Molecular characterization of Babesia caballi and Theileria equi, the aetiological agents of equine piroplasmosis, in South Africa

In an attempt to develop quantitative real-time PCR (qPCR) assays for the detection of equine piroplasms, sequence heterogeneity in the V4 hypervariable region of the 18S ribosomal RNA (rRNA) gene sequences within both Theileria equi and Babesia caballi from South Africa was discovered. A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was therefore carried out using horse and zebra samples from different geographical locations around South Africa. We evaluated the ability of a recently developed T. equi-specific qPCR assay in detecting all T. equi 18S rRNA variants identified in South Africa. We further present the first report on the development and application of a TaqMan minor groove binder (MGB™) qPCR assay, targeting the 18S rRNA gene, for the detection of B. caballi infections in equine blood samples. Despite the ability of the 18S rRNA T. equi- and B. caballi-specific qPCR assays to detect all known 18S rRNA gene sequence variants thus far identified in South Africa, the existence of as yet undetected variants in the field cannot be overlooked. Other qPCR assays targeting alternative genes could be developed which, used in conjunction with the 18S rRNA qPCR assays, may provide better confirmation of test results. A T. equi-specific qPCR assay targeting the equi merozoite antigen gene (ema-1) was recently developed for the detection of T. equi parasites in the midgut of Rhipicephalus (Boophilus) microplus nymphs. This assay was not able to detect T. equi in all South African samples that were confirmed positive by other molecular and serological assays. Sequence characterization of the ema-1 gene from South African isolates revealed the existence of variation in the regions where the qPCR primers and probes had been designed. Based on these observations, a conserved region of the ema-1 gene was selected and targeted in the development of an ema-1-specific TaqMan MGB™ qPCR assay, which was shown to have a higher sensitivity than the previously reported ema-1 qPCR assay. The rhoptry-associated protein (rap-1) gene from South African B. caballi isolates was also characterized following the failure of a B. caballi-specific competitive-inhibition enzyme-linked immunosorbent assay (cELISA) to detect B. caballi antibody in the sera of infected horses from South Africa. The genome walking PCR technique was used to amplify the complete rap-1 gene sequence from two South African B. caballi isolates. Significant heterogeneity in the rap-1 gene sequences and in the predicted amino acid sequences was found. Marked amino acid sequence differences in the carboxy-terminal region, and therefore the probable absence of the monoclonal antibody binding site, explains the failure of the cELISA to detect antibody to B. caballi in sera of infected horses in South Africa. This is the first comprehensive molecular study of the parasites that cause equine piroplasmosis in South Africa. Our results add further to the existing knowledge of piroplasmosis worldwide and will be invaluable in the development of further molecular or serological diagnostic assays.Thesis (PhD)--University of Pretoria, 2009.Veterinary Tropical Diseasesunrestricte

Obstetric outcomes of grand multiparous women in Soweto

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Medicine in Obstetrics and Gynaecology MMed (O&G) Johannesburg, October 2014Background Grand multiparous women, defined as women who have had five or more deliveries, have historically been considered to be at risk for maternal and fetal complications. Over the years, these complications have been attributed to physiological changes as a result of high parity, maternal age, age-related medical conditions and socioeconomic status. Recent research has indicated a strong relationship between access to health care, especially in the antenatal phase, and outcomes. This work aimed to describe maternal, obstetric and fetal complications occurring in GM women, to determine their attendance at antenatal clinic, to review their modes of delivery and to identify any demographic characteristics related to GMP. Methods This was a prospective, descriptive study undertaken at Chris Hani Baragwanath Academic Hospital, a tertiary and regional hospital situated in Soweto that serves approximately two million people within its jurisdiction. In excess of 23 000 deliveries take place there each year. The labour ward attends mostly to high-risk women and approximately 20 % low-risk walk-ins. Another 10 000 births are conducted at midwife obstetric units in Soweto. This study surveyed a sample of pregnant women presenting at Chris Hani Baragwanath and the referring midwife obstetric units who had had five or more viable deliveries, including the current birth, and was conducted over four months in 2011. Results A total of 122 women were included with 124 deliveries as there were two twin pregnancies. Detailed data were available for 98 of these women. The study group were largely of advanced maternal age and were generally healthy. The attendance rate at antenatal care was high (91.35%). Antepartum and postpartum complications were infrequent and there were no intensive care unit admissions or maternal deaths. The CS rate was high (32.79 %), with more emergency CSs performed than elective CSs. The majority of the emergency CSs performed was as a result of fetal distress. There were four stillbirths (3.23%), and 25 (20.16%) of infants weighed <2500g at birth. Conclusion This study showed good maternal and fetal outcomes in a group of GM women who have access to and who largely attended antenatal care facilities. The results, albeit from a small sample, do not support traditional views that GM women are at risk of poor outcomes due to advanced maternal age, physiological changes as a result of high parity or low socioeconomic status. GM women who are generally healthy and are afforded access to adequate health care facilities should have good pregnancy outcomes

