Dissertations / Theses on the topic 'Dairy cattle Dairy cattle Motivation in animals'

Thesis (M.S.) University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 21, 2007) Vita. Includes bibliographical references.

Experiments were conducted to investigate the effects of formulating rations using different ingredients and of manipulating the cation-anion balance of the rations on production, acid-base status, metabolism of macrominerals and renal function of dairy cows. The effects of dietary cation-anion balance were studied during early, mid and late lactation as well as during the dry period. Feeding an alfalfa-haylage based diet vs a corn silage based diet, both having the same cation-anion balance did not affect the voluntary consumption, milk yield or milk composition of cows in early lactation. The acid-base status of the animals was not affected by dietary treatment. The lower urinary bicarbonate concentration observed with the alfalfa haylage-based diet was not associated with a lower total urinary bicarbonate excretion since urine volume was significantly higher than when cows were fed the corn silage-based diet. Manipulating dietary cation-anion balance, however, resulted in changes in acid-base status at all stages of lactation studied. Urinary bicarbonate concentration and total daily excretion were increased by a higher dietary cation-anion balance at all stages of lactation. Similar effects of dietary cation-anion balance on urinary bicarbonate did not, however, translate into similar changes in production or intake by cows at differing stages of lactation. Cows in early and mid lactation seemed to have benefited more from a highly positive dietary cation-anion balance than cows in late lactation or dry period.

Brucellosis is a disease of socio-economic and zoonotic importance worldwide. In animals it is associated with the ingestion of feed that is contaminated with cyetic material from aborting herd-mates, while in humans it is associated with the consumption of unpasteurised milk and dairy products from infected animals. It may also be acquired from contact with infected material of animal origin by farmers, veterinarians, and abattoir and laboratory workers. Brucellosis was first reported in South Africa in the late nineteenth century. It is still present in the country today, with reported annual losses of at least R 300 million, and a national annual incidence of 5 000 cases in humans. The global incidence of human brucellosis is about half a million infections annually. As the incidence of human brucellosis is directly associated with prevalence in animals, control of animal brucellosis is emphasised. Veterinary control is compromised by the chronic nature and the variable incubation period of the disease, with an estimated up to 15% of cattle in infected herds aborting before sero-conversion. Latency, which involves about 5% of calves born from infected dams, is also problematic as these infected animals often test seronegative, only to seroconvert in the peri-parturient period, thus allowing opportunity for disease spread within and between herds before diagnosis is made. In addition, the currently used serological tests are at times unable to distinguish brucellosis from cross-reacting antibodies from other infections or brucellosis vaccines. x It was the objective of this study to investigate, under South African conditions, the value of the brucellin skin test (BST) in improving the sensitivity and specificity of the currently used serological tests. It has proved a valuable additional test in diagnosing early and latent infections as well as in differentiating brucellosis from cross-reacting organisms in unvaccinated cattle in Europe. The study also evaluated the benefit of replacing some of the currently used serological assays with the fluorescence polarisation assay (FPA). The FPA, a rapid and homogenous serological test with only a few operational steps, has been validated and is in current use in Canada. The study was carried out in Mpumalanga Province, on herds selected to reflect prevailing South African farming conditions. These herds were divided into certified Brucella abortus-negative herds (608 head) for the estimation of BST specificity, and confirmed B. abortus-infected herds (845 head) for the estimation of BST sensitivity. The results obtained indicated the BST had a specificity of 99.18%, and a relative sensitivity of 42.86%. However, 65.38% of BST-positive animals were negative on serology. When the high specificity is considered, together with the experiences of other researchers who found that the skin test became positive earlier than serological tests, these animals may be assumed infected. It is concluded that the BST is a valuable addition to the panel of diagnostic tests currently used to identify infected herds and individuals in South Africa. The FPA, with a relative sensitivity of 93.65% and a specificity of 98.85%, can potentially be of use as a screening test under South African conditions.
Dissertation (MSc)--University of Pretoria, 2012.
gm2014
Veterinary Tropical Diseases
unrestricted

