Theses and Dissertations (Veterinary Tropical Diseases)

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Prevalence of Toxoplasma gondii in commensal pest rodents at the National Zoological Garden in South Africa
(University of Pretoria, 2023-10) Morar-Leather, Darshana; Bokaba, Refilwe; De Bruin, Madeli; Lewis, Christian; u29041092@tuks.co.za; Thovhakale, Ndidzulafhi Terrence
Toxoplasmosis is a zoonotic disease caused by the ubiquitous Apicomplexan protozoa Toxoplasma gondii (T. gondii). The overall epidemiology of T. gondii in Southern Africa is understudied. Although a few studies have documented its circulation in humans, domestic animals, and wild animals, these studies were limited in species diversity and geographical location. Rodents are intermediate hosts and are recognised as key reservoir hosts for T. gondii. Rodents play an important role in the maintenance and transmission of the parasite as they are preyed on by cats, the definitive hosts. Toxoplasma gondii infection rates in the local rodent population may reflect infection rates in cats. The aim of this study was to determine the prevalence of T. gondii in pest rodents within the South African National Biodiversity Institute National Zoological Garden (SANBI NZG). Furthermore, an attempt was made to confirm the presence of T. gondii DNA in the various rodents’ tissues (brain, tongue, muscle, diaphragm, and heart) using quantitative polymerase chain reaction (qPCR). A total of 138 sera were tested for T. gondii antibodies using a commercial latex agglutination test. A cut-off titre ≥ 64 was used to distinguish between positive and negative cases. Ten samples were positive for T. gondii antibodies, bringing the overall prevalence to 7.25% (95%, CI= 3.53 – 12.92). Using the generalised linear model, there was a statistically significant (p<0.00432) positive correlation between presence of T. gondii antibodies and rodent body weight. No T. gondii DNA amplification was observed on the tissue samples from the ten T. gondii antibody positive rodents. The results of this study provide baseline knowledge about the role of rodents in the epidemiology of T. gondii natural infections, particularly in the human-wildlife interface.
Nematodes diversity in Mastomys rodents (Rodentia: Muridae) and molecular characterization of Trichuris species in the Mnisi Community, South Africa
(University of Pretoria, 2023) Marufu, Munyaradzi Christopher; Matthee, Sonja; Byaruhanga, Charles; jessemukisabryt@gmail.com; Mutesasira, Jesse Mukisa
Nematodes comprise of many species with diverse life histories and zoonotic potential. Understanding the distribution and diversity of nematodes in the commensal rodent genus Mastomys is crucial for assessing their impact on wildlife and livestock, and potential of zoonotic disease transmission. The current study investigated the nematode diversity in Mastomys species rodents in three habitats and characterized the recovered Trichuris sp. using morphometric and molecular techniques at a wildlife-human/domestic animal interface in the Savanna biome in Mnisi communal area, Mpumalanga, South Africa. Nematodes were recovered and identified in the gastrointestinal tracts of 68 M. natalensis and 27 M. coucha rodents which were trapped in crop, village and natural habitats in the Mnisi communal area in October 2020. Nematodes were microscopically identified using morphometric measurements. Molecular characterization of Trichuris sp. was achieved through deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR), Sanger sequencing and phylogenetic analyses of three genes: internal transcribed spacers (ITS) 1, ITS 2 and cytochrome B (CytB). Data were analyzed using descriptive statistics, univariate models, a zero-inflated negative binomial generalized linear model, and a binomial generalised linear model, to establish the frequency, measures of central tendency and the relationships between nematode counts or occurrence and predictor variables using R statistical software. Nematodes were recovered in 20% of the examined rodents, with a total of 46 nematodes recovered, representing two species: Trichuris sp. (mean abundance of 0.31± 0.22) primarily from the caecum and Abbreviata sp. (mean abundance of 0.15±0.14) primarily from the stomach. Almost all the rodents were infected with only one nematode species, while one rodent exhibited mixed infection of both nematode species. No significant differences (p>0.05) in nematode prevalence were observed between male and female Mastomys spp. Univariate and multivariable analysis confirmed a lack of significant differences (p>0.05) in nematode abundance concerning habitat type, rodent species, and sex. The obtained novel Trichuris sp. ITS1, ITS2 and CytB sequences, clustered in a distinct clade from published sequences, but showing genetic relationships with known Trichuris spp. The current study emphasizes the importance of integrating morphometric identification and molecular analysis to accurately categorize Trichuris spp. and suggests a need for a larger sample size per habitat type in future research on nematode diversity.
Knowledge, attitudes and practices analysis of farmers on the risk of introducing peste des petits ruminants from Angola into northern communal areas of Namibia
(University of Pretoria, 2023-11) Crafford, Jan Ernst; bbbgore@gmail.com; Gorejena, Brighton
Peste des petits ruminants (PPR) is a highly contagious viral disease primarily affecting goats, sheep, and some wild small ruminants. It is characterized by fever, necrotic stomatitis, gastroenteritis, pneumonia, and mortality. Namibia is officially free of PPR in one zone, not the entire country. The national herd has not been exposed to PPR and is naïve. Thus, an outbreak of the disease is potentially devastating on a socioeconomic level. The closest PPR outbreak was in Cabinda province in Angola. To better understand the risk factors for introducing PPR from Angola, a study was conducted using a knowledge, attitudes, and practices (KAP) survey. The research employed a qualitative descriptive survey design consisting of questionnaires and interviews with 376 communal farmers residing within 10-20 km of the Namibia/Angola border in Namibia's Omusati and Ohangwena regions. The results showed that 84% of the farmers surveyed had insufficient knowledge regarding PPR, while 89% were unaware of its clinical symptoms. Nevertheless, the farmers showed good comprehension of general disease prevention techniques, including vaccination (99%), livestock isolation (85%), quarantine (72%), and regulated animal movements (94%). Additionally, the farmers exhibited awareness of the detrimental effects of disease outbreaks (90%). It was concluded that farmers' knowledge, attitudes, and practices (KAP) in Namibia's surveyed northern communal areas present a moderate risk of PPR incursion. The current surveillance strategies the competent authority implements are deemed sufficient and can be sustained. However, the study recommends enhancing PPR awareness among northern communal farmers, particularly those living near the Namibia/Angola border.
Molecular detection of tick-borne haemoparasites in cattle and buffalo samples from Mashonaland West and Masvingo Provinces, Zimbabwe
(University of Pretoria, 2022) Sibeko-Matjila, K.P. (Kgomotso Penelope); Bhoora, Raksha V.; annicky.modirwa@up.ac.za; Modirwa, Annicky A.R.
Tick-borne haemoparasite diseases caused by Babesia, Theileria, Anaplasma and Ehrlichia species are a major constraint to the beef and dairy cattle industry, causing the most economic losses of cattle in sub-Saharan Africa. The cattle industry in Zimbabwe is continuously threatened by the spread of tick-borne diseases, which significantly affect the economy not only through morbidity and mortality but also through the costs involved in the control of diseases and treatment of sick animals. However, there is a lack of current data on the distribution of tick-borne diseases in Hurungwe district, Mashonaland West Province. The current study used molecular tools to investigate the occurrence of haemoparasites in cattle from Hurungwe district in Mashonaland West Province and buffalo from Gonarezhou National Park in Zimbabwe. DNA was extracted from 87 whole blood samples including 80 cattle and seven buffalo. The DNA samples were subjected to the Reverse-line blot hybridization (RLB) and quantitative real-time polymerase chain reaction (qPCR) analyses. Haemoparasite infections were detected in 58 samples (67 %) by RLB, and 55 % of these only hybridized to the genus-specific probes. Tick-borne haemoparasites detected by RLB included three Theileria species (T. mutans, T. velifera, and Theileria sp. sable), detected in single and mixed-parasite infections. Anaplasma centrale (3 %) and Babesia bigemina (1 %) were also detected by the RLB assay. The most commonly occurring tick-borne pathogens in cattle detected by qPCR assays were A. marginale (28 %) and B. bigemina (9 %); followed by A. centrale (8 %) and B. bovis (3 %). While in buffalo A. marginale (86 %), followed by A. centrale (14 %) were mostly detected. The results of the current study indicated that the species-specific qPCR assays used were more sensitive in detecting haemoparasites than the RLB assay. Anaplasma marginale and Babesia bovis were only detected by the species-specific qPCR assays and not by the RLB assay, which suggests that these haemoparasite infections were present at low levels thus could not be detected by RLB assay. The RLB assay suffers lower sensitivity when a sample is infected with more than one haemoparasite, especially when the levels of infection vary; the high infection will be preferentially detected over low infections of the same genus due to primer competition. Notably, T. parva or E. ruminantium was not detected from the investigated samples. The amplification and sequencing of the 16S and 18S rRNA genes from samples that hybridized exclusively to the RLB genus-specific probes yielded nine and one good quality sequences, for the 16S and 18S rRNA genes respectively. However, BLASTn analysis did not reveal hits to any haemoparasites expected to occur in cattle and buffalo. Our results did not follow the common trend for the prevalence of tick-borne diseases of cattle in Zimbabwe. Bovine theileriosis has recently been reported to be responsible for most cattle mortalities in Zimbabwe, followed by babesiosis, heartwater, and then anaplasmosis. Our results therefore suggest that the trend of occurrence of tick-borne diseases depends on the vector-parasite-host-environment dynamics for each province, thus may vary between provinces. Finally, this study confirms that buffalo in the sampled area are carriers of tick-borne diseases that pose risk to the cattle population.
