Giardia lamblia is a eukaryotic parasite that causes diarrhea in humans worldwide. Diarrheal diseases cause stunting and mental retardation in children in developing nations, therefore it is important to understand the molecular epidemiology of G. lamblia. Compounding this, it is not clear if companion animals such as dogs contribute to infections in humans through zoonotic transmission. The genotypes of G. lamblia that have been found in humans are A1, A2 and B, while those in dogs have been on rare occasions all three human genotypes, but largely C and D, which have only been reported in dogs and appear to be species-specific. The molecular epidemiology of G. lamblia in humans and dogs was assessed in an endemic region of Lima, Peru. With one exception, dogs were found to harbor the C and D dog genotypes of G. lamblia. A single family dog was found to harbor a human genotype of G. lamblia. A2 and B genotypes of G. lamblia, but not A1, were found in humans in the endemic region. Previous literature reported that A2 and B typing within genotype tools were available, however the A2 samples from the endemic region could not be distinguished from one another through nucleotide polymorphism sequence analysis. A molecular typing technique was developed to type A2 samples. The extensive sequence analysis performed on two chromosomes of G. lamblia, yielded different phylogenetic tree groupings for the same samples. This lead to algorithmic analysis, which demonstrated a significantly high probability that meiotic recombination is occurring in the A2 samples of G. lamblia. As G. lamblia is largely believed to be asexual, the conclusion of doctoral research performed in this study yielded controversial, yet significant evidence that sex in G. lamblia A2 genotype samples is indeed occurring.

Taenia solium is a zoonotic helminth which is thought to be one of the leading causes of acquired epilepsy in the developing world. T. solium cysticercosis infections in pigs and humans and human taeniasis were diagnosed using antigen-capture ELISAs. The parasite was found to be endemic in the study site, with cysticercosis being detected by HP10 Ag-ELISA in 6.6% of human samples (95% C.I. 5.6-7.8%) and 17.2% (95% C.I. 10.2-26.4%) of porcine samples. Human taeniasis was detected by Copro-Ag ELISA in 19.9% (95% C.I. 18.2-21.8%) of faecal samples. The study site was found to be co-endemic with a large selection of other neglected tropical diseases, including soil transmitted helminthiasis, schistosomiasis, strongyloidiasis and amoebiasis. Potential control measures for this parasite have been modeled and the exclusion of infective pork from the food chain through the use of a pre-slaughter test for pig farmers, traders and slaughtermen was found to have the potential to avoid 72.6% (95% C.I. 62.1-80.9%) of infective meals consumed in the area at an incremental cost-effectiveness ratio (ICER) of $0.25 (0.2-0.35). Such a diagnostic tool is currently under development and its performance was evaluated as part of this thesis. The novel, user-friendly lateral flow assay, utilising the HP10 monoclonal antibody, was evaluated using a Bayesian framework and was estimated to perform with a Sensitivity of 82.7% (95% B.C.I. 72.5-91.9%) and Specificity of 87% (95% B.C.I. 80.2-93.4), results which demonstrate the potential utility of this test in epidemiological studies and in control strategies. Free-ranging pig production has been previously demonstrated to be a key risk factor for porcine cysticercosis and is commonly practised in this study region. A study carried out as part of this thesis found that these pigs have a home range of 15,085m2 which is almost 10 times the average area of a homested. This work indicates that pigs can be exposed to infective eggs from any human T. solium carriers within that homerange area, greatly assisting transmission of this parasite. Western Kenya is a severely deprived region where pig production is becoming hugely popular and is seen as a major tool for economic development, yet the data presented in this thesis indicates an area with endemic status for the harmful parasite T. solium, for which effective control strategies are desperately required.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The human population in sub-Saharan Africa has been increasing due to decreases in mortality rates and increases in average human age; in turn increasing poverty and pressure placed on agriculture and agricultural production. However, livestock production in South Africa, and globally, is declining due to disease and parasite prevalence, lack of feed, poor breeding, marketing management, change in nutrition in both livestock and humans, rapid urbanization, encroachment on wildlife and unfavourable climatic conditions brought about by global change. One unintended consequence has been the emergence and spread of transboundary animal diseases and, more specifically, the resurgence and emergence of zoonotic disease. Zoonotic diseases are sicknesses transmissible from animals to humans, resulting from direct contact or environmental reservoirs. Previous studies have identified small-scale farmers as the group most prevalent to contracting zoonotic diseases, especially those working in a communal dispensation. Therefore, this study focused on the communal farming area of Steinkopf in the semi-arid Namaqualand region of South Africa. Steinkopf is one of the largest Act 9 areas, with communal land tenure and a mixed farming system, sheep and goats, on about 759 ha. Steinkopf is divided into two rainfall regions, the Succulent Karoo (winter rainfall region) and the Nama Karoo (summer rainfall region). This study aims to identify and characterise the bacterial microbial communities found in the topsoil layer and faecal matter (dung) within the winter and summer rainfall regions of Steinkopf communal rangeland using Next-generation sequencing. Further, the aim is to assess whether pathogenic bacteria are present within the rangeland and what their potential impact on the local farming community might be if present. A high-throughput sequencing technique (Next-generation sequencing) was used to amplify 16S rRNA targeting the V3-V4 hypervariable regions. The phylotypes produced were 37 phyla, 353 families and 634 genera of which the most abundant bacterial phyla were Planctomycetes, Firmicutes and Bacteroidetes and the most abundant genera were Gemmata, Akkermansia and Arthrobacter. Alpha diversity indices showed a variation in species diversity, evenness and richness between soil and dung samples, it shows a higher species richness, evenness and unique OTUs detected in summer soil samples and at natural water holes. Through these analysis soil samples were regarded as superior to dung samples within this particular environment and for this particular study. Natural water holes were identified as a safer option when compared to man-made water holes as there are natural systems in place that combat the spread and growth of harmful bacterial microbes. It was found that seasonality has a great impact on the development and growth of environmental bacterial microbiota and that the current randomness of grazing routes and migrations within the Steinkopf communal rangeland is not a detriment but instead acts as a benefits to environmental and livestock health. Furthermore, a total of three pathogenic bacteria were identified however, they occurred at relatively low abundances. It can thus be concluded that this study thoroughly describes the usefulness of using a high-throughput sequencing technique such as Next-generation sequencing when amplifying a small sample size in order to achieve a large volume of information; and that currently the Steinkopf communal rangeland is not subjected to or at risk of a potential zoonotic threat.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