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.
Mastitis is considered to be the most costly disease affecting the dairy industry. Management strategies involve the extensive use of antibiotics to treat and prevent this disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select for strains with resistance to antibiotics. In addition, a strong drive towards reducing antibiotic residues in animal food products has lead to research in finding alternative antimicrobial agents. Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, produces the bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositive bacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis as well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, according to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KM remained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. The peptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin and trypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode of action does not depend on glycosylation. Precipitation with 60 % saturated ammonium sulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM. Amplification of the genome of strain ST91KM with primers designed from the sequence of the macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220 bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicus ACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragment amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC 198, revealed 99 % homology to the mcdA of S. macedonicus ACA-DC 198 (accession number DQ835394). Macedocin ST91KM may thus be a related bacteriocin described for S. macedonicus. The peptide adsorbed equally well (66 %) to L. sakei LMG13558 and insensitive cells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinus ATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °C and at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that the receptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI and Na2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possibly due to competitive ion adsorption on the bacterial cell surface. The peptide has a bacteriocidal mode of action, resulting in lysis and the release of DNA and β-galactosidase. Atomic force microscopy of sensitive cells treated with macedocin ST91KM have shown deformation of the cell structure and developing of irregular surface areas. Antimicrobial susceptibility patterns were evaluated against eighteen mastitis pathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimum inhibitory concentrations (MICs) falling in the intermediate and susceptible range against erythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocin ST91KM. A teat seal preparation containing macedocin ST91KM effectively released bacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could form the basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as it is effective against pathogens that display resistance to conventional antibiotic therapy.

A cross-sectional study was conducted from February 2011 to August 2012 to investigate the status and identify risk factors for Bovine Tuberculosis (BTB) and cattle Brucellosis. A total of 1277 cattle from 6 selected dairy farms were tested. The farms were purposively selected on suspected risk. Samples were analysed using Rose Bengal Test (RBPT) and Complement Fixation Test (CFT) for Brucellosis and Comparative Intradermal Tuberculin Test (CITT) for BTB. The results revealed the overall prevalence of 1.6% for Brucellosis and no BTB was found. Logistic regression analysis revealed that age of cattle, herd size and number of calves a cow has had significant association with Brucellosis with p-values 0.0071, 0.0490 and 0.000 respectively. The role of location (p>0.05), gender (p>0.05) and breed (p>0.05) in the occurrence of the disease was not statistically significant at α=0.05, although higher rates were obtained in females and Jersey breed. Questionnaires were also administered to 47 individuals a total of 12 females and 35 males. Most respondents were not aware about cattle brucellosis and cattle tuberculosis. Consumption of raw or unpasteurized milk still occurs with 74.75% of respondents revealed.

