individuals, such as Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) patients, chronic diarrhoea may last for more than 2 months and can lead to mortality (Chalmers and Davies, 2010).
Since Cryptosporidium is a diarrhoeal disease of worldwide importance in humans, especially young children and immunosuppressed individuals, it is part of the complex group of parasitic, bacterial and viral diseases included in the World Health Organisation’s Neglected Disease Initiative (WHO, 2004). In developing regions of the world, these diseases are not adequately addressed nationally and internationally and cause considerable socio- economic burden in poor populations, particularly in remote and rural areas (Putignani and Menichella, 2010; Savioli et al., 2006). The life cycle of most Cryptosporidium species is completed within the gastrointestinal tract of the host.
Infective (thick-walled) oocysts are excreted in the faeces and are able to survive for long periods outside the host in a moist ambient environment, and may remain viable in water for 140 days (Putignani and Menichella, 2010; Ramirez et al., 2004) . In addition, they are very resistant to the most common disinfectants, making them difficult to destroy by chlorination treatment (Ramirez et al., 2004). A new host becomes infected by ingesting these oocysts, which release sporozoites, primarily in the small intestine and colon. At this point the sporozoites invade the epithelial cells and undergo asexual and sexual multiplication to produce thin-walled and thick-walled oocysts. The thin-walled oocysts have the ability to excyst endogenously and infect new cells, which leads to autoinfection. Due to the rapidity of the life cycle, along with the auto-infective cycle, huge numbers of organisms can colonize infect the enterocysts the intestinal tract within several days. The excreted thick-walled oocysts are fully sporulated and immediately infective (Chako et al., 2010). The duration of oocyst shedding can range from several days to months or years, depending on the host’s immunocompetence (Ramirez et al., 2004).

DIAGNOSIS OF CRYPTOSPORIDIOSIS

Conventional methods for diagnosis of Cryptosporidium infection in humans and animals are based on microscopic examination of stained faecal samples. Modified Ziehl-Neelsen (MZN) staining technique is still considered the gold standard for the detection of Cryptosporidium oocysts (Potters and Van Esbroeck, 2010). However, the sensitivity and specificity of the MZN technique in faecal samples from humans are reported to be 83.8% and 98.9%, respectively (Fayer et al., 2000; Morgan et al., 1998; Paul et al., 2009), and 79.3% and 67.7%, respectively in samples from cattle and pigs (Quílez et al., 1996a). The concentration of stool samples, using Sheather’s sugar flotation or the formalin-ethyl acetate sedimentation method, prior to staining has been described as essential since it allows the detection of seven times fewer oocysts than unconcentrated stool samples (Paul et al., 2009; Quílez et al., 1996a). The MZN technique is cost-effective; however, staining is a time consuming procedure (about 30 to 45 minutes) that requires intensive training and experience to interpret the results. A common problem is distinguishing Cryptosporidium oocysts from other elements, such as moulds and yeast (Potters and Van Esbroeck, 2010).
Monoclonal antibody-based immunofluorescence assays (IFA) offer an alternative method to conventional staining techniques and have been shown to be more sensitive for the detection of Cryptosporidium oocysts, especially in human and animal stools that contain few parasites and large amounts of debris (Garcia and Shimizu, 1997; Quílez et al., 1996a). In general, microscopic techniques have limited sensitivity and specificity. Therefore, their application for generation of prevalence data in epidemiological investigations has been questioned, whereas for clinical purposes microscopy techniques are believed to be sufficient, since the number of oocysts excreted by symptomatic patients is high (Quílez et al., 1996a). Molecular techniques, such as polymerase chain reaction (PCR), can ensure specific diagnosis to species/genotype (Coupe et al., 2005; Xiao et al., 1999) and subtype level (Xiao, 2010).
Only a few genes have been characterized for various Cryptosporidium spp., such as the small subunit rRNA (SSU rRNA) or 18S rRNA gene, 70kDa heat shock protein (HSP70) and the oocyst wall protein (COWP). Use of the SSU rRNA gene has some advantages over other genes because of the higher copy numbers and the presence of conserved regions interspersed with highly polymorphic regions, which facilitate the design of PCR primers (Xiao et al., 2004). Several PCR-restriction fragment length polymorphism (RFLP) techniques have been described for the differentiation of Cryptosporidium spp., all based on the SSU rRNA (Xiao and Ryan, 2004). Subgenotyping tools have been developed for C. hominis and C. parvum: microsatellite tools, HSP70, a double stranded (ds) RNA, and 60 kDa glycoprotein (GP60) gene tools (Xiao et al., 2001). The GP60 gene has a high variation in the number of trinucleotide repeats with extensive sequence differences in the non-repeat regions, which categorizes C. parvum and C. hominis into several subtype families, namely Ia, Ib, Id, Ie, If and Ig for C. hominis and IIa –IIi, IIk and IIl for C. parvum (Xiao, 2010).

