Low temperature-processed, porous sol-gel glasses represent a new class of materials for the immobilisation of biomolecules. If used to entrap biological recognition elements, these transparent and chemically inert glasses offer a new approach in the development of optical biosensors.

DNA profiling of exhibits that originate from forensic stock theft cases is routinely used as a tool to link suspects to the crime or scene. DNA derived from aged or degraded samples is often highly fragmented which compromises the efficiency for obtaining a complete genotypic profile using PCR. Conventional polymerases such as Taq, lack certain repair mechanisms for use on degraded DNA templates. New generation polymerases are known to have high fidelity characteristics. The aim of this study was to determine the efficiency of Restorase®, a novel DNA polymerase blend that is known to repair damaged DNA and the FastStart High Fidelity PCR System enzymes, on degraded forensic bovine samples using PCR-based methodology. Bovine meat samples were subjected to different degrees of degradation in the sun and in the shade during summer and winter seasons. DNA was extracted, subjected to PCR amplification using 16 bovine microsatellites and genotypes were generated for analyses. Rapid degradation of samples was observed during winter while during summer samples tend to dry out. Restorase® exhibited high enzyme activity on degraded samples as compared with FastStart and Taq DNA polymerase. Some of the markers that failed to be successfully amplified by Taq polymerase, such as ETH10 and SPS115 were recovered using Restorase®. Markers such as BM1818, BM2113, ETH3, INRA23 and TGLA227 remained active throughout the experiment using all the enzymes, and therefore can form a basis of the bovine marker panel. Restorase® was found to be an alternative enzyme for use in bovine forensic analysis. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

The diagnostic performance of four serological assays for bovine brucellosis in African buffaloes, namely Rose-Bengal test (RBT), complement fixation test (CFT), indirect enzyme-linked immunosorbant assay (iELISA) and fluorescence polarisation assay (FPA) were evaluated and compared in a case-control study. The study followed the OIE assay validation pathway for validation of diagnostic tests applicable to wildlife species where there is a validated test available in a taxonomically closely related species. Two uninfected and four infected herds were recruited and an uninfected composite reference panel of 107 sera and infected composite reference panel of 93 were selected using composite reference standards. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were calculated for individual tests and for different combinations of two tests in series and in parallel. Cut-off points were adjusted using receiver operating characteristics (ROC) analysis. Using these cut-off values, the index tests performed as follows: RBT DSe of 98.9% (95% CI 96.83% - 100%) and DSp of 98.1% (95% CI 95.6% - 100%), iELISA (cut-off >40.5%) DSe 98.9% (95% CI 94.2% - 100%) and DSp 100% (95% CI 96.6% - 100%), CFT (cut-off >0 iU/ml) DSe 74.2% (95% CI 64.1% - 82.7%) and DSp 100% (95% CI 96.6% - 100%) and FPA (cut-off >16 mP) DSe 97.9% (95% CI 94.2% - 99.7%) and DSp 100% (95% CI 96.6% - 100%). Based on performance index and area under the ROC curve, the iELISA performed best (198.9% and 1.0), followed closely by the FPA (197.9% and 0.989) and the RBT (197.0%). The CFT s lower performance (174.2%, and 0.871) was due to low DSe. Kappa values for test agreement between the index tests was above 0 for all combinations, and varied from unweighted Kappa of 0.685 (95% CI 0.608 0.762) between FPA and iELISA to 0.26 (0.136-0.383b) between CFT and RBT. Consideration of the indices for positive and negative test agreement between the index tests supported the differential specificity of tests for different immunoglobulin classes and higher in line with the findings in cattle. Positive predictive value in herd C and E were 100% for the iELISA, CFT and FPA, 97.3% in herd C and 98.4% in herd E for the RBT. Negative predictive values in herd C ranged from 89% for the CFT to 99.2% for the RBT and in herd E 73.1% for the CFT to 98.7% for the RBT. Overall repeatability was satisfactory, except for the FPA, which was considered the result of sample quality related to prolonged storage in a freezer. The index tests were all found fit for use to detect or confirm brucellosis in populations and individual animals. The values for DSe and DSp that were estimated will be of use in the interpretation of serological results and determination of diagnostic strategies in different circumstances. Different combinations of tests in series and parallel increased the DSp and DSe. Using the RBT in combination with the CFT/FPA/iELISA interpreted in series or in parallel in relation to the epidemiological setting and objective of testing is recommended. / Mini-dissertation (MSc)--University of Pretoria, 2015. / tm2016 / Veterinary Tropical Diseases / MSc / Unrestricted