As práticas de manejo das pastagens tropicais recomendadas para utilização intensiva da planta permitem ao animal colher material de boa qualidade. O objetivo do trabalho foi avaliar se a proteína bruta (PB) proveniente do capim seria suficiente para atender as exigências de vacas leiteiras no terço médio da lactação, tornando desnecessária a utilização de suplementos protéicos no concentrado. Buscou-se também a caracterização da PB do capim e a adequação protéica da dieta, visando otimizar a eficiência de utilização do nitrogênio. No primeiro estudo, 33 vacas holandesas e mestiças (HPBxJersey), no terço médio da lactação, produzindo em média 20 L dia-1, foram agrupadas em 11 blocos, de acordo com composição racial, dias em lactação e produção de leite e receberam suplemento concentrado com três níveis de PB durante 76 dias. Os concentrados continham milho moído fino, minerais, vitaminas e os três níveis de proteína (T1: 8,7%; T2: 13,4%; T3: 18,1% MS) foram obtidos substituindo-se parte do milho por farelo de soja. O capim elefante foi adubado com 50 kg N ha-1 por pastejo e apresentou teor médio de PB de 18,5%. As produções de leite e de leite corrigido para 3,5% de gordura, os teores e produções de gordura, proteína e caseína no leite, a síntese de proteína microbiana e o consumo de matéria seca (CMS) não foram influenciados (P>0,05) pelos teores de PB dos concentrados. A concentração de nitrogênio uréico no leite aumentou linearmente (P<0,01) com o aumento dos teores de PB dos concentrados. A excreção de N pela urina foi maior (P<0,05) para os concentrados contendo farelo de soja (T2 e T3) em relação ao concentrado contendo apenas milho (T1). No segundo estudo, 4 vacas holandesas secas, fistuladas no rúmen, foram distribuídas em um quadrado latino 4x4 e submetidas aos mesmos tratamentos do primeiro estudo, acrescidos de um quarto concentrado (T4) com 13,4% de PB, tendo uréia como fonte de nitrogênio. O CMS foi maior para o T4 (P<0,05), sugerindo deficiência de PDR nos outros tratamentos. Entretanto, essa hipótese não foi confirmada pelos parâmetros ruminais (pH, e ácidos graxos de cadeia curta) e síntese microbiana, que não sofreram efeito dos teores e fontes de PB no concentrado. A concentração de N amoniacal no rúmen foi menor (P<0.05) para T1 do que para os demais tratamentos. A excreção total de N no ambiente acompanhou numericamente o teor de PB dos concentrados, mas as diferenças não foram detectadas pela análise estatística. O terceiro estudo utilizou os mesmos animais e tratamentos do segundo para avaliar a cinética de degradação ruminal da MS, PB e FDN do capim por meio de ensaio in situ. Os concentrados protéicos (T2, T3 e T4) não melhoraram a degradação ruminal do capim, em relação ao concentrado energético (T1), confirmando que a proteína do pasto foi suficiente para maximizar a fermentação ruminal. O fornecimento de farelo de soja no concentrado de vacas em terço médio de lactação, mantidas em pastagens tropicais manejadas intensivamente, não resultou em nenhum benefício produtivo e aumentou a excreção de N no ambiente.
Grazing management techniques are improving the quality of the forage consumed by grazing dairy cows. The objective of this study was to determine if forage crude protein (CP) would be sufficient to achieve the nutritional requirements of midlactating dairy cows, making unnecessary the use of protein supplements on the concentrate. It also aimed to characterize the forage CP fractions and to balance the diet protein content to optimize nitrogen use efficiency. In the first trial, 33 Holstein and crossbred (Holstein X Jersey) mid-lactating dairy cows, producing 20 L d-1, were grouped in 11 blocks, according to breed, days in milk and milk yield and were fed three levels of CP on the concentrate supplement for 76 days. Concentrates contained fine ground corn, minerals, vitamins and three CP levels (T1:8.7, T2:13.4 and T3:18.1% DM) that were achieved by replacing corn with soybean meal. Pasture was fertilized with 50 kg N ha-1 after each grazing cycle and averaged 18.5% CP. There was no difference (P>0.05) in milk yield, fat corrected milk, fat, protein and casein content and yield, microbial synthesis and dry matter (DM) intake among treatments. Milk urea nitrogen increased linearly (P<0.01) as the concentrate CP content increased. The N urine excretion was greater (P<0.05) with the soybean meal concentrates (T2 and T3) than with the corn concentrate (T1). In the second trial, 4 rumen cannulated Holstein dry cows were used in a 4x4 Latin square and assigned to the same treatments used in the first study plus a fourth concentrate (T4) with 13,4% CP, containing urea instead of soybean meal as the N source. Dry matter intake was greater for T4 (P<0.05), suggesting a PDR deficiency in the other treatments. However, this hypothesis was not confirmed by ruminal parameters (pH and short chain fatty acids concentration) neither by microbial synthesis, that were not affected by the levels or sources of CP on the concentrate. Ruminal ammonia N content numerically increased as the concentrate CP content increased, but no differences were detected by the statistic analysis. The third study used the same animals and treatments of the second study to evaluate the ruminal kinetics of DM, CP and NDF of Elephant grass by an in situ assay. Protein concentrates (T2, T3 and T4) didnt improve forage ruminal degradation in relation to the energy concentrate (T1), confirming that the forage protein content was sufficient to optimize ruminal fermentation. The inclusion of soybean meal in the concentrate of midlactating dairy cows grazing intensively managed tropical grasses didnt result in productive advantage and increased the N excretion to the environment.

O presente relatório descreve as atividades desenvolvidas durante o estagio curricular na ANAVET Serviços Veterinários e na Segalab S.A., com uma duração de 6 meses. Numa primeira parte será abordada a casuistica acompanhada durante o estagio, e na segunda parte apresenta-se a revisão bibliográfica referente a diarreia viral bovina (BVD), seguida da apresentação do estudo realizado no âmbito da referida afeçao. A BVD e uma doença bovina com expressao a nivel mundial. Os animais persistentemente infetados (PI) constituem a principal fonte de disseminação e manutenção do virus e não existem limitações a sua comercialização ou troca entre explorações. Temos como objetivo mostrar a dinâmica de 85 animais PI, com uma prevalência de 0,9%, desde o nascimento e deteção até a morte, abate ou venda. Estes permaneceram nas explorações de origem em media 7,3 meses, tendo 48,2% sido vendidos a outras explorações, comprometendo esforços para o controlo e erradicação da doença. Abstract: Dairy cattle medicine and surgery The following report describes the activities developed during the internship at ANAVET Serviços Veterinários and Segalab S.A., with duration of 6 months. In the first part will be described the assisted case load during the internship, while the second part consists on the literature review on bovine viral diarrhea (BVD), followed by a study on the disease. BVD is a worldwide spread cattle disease. Persistently infected (PI) animals are the main source of virus spread and maintenance, and there are no restrictions for their trade or exchange between herds. Our study evaluated the movement dynamics of 85 PI animals, with a prevalence of 0,9%, since their birth and detection until they were sold, slaughtered or died from natural causes. They were kept in the herd in average during 7,3 months and 48,2% of them were sold to other herds putting at risk the efforts to control and eradicate the disease.

