Arshad Mather

Introduction: Angora goats are a distinct breed that differs significantly from common goats and shares a similar appearance to sheep. In Angora goats, only the level of glutathione (GSH) is elevated during under-stimulated conditions, as well as after the period of hypoxic stress; however, no changes are found in 2,3-diphosphoglycerate (2,3-DPG) levels, which are commonly present in the red blood cells (RBCs) of most mammals. We chose the Angora goat for our investigation because no previous studies have been conducted on the structural and functional aspects of hemoglobin (Hb). In addition, no sequence or structural information is currently available in any database. Methods: Angora goat Hb was isolated and purified by anion-exchange chromatography, followed by crystallization using various methods. X-ray data collection for Angora goat Hb was performed under a liquid nitrogen cryo-stream using a Bruker D8 Venture Bio Photon III 28-pixel array area detector system. Results: Good diffracting crystals were obtained using the hanging-drop vapor-diffusion method with polyethylene glycol (PEG) 3350 as the precipitant in water, without the addition of any salt or buffer. The Angora goat Hb diffracted to a resolution of 1.85 Å, and the structure solution was obtained by the molecular replacement method, using the structure of domestic goat Hb as the starting model. Discussion: The solved structure of Angora goat crystallized in the monoclinic space group P21, consisting of one whole biological molecule in the asymmetric unit, with unit cell dimensions of a = 52.08 Å, b = 76.70 Å, c = 74.08 Å, and β = 91.77 °. The solvent content and Matthews coefficient (Vm) for the Angora goat Hb are 49.05% and 2.41 Å3/Da, respectively, and are within the normal range for protein crystals. Conclusion: Purification, crystallization, and preliminary X-ray diffraction studies of Angora goat Hb were performed successfully. Structural refinement and biophysical characterization of Angora goat Hb are in progress in the absence and presence of GSH and 2,3-DPG.

Capripoxviruses are the causative agents of sheeppox, goatpox, and lumpy skin disease (LSD) in cattle, which cause economic losses to the livestock industry in Africa and Asia. Capripoxviruses are currently controlled using several live attenuated vaccines. It was previously demonstrated that a lumpy skin disease virus (LSDV) field isolate from Warmbaths (WB) South Africa, ORF 005 (IL-10) gene-deleted virus (LSDV WB005KO), was able to protect sheep and goats against sheeppox and goatpox. Subsequently, genes encoding the protective antigens for peste des petits ruminants (PPR) and Rift Valley fever (RVF) viruses have been inserted in the LSDV WB005KO construct in three different antigen forms (native, secreted, and fusion). These three multivalent vaccine candidates were evaluated for protection against PPR using a single immunization of 10⁴ TCID₅₀ in sheep. The vaccine candidates with the native and secreted antigens protected sheep against PPR clinical disease and decreased viral shedding, as detected using real-time RT-PCR in oral and nasal swabs. An anamnestic antibody response, measured using PPR virus-neutralizing antibody response production, was observed in sheep following infection. The vaccine candidates with the antigens expressed in their native form were evaluated for protection against RVF using a single immunization with doses of 10⁴ or 10⁵ TCID₅₀ in sheep and goats. Following RVF virus infection, sheep and goats were protected against clinical disease and no viremia was detected in serum compared to control animals, where viremia was detected one day following infection. Sheep and goats developed RVFV-neutralizing antibodies prior to infection, and the antibody responses increased following infection. These results demonstrate that an LSD virus-vectored vaccine candidate can be used in sheep and goats to protect against multiple viral infections.

Background: Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (Mmm SC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of Mmm SC. Since the production of IgG2 and IgA are associated with a Th₁ cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine. Results: A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG) were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease. Conclusion: Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for Mmm SC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with Mmm SC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in E. coli.