Feral swine (Sus scrofa domesticus) are spreading across North America at an alarming rate. Four Canadian provinces and 39 states within the continental United States now report standing populations of feral pigs. Estimates place the number of feral hogs in Texas >2M, accounting for more than half of the United States population. It is known that feral swine impact local ecology following establishment, with regard to shifts in local flora and fauna. The overall objective of this research was to investigate the role of feral swine in tick-borne pathogen transmission in Texas. The underpinning objectives were to establish host records for tick species parasitizing feral swine, determine the species assemblages associated with feral swine among different ecoregions of Texas, determine by immunoassay to which tick-borne bacteria feral pigs were being exposed, and detect the DNA of tick-borne bacteria by polymerase chain reaction assay in the event of poor or early immune response by the host. Feral pigs (N=432) were harvested from June 2008 to June 2010 using box and corral traps and by aerial gunning. Seven species of ticks, Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor albipictus, D. halli, and D. variabilis; and Ixodes scapularis, were collected. Immature stages of A. cajennense and A. americanum were collected as well. All classes of feral pigs, gender by age, were infested with ticks. Serum was collected through a multi-organizational effort from 2006 to 2010 and tested by ELISA for previous exposure to tick-borne pathogens in the genera Rickettsia and Ehrlichia (N=888) and Borrelia (N=849). Prevalence percentages by immunoassay were 27.59 percent, 13.18 percent and 2.12 percent for Rickettsia, Ehrlichia, and Borrelia, respectively. Samples positive by ELISA for exposure to Borrelia were further screened by Western Blot for exposure to Borrelia turicatae. The results were equivocal in most cases. Blood samples (N=233) were collected from 2008 to 2010 and analyzed by polymerase chain reaction for the detection of the DNA of these same three genera of bacteria. Two of the samples were positive by PCR for the presence of Borrelia DNA. Texas feral swine are serving as hosts for at least seven species of ticks and are interacting with tick-borne pathogen transmissions cycles in Texas.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. Escherichia coli which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49]. Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy. To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyer’s patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation in vitro. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2. To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

No description available.