Knowledge, attitudes and practices analysis, prevalence and molecular detection of Neospora caninum and Toxoplasma gondii in livestock in the Khomas region of Namibia
(University of Pretoria, 2023-12) Matjila, P.T. (Paul Tshepo); Neves, Luís C.B.G.; alastersamkange@gmail.com; Samkange, Alaster
This study chiefly focussed on the Khomas region of Namibia, and the research areas were: (1) the knowledge, attitudes and practices regarding neosporosis and toxoplasmosis among livestock farmers in the Khomas region and animal health practitioners in the whole country; (2) the seroprevalence and associated risk factors of Neospora caninum in cattle; (3) the seroprevalence and associated risk factors of Toxoplasma gondii in sheep and goats; (4) molecular investigation of N. caninum DNA in cattle and, (5) the molecular detection of T. gondii in sheep and goats in abattoir samples. Only 15.9% (10/63) of the livestock farmers had heard about neosporosis or toxoplasmosis or knew how animals get infected (p<0.0001). Five per cent (3/63) of the farmers knew the risks associated with keeping dogs and cats concerning neosporosis and toxoplasmosis, respectively (p<0.0001). None of the animal health practitioners (n=51) routinely requested N. caninum or T. gondii laboratory tests in cases of cattle, sheep or goat abortions. Although all animal health practitioners indicated they routinely interacted with livestock farmers, none regularly discussed neosporosis or toxoplasmosis. Five point seven per cent (42/736) of the bovine sera were seropositive to N. caninum. Eight of the 32 establishments had at least one positive animal, giving a herd-level seroprevalence of 25%. There was no significant association between N. caninum seropositivity in cattle and the presence of dogs, jackals, history of abortions, farm size, and the number of cattle or average annual rainfall. The establishments with moderate to high numbers of Feliformia were 9.8 times more likely to be seropositive to N. caninum than those with none to low levels of the former (p=0.0245). Overall, 3.68% (11/299) of the sheep sera were seropositive to T. gondii, and 61.54% (8/13) of the sheep flocks tested had at least one positive animal. Only 0.29% (1/345) of the goat sera were seropositive to T. gondii, and one of the 19 goat flocks had at least one positive animal, giving a herd-level prevalence of 5.26%. Sheep flocks had significantly greater animal-level and flocklevel prevalences than goats (p<0.05) and were 13.14 times more likely to be seropositive (OR = 13.14; CI 95%: 1.686-102.382) than goat flocks. Seropositivity to T. gondii was positively associated with the total number of sheep at the farming establishment, history of abortions and farm size (p<0.05), but not goats. The study concluded that sheep were probably more exposed to T. gondii infection than goats and that the T. gondii seroprevalence level in the Khomas region was very low compared to other countries. One hundred and ninety-nine bovine abattoir samples were collected from different animals, comprising 110 brain samples and 75 heart muscle samples. In addition, there were 14 whole blood samples from N. caninum seropositive cattle. The collected samples were tested using a conventional PCR targeting the pNc5 gene. All the samples tested were negative. The authors concluded that the negative results could be due to the low prevalence of N. caninum infection caused by adverse weather conditions and that a future study targeting aborted fetuses over a more extended period could yield positive results. The T. gondii molecular study analysed 174 brain and heart tissue samples from sheep and goats for the presence of T. gondii DNA using nested PCR targeting the B1 gene. The tissue samples were obtained from animals at abattoirs designated for human consumption. The study found that 16.7% of the samples tested positive for T. gondii DNA, with a higher prevalence in sheep (17.4%) than in goats (7.7%). Eight of the 29 positive samples were successfully sequenced using the Sanger method. All isolates identified were closely related to T. gondii type III genotype, exhibiting alignment scores ranging from 96.44% to 100%. This study emphasizes the public health hazards of consuming undercooked sheep and goat meat and highlights the pressing need to introduce control measures to mitigate human exposure.
One Health investigation at abattoirs in South Africa
(University of Pretoria, 2023-12) Van Heerden, Henriette; Kolo, Francis B.; Jaja, Ishmael F.; desireemazwi@gmail.com; Mazwi, Koketso Desiree
One Health is a collaborative, multisectoral, and transdisciplinary strategy/approach with the aim of attaining the best possible health outcomes that acknowledge the connections between humans, animals, plants, and their common environment at the local, national, regional, and global levels. Zoonoses pose a threat to environmental integrity, animal and human welfare, and economic development. In this One Health project, zoonotic diseases were investigated that influence the health of abattoir workers and domestic animals slaughtered in this environment. Zoonotic diseases such as brucellosis and coxiellosis (Q-fever) are well known infections of humans and domestic animals while toxoplasmosis is zoonotic from cats to humans and thus investigated for its infectious potential in this study. Brucella abortus and B. melitensis have been reported in South Africa, first in small ruminants and later in cattle as well as humans since the turn of the century. Similar to brucellosis, humans are infected with Q-fever through contact with infected animals which may be asymptomatic. Their secretions result in occupational public health exposure potential for humans in contact with animals such as abattoir workers, animal handlers and veterinarians. Toxoplasma gondii is a ubiquitous protozoan parasite of warm-blooded animals. The organism causes infection in humans, wildlife, and domestic animals, including birds, cats, sheep, goats, cattle, and pigs. This study aimed to determine the knowledge, attitudes and practices (KAP) on zoonotic diseases of the abattoir workers at Eastern Cape province; assessing seropositivity of Coxiella burnetii, Toxoplasma gondii and Brucella spp. antibodies in African abattoir workers with meta-analysis; detect antibodies to zoonotic pathogens (C. burnetii, T. gondii and Brucella spp.) and characterize Brucella spp. in livestock slaughtered at these abattoirs using serology, bacteriological and molecular methods; and characterize B. abortus and B. melitensis isolated from cattle, goat and human in South Africa using whole genome sequencing single nucleotide polymorphism (wgSNP) analysis to determine the diversity of isolates from different hosts. Semi-structured questionnaires were administered to the abattoir workers regarding their KAP on zoonotic diseases. Of the 76 respondents who participated in the KAP questionnaire, it was discovered that 18,4%, 19.2% and 47.4% of abattoir workers had knowledge of Q-fever, toxoplasmosis, and brucellosis, respectively. Consumption of undercooked meat was reported by 11,8% while 31,6% of the abattoir workers drank unpasteurized milk. Most abattoir workers (84%) wore PPE, however, some of them explained that the use of PPEs was to protect their clothes from blood stains. Abattoir workers (75,3%) also reported hand injuries which occurred whilst at work. Lack of knowledge regarding micro-organisms and improper use of PPE were identified as important risk factors in the abattoirs. A total of ninety-two abattoir workers with known brucellosis seropositivity were evaluated for C. burnetii using the C. burnetii ELISA IgG and IgM assay. The overall C. burnetii seropositivity was 61.90% (95%Cl: 51.3 – 71.9) in abattoir workers with 61.96% IgG antibodies and 1.09% had IgM and IgG antibodies. The African abattoir worker meta-analysis indicated brucellosis seroprevalence ranging from 1.6-33.5% with two test or more done in 12 African countries, toxoplasmosis seroprevalence detected with different single test ranging from 39.1-84.0% in 8 countries and Q-fever seroprevalence done in 3 countries ranging from 6.5-37.1% which was much lower than the 61.9% observed in abattoir workers in this study. Livestock slaughtered at abattoirs from Eastern Cape Province included a total of 565 animals (280 cattle, 200 sheep and 85 pigs) sampled from 5 abattoirs including both high-throughput and low-throughput. Blood/serum and tissue (kidney, liver, spleen, lymph nodes and tonsils) samples were collected from corresponding animals. The objective of the study included determining the seropositivity and co-infection of infectious/zoonotic pathogens in slaughtered animals. This was achieved by serum screening using Rose Bengal test (RBT) and followed by confirmation for antibodies against smooth Brucella using indirect enzyme-linked immunosorbent assay (iELISA), Compliment Fixation Test (CFT). The Mast® Toxoreagent test and iELISA were used for the detection of antibodies against T. gondii and C. burnetii, respectively. The overall Brucella positivity based on at least two tests using RBT, CFT, iELISA and PCR was 4.3%, 1.0% and 0.0% in cattle, sheep, and pigs respectively. T. gondii seropositivity of 37.9%, 1.5% and 7.1% was observed in cattle, sheep, and pigs, respectively. Coxiella burnetii seropositivity of 26.4%, 15% and 2.4% was observed in cattle, sheep, and pigs, respectively. Co-exposure was detected in cattle for antibodies against C. burnetii and T. gondii (40.54%), Brucella