An evaluation of the capital budgeting process for a multinational firm.

Thesis (MBA)-University of Natal, Durban, 2001.For the purposes of this study I have adopted a case study approach, based on a Multinational company in the UK, with business units geographically spread throughout the world, including South Africa. I intend to provide a detailed analysis of all aspects of the Capital Budgeting process. The dissertation will cover the follow ing areas : • The capital appraisal techniques used to evaluate capital projects. • The determination of a cost of capital. • Adjustments to the cost of capital in a multinational context. The approach in this study will be to divide Capital Budgeting into the three specific areas as detailed above, discuss the theory associated with the subject, analyse empirical research on the topic and critically evaluate the findings of the practices at the Multinational chosen for this study. Due to confidentiality reasons I shall refer to the company as "PLC" for the purposes of this study. 1.3. Objectives of the Study The objective of the study is to evaluate the capital investment appraisal process of "PLC", in the light of theoretical and empirical literature on the subject, leading either to suggestions for improvement or acknowledging the merit of the current practice. It is expected that "PLC" utilises sophisticated methods for investment appraisal but does allow room for improvement

The role and potential of Isipingo as an inter-modal transport node within the Durban metropolitan area.

Thesis (M.T.R.P.)-University of KwaZulu-Natal, Durban, 2009.No abstract available

Evaluation of serological assays for the diagnosis of HIV infection in adults

Serological tests based on the enzyme immunoassay (EIA) are the primary tool for the diagnosis of human immunodeficiency virus (HIV) in adults and have rapidly evolved to quicker, affordable and more accurate test formats to detect early HIV infection. Secondand third-generation HIV rapid tests detect the immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to the HIV and are used at the point of care and in HIV self-testing. The tests are affordable and accessible in state and private diagnostic laboratories. The presentday fourth- and fifth-generation EIAs can detect both p24 antigen and IgG and IgM HIV antibodies and thereby diagnose early HIV infection at approximately 2 weeks. The fourthand fifth-generation EIAs also report sensitivity and specificity of more than 99%. The correct interpretation of HIV diagnosis of false-positive and false-negative EIA test results requires collaborative scrutiny of patient factors and laboratory test methodologies.https://safpj.co.za/index.php/safpjChemical PathologyMedical Virolog

Flow cytometric analysis of apoptotic biomarkers in actinomycin D-treated SiHa cervical cancer cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 °C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.The National Research Foundation (NRF) and the South African Medical Research Council (SAMRC).https://www.jove.com/journalam2022Chemical Patholog

Development of cathepsin L-like real-time PCR assays for the detection of African animal trypanosomosis (AAT) in South Africa

African animal trypanosomosis (AAT), is an infectious parasitic disease of wildlife and livestock caused by multiple species and strains of Trypanosoma. In South Africa, it is restricted to northern KwaZulu-Natal (NKZN) and caused by Trypanosoma congolense and Trypanosoma vivax. A cross-sectional study was done to determine AAT prevalence in 384 goat samples and identify trypanosome species circulating in 60 cattle at dip tanks that are on the interface with the HluhluweuMfolozi game reserve in NKZN. Both cattle and goat samples were analyzed using the buffy coat technique (BCT) and a polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 1 (ITS) region. Cattle samples were further analyzed using an ITS quantitative real-time PCR (qPCR) assays designed for the detection of T. congolense, T. vivax, and T. brucei. None of the goat samples tested positive for Trypanosoma infections. The ITS qPCR assay detected Trypanosoma DNA in 30% of the cattle samples, while only 8.3% were positive with the ITS PCR and 11.7% were positive using BCT. Quantitative real-time PCR assays were designed to amplify a 98 bp, 137 bp, and 116 bp fragment of the cathepsin L-like (CATL) gene from T. brucei, T. theileri, and T. congolense, respectively. Each assay was shown to be efficient (>94%) and specific (109 to 102/101 copies/reaction) in the detection of Trypanosoma species. The CATL qPCR assays detected T. congolense and T. theileri infections in 33.3% of the cattle samples. The CATL qPCR assays also detected T. congolense infections in goats (23.1%) that were neither detected by BCT nor the ITS PCR. The CATL qPCR assays provide an additional, sensitive, and specific tool for Trypanosoma diagnostics. The presence of trypanosomes in goats suggests they might be potential reservoirs of infections to other livestock.The Belgian Directorate-General for Development Co-operation Framework Agreement.https://www.mdpi.com/journal/pathogensam2023Veterinary Tropical Disease