In vitro production and transfer of embryos has become a common practice within the dairy industry to efficiently breed superior animals and meet the consumption demand of the growing population. Cyropreservation is necessary for the application of commercialized embryo transfer, however, in vitro-produced embryos show morphological and physiological defects which negatively impact their ability to withstand cryopreservation in comparison to their in vivo counterparts. These artifacts result from culture conditions that cause stress to the embryo during development, leading to an accumulation of intracellular lipids, mitochondrial dysfunction, and ultimately poor ability to withstand freezing and thawing. The objective of these studies was to examine the effects of various metabolic regulators on the viability and cryotolerance of in vitro-produced embryos. Pilot studies revealed that evaluating early (stage 6) versus late (stage 7) blastocysts did not affect the trend seen in results, nor did culturing embryos in continuous versus sequential media. From the main experiment performed, it was concluded that a combination of metabolic regulators decreased lipid content, improved cryopreservation survival, and lowered the percentage of apoptotic cells present after thawing. Conditioned media increased the blastocyst percentage, but did not produce superior quality embryos as measured by cryotolerance. Research concerning the metabolic needs of the preimplantation embryo must continue to determine more relevant markers of embryo quality in vitro.

Brucella spp. are zoonotic infectious agents, primarily of livestock, that cause the disease brucellosis. Bovine brucellosis, caused by Brucella abortus, is of greatest concern due to the disease’s significant economical and public health impact. Canada fully eradicated bovine brucellosis from domesticated cattle herds in 1985, however, continued surveillance through screening for B. abortus exposure is paramount to the maintenance of bovine brucellosis eradication nationwide. The Canadian Food Inspection Agency (CFIA) is responsible for the surveillance of bovine brucellosis outbreaks in Canada and the maintenance of eradication. Current B. abortus serodiagnostics and serological screening is mostly based on the detection of antibodies against Brucella lipopolysaccharide (LPS), a highly immunogenic component of the outer cellular membrane. Such tests face difficulties with false positive results due to cross reactivity with other Gram-negative bacteria that produce LPS. The purpose of the research presented here was to address this issue through identifying new B. abortus protein antigens for the improvement of serological test specificity. In this study, 101 candidates were identified through predictive bioinformatic analyses and selected for immunogenic evaluation. While none of the expressed candidates displayed positive serological activity with in-house brucellosis positive bovine serum panels, the workflow presented here can be used for continued research and the assessment of more proteins from B. abortus and other bacterial pathogens.