spp. and T. gondii (1.35%), and Brucella spp. and Coxiella burnetii (4.05%). Co-exposure to Brucella spp., Coxiella burnetii and Toxoplasma gondii (4.05%) was detected in cattle. Co-exposure to Brucella spp. and Coxiella burnetii (6.67%) was detected in sheep. The 16S-23S ribosomal DNA interspacer region (ITS) PCR was used for the direct screening of Brucella spp. DNA from the tissues of slaughtered animals. The positivity frequency of 33.57% (94/280) cattle, 14.5% (29/200) sheep and 4.71% (4/85) pigs were observed. Suspect Brucella cultures from tissues were recovered from 43.6% (41/94) cattle, 51.7% (15/29) sheep, and 50% (2/4) pigs. The AMOS-PCR characterised B. abortus in 38/41 cattle,11/15 sheep and 2/2 pig cultures. A mixed infection of B. melitensis and B. abortus was observed in 3/41 cattle and in 4/15 sheep cultures. In cattle, the highest number of isolates were recovered from lymph nodes (33%, 31/94), followed by liver (26.6%, 25/94), while the lowest isolation was recovered from tonsils (10.6%, 10/94). In sheep, the highest number of isolates were recovered from liver and kidney (38%, 11/29), while the lowest isolation was recovered from lymph nodes (27.6%, 8/29). The isolation in pigs was only observed from tonsils (50%, 2/4). Brucella abortus and B. melitensis isolates from humans and animals in South Africa were characterised by whole genome sequencing single nucleotide polymorphism (wgSNP) analysis. The wgSNP analysis of B. abortus isolated from South African cattle showed diversity as well as transmission amongst livestock and humans since the B. melitensis isolated from humans and goats from the same outbreak clustered together. In conclusion, education of abattoir workers is essential as these workers are at risk of zoonotic diseases detected at these abattoirs. Lack of knowledge observed regarding zoonotic diseases, as well as failure to understand the correct use of PPE raises a concern since abattoir workers have high exposure potential to different zoonotic infections. This study reports the serological identification of Brucella spp., C. burnetii and T. gondii infections from apparently healthy slaughtered livestock. Mixed infections of B. abortus and B. melitensis were reported from cattle and sheep tissues that need to be further investigated together with B. melitensis in cattle and sheep as well as B. abortus in pigs. We recommend that training on PPE is done together with awareness of microorganisms to emphasize this unseen treat to workers. Furthermore, education to farmers on zoonotic disease and their control will also help to strengthen the health aspect in both animals and humans as animal health influence human health and the environment. Animals slaughtered at abattoirs provide ideal samples for investigation of serological and molecular detection of zoonotic and infectious pathogens that can be used for surveillance and better understanding of prevalence of these diseases in South Africa.
Diagnosis of anthrax and the roles of host and environment in the transmission of anthrax in Kruger National Park
(University of Pretoria, 2023) Van Heerden, Henriette; Turner, Wendy C.; Archer, Emma; jaymn4u@yahoo.com; Ochai, Sunday Ochonu
Over the years, various techniques have been employed to diagnose infectious zoonoses, such as anthrax caused by Bacillus anthracis. These methods encompass microscopic examination of blood smears, bacterial culture, molecular diagnostics targeting genetic markers of the pathogen, and serological tests to identify antibodies against pathogen antigens. The accuracy of these techniques largely depends on the specificity and sensitivity of the tests used. Disease monitoring in free-roaming wild animals is challenging, often relying on passive surveillance. However, proactive surveillance, which involves detecting specific antibodies, can provide more reliable and timely insights into disease presence and prevalence within populations, especially when disease signs are below passive surveillance detection thresholds. Nevertheless, primary binding assays, such as the indirect enzyme-linked immunosorbent assay (ELISA) used for detecting antibodies in wildlife, face challenges due to the absence of species-specific conjugates. Also, the diagnosis of anthrax, remains a matter of concern due to the challenges posed by the identification of closely related species that carry regions on plasmids (pXO1 and pXO2) and chromosome that highly resembling those found in B. anthracis. As mentioned, traditional methods for diagnosing anthrax include microscopy, identifying isolates through culture, and using genetic markers such as B. anthracis protective antigen (pagA also known as BAPA on pXO1), lethal factor (lef on pXO1), chromosomal (Ba-1), and capsule (capB on pXO2) genes for molecular detection. Because anthrax is not contagious, the exposure of herbivorous mammalian hosts to B. anthracis is greatly influenced by environmental and climatic factors, as well as host demographics and behaviour. In Kruger National Park (KNP), the most impacted host species used to be kudu (Tragelaphus strepsiceros) until the early 1990s, and outbreaks were more common during the dry season. However, there has been a shift in this pattern, and impala (Aepyceros melampus) is now the most affected species, with outbreaks occurring more frequently during the wet season. In this study, we first developed anti-kudu and anti-impala immunoglobulin-specific conjugates in chickens to compare their binding efficiency with that of commercially available protein-G and protein-AG conjugates. This was done using an ELISA-based avidity index to enhance the serological diagnosis of anthrax. Second, we investigated the complications posed by the presence of atypical B. cereus and other closely related species in diagnosing anthrax with genetic markers and qPCR. For this purpose, we analyzed blood smears from wildlife mortalities in Kruger National Park (KNP), South Africa, comparing the outcomes of anthrax diagnostics using qPCR, microscopy, and culture methods. Finally, we explored the transition of the primary anthrax host from kudu to impala within KNP. Our focus was on identifying potential links between environmental factors—such as precipitation, soil moisture, temperature, and the Normalized Difference Vegetation Index (NDVI)—and the patterns of anthrax mortality occurrences and frequencies. Additionally, we examined the variations in environmental factors and the population densities of various host species over time, aiming to identify any correlations between the densities of host species and the rates of anthrax mortalities. The developed conjugates had a high avidity of >70% against kudu and impala sera. The commercial conjugates (protein-G and protein-AG) had significantly low relative avidity (<20%) against these species. Eighteen additional wildlife species exhibited cross-reactivity, showing a mean relative avidity of over 50% with the developed impala and kudu conjugates, compared to less than 40% with the commercial conjugates. This study underscores the value of species-specific conjugates as crucial tools for developing and validating immunoassays in wildlife, and for monitoring zoonotic diseases across the livestock-wildlife-human interface. In our analysis of 1,706 blood smears from wildlife mortalities, 890 samples were positive for B. anthracis, detected either through genetic markers or microscopy. Specifically, 15.2% of these samples tested positive for the lef marker, and 12.6% for BAPA. The use of both BAPA and lef markers together identified 24.4% of samples as positive, which increased to 44.4% when combined with microscopy, indicating strong concordance between molecular and microscopic methods (p<0.0001). Out of 506 cultured isolates, 24.7% tested positive by either genetic markers or microscopy, but only 4 samples were definitively confirmed as B. anthracis through culture, microscopy, and sensitivity testing to penicillin and gamma-phage. The lef marker was found to have the lowest specificity and accuracy. Conversely, combinations such as Ba-1/capB, BAPA/capB, Ba-1/BAPA/capB/lef, and BAPA/lef/capB achieved 100% specificity and accuracy, with a sensitivity of 75%. The combination of BAPA/lef/Ba-1 also reached 100% in specificity, sensitivity, and accuracy. The findings emphasize the need to identify precise markers for B. anthracis in southern Africa to enhance anthrax diagnosis. The strategic use of both microscopy and multiple markers can significantly reduce false positives. The study also noted distinct trends in anthrax mortality over different years and regions, with a notable shift in the primary host species from kudu to impala. Furthermore, significant correlations were found between anthrax mortality in kudu and environmental factors such as NDVI, average temperature, and standardized precipitation indexes (SPI-6 and SPI-12). In contrast, impala mortality was associated with changes in SPI-3, temperature increases, and higher mortality rates during the rainy season. Interestingly, elephant density was negatively correlated with kudu mortality but positively correlated with impala mortality and density. These observations suggest that environmental conditions and the density of species play significant roles in determining the frequency and variety of hosts exposed to B. anthracis. The study concludes that over time, climate extremes could amplify the severity of anthrax outbreaks by affecting species susceptibility and exposure chances.