Equine piroplasmosis status in the UK: an assessment of laboratory diagnostic submissions and techniques

Equine piroplasmosis (EP) has historically been of minor concern to UK equine practitioners, primarily due to a lack of competent tick vectors. However, increased detection of EP tick vector species in the UK has been reported recently. EP screening is not currently required for equine importation, and when combined with recent relaxations in movement regulations, there is an increased risk regarding disease incursion and establishment into the UK. This study evaluated the prevalence of EP by both serology and PCR among 1242 UK equine samples submitted for EP screening between February and December 2016 to the Animal and Plant Health Agency and the Animal Health Trust. Where information was available, 81.5 per cent of submissions were for the purpose of UK export testing, and less than 0.1 per cent for UK importation. Serological prevalence of EP was 8.0 per cent, and parasite DNA was found in 0.8 per cent of samples. A subsequent analysis of PCR sensitivity in archived clinical samples indicated that the proportion of PCR-positive animals is likely to be considerably higher. The authors conclude that the current threat imposed by UK carrier horses is not adequately monitored and further measures are required to improve national biosecurity and prevent endemic disease

The significance of viral, bacterial and protozoan infections in zebra : a systematic review and meta-analysis of prevalence

TABLE S1 : PRISMA Checklists.TABLE S2 : Checklists based on Migliavaca et al. (2020).TABLE S3 : JBI critical appraisal checklists.TABLE S4 : Clinical signs, influencing factors and genotype/serotypes associated with microbial infections in zebra.Wild equids can harbour multi-host infectious agents that are able to affect other wildlife species, but also domestic animals and humans. The direct and indirect contact between wild and domestic equids is constantly increasing due to global movement of horses and equine products, the depletion of natural areas and climate and land-usage changes, which could result in burdensome epidemics. Nevertheless, currently there is a lack of adequate epidemiological data from zebra. Three electronic databases were searched from 10 to 20 March 2021 for publications reporting bacterial, viral and protozoan infections in zebra. Data for a total of 12 relevant variables were extracted from reviewed papers to undergo a qualitative analysis. Prevalence-reporting studies were subjected to meta-analysis for estimating the pooled prevalence and seroprevalence of microbials in wild zebra populations. We identified 30 pathogen species and the most represented were equine Herpesvirus 1 and 9, Bacillus anthracis, African horse sickness virus and Theileria equi. They were reported from all the three zebra species, both in captivity and wilderness. Pooled seroprevalences were estimated for the equine Orbiviruses AHSV (70%; 95% CI: 35–96%) and EEV (21%; 95% CI: 8–38%) and for the equine α-Herpesviruses EHV-1 (72%; 95% CI: 43–93%), EHV-4 (40%; 95% CI: 0–100%) and EHV-9 (58%; 95% CI: 9-98%), and pooled prevalences for the equine piroplasms T. equi (100%; 95% CI: 94–100%) and B. caballi (8%; 95% CI: 0–28%). Zebra is most probably a component of the reservoir from which AHSV, EHV-1 and T. equi can be directly or indirectly transmitted to horse populations, potentially causing disastrous epidemics. Zebra can also harbour zoonotic pathogens like B. anthracis, Brucella spp., A. phagocytophylum, CCHFV and T. brucei. Other agents like EHV-9, BPV-1 and BPV-2 have the potential to spread from zebra to other wild endangered animal species. We conclude that zebra is an important host of multiple and dangerous pathogens for both animals and humans. Comprehensive studies focused on the prevalence of infectious agents present in zebra populations and the associated risk factors are required.http://www.italian-journal-of-mammalogy.ithj2023Veterinary Tropical Disease