This thesis aimed to examine the impact of environmental tobacco smoke on biological markers of ageing in pet dogs. In order to achieve this, community-based dogs and owners were recruited, approximately half of whom lived in smoking homes and half non-smoking homes. Owners were asked to attend 2 appointments, 12 months apart. Questionnaire assessments of dog environmental tobacco smoke exposure were compared to biomarkers in hair of the dogs (Chapter 3). This gave an objective measure of exposure and hair nicotine concentrations reliably reflected information provided by owners. A qPCR technique was optimised to measure telomere lengths (Chapter 4). This was utilised to measure telomere lengths in leukocytes, buccal cells, cremaster muscle, vas deferens and epididymis samples from the study dogs. Owners were offered free-of-charge neutering for their pet, and the spare tissues used for these analyses. A negative relationship in leukocyte telomere length with hair nicotine was observed, among other changes (Chapter 5). mRNA levels of further biomarkers of ageing were measured in testes, as well as leukocyte global DNA methylation percentage (Chapter 6). Again, several significant relationships were found between tobacco smoke exposure markers and the biomarkers of ageing, including a significant negative relationship with CDKN2A expression with increased tobacco smoke exposure. Plasma testosterone and hair cortisol concentrations were measured. In addition, factors which related to weight gain after neutering were examined (Chapter 7). Increased cotinine concentrations in fur were significantly related to increased percentage weight gain. Several avenues for future research were generated, and many areas warrant further investigation.

Acquired urinary incontinence in the canine is a distressing and debilitating condition affecting up to 20% of neutered bitches, whilst less than 1% of entire bitches and males suffer from this condition. Although a number of medical and surgical therapies exist for treatment of acquired urinary incontinence, none are able to cure the condition and many animals become refractory to treatment over time. It has long been thought that a decrease in resting tone within the urethra of a bitch following neutering is responsible for the development of acquired urinary incontinence; however, recent studies show that low urethral tone does not always lead to urinary incontinence, suggesting further factors must be involved. Although the exact aetiology and pathophysiology of the condition in the neutered bitch is unknown, it is thought to have many similarities to that of post menopausal urinary incontinence in women. In this condition, urinary incontinence is known to be mediated primarily by changes in the structure and function of the urinary bladder post menopause. The present study looks at the structure and function of the canine urinary bladder in vitro to determine if changes occur post neutering that could lead a bitch to develop acquired urinary incontinence and which may provide novel therapeutic targets for treatment of this disease. Contractility in response to carbachol (muscarinic) and electrical field stimulation was assessed in isolated strips of detrusor muscle from male and female, intact and gonadectomised canines. The potential role of non-adrenergic, non-cholinergic mediated contraction of the detrusor muscle was also examined and this system does not appear to be significantly altered by gonadectomy. Maximal contractile responses were, however, decreased in detrusor strips from neutered compared to entire canines regardless of gender, with detrusor strips from incontinent bitches having some of the weakest responses. Sensitivity to carbachol was also decreased in detrusor strips from neutered compared to entire canines. This suggests a decrease in contractile function of the urinary bladder in neutered canines and is similar to that seen in the bladders of women suffering from urinary incontinence post-menopause due to impaired contractility of the bladder and idiopathic detrusor instability. This suggests that changes in the function of the bladder post neutering may be partly responsible for the development of acquired urinary incontinence in the bitch. Post-menopausal urinary incontinence in women is hypothesised to be a linked to an increase in the collagen to smooth muscle ratio within the wall of the urinary bladder which is thought to impair bladder contractility and lead to the development of detrusor instability. Morphometric analysis of the urinary bladder wall of canines showed that the percentage of collagen within this organ was significantly increased in neutered compared to entire bitches, with incontinent bitches having some of the highest percentage collagen. The percentage of collagen was unchanged in neutered compared to entire males which were similar to entire bitches. These results support the long postulated theory that a decrease in oestrogen following gonadectomy / menopause is involved in the increase of collagen within the bladder. Results describing the pharmacological characterisation of muscarinic receptors (Schild analysis of pKB values) in strips of canine detrusor muscle suggest that the M3 receptor is the primary receptor responsible for bladder contraction in entire canines in vitro but that the M2 receptor predominates in neutered canines. This previously unreported finding could be significant in providing a novel therapeutic target to treat this debilitating disease. Studies that looked at mRNA expression for the muscarinic as well as the LH and GnRH receptors in canine bladder wall showed that there was an increase in expression of all receptors in tissue from neutered compared to entire canines and that tissue from females had higher expression levels than that from their male counterparts. It is known that gonadotrophin levels in the blood increase post neutering, and that decreasing these levels can