Identification of tick species and their bacterial pathogens from cattle in two provinces of South Africa
(University of Pretoria, 2024) Mnisi, Zamantungwa; Oosthuizen, Marinda C.; bongekilel.khoza@gmail.com; Khoza, Bongekile Lungile
In South Africa, resource-poor farmers are negatively affected by death and ill health of livestock due to high tick infestations. Tick infestations are associated with tick- borne pathogens causing various diseases that are a major constraint to cattle farming, a threat to human health and consequently the economy. This has been an ongoing concern for resource-poor farmers, mostly influenced by the inability to access veterinary care or proper education on the usage of veterinary products. This study sought to investigate the presence of ticks and their associated pathogens at three study sites, namely Harrismith and Phuthaditjhaba in the Free State province as well as Bergville in KwaZulu Natal. These are three neighbouring towns, where the point of intersection for livestock is the Drakensberg Mountains, which serve as a source of vegetation for grazing livestock. Between these three study sites there is uncontrolled translocation of livestock due to traditional practices and trade and thus the introduction of several tick species. Ticks are recognised worldwide as major vectors of several disease-causing pathogens and are good indicators of pathogen distribution and epidemiology. However, global warming has result in climate change and consequently expanded tick distribution. Consequently, growing incidences of emerging and re-emerging tick- borne pathogens capable of causing tick-borne diseases (TBDs) of veterinary and economic importance. These TBDs are major hindrances that constrain cattle farming, thus culminating in significant losses: threatening food security, global trade, eco- tourism, and affecting human and livestock health. Therefore, this study sought to identify ticks and detect bacterial tick-borne pathogens in the three neighbouring towns: Harrismith, Phuthaditjhaba and Bergville using a 16S rRNA next-generation sequencing (NGS) approach based on the PacBio sequencing platform. A total of n=50 blood samples were collected from cattle in each study site and n=418 ticks were collected from these cattle, comprising n=126 ticks from Harrismith, n=160 from Phuthaditjhaba and n=132 from Bergville. Ticks infesting cattle were identified morphologically to belong to the genera Rhipicephalus with six species and Hyalomma with only two species. Harrismith had Rhipicephalus decoloratus, R. microplus, R. evertsi evertsi, Hyalomma truncatum, H. rufipes, Phuthaditjhaba: R. appendiculatus R. simus, R. evertsi evertsi, R. afranicus, H. rufipes and Bergville: R. evertsi evertsi, R. appendiculatus, H. truncatum. Out of n=418 ticks collected, R. evertsi evertsi with n=332 was the most dominant tick species in the three study sites, whereas R. decoloratus and R. microplus tick species were only present in Harrismith. A full-length 16S rRNA gene was amplified and sequenced using PacBio technology for the identification of bacterial pathogens associated with these ticks. A total of 7,687,581 reads were obtained. Bacterial pathogens identified belonged to the genera Anaplasma, Mycoplasma and Ehrlichia. Anaplasma species detected were A. marginale, A. centrale, A. phagocytophilum, A. platys and A. bovis. Mycoplasma species were M. wenyonii and M. bovis. Ehrlichia species detected were E. ruminantium and E. canis. Anaplasma marginale, with a relative abundance of 43.5% in Harrismith, 54.2% in Phuthaditjhaba and 56.2% in Bergville, was the most abundant, followed by A. platys with 31.5% in Harrismith, 32.9% in Phuthaditjhaba and 22.6% in Bergville. Mycoplamsa wenyonii was 19.6% Harrismith, 7.8% in Phuthadijthaba and 14% in Bergville. The bacterial composition at the three sites aligned with the tick vectors identified at the three-study sites. The presence of R. microplus and R. decoloratus was reported for the first time in Harrismith, while R. turanicus was identified for the first time in Phuthaditjhaba. This shows that there has been an expansion in tick distribution because of climate change and possibly other ecological and anthropogenic factors.
Resistance of Trypanosome species isolated from cattle populations in Lambwe Valley, Kenya, to diminazene aceturate
(University of Pretoria, 2023-11) Neves, Luís C.B.G.; Masiga, Daniel K.; Vorster, Ilse; kimathibos@gmail.com; Kimathi, Boscoh Odhiambo
Trypanosomosis is a parasitic disease of humans and animals that occurs mainly in sub- Saharan Africa where it negatively affects livelihoods. The control of trypanosomosis in animals has for decades relied on the use of trypanocidal drugs that have increasingly reported resistance. A cross-sectional study was conducted in Kigoto, Wiga and Gendo villages of Lambwe Valley in South-West Kenya to determine the point prevalence of trypanosomosis and to investigate the presence and level of resistance to diminazene aceturate (DA), a commonly used trypanocidal drug in the study area. Three hundred and ninety-five cattle were microscopically screened for trypanosomosis using the buffy coat technique (BCT). To test treatment efficacy, trypanosome positive cattle were recruited into a block treatment experimental design, with DA at 3.5mg/Kg body weight. They were monitored on days 7 and 28 and screened using the BCT and internal transcribed spacer 1-polymerase chain reaction (ITS1-PCR). Data were entered in Microsoft Excel 2016, coded and cleaned. Statistical analysis was carried out using statistical package for social sciences (IBM SPSS) version 2020. The results were presented as mean with their standard deviations (mean ± SD). The T-test was used to compare differences in packed cell volume (PCV) between infected and non-infected cattle while the Pearson Chi-square was used to compare statistical differences in trypanosome infection based on villages, sex and age categories. Analysis of variance (Ivanova et al.) provided statistical differences in mean PCVs across the treatment group. The study did not find any significant statistical difference on the prevalence of trypanosomosis across villages, cattle ages and sexes. On day 0, 4.94% (19/395) of the cattle tested positive for one or more species of trypanosomes. Trypanosoma vivax was the most prevalent species at 73.6% (N=19) followed by Trypanosoma congolense at 24.4%. There was however no significant difference in prevalence between the Trypanosoma species isolated. On day 7, no cattle tested positive on both BCT and ITS1-PCR. On day 28, 3 cattle tested positive by BCT while on PCR, 4 tested positive. The relapses in cattle 4111, 4116 and 4118 encountered on day 28 were either a result of new infections or probable resistant parasites that were not detected in the initial days. The T. vivax of animal 4102 isolated on day 28 could be a relapse due to a possible resistance or appearance of parasites previously sequestered in parts of the body that are not easily accessible by DA such as Central Nervous System, adipose tissue and eye globe. The findings from this study suggest a likelihood of resistance to diminazene aceturate by Trypanosoma species in cattle populations of Lambwe Valley a finding that could not be absolutely confirmed. Further molecular analysis of day 28 infections or drug efficacy experimental trials in goats are therefore recommended to confirm/rule out resistance. Incorporating pyrethroid insecticide treatment of cattle in block treatment program, monitoring on day 14 and extension of monitoring beyond day 28 would improve outcomes for future research deploying block treatment. Community training and sensitization on appropriate use of trypanocides, insecticides and other veterinary drugs to avert the development of resistance against veterinary drugs are recommended.
