A web-based and mail-out survey instrument was created to gather information on camelids in Texas. Information on management, nutrition, diseases, and reproductive problems was collected. The objectives of this research study were: (1) to establish prevalence of various diseases in alpaca and llama populations in Texas; (2) to evaluate association between potential management/nutrition risk factors and specific diseases/reproductive problems; (3) to determine how many camelids are kept in Texas and what their use is; (4) to determine possible disease clustering through spatial analysis. The survey results included 2,079 camelids on 125 farms within Texas. The top five camelid diseases in this sample were intestinal parasites, incisor overgrowth, mites, heat stress, and colic. Univariate analysis and multivariable modeling found associations between potential risk factors and these diseases.

A web-based and mail-out survey instrument was created to gather information on camelids in Texas. Information on management, nutrition, diseases, and reproductive problems was collected. The objectives of this research study were: (1) to establish prevalence of various diseases in alpaca and llama populations in Texas; (2) to evaluate association between potential management/nutrition risk factors and specific diseases/reproductive problems; (3) to determine how many camelids are kept in Texas and what their use is; (4) to determine possible disease clustering through spatial analysis. The survey results included 2,079 camelids on 125 farms within Texas. The top five camelid diseases in this sample were intestinal parasites, incisor overgrowth, mites, heat stress, and colic. Univariate analysis and multivariable modeling found associations between potential risk factors and these diseases.

The purpose of the studies reported in this thesis was to provide further evidence in support of the hypothesis that ovulation-inducing factor (OIF) is a component of seminal plasma which is conserved amongst mammals. Based on studies conducted in vivo, the results indicate that males ejaculate a substance during copulation which is responsible for the ovulatory and luteotrophic effect in female camelids. In our lab we have developed an <i>in vivo</i> llama bioassay to study the presence and biological effects of OIF in seminal plasma from different species.<p> The objective of the first experiment within the first study was to determine if llama seminal plasma would stimulate ovulation in prepubertal mice. Mice were treated with a single 0.1 mL intraperitoneal dose of 1) phosphate-buffered saline (negative control), 2) 5 µg gonadotropin-releasing hormone (GnRH), 3) 5 IU of human chorionic gonadotropin (hCG) or 4) llama seminal plasma. Results indicate that prepubertal mice treated with GnRH, hCG or llama seminal plasma stimulated similar proportions of mice to ovulate, which were all higher than the proportion of mice that ovulated after saline treatment. The number of oocytes observed under a stereomicroscope was also higher in all treatment groups than in mice treated with saline. However, the number of oocytes observed was lower in mice treated with seminal plasma than those treated with GnRH, both of which were similar to the number of oocytes observed in hCG treated mice.<p> In a second part of this study the corollary that OIF is present in the seminal plasma of horses and pigs was examined. Seminal plasma from horses or pigs was administered intramuscularly to female llamas and ovulation was monitored using transrectal ultrasonography. Llamas were treated with an intramuscular dose of 1) phosphate buffered saline (negative control), 2) llama seminal plasma (positive control), 3) equine seminal plasma or 4) porcine seminal plasma. Ovulations were detected in llamas treated with seminal plasma while none were observed in saline-treated llamas. The proportion of llamas that ovulated when treated with equine seminal plasma was higher than llamas treated with saline. The proportion of llamas that ovulated after porcine seminal plasma tended to differ from negative control groups, but did not reach statistical significance. The proportion of llamas that ovulated after equine or porcine seminal plasma treatment was lower than animals treated with llama seminal plasma which indicates that either OIF is not present in equal concentration among mammals, or that OIF is not structurally the same across mammals.<p> The second study was carried out to test the hypothesis that OIF stimulates LH secretion at the level of the anterior pituitary gland. The second objective was to determine if the degree of LH release was related to the dose of OIF treatment. Anterior pituitary cells (2 x 10^6 cells/ well) from either llamas (reflex ovulator) or cattle (spontaneous ovulator) were incubated for 2 hours with either media containing no treatment (control), GnRH or OIF. In all experiments, GnRH and OIF stimulated more LH secretion than control groups. An effect of dose was evident in the llama pituitary cell culture where mean LH concentrations were greater in wells treated with a higher dose of OIF in comparison to wells treated with a lower dose, both of which were higher than in wells with no treatment. Although OIF stimulated LH release in bovine cell cultures, an apparent dose response was not detected. Results indicate that the preovulatory LH surge observed after OIF treatment in camelids may be the result of OIF directly stimulating LH release from gonadotrope cells within the anterior pituitary gland. In conclusion these results illustrate that the presence and the response to OIF is conserved among species that share no relation or common reproductive strategy.