provide continence in a number of animals. It is therefore possible, that an up-regulation of mRNA expression for these receptors is involved in the changes at the level of the detrusor that could lead to development of acquired urinary incontinence. It is also acknowledged that the muscarinic pathway is the primary pathway responsible for bladder contraction and emptying, therefore, a change in the expression of muscarinic receptors has the potential to alter bladder contractility as demonstrated previously. In conclusion these studies have shown that the structure and function of the urinary bladder of a neutered canine is altered compared to that of an entire canine, and that these changes have the potential to be involved in the development of acquired urinary incontinence in the bitch. Many of these changes mimic those seen in the bladders of post menopausal women suffering from urinary incontinence, thus suggesting that there may be commonality of disease process between the two species which may allow the use of the canine as a model of human urinary incontinence. This data, the first to include male animals in the study of bladder function and structure, suggest that the loss of oestrogen in the female and the concurrent increase in percentage collagen within the urinary bladder are not significant factors in the development of decreased detrusor contractility per se. On the contrary these results suggest that the muscarinic receptor effector pathway may play a crucial role in the development of altered bladder contractility and acquired urinary incontinence, and may provide a therapeutic target for effective treatment of this disease.

The aims of this study were to (1) evaluate cellular senescence in chondrocytes from osteoarthritic articular cartilage, (2) investigate the hypothesis that oxidative stress is a feature of canine OA chondrocytes and that oxidative stress contributes to cellular senescence in canine chondrocytes, (3) investigate the hypothesis that osteoarthritic chondrocytes alter the gene expression of adjacent normal chondrocytes in OA joints leading to modulation of genes known to play a role in the pathogenesis of OA and (4) evaluate the presentation of dogs undergoing femoral head excision in veterinary referral practice in the UK as a treatment for osteoarthritis of the coxofemoral joint, and to categorise the distribution and severity of associated pathological lesions. Chondrocytes from osteoarthritic and normal cartilage were examined for levels of senescence. Initially chondrocytes were cultured using an alginate bead culture system, thought to mimic the extracellular matrix of articular cartilage. However, these chondrocytes showed almost no growth as compared to monolayer culture where they grew rapidly. OA chondrocytes entered the senescent state after 1.5 to 4.9 population doublings in monolayer culture, while normal chondrocytes underwent 4.8 to 14.6 population doublings before entering the senescent state. Osteoarthritic chondrocytes had increased levels of markers of cellular senescence (senescence associated beta-galactosidase accumulation and p16 protein accumulation) as compared to normal chondrocytes, suggesting that chondrocyte senescence is a feature of canine osteoarthritis. An experimental model for the induction of oxidative stress in chondrocyte cell culture was developed using tert-Butyl hydroperoxide and total cellular glutathione was measured as an indicator of cellular oxidative stress levels. Experimental induction of oxidative stress in both normal and osteoarthritic chondrocytes in cell culture resulted in increased amounts of cellular senescence, shown by an increase in levels of senescence associated beta-galactosidase accumulation and decreased replicative capacity. Experimental induction of oxidative stress also resulted in altered gene expression of three genes important to the degradation of the extracellular matrix; MMP-13, MMP-3 and Col-3A1, measured by RT-PCR, in normal canine chondrocytes in monolayer cell culture. MMP-3 showed the greatest relative expression change, with a fold-change of between 1.43 and 4.78. MMP-13 had a fold change of 1.16 to 1.38. Col-3A1 was down regulated, with a fold-change of between 0.21 and 0.31. These data demonstrate that experimentally induced oxidative stress in chondrocytes in monolayer culture increases levels of cellular senescence and alters the expression of genes relevant to the pathogenesis of canine OA. Coculture of osteoarthritic chondrocytes with normal canine chondrocytes resulted in gene modulation in the normal chondrocytes. Altered gene expression of ten genes known to play a role in the pathogenesis of osteoarthritis was detected in the normal chondrocytes (fold change shown in brackets); TNF-alpha (11.95), MMP-13 (5.93), MMP-3 (5.48), IL-4 (7.03), IL-6 (5.3), IL-8 (4.92), IL-F3 (4.22), COL-3A1 (4.12), ADAMTS-4 (3.78) and ADAMTS-5 (4.27). In total, 594 genes were significantly modulated suggesting that osteoarthritic chondrocytes contribute to the disease propagation by altering the gene expression of adjacent normal chondrocytes, thus recruiting them into the disease process. Gene expression changes were measured by microarray analysis and validated by RT-PCR and Western blot analysis. An epidemiological study of femoral heads collected from dogs undergoing total hip replacement surgery as a treatment for osteoarthritis of the coxofemoral joint secondary to canine hip dysplasia revealed that there was no characteristic pattern of cartilage lesion for canine hip dysplasia. Severe pathology of the femoral head with cartilage erosion occurred in 63.9% of cases and exposure of subchondral bone in 31.3% of cases. The work presented in this thesis has demonstrated that cellular senescence is a feature of chondrocytes from canine osteoarthritic cartilage and suggests that cellular senescence and oxidative stress play an important role in the pathogenesis of osteoarthritis in dogs.