Development and evaluation of a RT-qPCR assay for the detection of Orthoflavivirus wesselsbronense from formalin-fixed, paraffin-embedded tissues from sheep and cattle
(University of Pretoria, 2024-06) Odendaal, Lieza; Quan, Melvyn; pbsetlhodi@gmail.com; Setlhodi, Palesa
Wesselsbron disease (WSL) is a neglected zoonotic disease caused by Wesselsbron virus (WESSV) that belongs to the species Orthoflavivirus wesselsbronense in the family Flaviviridae, genus Orthoflavivirus. It is an economically and veterinary important viral mosquito-borne disease primarily affecting ruminants of young age. The most significant differential diagnosis for WSL is Rift Valley fever (RVF), also an important zoonotic disease. Both diseases have similar geographic distributions and cause similar clinical outcomes. However, WSL disease may be underreported during a large-scale RVF outbreak and human cases possibly also overlooked. This first aim of the study was to develop and optimize a reverse transcription quantitative real-time PCR (RT-qPCR) assay for nucleic acid detection of WESSV in formalin-fixed paraffin-embedded (FFPE) tissue samples. Virological and serological methods have been described for the definitive diagnosis of WSL, however, this study reports on the development of an RT-qPCR assay that is sensitive and specific for the detection of WESSV RNA extracted from FFPE tissue samples. This is because FFPE tissues are often the only sample available by the time diagnosis is suspected. An alternative test using immunohistochemistry (IHC) has been in use at the Faculty of Veterinary Science, University of Pretoria, since 1996. However, this test often yields what is termed ‘non-specific staining’ by pathologists. This is when cells or other material stain with a chromogen in a non-typical way. In the case of WSL, this is difficult to interpret and an alternative test was considered necessary. The newly developed test was used to test for WESSV from sheep (n = 100) and cattle (n = 99) tissue samples selected from the 2010 - 2011 RVF outbreak across different districts within South Africa. The purpose of the selected cases was to determine if WESSV was present during the 2010 - 2011 RVF outbreak. Three cases that were classified as having non-specific labelling using IHC were also tested. To achieve the first aim of the study, flavivirus sequences available on GenBank were downloaded and aligned. The assay was designed to avoid non-specific cross-reactions with other flaviviruses. It employed WESSV-specific primers targeting the conserved region of the WSL envelope (E) protein and a WESSV minor groove binding probe. FFPE-positive and negative controls were produced by spiking different virus titres (1 x 106, 3 x 106, 5 x 106) of WESSV in cell cultures, fixing the cells in formalin and subsequently embedding the cells in paraffin wax. Tissues from ten animals that were experimentally infected with Bluetongue virus (BTV) in a previous study, were included as negative controls. FFPE blocks containing tissues from natural field cases, that previously tested positive for WESSV, were included to confirm the amplification of the target RNA. Nucleic acid was purified using two methods in parallel: a modified method employing MagMAX™ CORE Nucleic Acid Purification Kit using Qiagen reagents to deparaffinise samples, and a MagMAX™ FFPE DNA/RNA Ultra Kit using xylene to deparaffinise samples. Results from the MagMAX™ FFPE DNA/RNA Ultra Kit data showed improvement in RNA quality extracted as it gave lower CT values with steeper amplification curves. Due to this, it was selected as the method of choice for RNA purification as it produced better PCR results. The optimal primer and probe concentrations of the assay were 400 nM and 150 nM respectively. VetMAX™-Plus One-Step RT-PCR Kit reagents were used to amplify the WESSV target with one-step RT-qPCR format on the Applied Biosystems StepOnePlus system. Results of tissue sections previously tested with antibodies to WESSV using immunohistochemistry (IHC) (n = 8) were compared with the results of the RT-qPCR assay and found to be congruent. The efficiency of the assay was calculated to be 87.41% by performing serial dilutions (10-1 to 10-7) of the WESSV RNA in triplicates. The established sensitivity of the assay was 88.89% using natural field cases which were previously determined IHC-positive for WESSV. The assay was confirmed as a highly specific method for identifying WESSV, given that no traces of the virus were found in any of the negative control FFPE tissue samples from sheep (n = 10) that were previously experimentally infected with BTV. The second aim of the study was to determine if WESSV can be detected in field cases that were sampled during the 2010-2011 RVF outbreak. Tissues from 100 sheep sampled during the outbreak were tested and only one case tested positive for WESSV (CT value = 34.66). Out of a total of 99 cattle tissue samples tested, 3 samples were positive (CT values = 20.58, 26.58, 31.88). Among these positive cases, one was a foetal brain FFPE sample. Additionally, one weak positive cattle case (CT value = 34.17) was detected. Although the limit of detection could not be determined, these findings from this study suggest that field tissue samples were either infected at levels that were too low to be detected by the developed assay, or they were not infected. If the latter, it might be possible that WSL disease occurred infrequently during the RVF outbreak in 2010-2011. However, more cases would need to be studied to determine the occurrence of WSL disease. Overall, the developed WESSV RT-qPCR assay was efficient, sensitive, and specific and is a useful additional test to confirm a diagnosis of WSL in cases where only FFPE samples are available and results for IHC are inconclusive.
Evaluation of five housekeeping genes for genotyping southern African Ehrlichia ruminantium strains
(University of Pretoria, 2024-04) Morar-Leather, Darshana; Liebenberg, Junita; u19017252@tuks.co.za; Labuschagne, Martinet
Heartwater is a tick-borne disease caused by the bacterium Ehrlichia ruminantium. It affects domestic animals such as cattle, sheep and goats as well as a range of wild animals. The disease is characterised by various clinical signs including high fever, exaggerated blinking, a stiff gait, convulsions, loss of appetite, heavy breathing, hanging head, depression, anorexia, hyperaesthesia, lacrimation and recumbency. The pathogen is transmitted to hosts by tick vectors in the genus Amblyomma and disease distribution corresponds to vector distribution. The disease is endemic to the majority of sub-Saharan Africa, excluding only the very dry south-west, as well as the islands of Grande Comore, Madagascar, Mauritius, Réunion, and São Tomé. Heartwater is also present in the Caribbean islands of Guadeloupe, Antigua and Marie-Galante. Measures to control heartwater include tick control, immunisation, farming with resistant stock and the use of antibiotics. Multiple serological and molecular tests have been developed for diagnostic and phylogenetic purposes but genotyping of the E. ruminantium genome is limited to ftsZ, gltA, groEL, groESL, lepA, lipA, lipB, nuoB, pCS20 gene region, secY, sodB and sucA, with little success in observing a rich genetic constitution. These genes demonstrate genetic variation, but not enough to adequately differentiate between E. ruminantium field strains in southern Africa. This gap in knowledge establishes the need to identify new genes that can provide us with a more in-depth and comprehensive understanding of said genetic diversity. Therefore, the aim of this study is to investigate the genetic constitution of five housekeeping genes of southern African E. ruminantium isolates by evaluating their capability to differentiate between strains. Gene alignments were made, primers were designed in a nested approach, PCR was conducted, and sequencing results of PCR amplicons were phylogenetically analyzed individually and via MLST for the genes carA, glnA, guaB, hemC and pyrD. The results indicate that carA, glnA and guaB are most efficient at differentiating at strain level; hemC does not adequately differentiate at strain level; and pyrD failed at sequencing and could therefore not be analyzed phylogenetically. The concatenated sequence tree of genes carA, glnA, guaB and hemC gave an overall better understanding of the differentiation of southern African strains as it delivers 18 different genotypes from the 18 field samples.
Prevalence of Cryptosporidium infection and associated risk factors in ruminants in Gauteng Province, South Africa
(University of Pretoria, 2024-03) Marufu, Munyaradzi Christopher; Korsten, Lise; Byaruhanga, Charles; seanego.tebogo23@gmail.com; Seanego, Tebogo Atlivia
Cryptosporidiosis, an emerging enteropathogenic disease with negative implications for public and livestock health, remains poorly investigated in Africa, necessitating focused epidemiological studies. To determine the prevalence and risk factors associated with Cryptosporidium infections in domesticated ruminants, a longitudinal study was conducted on eight farms in Rust de Winter, Gauteng Province, South Africa. During winter (2022) and summer (2023), 370 faecal samples were collected from cattle (n=146), sheep (n=105), and goats (n=119) kept in extensive and semi- intensive production systems. Microscopic analysis using the gold standard Modified Ziehl Neelsen (MZN) test was employed, followed by screening of the positive samples for Cryptosporidium parvum using quantitative real-time PCR (qPCR). Semi- structured interviews were conducted with the farmers to establish demographic, behavioural and management practices that could pose a risk for Cryptosporidium infection between animals and humans. The microscopic analysis identified Cryptosporidium oocysts in 57 (15.4%) of the 370 faecal samples. However, the qPCR detected Cryptosporidium DNA in only one (1.8%) of the 57 MZN-positive samples, possibly because of low oocyst concentration, below the detection limit of the qPCR, or the presence of Cryptosporidium species other than C. parvum. The Cryptosporidium data from the MZN test revealed that infection was significantly higher (p=0.0133) in summer (19.4%) than in winter (11.6%). No statistically significant differences (p>0.05) in infection were observed between male (16.2%) and female (14.0%) animals and across ruminant species, although cattle (11.6%) tended to have lower infection rates than sheep (20.0%. OR=1.9; p=0.075) and similar infection rates to goats (16.0%; OR=1.16; p=0.695). All interviewed farmers (n=13) were unaware of cryptosporidiosis, highlighting the importance of awareness and training to reduce the potential risk for disease transmission to humans and other animals. Despite the limitations associated with the qPCR method, this research provides valuable insights into the epidemiology of Cryptosporidium infections in ruminants in Gauteng Province. The observed seasonal variation and the need for farmer education underscore the significance of proactive measures to mitigate livestock mortality and prevent the potential spread of infection to humans. Further investigation involving more animal farms and assessment of the occurrence of infections in humans will contribute to better understanding of Cryptosporidium infections. There is also a need to evaluate clinical vs sub-clinical cases on farms with regards to the detection limit of nucleic acid- based methods.
