Tissue Culture Laboratory
A well equipped tissue culture laboratory was established to support the research studies in aquatic pollution, veterinary pharmaceuticals and phytomedicine at the cellular and sub-cellular level, thereby reducing the number of animals required in experimental studies. As xenobiotics and pharmaceuticals are mainly metabolized in the liver, great emphasis is placed on establishing primary hepatocyte cultures and hepatocyte cell lines of different species in vitro. Liver specific functions are maintained in vitro in primary hepatocyte cultures for about 10 days. Cellular and sub-cellular morphological changes, basal cell functions (e.g. viability in terms of mitochondrial activity) and organ specific functions (e.g. biotransformation in terms of enzyme induction (CYP) and protein synthesis, e.g. vitellogenin) are used as biomarkers. Primary liver cell cultures of the Nile crocodile (Crocodylus niloticus) and Sharptooth catfish (Clarias gariepinus) were established and are being validated for use in aquatic pollution studies. Cell lines (liver, muscle, fat, macrophage, pancreas and kidney) have been established in the laboratory and are used for cytotoxicity, genotoxicity and diabetes studies of plant extracts and purified plant chemicals by researchers in the Phytomedicine Programme. It is essential to gain an indication of the cellular toxicity of a plant extract prior to continuing research on the compounds responsible for selective biological activity. A variety of assays measuring different parameters of cytotoxicity are used. Genotoxicity assays include the comet and micronucleus assays as well as the Ames test. Antidiabetic assays are conducted to evaluate the inhibitory activity of plants against enzymes responsible for breaking down carbohydrate, namely alpha-amylase and alpha-glucosidase. The effects of samples on glucose uptake and insulin release are also measured in appropriate cell lines. Future studies in the tissue culture laboratory include first the separation of the different crocodile liver cells and then the recombination of the different cell types in culture to study the effect of homologous and heterologous cellular interactions on the expression of organ specific cellular functions. The detrimental effect of acid mine drainage on the aquatic environment is a big concern and therefore the establishment of primary Sharptooth catfish gill epithelial cell cultures for metal cytotoxicity studies are being planned. Understanding the functionality of the mammary gland and subsequent drug transport into milk are important factors in determining milk withdrawal periods and drug residues in milk intended for human consumption. Caprine mammary epithelial cells from lactating udders of the Boer goat will be established in culture (tight cellular monolayers to resemble the in vivo blood-udder-barrier).Transporter protein expression (responsible for transportation of drugs) will be evaluated by means of Western blotting.
Diagnostic Pathology Reference Laboratory
The laboratory has a huge diagnostic workload and provides a diagnostic service to the Onderstepoort Veterinary Academic Hospital at the Faculty as well as to all the other Departments. This service is also available to private clients including pharmaceutical companies. The main purpose of the diagnostic service is to generate material for under- and post-graduate student training and it is not the intention to compete with the private sector for cases. This is well illustrated by the fact that IDEXX Vet Path, a private pathology laboratory, is renting space within the Pathology Complex. The necropsy service involves a thorough post mortem examination (usually done by final year students or a clinical assistant (pathologist-in-training) under the supervision of a pathologist), with the recognition of macroscopic lesions in organs and tissues, the ability to give appropriate morphological diagnoses, judge the significance of lesions and collection of appropriate samples for confirmation/exclusion of diagnoses/differential diagnoses (DDs).
Annually approximately 1 100 post mortems are performed and 4000 cases processed for microscopical examination. The histopathology service offers morphological, aetiological (where possible) or differential diagnoses, based on microscopic morphology. Haematoxylin-and-Eosin (H&E)-stained tissue sections from a wide variety of animals (submitted by clinicians at the OVAH, veterinarians in private practice in South Africa and in neighbouring countries, e.g. Zambia, and private pathologists for a second opinion), are interpreted in conjunction with information supplied by the history, macroscopic findings and results of other investigational procedures, e.g. electron microscopy (EM), polymerase chain reaction (PCR), virus isolation, etc. Special diagnostic techniques used include special stains for fungi etc, immunohistochemical labelling for a variety of conditions/diseases, immunofluorescent staining, and negative staining electron microscopy. The histopathology laboratory is accredited by the South African Veterinary Council.
The real strength of IHC lies in the association of infectious disease agents with specific microscopic lesions, when the latter are present, in formalin-fixed, paraffin-embedded/FFPE tissues (which also allows for retrospective studies). In addition, IHC is an extremely useful tool for the identification of target organs/tissues and cells for infectious disease agents (especially viruses). We have worked on the tissue and cell tropism of African horse sickness and Rift Valley fever viruses. Furthermore, we have done work on growth factor expression and characterization of immuno-inflammatory cell infiltrates in oesophageal nodules in dogs with spirocercosis (another re-emerging disease in Africa), in an attempt to understand mechanisms underlying the neoplastic transformation of these nodules in dogs. Currently, the IHC laboratory is also involved in a collaborative research project with the on canine babesiosis whereby the pathogenesis of the disease is being investigated using IHC (amongst other things) to detect different cells and adhesion molecules in a variety of FFPE tissues. We are also currently busy with the validation of an indirect IHC test for rabies virus, a crucially important disease in Africa, using FFPE sections of brain from a wide variety of domestic and wildlife species in Southern Africa. At this stage, not a lot is known about rabies virus (for example viral distribution) in wild animals from the point of view of pathology. In addition to these projects, there are a number of other projects involving the immunohistochemical identification of cells and other markers in neoplasia in domestic and wild animal species.
Ultimately, we would like to be recognized as the IHC reference laboratory for African infectious diseases in a wide variety of domestic and wild animal species. In order to realize this ambition we will endeavour to encourage multidisciplinary, collaborative research between departments, Faculties and Universities in South Africa and abroad. We will continue to encourage and support studies on the pathogenesis of diseases, particularly those that are endemic to or emerging in Africa.