The extensive use of antibiotics has led to the emergence of methicillin-resistant Staphylococcus pseudintermedius, a bacterium causing difficult-to-treat canine skin infection (pyoderma). The administration of bacteriophages (phage therapy) can be an alternative to antibiotic therapy. Lytic phages, which lyse their host, are considered the only appropriate type of phages for phage therapy as opposed to temperate phages, which can survive within their host (lysogeny). However, it is possible to mutate temperate phages so that they cannot establish lysogeny anymore. Phage λ virulent (Vir) mutants have lost the operator to which the CI repressor binds to inhibit the expression of lytic genes. As a result, Vir mutants are strictly lytic. The work presented in this thesis was undertaken to isolate S. pseudintermedius phages and gain knowledge about their biology with the aim to develop phage therapy to treat pyoderma. The work was novel; very few data were available on S. pseudintermedius phages and no data have been published on phage therapy to treat canine skin infection. Four temperate phage candidates were selected after phenotypic and genotypic characterisation. No lytic phages were found. Random mutagenesis approaches were unsuccessful for the isolation of Vir mutants. An operator and three point mutations leading to the absence of CI repressor binding to this operator were identified through gel shift assay. These mutations should lead to a virulent phenotype if introduced in the relevant phage genome through site-directed mutagenesis. A PCR-based assay was performed to explore how widespread lysogeny was in S. pseudintermedius: 11 out of 45 tested strains were positive for the presence of prophage genes. Bioinformatic analyses revealed some of the genetic characteristics of S. pseudintermedius phages: genomic circular permutation and the presence of a genetic switch similar to that of phage λ. The work reported in this thesis represents a first step towards understanding the biology of S. pseudintermedius phages and developing phage therapy.

Lymphoma is the most common haematopoietic malignancy of dogs, but little is known about its aetiology and pathogenesis. Canine lymphoma is composed of a clinically heterogeneous group of tumours, as in human non-Hodgkin lymphoma (hNHL), which shares many similarities with the canine disease. In humans, lymphoma is classified into many different subtypes, each with its own clinicopathological characteristics, prognosis, and in many cases, treatment regime. In dogs, diagnosis is often rudimentary, and many cases are not routinely or robustly subtyped. This has hampered efforts to develop accurate prognostic indicators and novel therapies, as heterogeneous groups of cases are analysed. More accurate classification of canine lymphomas is required before real progress can be made. In the work described in this thesis, molecular techniques were used to develop diagnostic tests to aid diagnosis of canine lymphoma, with a view to improving disease classification, and gaining a greater understanding of disease pathogenesis. The diagnosis of canine lymphoma can be assisted by PCR for antigen receptor rearrangements (PARR), which can determine clonality and tumour cell lineage. Diagnostic samples from suspected lymphoma patients were screened with multiple published PARR primers to establish a panel of assays for routine use. Primer coverage was assessed, and modifications to both primer sequences and primer combinations made where necessary. The optimised PARR assay was robust, highly sensitive and specific, comparable with published studies and provided useful diagnostic information unavailable from other tests. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it has been suggested that EBV or an EBV-like virus is circulating in dogs. Serological and molecular techniques were used to investigate whether EBV, or a novel herpesvirus, is associated with canine lymphoma. In an assay designed to detect antibodies to EBV viral capsid antigens, 41% of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridisation or PCR and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. No evidence that herpesviruses are directly involved in common types of canine lymphoma was found, although the presence of an EBV-like virus in the canine population cannot be excluded. Gene expression profiling has been used to subtype human diffuse large B-cell lymphoma, and to provide insight into pathogenesis. Next-generation sequencing was used to generate transcriptome data from a panel of canine B- and T-cell lymphomas. Gene expression analysis showed clear clustering of B-cell and T-cell types, and suggested that differentiation within the B-cell group was possible. Pathway analysis demonstrated up-regulation of genes in pathways appropriate to the tumour type. Sequence data were also explored to identify variants in important genes and pathways which may have functional consequences. Many affected genes were identified, and comparison with hNHL suggested that common driver mutations are present. By identifying novel targets, such studies may pave the way for development of new therapies to benefit both man and dog.

