Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for trypanosomosis are unsatisfactory and vaccine development has been hampered by antigenic variation. An anti-disease vaccine is based on the idea that disease is caused by the pathogenic factors released by the parasite, rather than by the parasite itself. Therefore, if these pathogenic factors could be neutralised by antibodies produced by vaccination, the disease could be circumvented. The method used here for identification of novel pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate the disease by generating a specific immune response against a few key antigens (pathogenic factors). Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected sera and a second column containing IgG from trypanotolerant N’Dama cattle serially infected with T. congolense. The differential binding of antigens to the two columns allowed identification of antigens specifically recognised by the immune system of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens identified included several variant cathepsin L-like cysteine peptidases (CPs) and the Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic organisation was conducted in order to further understand the variability present in this gene family. To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in which genes as different as possible from congopain (the major CP of T. congolense) were selected, and a second which contained all possible genes present in the congopain array. Analysis of the sequences obtained in these two mini-libraries showed that there was significant variability of the genes within the congopain array. Two variants of CPs, chosen for differences in their catalytic triads, were cloned for expression. The recombinantly expressed CP variants differed in substrate preferences from one another and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli system, and subsequently refolded. The refolded APs showed a substrate preference for H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by puromycin. The two APs were not developmentally regulated and present in procyclic, metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of BALB/c mice with the APs did not provide protection when challenged with T. congolense. For an anti-disease vaccine to be protective, it would possibly have to include all pathogenic factors, including the two APs and at least one CP described in the present study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Two studies were conducted to determine the effects of zinc (Zn), copper (Cu) and/or selenium (Se) on the humoral immune response of weaned steers fed diets deficient in these minerals. Steers were bled weekly for eight weeks to measure plasma mineral levels. Humoral immune response was measured as antibody response to Keyhole limpet hemocyanin (KLH) over seven weeks of the trials. Steers were injected with 0.5 ml KLH vaccine on weeks 2 and 5 of the Zn study. Ten steers (5 per treatment) were randomly assigned to either a Zn supplemented (z), 3.25 ml injectable zinc-oxide suspension (100 mg/ml oil) or a control (c) treatment. Plasma Zn was maintained at higher (P<.05) levels in supplemented steers from week 3 through week 8. Humoral immune response was not different (P>.10) between treatments. In 1988, 20 steers were stratified by weaning weight and randomly assigned to one of four treatments: 1) Injectable Se 1 ml Mu-Se/ 90.9 kg body weight, 2) Injectable Molycu 2 ml, 3) control, or 4) Se + Cu. Plasma Cu was higher (P<.05) in treatments 2 and 4 from week 2 through 8. Steers were injected with 0.5 ml of KLH vaccine on weeks 2 and 6. Immune response was not different (P>.10) between Cu supplemented steers and other treatments. Plasma Se was higher (P<.05) in treatment 1 as compared to 2 and 3, but not different (P>.10) from 4. Immune response was highest (P<.05) in treatment 1 with others not different(P>.10). In 1990, a second trial was conducted with 48 steers stratified by weight and randomly assigned to one of the same four treatments. Plasma Cu was higher (P<.05) on week 3 for both groups receiving Cu. Plasma Se was higher (P<.05) on weeks 2 through 5 for both groups receiving Se. Humoral immune response was higher (P<.05) on weeks 7 and 8 for both groups receiving Se. Humoral immune response was uneffected by Zn status of weaned beef steers. Selenium had a positive effect and Cu had no effect on humoral immune response of weaned beef steers. Copper also tended (P=.07) to reduce the positive response from Se. / Graduation date: 1991

Calcium (Ca$ sp{2+}$) and protein kinase C (PKC) are believed to act as intracellular signals triggering the activation of NADPH oxidase in neutrophils leading to superoxide generation. This was tested on bovine neutrophils by chelating extracellular and/or intracellular free Ca$ sp{2+}$ and by measuring PKC activity when the cells were stimulated by phorbol myristate acetate (PMA) or opsonized zymosan (OZ). Chelation of extracellular Ca$ sp{2+}$ with EGTA did not alter O$ sb2 sp{-}$ production from PMA stimulated cells. However, it did cause a 64% decrease in O$ sb2 sp{-}$ production in the neutrophils when stimulated with OZ. When intracellular Ca$ sp{2+}$ was chelated with BAPTA/AM, there was a significant decrease in O$ sb2 sp{-}$ generation following PMA activation. Yet, OZ activated cells, pre-treated with BAPTA/AM, showed an increase in the respiratory burst proportional to the chelator's concentration. Moreover, although OZ was previously shown to increase O$ sb2 sp{-}$ generation by neutrophils, no significant changes in PKC activity were observed. PMA stimulation led to an increase in PKC activity at the membrane level. Furthermore, treating the cells with calphostin C, a PKC activity inhibitor, caused a 69% decrease in O$ sb2 sp{-}$ production demonstrating the involvement of PKC in PMA-stimulated cells. However, no differences were observed between the OZ activated cells incubated with the inhibitor and the control cells. These data provide evidence that activation of NADPH oxidase can be achieved by either a PKC-dependent or a PKC-independent pathway depending on the stimulatory agent.