The purpose of the studies reported in this thesis was to provide further evidence in support of the hypothesis that ovulation-inducing factor (OIF) is a component of seminal plasma which is conserved amongst mammals. Based on studies conducted in vivo, the results indicate that males ejaculate a substance during copulation which is responsible for the ovulatory and luteotrophic effect in female camelids. In our lab we have developed an <i>in vivo</i> llama bioassay to study the presence and biological effects of OIF in seminal plasma from different species.<p> The objective of the first experiment within the first study was to determine if llama seminal plasma would stimulate ovulation in prepubertal mice. Mice were treated with a single 0.1 mL intraperitoneal dose of 1) phosphate-buffered saline (negative control), 2) 5 µg gonadotropin-releasing hormone (GnRH), 3) 5 IU of human chorionic gonadotropin (hCG) or 4) llama seminal plasma. Results indicate that prepubertal mice treated with GnRH, hCG or llama seminal plasma stimulated similar proportions of mice to ovulate, which were all higher than the proportion of mice that ovulated after saline treatment. The number of oocytes observed under a stereomicroscope was also higher in all treatment groups than in mice treated with saline. However, the number of oocytes observed was lower in mice treated with seminal plasma than those treated with GnRH, both of which were similar to the number of oocytes observed in hCG treated mice.<p> In a second part of this study the corollary that OIF is present in the seminal plasma of horses and pigs was examined. Seminal plasma from horses or pigs was administered intramuscularly to female llamas and ovulation was monitored using transrectal ultrasonography. Llamas were treated with an intramuscular dose of 1) phosphate buffered saline (negative control), 2) llama seminal plasma (positive control), 3) equine seminal plasma or 4) porcine seminal plasma. Ovulations were detected in llamas treated with seminal plasma while none were observed in saline-treated llamas. The proportion of llamas that ovulated when treated with equine seminal plasma was higher than llamas treated with saline. The proportion of llamas that ovulated after porcine seminal plasma tended to differ from negative control groups, but did not reach statistical significance. The proportion of llamas that ovulated after equine or porcine seminal plasma treatment was lower than animals treated with llama seminal plasma which indicates that either OIF is not present in equal concentration among mammals, or that OIF is not structurally the same across mammals.<p> The second study was carried out to test the hypothesis that OIF stimulates LH secretion at the level of the anterior pituitary gland. The second objective was to determine if the degree of LH release was related to the dose of OIF treatment. Anterior pituitary cells (2 x 10^6 cells/ well) from either llamas (reflex ovulator) or cattle (spontaneous ovulator) were incubated for 2 hours with either media containing no treatment (control), GnRH or OIF. In all experiments, GnRH and OIF stimulated more LH secretion than control groups. An effect of dose was evident in the llama pituitary cell culture where mean LH concentrations were greater in wells treated with a higher dose of OIF in comparison to wells treated with a lower dose, both of which were higher than in wells with no treatment. Although OIF stimulated LH release in bovine cell cultures, an apparent dose response was not detected. Results indicate that the preovulatory LH surge observed after OIF treatment in camelids may be the result of OIF directly stimulating LH release from gonadotrope cells within the anterior pituitary gland. In conclusion these results illustrate that the presence and the response to OIF is conserved among species that share no relation or common reproductive strategy.

No description available.

The increasing incidence of disc degeneration and its correlation with lower back pain is an alarming trend in modern society. The research of intervertebral disc degeneration and low back pain would greatly benefit from additional methods to study its etiology and possible treatment methods. A large animal model that maintains the biological and mechanical environment that is most similar to the human lumbar spine could provide substantial improvements in understanding and resolving the problem of intervertebral disc related low back pain.This dissertation presents my doctoral work of investigating the potential for the camelid cervical spine to serve as a suitable animal model for advancing biomechanical research of low back pain and intervertebral disc degeneration in the human lumbar spine. Specifically, this work identifies the cellular, morphological and biomechanical characteristics of the camelid cervical spine and intervertebral disc as compared to the human lumbar spine. My results demonstrate that there are remarkable similarities in all aspects. Many of the similarities with respect to the cellular environment of the intervertebral disc are a consequence of the camelid status as a large mammal. Additional testing of the cellular makeup of the camelid intervertebral disc cells revealed that many human qRT-PCR primers associated with disc degeneration are suitable for use in alpacas without modification. From a biomechanics standpoint, the camelid cervical spine also has a vertically oriented spinal posture and is unsupported near the end in an open kinetic chain, providing a mechanical parallel with the human lumbar spine. The camelid cervical intervertebral disc size is closer to the human lumbar intervertebral disc than all other currently used animal models available for comparison in the literature. Average flexibility (range of motion) of a camelid spinal motion segment showed similarities in all modes of loading. Based on magnetic resonance imaging and radiologic grading of the intervertebral disc, almost 90% of elderly camelids exhibited advanced degeneration (Pfirrmann grade 3 or higher) in their cervical spine, and about half of aged camelids have developed severe degeneration (Pfirrmann grade 4 or higher) in at least one or more of their cervical segments, most commonly within the two lowest cervical segments (e.g. c6c7 and/or c7t1). Thus, while there remain differences, the remarkable similarities between the camelid and human spine strengthen the case for using camelids as a model for human disc degeneration, normal and pathological biomechanics and fluid transport, and potentially as a pre-clinical model for investigating the efficacy of novel spinal devices.

The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.