Pedigree dog welfare in the United Kingdom is a controversial subject. This PhD aimed to investigate small animal veterinary surgeons’ (VS), pedigree dog breeders’ (PDB) and owners’ perceptions surrounding canine health, their roles, information acquisition and the quality of online resources available to new dog owners. Via a mixed-methods approach VS and PDB were asked about their perceptions of pedigree dog welfare using online questionnaires. Focus groups were then used to explore these parties’ opinions on advising dog owners and buyers. Dog owners were administered an online questionnaire to explore drivers of dog acquisition and sourcing of information about canine health. A further sample of dog owners provided possible internet search terms they would use to find information on how to care for a new dog. These terms were used for a scoping review to examine the quality of available information. The VS and PDB exhibited varied perceptions relating to pedigree dog breeding, inherited disease and health screening programmes. Each group considered themselves the most knowledgeable on these topics and lacked clarity on how their roles interrelated. Focus groups showed conflicting views on advising dog owners and buyers. Dog owners varied in their drivers for ownership, from whom they obtained information and the internet search terms used when looking for information online. The scoping review identified a lot of easy to understand, poor quality information. VS and PDB need to better understand and appreciate each others’ roles, perspectives and knowledge to instigate effective collaboration and improve pedigree dog health. Owners are diverse and require a multifaceted educational approach to improve knowledge. The quality of online information for owners needs to be made clear and a coordinated approach involving all stakeholders is required to enable dissemination of practical, evidence-based information on pedigree dog health.

Little has previously been described about how or why owners, veterinary nurses or veterinary surgeons make decisions about pets under their care. The Animal Welfare Act (2006) and the Royal College of Veterinary Surgeons’ oath should ensure the health and welfare of pets in the United Kingdom (UK) is the central focus of those decisions. The aim of this thesis was to characterise the nature and basis of decisions made about the treatment and welfare of osteoarthritic dogs by owners, veterinary surgeons and veterinary nurses. Three studies were performed. Firstly, two rapid reviews identified and appraised the outcome measures used in the peer-reviewed literature to assess canine quality of life and canine osteoarthritis. Secondly, thirty-two interviews were performed with owners of dogs with osteoarthritis and five focus groups were performed with veterinary surgeons and veterinary nurses who manage osteoarthritic dogs. Thematic analysis performed on transcripts of those interviews and focus groups identified key themes. Thirdly, a prospective study was performed to test a novel home monitoring outcome measure for use by owners of osteoarthritic dogs, developed using data gathered in the previous studies. The first study found outcomes assessed in the peer-reviewed literature focus predominantly on physical health. Those assessments are frequently unvalidated, may be subject to bias and neglect other aspects of the dogs’ welfare impacted by osteoarthritis and its management. The second study identified four important themes in the interviews and focus group data in relation to decision making. Most owners were highly motivated to make good decisions about their dogs’ welfare. However, many barriers to dog-focused decisions were recognised including: incorrect prior knowledge; ineffective veterinary consultations, in part due to different language used by owners and veterinary surgeons; the lack of available, relevant evidence on which to base decisions; an inability to reliably interpret canine behaviour; and risk aversion. A wide range of impacts of canine osteoarthritis on the welfare of the dogs, their owners and the veterinary professionals caring for those dogs were described. The third study identified several significant deficits in existing outcome measures designed for owners to assess their osteoarthritic dogs. Dogs with osteoarthritis may have day-to-day variations in their physical health and demeanour and owners appear to assess a complex mix of inputs to, and indicators of, their dogs’ welfare when decision making. More work is needed to develop outcome measures that are relevant to owners and more accurately reflect all aspects of canine welfare. This thesis is the first in-depth body of work using evidence synthesis and qualitative methods to characterise how decisions are made about osteoarthritic dogs under veterinary care. Most decisions about osteoarthritic dogs are made by owners using unvalidated assessments with little veterinary guidance. Valid, relevant and practical outcome measures are needed to collect information on which decisions can be based. Evidence does not exist to guide the majority of decisions made; the evidence that does exist appears to be poorly disseminated, particularly amongst owners. Relevant evidence must be created through well designed clinical trials to support those decisions then widely disseminated. Veterinary consultations are not always effective in making decisions focused on the best interests of osteoarthritic dogs, particularly in relation to their welfare; differences in language and perspectives may play a significant part in this. Future work in this field should involve collaboration between owners, veterinary professionals in general and specialist practice, animal welfare scientists and experts in dog behaviour. Methodological approaches taken and conclusions drawn from this thesis may be relevant to many other veterinary diseases.