Dissertação de Mestrado Integrado em Medicina Veterinária
As mastites são tidas como a doença mais frequente e com maior impacto económico na produção leiteira. Porém, os produtores nem sempre têm a percepção da dimensão do seu impacto. A circunstância em que a doença se apresenta nas explorações é bastante díspar, evidenciando-se a importância de realizar uma análise económica para cada realidade produtiva. Através da aplicação de um modelo económico, pretendeu-se aferir o custo das mastites em três bacias leiteiras (S. Miguel, Entre-Douro-e-Minho e Centro do país), bem como caracterizar as práticas de maneio em cada região e procurar uma relação entre ambos. Os resultados alcançados demonstraram que o custo de um caso clinico foi de, em média, 118 €, com diferentes valores entre regiões (p<0,05). O impacto económico anual foi de, em média, 147 €/vaca, todavia com grande amplitude de resultados entre explorações. O maneio praticado revela padrões distintos por região, contudo não se comprovou a existência de uma relação entre este e o custo da doença. A análise do maneio das explorações e das consequências económicas da doença, bem como a motivação do produtor para a melhoria contínua da saúde do úbere, são factores cruciais na redução da incidência das mastites e do seu impacto económico.
ABSTRACT - Milk quality characterization and economic impact of mastitis in 3 areas of Portugal – S. Miguel island, Entre-Douro-e-Minho e central area of continental Portugal - Mastitis is considered as the most frequent and costly disease affecting dairy farming. Farmers are not always aware of the full dimension of its impact. The circumstances in which the disease manifests itself on different farms may be quite different, leading to the need for an economic analysis to be performed for each production setup. Through the use of an economic model, we aimed at assessing mastitis costs in three dairy areas (S. Miguel, Entre-Douro-e-Minho and e central area of continental Portugal), as well as to characterize management practices in each region, trying subsequently to search for possible relations between them. The results obtained showed that the cost of a clinical case of mastitis was 118€ on average, with different costs per region (p<0.05). The annual economic impact was 147€/cow/year on average, with a large distribution of results between farms. The management practices showed distinct patterns for each region, with no relationship between management and mastitis cost being shown. Farm management procedures and economic consequences of mastitis, as well as farmer motivation for the continual improvement of udder health, are crucial in the reduction of mastitis incidence and its economic impact.