Between 60-70% of Namibia’s population practice subsistence agro-pastoralism on communal land that constitutes 41% of the total land area. Cysticercosis bovis is found worldwide, but most often in rural developing countries, where unhygienic conditions are coupled with poor cattle management practices and lack or absence of meat inspection. Because livestock is so important to the economy and social structure of the majority of people in Namibia, risks from zoonotic diseases transmitted from domestic animals to humans are a constant threat and are therefore of major concern. In addition, C.bovis is also emerging as a public health risk not only in these rural communities but also in urban areas where many infected cattle are transported, informally slaughtered and consumed. This disease has a negative impact on food safety, and thus is related to Veterinary Public Health (VPH) strategies in Namibia. Over the period from 2000 to 2004, 3232 (8%) measles detections were recorded from the 40 373 cattle slaughtered at Meatco Oshakati Abattoir. C.bovis is considered important from both an economic (loss of income to cattle owners) and human health (it is a zoonosis) point of view. The aim of the study was to do an analysis of the level of C.bovis in bovine carcasses at the abattoir, in order to identify geographical areas where the disease is prevalent in cattle in North Central Namibia and to develop a risk communication strategy, to improve the control of this disease in the target population (subsistence cattle farmers in the study area). Meat inspection was carried out for a period of 12 months and the abattoir records examined covered two years (2004 –2005). Results showed that incidence for C.bovis of cattle originating from Oshikoto Region were high: (12%). Omusati and Oshana Regions had an incidence of 7% and a much lower incidence of C.bovis (5%) was reported in cattle from the Ohangwena Region. Structured interviews with 95 farmers (99% male and 1% female) in all the four Regions of the study area, were carried out using a set of questionnaires (Appendix A). The questionnaires had provision for numerical data and comments concerning changes, constraints and suggestions for improving dissemination of information and extension services in the target areas. It was noted that between 5% and 13 % of respondents had neither pit latrines nor waterborne sewage. This leads to the conclusion that a significant proportion of the rural population is defecating in an area which is available to the cows grazing close to the homestead or cattle post. In addition, 61% of cattle from this area are marketed through informal marketing and many of them are slaughtered informally with no meat inspection taking place. This provides a high risk of infestation for the consumer, which perpetuates the parasite in the rural population. The educational level showed that 24% had no formal education and 33% had only primary school education. The language spoken by 93% of respondents is Oshiwambo and although only 20% speak English or Afrikaans, extension materials, including visual material, is not available in the vernacular. Between 58% and 96% of the respondents from the four Regions had no knowledge of the disease and how it could be diagnosed and controlled in both people and cattle. In Oshikoto Region, where the incidence in slaughtered cattle was the highest, only 4% of the respondents knew about C.bovis in cattle. The results obtained for language proficiency advocates for extension materials to be developed in Oshiwambo (which 93% of the target population are able to read and write). Skills training using visual aids and personal communication in Oshiwambo would be needed for other 7% who are illiterate. From the above, it was concluded that extension is needed to reduce the incidence of C.bovis in cattle. The most important extension messages, as determined by an expert opinion survey of veterinarians in Namibia, were firstly that families should be treated for tapeworms and secondly that they should only buy meat that has been inspected after slaughter at an abattoir. It was recommended that stakeholders in the livestock industry, and the state as well as the Department of Health should be made aware of the high level of cysticercosis and the equally high level of ignorance about the disease in rural areas of North Central Namibia. Veterinary and agriculture staff should be motivated to communicate and combine efforts to assist each other, as transport is expensive and one of the main constraints to successful extension because of the great distances in North Central Namibia. Funding should also be found for production of extension materials in the vernacular. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Paraclinical Sciences / MSc / unrestricted

Torque-teno viruses (TTVs) are small ubiquitous non-enveloped single-stranded circular DNA viruses. Since their discovery in a post-transfusion hepatitis patient, they have been isolated in several vertebrate hosts with over 90% prevalence, including swine. They have been detected in the environment, water sources, human drugs, vaccine and blood product as contaminants. Intriguingly, the role of TTVs in human disease causation is still not fully understood. Several epidemiological studies have associated TTVs to human diseases, like cancers, hepatitis, and autoimmune diseases, but no clear link between infection and clinical disease has been demonstrated yet. In contrast, experimental studies done in pigs demonstrated that swine TTVs (TTSuVs) could an act as sole pathogens. Other studies also demonstrated that TTSuVs could exacerbate symptoms of other viral pathogens in coinfections. Here, we showed that TTSuV1 could be zoonotic, as we detected TTSuV1 DNA in human serum samples. We also showed that TTSuV1 could replicate in human immune cells, and consequently suppress their ability to respond to immune stimuli. Further in-vivo studies, to elucidate host immune regulation by TTSuVs, showed a delayed antibody response and minimal viremia. Also, we found that viral sensing could be limited to interferon-inducing sensors (DHX36), while upregulation of PD-1 could demonstrate how these viruses may establish chronic infections. In another study, we showed the use of our novel recombinant TTSuV1 culture system to study the synergistic interactions between TTSuV1 and porcine circovirus 1 (PCV1). When both viruses were cultured together in-vitro, their respective viral titers were increased, compared to the single virus infections. We also demonstrated that increased in-vitro replication of TTSuV1 could be relying on expression of PCV1 replicase. In addition, molecular mechanisms were used to explain this synergistic relationship; a strong promoter activity by the putative major promoter of TTSuV1 was shown to be blocked PCV1 and TTSuV1 replicase proteins, but protein-DNA interaction assays need further optimizations to demonstrate physical interaction between these viruses. In conclusion, our result showed new information about TTSuV1 transmission, pathogenesis, host innate immune regulation, and their role in coinfections.