Diapedesis, phagocytosis and microbicidal activity are important parameters of neutrophil functionality and thus outcome of mastitis. An in vitro model of an "alveolar pavement" using the MAC-T3 bovine mammary epithelial cell line was developed to assess neutrophil diapedesis. Features of this biologically-meaningful barrier include: characteristic transepithelial resistance, tight junction complexes and polarity. Continuous transepithelial resistance measurements showed no significant changes throughout the assay period. Neither a Staphylococcus aureus challenge ($1 times10 sp7$ and $2 times10 sp9$ cfu/ml), or the presence of neutrophils, both resting and challenged had any deleterious effects on monolayer integrity over a short term (1-2 h) exposure. Neutrophils, both resting and challenged gave no indication of causing damage to the epithelium over the short term. Neutrophils isolated from proven sires and evaluated for phagocytic activity were found to differ significantly (p $<$ 0.05) in activity, rate and capacity to uptake particles. Correlations between phagocytic parameters and production traits were negative and small in magnitude. Microbicidal activity of neutrophils isolated from proven sires showed a highly significant variation between animals due to test day (p $<$ 0.001), however variation due to source of cells (i.e. animal) was not significant. in vitro analysis of diapedesis and phagocytosis is promising as a tool for the assessment of resistance or susceptibility to mastitis.

Bovine mastitis due to bacterial infection is one of the most costly diseases affecting the dairy industry. The polymorphonuclear neutrophils (PMNs) present in milk have a central protective role against invading pathogens, However, the manner by which PMNs traverse the secretory epithelia and the relationship between PMN diapedesis and the epithelial damage are unclear. This in vitro study investigated the process and rate of bovine PMN transepithelial migration. The bovine mammary epithelial cell line, MAC-T, formed a confluent monolayer with characteristic tight junctions, polarity and functional barrier to the dye trypan blue. In the first series of experiments, neutrophils were added into the upper compartment of the culture insert and stimulated to migrate across the epithelium in an apical-to-basal direction by the addition of Staphylococcus aureus to the lower compartment. Light and transmission electron microscopy revealed the following series of events for PMN transmigration: (1) adherence of PMNs to the surface of the epithelium; (2) projection of pseudopods toward the intercellular junction; (3) migration between adjacent epithelial cells; and (4) re-approximation of epithelial cell membranes and reformation.

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The purpose of this study was to investigate which type(s) of somatic cells release nitric oxide (NO) in response to Escherichia coli lipopolysaccharide (LPS) and cytokines in vitro and how NO affects superoxide anion (O2-) production by bovine neutrophils and blood monocytes. Mammary epithelial cell line (FbE) released NO after stimulation with recombinant bovine interleukin-1beta (rBoIL-1beta). Moreover, monocytes produced NO in response to recombinant bovine interferon gamma (rBoIFN-gamma) alone or in combination with LPS in a dose- and time-dependent manner. Nitric oxide production was diminished by addition of inducible nitric oxide synthase (iNOS) inhibitors L-N 6-(1-Iminiethyl)lysine or aminoguanidine. However, NO release could not be induced in freshly isolated bovine neutrophils under the experimental conditions used, even after 96 h of incubation. Interestingly, when reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers for iNOS was performed to study mRNA expression, iNOS expression was observed in both monocytes and neutrophils in response to LPS and rBoIFN-gamma. / Unlike neutrophils, monocytes were poor producers of superoxide anion under the experimental conditions. A neutrophil-monocyte co-culture system was set up to study the effect of monocyte derived-NO and iNOS inhibitors on superoxide anion production by neutrophils. Neither NO derived from activated monocytes nor iNOS inhibitors seemed to have an effect on bovine neutrophil ability to release O2-. These results suggest that mammary epithelial cells and mononuclear phagocytes are among the cell types responsible for the important quantities of NO released by somatic cells recovered from LPS-infused mammary quarters during endotoxin-induced bovine mastitis. In addition, NO or iNOS inhibitors have no effect on the ability of activated bovine neutrophils to produce superoxide anions.

The objective of this study was to investigate the effect of recombinant human interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration and degranulation. An in vitro co-culture system was used to study bovine neutrophil migration. This simulative system allowed studying neutrophil migration across endothelium (bovine aorta endothelial cells), extracellular matrix (ECM), and epithelium (MAC-T) in the correct sequences and directions. Quantification of neutrophil migration was carried out by assaying the activity of myeloperoxidase, a major enzyme of neutrophils. Degranulation of azurophilic, specific, and tertiary granules was studied by measuring releases of myeloperoxidase, lactoferrin, and gelatinase, respectively. The results showed that bovine neutrophils were able to migrate across the simulative co-culture system in response to zymosan activated serum. Recombinant HIL-8 was demonstrated to have a dose-dependent effect on bovine neutrophil migration. Furthermore, rHIL-8 had a dose-dependent effect directly on degranulation of azurophilic and specific granules, but not on tertiary granules. On the other hand, rHIL-1beta only had a significant effect on degranulation of azurophilic granules when the concentration of 100 ng/ml was used. The dose effect of rHIL-1beta on specific degranulation was much stronger. Moreover, the effect of 100 ng/ml rHIL-1beta was augmented when the rHIL-1beta containing solution was preincubated with MAC-T monolayers for four hours. This indicated that MAC-T cells might generate other degranulating factors in response to the stimulation of rHIL-1beta. These MAC-T-derived degranulating factors did not have effect on the release of tertiary granule contents.