The cost of drug development is high with many drugs failing during toxicity testing. This is a particular problem in veterinary medicine where the pharmaceutical market size is so small that it may not be economically viable to develop drugs. The liver and specifically hepatocytes have a crucial role in drug metabolism via oxidation by cytochrome enzymes (CYP), conjugation and excretion into the biliary system. This drug metabolism is unpredictable between species as each has unique CYP profiles. Furthermore there is breed variation of CYP profiles within the canine species. The ability to produce an in vitro source of canine hepatocytes to model drug metabolism in this species and in different breeds would greatly reduce the expense of candidate drug testing. If an unlimited supply could be produced in vitro this would reduce the number of animals required in pre-clinical testing. The aim of this thesis was to produce an in vitro supply of canine hepatocyte-like cells from stem cell sources, namely hepatic progenitor cells (HPC), mesenchymal stem cells (MSC) or induced pluripotent stem cells (iPSC). Cultures of canine primary hepatocytes were produced to use as a gold standard, but also to develop and refine tests of hepatocyte characterisation and function. A panel of primers was developed for use in real time polymerase chain reaction (PCR) as well as optimising tests for low density lipoprotein (LDL) and indocyanine green uptake, albumin production, periodic acid- Schiff staining for glycogen and CYP activity using a luciferase-based system. As primary hepatocytes rapidly lost their defining characteristics and function in vitro, methods of maintaining function using CYP inducers and culture substrates were assessed. Isodensity centrifugation and magnetic-activated cell sorting was employed to isolate HPCs. Selection of cells from the non-parenchymal cell fraction with stem cell marker Prominin 1 demonstrated that these were keratin 7 positive, a HPC marker. Cells morphologically consistent with HPC appeared and expanded in culture after 2 weeks. On passaging, these cells failed to continue expanding, despite plating onto collagen, laminin, SNL feeder cells or using Kubota’s medium (known to allow rapid expansion of rodent and human HPCs). Canine adipose (Ad-MSC) and bone marrow-derived mesenchymal stromal cells (BM-MSC) were isolated post mortem. These were characterised as CD45, 105 and STRO-1 positive, CD11b, 19 and 45 negative cells which could be differentiated into adipocytes, chondrocytes and osteocytes based on staining characteristics and relative gene expression. Protocols published for other species were used to differentiate both Ad-MSC and BM-MSC towards a hepatocyte phenotype. Although a dramatic change in morphology and a reduction in vimentin gene expression were noted, suggesting a loss of mesenchymal phenotype, these protocols did not induce a hepatocyte phenotype. Pre-treatment with 5-Aza-2′-deoxycytidine to cause DNA demethylation and valproic acid to inhibit histone deacetylation also failed to allow transdifferentiation. A polycistronic vector containing Oct-4, c-Myc, Sox2 and Klf4 was successfully transfected into canine epidermal keratinocyte progenitor cells which became alkaline phosphatase positive and assumed a morphology consistent with iPSC. After colony selection and expansion, PCR evidence of plasmid presence was lost, colony morphology changed, and alkaline phosphatase activity reduced, consistent with vector expression factor and pluripotency loss. Canine iPSCs produced by lentiviral method were then differentiated towards hepatocyte phenotype using a published protocol for mouse and human iPSC. These cells were then assessed for hepatocyte characteristics using the developed reagents and primers. These cells demonstrated increased gene expression and morphology consistent with differentiation towards a hepatocyte-like phenotype. This thesis demonstrates successful culture of canine primary hepatocytes and validation of tests of hepatocyte phenotype. This provides a basis for optimising primary hepatocyte function in vitro and assessment of the success of differentiation protocols on stem cell sources. Canine mesenchymal stromal cells do not appear to transdifferentiate towards a hepatocyte-like phenotype using published protocols for other species. Canine iPSC are a promising candidate for an in-vitro source of hepatocyte-like cells.