Trichomonosis is currently the most important venereal disease of cattle in South Africa with adverse economic implications to the beef production industry due to cow abortions, infertility and culling of carrier bulls. Once diagnosed in a herd, eradication is difficult due to financial and biological implications. Bulls are asymptomatic carriers and susceptibility increases with age. In infected females, clinical signs include embryonal death, abortion, pyometra, foetal maceration and uterine discharge. Diagnostic accuracy is one of the major clinical problems preventing easy eradication of trichomonosis from a herd and can be influenced by biological variance in the occurrence of the organism, sampling errors, sample degradation during sample transport and diagnostic laboratory inaccuracies. This study aimed to validate the accuracy of voluntarily enrolled private (n = 8) and state-owned (n = 5) laboratories that perform trichomonosis diagnostic tests by estimating the sensitivity (Se) and specificity (Sp) per laboratory. It was hypothesized that diagnostic laboratories in South Africa play an insignificant role in the inaccuracy of the diagnosis of trichomonosis. Laboratories performed either the culture method (n = 5), polymerase chain reaction (PCR) (n = 6) or a combination of culture and PCR (n= 2). Fresh preputial scrapings from four bulls with known negative status for trichomonosis were pooled in 200ml of phosphate buffered saline (PBS) to form the sample base for 12 subsamples of 13ml each. Duplicate subsamples were then contaminated with 2ml originating from four different laboratory cultures of Tritrichomonas foetus or 2ml of culture medium for four negative samples. Aliquots of the subsamples were transferred to an anaerobic transport medium, and the final concentration reached in these samples submitted to the laboratories, were categorised as follows: weak (<10 organisms/μl), moderate (10 – 30 organisms/μl) or strong (>30 organisms/μl). A total of 312 samples were sent by courier in two separate rounds: eight (4 duplicates) positive and four negative samples per round. Multiple logistic regression was performed on sensitivity, using sampling round, laboratory sector, diagnostic test type and sample concentration as independent variables, and removing variables in a stepwise manner based on the highest P-value. Two public laboratories only reported on one round of sampling, and one batch of 12 samples was severely delayed in reaching another public laboratory. The sample identifications of a further two batches were not recorded by the respective private laboratories. The results from these 60 unreported samples were not included in the analysis. Laboratories that performed the PCR assay (solely, or in addition to culture) were grouped for data analysis. The overall specificity (Sp) was 100% and the sensitivity was 88.7% (95% CI 83.9% - 93.5%). Laboratories using PCR recorded higher sensitivity than those using the culture method (95.5%; 95% CI 91.0% – 99.9% and 81.3%; 95% CI 72.5% - 90.0% respectively, P < 0.01), and private laboratories recorded higher Se than public laboratories (96.4%; 95% CI 92.9% - 99.9% and 73.2%; 95% CI 61.2% - 85.2%, P < 0.01). For laboratories using PCR, weak positive samples recorded a lower sensitivity than strong positive samples (86.4%; 95% CI 70.8% - 101.9% and 100%; 95% CI 100% - 100%, respectively, P < 0.01). One public and six private laboratories obtained 100% accuracy during the two sampling rounds. In the logistic regression model, private sector (compared to public), an increasing concentration of organisms in the sample and the second round of sampling (compared to the first round) were independent predictors of laboratory sensitivity for the detection of Tritrichomonas foetus. It is concluded that inaccuracies in the diagnostic laboratory contributes to the deficiencies in diagnostic sensitivity for trichomonosis in South Africa, but does not influence diagnostic specificity. It is further concluded that diagnostic sensitivity was independently influenced by the sector in which the laboratory operates (private vs public) and the concentration of Tritrichomonas foetus organisms in the sample. / Dissertation (MSc)--University of Pretoria, 2019. / Production Animal Studies / MSc (Veterinary Science) / Unrestricted