Eco-Epidemiology and Microbiological Evaluation of Poultry Salmonellosis in North Central Nigeria, and its Socioeconomics and Public Health Impacts
(University of Pretoria, 2024) Fasina, Folorunso Oludayo; Jonker, Annelize; drsao.epidem@gmail.com; Sanni, Abdullahi Ozomata
Nigeria is a country with a mid-2020 human population of approximately 209 million, and the poultry industry in Nigeria has rapidly expanded in recent years despite many health and economic challenges. Poultry production in the different agro-ecological zones of Nigeria are characterized by generalized and specific production and health-related challenges principal among which are: 1) low level of production, 2) inadequate scaling up and specialization, 3) antimicrobial use and resistance, and 4) a poor level of biosecurity implementation. Hence, there are a number of poultry-related zoonoses can be found in humans and animals in Nigeria. The Salmonella spp. is a Gram-negative enteric pathogen (bacterium), with potentials to infect almost all animals including humans. Though, only two species have been identified in this genus, vis the enterica and bongori, almost 2,700 serotypes (serovars) have been listed with approximately 10% isolated from birds. Most serotypes of Salmonella can infect several animal species including humans, such as Salmonella Typhimurium and Salmonella Enteritidis, which are primarily poultry- associated. Salmonellosis, as a bacterial zoonosis, causes an array of health conditions in humans and animals, and the non-typhoidal salmonellosis (NTS) is prevalent with substantial under- appreciated public health impacts. This work was set out with the objectives of conducting microbiological evaluation of NTS in North Central zone of Nigeria (NCN) using classical and molecular methods; conducting a comprehensive re-analysis of risk of introduction of NTS to poultry farms, determining the epidemiology of foodborne Salmonella among poultry farmers and consumers, determining the economic burden of food borne salmonellosis in humans and poultry, demonstrating the benefit of disease control measures against salmonellosis in poultry using validated tools, and map spatial heterogeneity of habitat suitability for salmonellosis in poultry farms in Nigeria to aid evidence-based support to decision makers. Using field sampling, laboratory methods and a semi-structured questionnaire (n = 1000) in poultry farms in NCN, the incidence and risk factors for the persistence of NTS infection in poultry were explored. Approximately 41.6% (95%CI: 38.58 to 44.68) of the farms had experienced NTS over the last 18 months and the awareness of salmonellosis was moderate. A number of risk factors for increased odds of NTS infection in poultry including increasing stock in smallholder farms, self-mixing of concentrate on the farm, usage of stream water, pen odour, non-adherence xxiii and partial adherence of farms to recommended poultry vaccination against pullorum and fowl typhoid and lack of and non-adherence to biosecurity were identified. Overall, 66 isolates including Salmonella enterica, Salmonella arizonae, S. paratyphi, and S. typhi, with 94.5% mixed infections with Klebsiella pneumoniae and Lactobacillus bulgarius, were obtained, and irrational antibiotic-use practice remains a major problem in the industry. Specifically, the study obtained a number of multi-drug resistant isolates, with likelihood of passing such resistant organisms through the human food chain. To evaluate the economic and social burden of NTS, poultry and human populations, economic and epidemiological data were retrieved from various sources and validated. A customized and validated Microsoft Excel® tool was utilized to conduct the economic analysis for the reference year 2020. The burden of NTS was 325,731 human cases and a total of 1,043 human deaths per year, at a disability-adjusted life year (DALYs) of 37,321. The cost associated with infection in humans was US$ 473,982,068, and for poultry, US$ 456,905,311 (the direct value of animal loss, US$ 224,236,769, loss from salvage slaughter and culling, US$ 220,386,556, and value of foregone production, US$ 12,281,987). Using Outbreak Costing Tool (OCT), the benefit-cost of multisectoral intervention against NTS was estimated. Approximately 4,835 technical officers and 3,700 non-technical staff (n = 8,535) with an annual investment of over 2.2 million work hours, and at a total cost of US$ 53,854,660.87 are needed for an annual NTS control programme in humans and animal. The non-labour-related cost was 89.21% of the total intervention costs, and major costs were incurred in medical countermeasures, travel and transports, and laboratory support. The overall intervention’s investment was 374.15% of the estimated national and subnational systems’ annual budget for diarrhoeal diseases, and the outbreak response period incurred the highest costs (53%) of the total intervention. The benefit– cost ratio (BCR) of intervention was 17.29. Through a cohort study, the cost-effectiveness of NTS in humans in Nigeria for the year 2020 was determined. Specifically, an Excel-based cost- effectiveness analysis tool was developed to compare structured (One Health) and unstructured (episodic intervention against NTS), with input data from various sources. The non-complicated and complicated cases were 309,444 (95%) and 16,287 (5%) respectively, and the overall programme cost was US$ 31,375,434.38. The current non-systematic episodic intervention costed US$ 14,913,480.36, indicating an additional US$ 16,461,954 to introduce the proposed intervention. The intervention averted 4,036.98 NTS DALYs in a single year. The non-complicated NTS case was US$ 60/person with significant rise in complicated cases. The cumulative costs of NTS with and without complications far outweighed the program cost for One Health intervention with an incremental cost-effectiveness ratio (ICER) of -US$ 221.30). The suitability map for continued infection in humans and poultry indicated that the disease would remain prevalent unless significant behavioural change communication is undertaken and intense control for NTS challenges are implemented. The identified risk practices must be mitigated intentionally, and biosecurity and hygiene must be improved to reduce the burden of NTS. Since the utilization of One Health approach to intervention is cost-effective and cost-beneficial, and they carry additional mitigative benefits for other diseases, multisectoral investigation and response against NTS in Nigeria is advocated. The health system should re-focus and re-prioritize, with coordinated collaborations and through the utilization of a more decentralized approach. Anticipatory planning, adequate resource allocation and more intense outbreak investigations to reduce critical response time are warranted. Identified limitations in this study must be improved to optimize benefits associated and to facilitate policy discussions. The outcomes of this work provide empirical evidence to support informed decisions and investments in the control and eradication of human and poultry salmonellosis (NTS) in Nigeria.
Brucella melitensis in wildlife in South Africa
(University of Pretoria, 2023) Van Heerden, Henriette; Abernethy, D.A.; akorfa1k@yahoo.co.uk; Glover, Barbara Akorfa
This thesis offers a comprehensive examination of Brucella melitensis in South African wildlife, with a particular focus on sable antelopes (Hippotragus niger). The research encompasses an extensive review of existing literature and empirical studies, aiming to deepen the understanding of the epidemiology, diagnosis, and management of B. melitensis in wildlife, particularly in the context of the South African wildlife ranching industry. Chapter 1 Summary: "Research Introduction and Literature Review" Chapter 1 of the thesis delves into the research background by outlining the historical outbreaks of B. melitensis in South Africa, tracing its evolution from affecting sheep to goats and, ultimately, humans. A notable outbreak on a farm in the Western Cape is emphasised, illustrating the cross-species transmission potential between livestock and humans. This section also highlights the growing practice of wildlife/game ranching in South Africa, drawing attention to its possible role in spreading zoonotic diseases such as brucellosis. The aim of the research is clearly defined: to investigate, analyse, and describe ongoing outbreaks of B. melitensis in South African wildlife, with a particular focus on the wildlife ranching industry. Objectives are set to include conducting retrospective studies on B. melitensis in sable, adapting diagnostic tests for wildlife, and performing genomic characterisation of the isolates in South Africa. The scope and approach of the research involve studying anonymised cases from wildlife farms and closely examining the role of wildlife/game ranching in rural South Africa, especially focusing on the ranching of sable antelopes. Discussing the interactions between wildlife and livestock and the critical importance of effective disease surveillance in these contexts complements this. The chapter offers an in-depth look at brucellosis, particularly B. melitensis, discussing its pathogenicity, zoonotic potential, clinical signs, diagnostic methods, and control measures in livestock and wildlife. It further explores the challenges of disease spillover to wildlife, transmission mechanisms, and environmental survival, stressing the need for comprehensive control and prevention strategies that consider wildlife's unique biological and ecological aspects. In conclusion, Chapter 1 lays a comprehensive groundwork for the thesis, providing a thorough understanding of the current state of knowledge regarding B. melitensis in wildlife, with an emphasis on the South African context, and frames the research within the broader objectives of wildlife conservation, public health, and the dynamics of zoonotic diseases in an evolving ecological and economic landscape.