TABLE OF CONTENTS :

• DECLARATION
• DEDICATION
• ACKNOWLEDGEMENTS
• SUMMARY
• TABLE OF CONTENTS
• LIST OF TABLES
• LIST OF FIGURES
• LIST OF ABBREVIATIONS
• CHAPTER 1: GENERAL INTRODUCTION
  • DIAGNOSIS OF CRYPTOSPORIDIOSIS
  • ZOONOTIC CRYPTOSPORIDIOSIS
  • CRYPTOSPORIDIOSIS IN WILDLIFE
  • CRYPTOSPORIDIOSIS IN CATTLE
  • CRYPTOSPORIDIOSIS IN HUMANS
  • OUTLINE OF THE THESIS
• CHAPTER 2:GENETIC CHARACTERIZATION OF CRYPTOSPORIDIUM SPP. IN DIARRHOEIC CHILDREN FROM FOUR PROVINCES IN SOUTH AFRICA
  • ABSTRACT
  • INTRODUCTION
  • MATERIALS& METHODS
  • SAMPLING
  • DNA EXTRACTION AND CRYPTOSPORIDIUM GENOTYPING AND SUBTYPING
  • RESULTS
  • DISCUSSION
• CHAPTER 3:THE PREVALENCE OF CRYPTOSPORIDIUM SPP. OOCYSTS IN WILD MAMMALS IN THE KRUGER NATIONAL PARK, SOUTH AFRICA
  • ABSTRACT
  • INTRODUCTION
  • MATERIALS& METHODS
  • STUDY AREA
  • FAECAL SAMPLING
  • LABORATORY ANALYSIS
  • STATISTICAL ANALYSIS
  • RESULTS
  • DISCUSSION
• CHAPTER 4: MOLECULAR CHARACTERIZATION OF CRYPTOSPORIDIUM SPECIES AT THE
  • WILDLIFE/LIVESTOCK INTERFACE OF THE KRUGER NATIONAL PARK, SOUTH AFRICA
  • ABSTRACT
  • INTRODUCTION
  • MATERIALS& METHODS
  • STUDY AREA
  • ETHICS STATEMENT
  • SAMPLING
  • QUESTIONNAIRE
  • DNA EXTRACTION
  • CRYPTOSPORIDIUM GENOTYPING AT THE 18s rRNA LOCUS
  • SEQUENCE ANALYSIS
  • RESULTS
  • CONTACT BETWEEN CATTLE AND WILDLIFE
  • CRYPTOSPORIDIUM GENOTYPING
  • DISCUSSION
• CHAPTER 5: PREVALENCE, GENOTYPES AND RISK FACTORS OF CRYPTOSPORIDIUM
  • INFECTION IN CHILDREN AND CALVES AT THE LIVESTOCK AND HUMAN INTERFACE OF THE KRUGER NATIONAL PARK, SOUTH AFRICA
  • ABSTRACT
  • INTRODUCTION
  • MATERIALS& METHODS
  • STUDY SITE
  • ETHICS STATEMENT
  • SAMPLING COLLECTION
  • LABORATORY ANALYSIS
  • DNA EXTRACTION
  • CRYPTOSPORIDIUM AMPLIFICATION AND GENOTYPING AT THE 18s rRNA
  • LOCUS
  • CRYPTOSPORIDIUM AMPLIFICATION AND SEQUENCING OF THE GP60 GENE
  • STATISTAL ANALYSIS
  • RESULTS
  • CHILDREN
  • CALVES
  • DISCUSSION
• CHAPTER 6: GENERAL DISCUSSION
  • PREVALENCE OF CRYPTOSPORIDIUM INFECTION IN WILDLIFE, CATTLE AND
  • HUMANS
  • PREVALENCE IN WILDLIFE
  • PREVALENCE IN CATTLE
  • PREVALENCE IN HUMANS
  • DIAGNOSTIC TESTS
  • DIAGNOSTIC TESTS IN WILDLIFE
  • DIAGNOSTIC TESTS IN CATTLE
  • DIAGNOSTIC TESTS IN HUMANS
  • SEASONALITY
  • SEASONALITY IN HUMANS
  • SEASONALITY IN ANIMALS
  • ZOONOTIC IMPORTANCE OF CRYPTOSPORIDIOSIS
  • POTENTIAL SOURCES OF INFECTION AND ROUTES OF TRANSMISSION AT THE
  • WILDLIFE/LIVESTOCK/HUMAN INTERFACE
  • CONCLUSION
  • FINDINGS WITH RESPECT TO RESEARCH QUESTIONS
  • LIMITATIONS OF THIS STUDY
  • RECOMMENDATIONS FOR FUTURE RESEARCH
  • REFERENCES

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