Research aimed at optimizing diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses resulting in reproductive losses due to foetal death or persistently infected (PI) calves that usually die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of BVDV infection. Persistently infected animals that survive calfhood are at risk of developing mucosal disease in later life which is a severe and usually fatal condition. In addition, persistently infected calves that become replacement heifers in the herd may experience significant morphological changes that occur in the ovaries which can result in impaired reproductive performance. This poses important challenges to overall animal/herd health and causes losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected (PI) with BVDV may affect the ability of diagnostic assays to efficiently detect the virus. This study assessed the effects of 1) long-term storage of formalin-fixed samples at room temperature to detect BVD viral antigen with the aid of immunohistochemistry (IHC), 2a) long-term storage of fresh ear notch samples kept at -20°C, and 2b) long term storage of phosphate-buffered saline (PBS) ear notch supernatant kept at -20°C on the ability of an antigen-capture ELISA (AC-ELISA) to detect viral antigen. Previous studies have verified 100% sensitivity for both AC-ELISA on ear notch supernatant and immunohistochemical testing of ear notches to detect BVDV provided that samples are properly collected and stored. In this study, ear notch samples from seven animals were subjected to prompt formalin fixation and fresh samples to prolonged storage at -20°C. Frozen ear notches and ear notch supernatant yielded positive results on AC-ELISA for the duration of the study, i.e. 6 months, and OD values remained significantly within range. There was no significant difference between storing fresh ear notch samples and PBS ear-notch supernatant at -20°C. However, positive IHC staining on formalin-fixed ear notches started to fade away between day 17 and day 29 when stored at room temperature. We conclude that fresh ear notches could safely be stored at -20°C for a period of 6 months for detecting BVD viral antigen at a later stage. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Rennet extract is an important commercial enzyme preparation which is indispensable to the cheese making industry, The term "rennet" is used to denote the enzyme system extracted from the fourth stomach (abomasum) of a suckling calf. Most of the enzymatic material in the extract is rennin. It also contains a small amount of pepsin and some other proteolytic enzymes. The amount of pepsin in rennet extract appears to depend upon the age of the calves at the time of slaughter. It has been suggested that some proteolytic enzymes from higher plants might be useful in cheese manufacture. Krishnaswamy et al. reported that Cheddar cheese made with ficin quickly developed a bitter flavor which decreased in intensity during curing. Cheddar cheese made with an extract from the flower petals of Cynara cardunculus developed extreme bitterness and a pasty body during 30 days curing at 10 C. An extract from the fruit of Withania coagulans was used by Kothavalla and Khubchandani as a coagulating agent in the manufacture of Surati and Cheddar cheese. No unusual flavors were reported, but they experienced rather high fat losses.

The study objective was to evaluate the effects of vaccination (respiratory and clostridial vaccination or no vaccination) and deworming (fenbendazole and levamisole or no deworming) of high risk stocker calves on-arrival on health and growth performance. Eighty sale barn origin calves were purchased three separate years (n=240) from local order buyer. Steers (n=61) and bulls (n=179) were received over three days (d -3 to -1). On d 0 calves were stratified by arrival BW and FEC into 20 pens of 4 calves each, and treatment was applied to pens in 2 x 2 factorial. Vaccination increased the likelihood of BRD 1.7 times (P=0.07) versus calves not vaccinated. Vaccination did not affect gain, but calves receiving dewormer had greater ADG than those not receiving dewormer. Calves that arrived uncastrated or with high fever (≥40.0°C) gained less and were 1.7 and 4.3 times more likely (P<0.10) to be treated for disease, respectively.

Bovine respiratory disease (BRD) complex is a multiactorial disease syndrome that results from various individual contributions and interactions of pathogen, host, and environmental/management factors. Despite the efforts in research, prevention and treatment, BRD remains a leading cause of economic loss in the cattle industry. While advances in therapeutics and new vaccines have been developed over the past 20 – 25 years, the incidence of respiratory disease does not appear to be on the decline, rather it is appears to be increasing. While bacterial and viral pathogens, and various stressors associated with BRD have been characterized, there are no animal models that can reproduce similar presentation of symptoms observed for BRD in the industry. Based on the etiology of BRD, a series of projects were designed to provide a better understanding of the individual and multiple contributions for the factors associated with the complex. It is believed that the viral pathogens or stressors can suppress immune defenses allowing opportunistic bacteria the ability to colonize and cause an infection. Therefore, trials investigating the individual contribution that varying doses of infectious bovine rhinotracheitis virus and transportation stress have on cattle were conducted. A final project investigating the combination effect of the bacterial pathogen M. haemolytica and activation of the hypothalamic-pituitaryrenal axis to elicit glucocorticoid release was evaluated. Ultimately, the research projects were designed to build upon each other to understand each component in the etiology of this disease.