There are increasing concerns that some pedigree breeding practices compromise canine welfare; with many breeds affected by inherited disorders likely due to adherence to breed standards that encourage exaggerated conformations. Consequently, calls for quantitative limits to be included in breed standards have been advocated to safeguard against conformations that are detrimental to canine welfare. This thesis explores the relationships between extreme conformation and disease-risk, to identify the most at-risk conformations currently being selected for, and recommend healthy 'limits'. Three conformation-related disorders of high welfare importance were investigated; brachycephalic obstructive airway syndrome (BOAS), intervertebral disc extrusion (IVDE) and corneal ulceration. Seven hundred dogs entering the RVe's small animal referral hospital were recruited over 14-months; their conformations quantified using established protocols and disease-status assessed. Generalised linear mixed-model analyses demonstrated for the first time that: (1) relatively shorter muzzles and relatively thicker necks increased the risk of BOAS; (2) relatively longer backs, miniaturisation and being overweight increased the risk of IVDE; and (3) the presence of nasal folds, relatively short muzzles, relatively larger eyelid openings and the presence of exposed sclera increased the risk of corneal ulcers. To test the risk estimates generated from this population, a second study of BOAS was carried out in a non-referral population. Short muzzles and thick necks were confirmed to be risk factors for BOAS, with two further lifestyle risk factors identified: obesity and neutering. A questionnaire-based study discovered an owner-perception of BOAS being 'normal for the breed' that may hamper efforts to reduce its prevalence in the future. As such, educational interventions to change attitudes may be required. The results of these studies are hoped to instigate and inform future breed standard revisions, and influence those within the breeding community to change current breeding practices to improve canine welfare by selecting for more moderate, lower-risk morphologies.

The colony-stimulating factor-1 receptor (CSF-1R) is expressed by the mononuclear phagocytic lineage, and is important for the development of these cells from their progenitors and also for promoting their survival and activation after maturation. The receptor has two ligands, CSF-1 and IL-34, which induce the formation of a stable dimer between two receptor monomers. This leads to intracellular autophosphorylation of tyrosine residues and subsequent signalling cascades, leading to rapid protein expression, cytoskeleton remodelling and cellular motility. Although CSF-1R signalling is crucial for normal embryogenic development and other physiological functions mediated by the phagocytic lineage, it has also been found to promote the pathogenic progression of cancer. Tumour-associated macrophages (TAMs) can comprise a large proportion of the cellular population in several solid tumours. These cells promote several hallmarks of cancer malignancy, such as increased neovascularization, tissue invasion, induction of metastases and immunosuppression. In this work, it was confirmed that CSF-1 had a prominent role in inducing cancer-promoting cellular phenotypes. Both canine cancer cells and macrophages respond to this cytokine, respectively increasing cancer cell proliferation and reducing inflammatory activation. Given the importance of CSF-1R signalling in the tumour microenvironment, antibodies were generated with the objective of blocking receptor function. Mice were immunized with either the extracellular region or the dimerization domain of the CSF-1R. Hybridomas were produced using the primed splenocytes, and monoclonal antibody (mAb) candidates were selected based on their performance in immunostaining and on their capacity to inhibit CSF-1R+ cells. The best antibodies were subjected to speciation. Chimeric antibodies maintained the ability of the parental mAbs to inhibit macrophage proliferation following CSF-1R stimulation. However, the mAbs possessed moderate affinity and specificity for their target, failing to stain monocytes and presenting a degree of cross-reactivity. The binding properties of one of such mAbs were altered by PCR-induced mutations, generating semi-synthetic antibody libraries. These were screened by phage display, yielding novel clones that show reduced cross-reactivity with unrelated proteins and retain the property of inhibiting macrophage survival. These results are a step in the development of therapeutic monoclonal antibodies for cancer treatment in dogs.