O objetivo deste estudo foi caracterizar o perfil de fermentação anaeróbia de colostro e determinar seu valor nutritivo, assim como avaliar o desempenho de bezerros leiteiros alimentados com silagem de colostro como dieta líquida. No primeiro experimento, colostro bovino de segunda e terceira ordenhas foi fermentado em garrafas plásticas tipo PET que foram cheias e ligeiramente pressionadas antes do seu fechamento, criando assim uma condição anaeróbia. As garrafas foram armazenadas em sala escura à temperatura ambiente e cinco garrafas foram abertas 0,1, 2, 3, 4, 5, 6, 7, 14, 21, 28 e 56 dias após, para determinação de pH, acidez titulável, temperatura, ácido lático, nitrogênio total, nitrogênio não-protéico (NNP), caseína, lactose, glicose, gordura e contagem de microorganismos. Os valores de pH, acidez titulável e concentração de ácido lático apresentaram variação durante o armazenamento (P<0,0001). A fração NNP apresentou aumento (P<0,0001), enquanto que os valores de caseína e lactose diminuíram (P<0,0001) e apresentaram baixos valores após 56 dias. O desenvolvimento de bactérias ácido-láticas (BAL) foi intenso durante o processo fermentativo, enquanto que enterobactérias e leveduras apresentaram declínio. No segundo experimento, colostro bovino de segunda e terceira ordenhas foi fermentado da mesma forma que no primeiro experimento, mas em diferentes condições de temperaturas ambientais. As garrafas foram armazenadas em incubadora BOD com temperatura controlada (32,5±1°C ou 22,5±1°C) ou em sala escura à temperatura ambiente, sendo abertas três garrafas após 0, 1, 7, 14, 21, 28 e 35 dias para avaliação dos mesmos parâmetros avaliados no primeiro estudo. A maioria dos parâmetros apresentou comportamento semelhante ao observado no primeiro experimento. O desenvolvimento de BAL foi intenso, principalmente quando o colostro foi armazenado em temperatura mais elevada. A temperatura também afetou os parâmetros nutricionais (P<0,0001) com acréscimos nos valores de NNP e redução da concentração de caseína e lactose. No terceiro experimento, dezoito bezerros Holandês foram alocados em abrigos individuais até a oitava semana de vida, com livre acesso à água e concentrado inicial, e passaram a receber 4L da dieta líquida, sucedâneo lácteo ou colostro fermentado sob condições anaeróbicas (silagem de colostro), diluído na razão de 1:1. Os animais alimentados com silagem de colostro apresentaram menor consumo de concentrado, ganho de peso diário e peso vivo (P<0,07) durante o período experimental, em comparação aos animais consumindo sucedâneo lácteo. As avaliações quanto à altura na cernelha, perímetro torácico e largura da garupa não apresentaram diferenças (P>0,07). Todos os parâmetros sanguíneos avaliados (glicose, N-uréico, ácidos graxos livres e -hidroxibutirato) foram afetados pelos tratamentos (P<0,07), exceto a concentração plasmática de proteínas totais (P>0,07). O escore fecal foi afetado pelos tratamentos durante a segunda semana de vida (P<0,07), com animais alimentados com silagem de colostro apresentando fezes anormais e muito secas. A dinâmica fermentativa observada mostra que o colostro pode ser conservado através de fermentação anaeróbia de forma eficiente. Entretanto, o fornecimento de silagem de 12 colostro como dieta líquida exclusiva durante o período de aleitamento não resulta em desempenho animal adequado, não sendo uma boa alternativa de substituto de leite.
The objective of this study was to characterize the fermentative dynamic of bovine colostrum, under anaerobic condition, and determine the nutritional value, as well as evaluate dairy calves´ performance receiving colostrum silage as liquid diet. In the first experiment, bovine colostrum from second and third milking was fermented at room temperature in PET plastic bottles that were filled and lightly pressed before its closure, thereby creating an anaerobic condition. The bottles were stored in the dark at room temperature and five bottles were opened on days 0,1, 2, 3, 4, 5, 6, 7, 14, 21, 28 and 56 after for determination of pH, titratable acidity, temperature, lactic acid, total nitrogen, non-protein nitrogen (NPN), casein, lactose, glucose, fat and counts of microorganisms. The pH, titratable acidity and concentration of lactic acid showed large variation during storage (P<0.0001). The NPN fraction increased (P<0.0001), whereas the values of casein and lactose decreased (P<0.0001) and showed very low values after 56d. The development of lactic-acid bacteria (LAB) was intense during the fermentation process, while enterobacteria and yeasts declined. In the second experiment, bovine colostrum from second and third milking was collected, mixed and stored in PET plastic bottles, in the same manner as it was for the first study. However, the bottles were stored in a dark room with temperature controlled by a BOD incubator (32.5±1°C or 22.5±1°C) or at room temperature. Three bottles were opened after 0, 1, 7, 14, 21, 28 and 35 days for evaluation of the same parameters done during the first study. Most parameters presented similar patterns as observed in the first trial. The development of LAB was intense, especially when colostrum was stored at higher temperature. Temperature also influenced the results of nutritional parameters (P<0.0001) with increases in NPN values and reduction of casein and lactose concentration. In the third experiment, eighteen Holstein calves were allocated to individual hutches until the eighth week of life, with free access to water and starter feed, and fed 4 liters of liquid diet: milk replacer or colostrum fermented under anaerobic condition (colostrum silage), diluted at a 1:1 ratio. Animals fed colostrum silage had lower intake of starter, average daily gain and body weight (P<0.07) during the experimental period, compared to animals consuming milk replacer. The values of withers height, heart girth and hip width showed no differences (P>0.07). All blood parameters measured (glucose, urea-N, free fatty acids and -hydroxybutyric acid) were affected by treatments (P<0.07), except the total protein plasma concentration (P>0.07). The fecal score was affected by treatments during the second week of life (P<0.07), with animals fed colostrum silage presenting abnormal and very dry feces. The dynamics of fermentation show that colostrum can be efficiently stored under anaerobic conditions. However, feeding colostrum silage as an exclusive liquid diet, during the whole milk-feeding period, results in poor animal performance, being considered as an inadequate alternative as milk replacer.

Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.