Lesions and cellular tropism of natural Rift Valley fever virus infection in sheep
(University of Pretoria, 2019-11) Venter, Estelle Hildegard; Davis, A. Sally; lieza.odendaal@up.ac.za; Odendaal, Lieza
Rift Valley fever (RVF) is a viral haemorrhagic disease caused by a segmented single stranded RNA virus of the Bunyavirales order, Phenuiviridae family. The virus causes severe disease in both humans and animals and epidemics have been reported in most countries in Africa and the Arabian Peninsula. The virus is primarily transmitted by mosquitoes, mainly of the genera Aedes and Culex. Characteristically RVF virus (RVFV) causes abortion storms in sheep during which approximately 10 to 20% of adult ewes die. In the summers of 2010-2011, South Africa had an extensive outbreak of RVF affecting livestock and humans. The diagnosis of RVF was confirmed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), and in selected cases also by immunohistochemistry (IHC). Sheep necropsied during the outbreak were examined by histopathology and IHC. The first chapter is a review of research conducted regarding RVF infection in sheep, including descriptions of the pathology and immunohistochemical findings. Relevant information concerning the pathogenesis of RVFV and other viral haemorrhagic diseases with a similar pathogenesis was also summarized. The aim of the second chapter was to describe the tissue tropism and target cells of RVFV in 99 adult sheep from the 2010 South African RVF outbreak. A further aim was to determine the extent to which virus could be detected in different organs and summarize gross, histopathological and immunohistochemical findings relative to results from previous research. Multifocal-random, necrotizing hepatitis was the most distinctive lesion of RVF cases in adult sheep. Of cases where liver, spleen and kidney tissues were available 45/70 (64%) had foci of acute renal tubular epithelial injury in addition to necrosis in both the liver and spleen. In some cases, acute renal injury was the most significant RVF lesion. Concerning the diagnostic specificity of lesions in the liver, early apoptotic hepatocytes (Councilman bodies) and late apoptotic bodies with nuclear fragments and condensed cytosol were diagnostically useful. In contrast, eosinophilic intranuclear inclusions were of limited diagnostic value since they were only identified in 7/99 (7%) adult sheep. Immunolabelling for RVFV was most consistent and unequivocal in liver followed by spleen, kidney, lung and skin. It was determined in this study that the minimum set of specimens to be submitted for histopathology and IHC to confirm or exclude a diagnosis of RVF are liver, spleen and kidney. Skin from areas with visible crusts and lung could be useful additional samples. RVF is much more acute in young lambs than in older sheep, and there appear to be important differences in the lesions and tropism. Therefore, the principle aim of the third chapter was to describe the quantitative and qualitative pathomorphology of RVF in 71 young lambs and contrast this with results for adult sheep. A further aim was to determine the diagnostic significance of different tissues and specific histologic features of RVF in young lambs. The liver lesion of RVF was much more severe in young lambs, where it caused a diffuse necrotizing hepatitis with widespread labelling of virtually every hepatocyte. A striking diagnostic feature in lambs was multifocal liquefactive hepatic necrosis (primary foci) against a background of diffuse hepatocellular death. Primary foci were present in 59 of 71 (86%) cases, with viral antigen noticeably sparse within them. Intranuclear inclusion bodies were also diagnostically useful, and present in 27 of 71 (38%) cases. Additionally, cell death morphology in the liver in RVFV infected lambs was more heterogeneous than previously suspected whereby many hepatocytes displayed histomorphological features of lytic cell death mechanisms (cell swelling, rupture of the plasma membrane and cellular collapse) as well as apoptosis. Whereas, apoptosis was demonstrated in RVFV infected mice using terminal deoxynucleotidyl transferase dUTP nick-end labelling, the involvement other cell death mechanisms in RVF, including pyroptosis and necroptosis, should be further explored. In young lambs, lymphocytolysis was present in all lymphoid organs except for the thymus. The kidney lesion was also much less severe than in adult sheep but viral antigen was easier to find and generally widespread. Histopathological lesions in the lungs of lambs mirrored that in adults. However, viral antigen was easy to find in lambs compared to adult sheep where viral antigen was very difficult to find in the lungs. As in adult sheep, splenic lymphocytolysis was a prominent feature, but due to a developmentally normal lack of germinal centres this was more prominent at the edge of the periarteriolar lymphoid sheaths and the red pulp in lambs. Labelling was also often seen in capillaries and small blood vessels either as non-cell-associated antigen, or as antigen in endothelial cells or intravascular cellular debris. Additionally, in the kidney viral antigen was in the peri-macular cells including the juxtaglomerular and granular extraglomerular mesangial cells. Liver and spleen specimens were the most consistently positive for RVFV antigen and were adequate to confirm or exclude a diagnosis of RVF in most cases in young lambs using IHC. Specimens from the lungs and kidneys were useful additional samples due to their characteristic histologic lesions. There is a perception that RVF disappears in the interepidemic periods and only reappears suddenly when heavy rainfall occurs. However, RVFV infection in pregnant ewes causes a wide variety of outcomes for both ewes and foetuses and it is difficult to exclude RVF from the list of possible differential diagnoses. Diagnosing the cause of abortions in endemic areas are further complicated by the observation that the virus seemingly does not always cross the placenta to cause lesions in the foetus. Additionally, the tropism and lesions of the placenta have not been thoroughly described. Therefore, the principle aim of the fourth chapter was to describe RVF in naturally infected foetal tissues and determine the diagnostic significance of the lesions. A total of 72 foetuses were studied of which 58 were confirmed positive for RVF.
Antimicrobial sensitivity of faecal Escherichia coli isolated from backyard broiler chickens in Chitungwiza, Zimbabwe
(University of Pretoria, 2024-04-19) Jonker, Annelize; nyarisamakonde@gmail.com; Samakonde, Nyaradzo Patricia
Antimicrobial resistance (AMR) is currently one of the greatest public health threats being faced by humanity. Although a lot of funding for research is being availed there are still a lot of gaps in understanding its dynamics as well as the actual degree of this problem. There is also the concept of using the One Health approach to fully understand this threat. The aims of this study were to isolate E. coli from backyard broiler chickens from selected households in Chitungwiza, Zimbabwe, to determine the antimicrobial sensitivity of the isolates and to assess the level of knowledge of the backyard poultry keepers with regards to AMR. One hundred and thirty-eight (138) cloacal samples were randomly collected from backyard poultry from randomly selected households. Escherichia coli isolation was done on MacConkey agar and biochemical tests were carried out to identify the isolates. Antibiotic sensitivity analysis was carried out on Mueller Hinton agar and the zones of inhibition were measured and interpreted in line with CLSI guidelines. A total of 98 (68%) of E. coli isolates were obtained. Antimicrobial susceptibility analysis revealed four (4%) isolates that were resistant to all six (6) antibiotics. Escherichia coli resistance to tetracycline was 93.6%, ciprofloxacin 44.7%, ampicillin 59.6%, ceftriaxone 9.6%, trimethoprim / sulphamethoxazole 79.8% and colistin sulphate 4.3%. Isolates were most often resistant to tetracycline. Fifteen (15) people answered the questionnaire and of these only two (13.3%) knew about withdrawal periods and practiced them. The overuse of poultry drugs and their wide availability could be the main driver of AMR in poultry. The results obtained in this study indicated that there is indeed AMR in E. coli from backyard broiler birds and the greater population is not aware of this. This poses a great public health threat since some of the drugs are used as first line drugs in the treatment of human infections.
Prevalence and characterization of brucellosis and tuberculosis in cattle and its zoonotic risk associated factors in Rwanda
(University of Pretoria, 2021) Van Heerden, Henriette; Kolo, Francis B.; Michel, Anita Luise; boscus2@gmail.com; Ntivuguruzwa, Jean Bosco
Bovine brucellosis (BB) and bovine tuberculosis (bTB) are endemic in Rwanda; however, little is known about the diseases. Before this study, there were only three serological studies on BB, two serological studies on human brucellosis, and two studies on bTB. The aims of this study were to determine with the following objectives: to determine the prevalence and characterize Brucella in cattle from six districts which included the wildlife-livestock-human interface (5 districts) and peri-urban area together with the zoonotic associated risk factors; to characterize Brucella spp., and other abortigenic pathogens in aborted tissues from cattle from selected districts in Rwanda; to assess the awareness and occupational exposure to brucellosis, bTB, and other zoonotic diseases among abattoir workers at the six slaughterhouses in Rwanda and; to characterize BB and bTB from tissue samples collected from slaughtered animals in Rwanda using culturing and molecular characterization. The BB prevalence was determined using Rose Bengal test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA) in series and the animal-level seroprevalence was 7.4% (141/1907) in cattle from the six districts, 8.3% (141/1691) in cattle farmed at the wildlife-livestock-human interface (5 districts), and 0.0% (0/216) in the peri-urban areas (1 district). The herd-level seroprevalence of BB was 28.9% (61/212) in herds from the six districts and 30.9% (61/198) in herds from the wildlife-livestock-human interface (5 districts). Multivariate analysis showed that old age (≥5 years), cattle from districts bordering national parks, history of abortions, and replacement animals were significantly associated with brucellosis (p < 0.05). Low awareness of zoonotic brucellosis transmission, assisting calving without biosafety protection, drinking raw milk, and manual milking were each observed in more than 21.7% of cattle keepers whose herds were seropositive. Whole blood (n=118), milk (41), and vaginal swabs (n=51) samples from brucellosis seropositive (n=183) and seronegative (n=27) cattle were cultured and Brucella cultures were identified using the 16S-23S ribosomal interspacer region (ITS) PCR assay. The culture prevalence determined by the gold standard (cultures and ITS-PCR) was 16.7% (35/210) and AMOS-PCR assay identified mixed B. melitensis and B. abortus (n=12) isolates, B. melitensis (n=3), B. abortus (n=19) while Bruce-ladder PCR assay identified B. abortus RB51 vaccine strain (n=2) amongst the B. abortus and B. melitensis cultures. Aborting livestock samples (19 aborted tissues from isolated cases for cattle and 1 aborted tissue and 3 vaginal swabs from an abortion outbreak for goats) were investigated for brucellosis using culture and PCR assays. Two aborting cattle (2/19) were infected by B. melitensis (n=1), and B. abortus (n=1) while mixed B. abortus and B. melitensis were isolated from goats. The Brucella negative samples from cattle (n=17) where further characterize using a PCR abortion panel (Anaplasma phagocytophilum, Bovine Herpes Virus Type 4, Campylobacter fetus, Chlamydophila spp., Coxiella burnetti, Leptospira spp., Listeria monocytogenes, and Salmonella spp.). Campylobacter fetus (n=7), and Leptospira spp. (n=4) were identified including co-infections (n=2) of C. fetus and Leptospira spp. BB seroprevalence using serological tests (RBT and i-ELISA) in parallel was 2.9% (8/300) from slaughtered cattle at six abattoirs in Rwanda. The culture prevalence determined by the gold standard method (culture confirmed by Brucella specific ITS-PCR) was 5.6% (11/300). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3), and B. melitensis (n=5) isolated from lymph nodes while Bruce-ladder PCR assay identified B. abortus and B. melitensis. The prevalence of bTB was 1.7% (5/300) and bTB was caused by M. bovis (n=4) and M. tuberculosis (n=1). Rifampicin-resistant (RR) M. tuberculosis (n=1) was recorded. The prevalence of non-tuberculous mycobacteria (NTM) was 12.0% (36/300).
Genetic diversity and geographic relationships amongst southern African Amblyomma ticks, Rickettsia africae and Ehrlichia ruminantium
(University of Pretoria, 2024-03-05) Neves, Luís C.B.G.; Makepeace, Benjamin Lawrence; Morar-Leather, Darshana; u14023190@tuks.co.za; Smit, Andeliza
Amblyomma, the third largest genus in the Ixodidae, are known for their vibrant decorative appearance and aggressive hunting behaviour. The genus comprises 24 species found across the African continent, with 17 identified as inhabiting various ecological niches within the southern regions of Africa. Of these species, Amblyomma hebraeum and Amblyomma variegatum have been well studied due to their widespread geographical range and their status as competent vectors of pathogens including Ehrlichia ruminantium, the causative agent of heartwater, and Rickettsia africae, the causative agent of African tick bite fever (ATBF) in humans. Heartwater is a lethal disease in ruminants, estimated to cause an economic loss of 44.4 million US$ annually in South Africa and 5.6 million US$ annually in Zimbabwe. ATBF is a non-lethal disease, mainly afflicting individuals travelling to southern Africa. In this study, Amblyomma ticks were collected from livestock in Angola, South Africa, Zambia and Zimbabwe and from both wildlife and livestock in Mozambique. The collected ticks were morphologically identified with identification keys and morphological characteristics were documented by photography. Primary screening for E. ruminantium was conducted, targeting the pCS20 gene fragment and strain characterization of 100 positives was conducted with Ampliseq technology. A subsample of ticks was screened with the use of the ompA gene for R. africae, while characterization was conducted on 50 positives with ompA, ompB, gltA and omp genes. Tick genetic diversity was evaluated using 12S, 16S, coi and ITS2 genes. In total, 7,773 Amblyomma ticks were collected and identified as belonging to four species: Amblyomma eburneum, A. hebraeum, Amblyomma pomposum and A. variegatum. The phylogenetical analysis of the four Amblyomma species illustrated clear separations between A. eburneum and A. hebraeum, while A. pomposum and A. variegatum clustered together. Little intraspecies variation was also observed, where ticks of the same species from different countries cluster together indiscriminately. Using phylogenetic, pairwise distance and automatic barcode gap discovery analyses, we are unable to molecularly distinguish between A. pomposum and A. variegatum. Ehrlichia ruminantium infection rates in ticks per country ranged from 7.7% to 36.5%. Genotyping analysis indicated the clustering of the sequences with several strains, including the Gardel, Grootvallei and Springbokfontein1. The Ampliseq analysis had amplification failures as documented with the genes used, emphasizing the requirement for the development of new markers for phylogenetic characterization. Infection rates of R. africae ranged from 11.4% to 35.6% per country. The phylogenetical analysis depicts little variation and illustrates that the genes used were not sufficient enough to distinguish between strains. This study is one of the largest performed in this region of Africa, depicting the genetic variation of E. ruminantium, R. africae and the four collected Amblyomma species. The infection rates obtained during this study provides new and current insight of the pathogen distributions ranges in southern Africa. Greater attention should be given to the neglected Amblyomma species and their role in the distribution and maintenance of pathogens.
Bacterial and fungal causes of abortion in domestic ruminants in South Africa
(University of Pretoria, 2023-11-10) Michel, Anita Luise; Thompson, P.N. (Peter N.); annelize.jonker@up.ac.za; Jonker, Annelize
Abortions in cattle, sheep and goats represent important economic losses in the agricultural industry. Determining causes of abortions is important for control efforts, but can be challenging. This study investigated detection methods for and significance of bacteria and fungi as agents of abortion in domestic ruminants in South Africa. Retrospective data was collected by searches of case reports (2006-2016) of participating pathology and bacteriology laboratories. During the prospective study (2017-2019), samples were analysed by a combination of conventional bacteriology methods. Quantitative real-time PCR assays for detection of the Chlamydiales, Chlamydia abortus, Chlamydia pecorum, Parachlamydia acanthamoeba and Waddlia chondrophila were created by combining primers and probes selected from literature. These assays were optimized and employed to analyse samples from 25 cases, with placentitis and/or pneumonia lesions, selected from the prospective study. The retrospective study reported 288 cases from six provinces. Diagnostic rate was 35.1%. The prospective study reported 135 cases from six provinces. Diagnostic rate was 42.2%. Brucella species were most commonly isolated in both the retrospective and prospective studies at 7.3% and 7.4% of cases, respectively. The qPCR assays detected Chlamydiales in 60% of cases with placentitis and/or pneumonia. Chlamydia abortus, P. acanthamoeba and W. chondrophila were detected in bovine; and C. pecorum and W. chondrophila in ovine and caprine cases. Chlamydiales were detected in three previously inconclusive cases. Identification was improved from genus to species level (C. pecorum). In conclusion, retrospective laboratory records yielded valuable passive surveillance data. Submission of placenta was an important factor in successful diagnosis. The most effective combination of conventional culture methods was aerobic culture together with selective Brucella, Campylobacter and fungal culture. This combination lead to improvement of the diagnostic rate in comparison with the retrospective study. Brucella abortus was the most common cause of bovine abortion over 12 years in the retrospective and prospective studies. Trueperella pyogenes was the second most common. Real-Time qPCR assays improved detection of Chlamydiales and differentiation to species level. The first detection of P. acanthamoeba and W. chondrophila in abortion cases in South Africa was reported indicating a potential role in abortions in this country.
A mixed-method evaluation of the One Health-ness at the Faculty of Veterinary Science, University of Pretoria
(University of Pretoria, 2021) Michel, Anita Luise; Aqil, Jeenah
One Health (OH) is a concept that emphasises the interconnected nature of human, animal and environmental health. To achieve complete health through a OH approach, transdisciplinary work is required to ensure that different fields of health are cognisant of the impact that factors in other fields have on each other and that risks are addressed holistically. This study intended to create an appropriate understanding of the OH-ness at the Faculty of Veterinary Science, University of Pretoria (FVS) to measure the impact of current and future strategic OH initiatives. The OH-ness refers to the overall orientation of the faculty towards the OH concept, through research and other academic activities. This study has provided an understanding of the current state of OH-ness at the FVS by examining three main areas that allowed for a comprehensive and diverse evaluation of OH at the FVS. These three areas were evaluated using a combination of quantitative and qualitative data. A systematic review conducted between January 2010 and March 2020 of scientific research publications from the FVS was used to determine if there was an improvement or digression in OH related research publications, as well as an improvement or digression in the focus areas of these publications. Semi-structured interviews were performed with various staff members involved in OH activities within the FVS in order to determine the drivers, objectives and barriers faced by the various OH activities. A quantitative assessment of the OH activities was performed to evaluate their OH orientation. Two baselines were created. Data related to the total number of OH-related research publications and the focus of these publications were collected. A second analysis was conducted on the OH orientation of activities at the FVS. These data sets provided a baseline that will allow for future studies to compare the progress of the OH orientation at the FVS. Over the period under review a total of 1670 articles was published, with 197 (12%) being OH-related. The research identified that while there was an improvement in diversity and transdisciplinary efforts of scientific publications over the last 10 years, the growth fell below the global growth of OH-related research. There was an overreliance of OH research from a single department within the faculty and a lack of focus on environmental health research. Five OH activities were identified through a review of scientific publications from the FVS. The project leaders of the OH activities were interviewed through a semi-structured approach in order to understand the reasons for initiating the project and potential barriers. Four of the areas were research- driven and did not involve undergraduate veterinary science students. The fifth areas was aimed at advancing the knowledge of undergraduate veterinary students about zoonotic diseases. Objectives of the OH activities varied from scientific gap to action. The FVS has the potential to grow its OH-ness because it has the experience, skills and knowledge. However, there was a lack of special OH funds or faculty level OH plans.

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