A quantitative competitive binding "triple sandwich" enzyme immunoassay was used to evaluate pathogen/class-specific antibody responses in Holstein-Friesian calves vaccinated against Clostridium perfringens B-toxin at various ages postpartum. Vaccination of dams at six weeks and again at two weeks prepartum increased pathogen-specific antibody levels in their colostrum and respective calf's serum. Calves initially vaccinated at three days produced both a primary and secondary pathogen-specific antibody response, whereas calves initially vaccinated at 12 and 21 days produced only secondary responses. Maternally-derived antibodies were found to suppress neonatal antibody production following primary immunization. They were also found to influence secondary humoral immune responses, although in a diminished capacity. Pathogen-specific IgG and IgM concentrations in dams' sera and colostra were found related to subsequent pathogen-specific IgG and IgM neonatal serum concentrations. Only pathogen-specific IgA in dams' colostra was correlated to neonatal levels, possibly owing to a different origin and role of this immunoglobulin class. All class-specific colostral immunoglobulin levels were related to subsequent neonatal concentrations. Based on results from this experiment, it is recommended that calves be vaccinated at three days postpartum with a booster administered at 63 days.

Orientador: Gilson Hélio Toniollo / Banca: Fabiana Ferreira de Souza / Banca: Antonella Cristina Bliska Jacinto / Banca: César Roberto Esper / Banca: Francisco Guilherme Leite / Resumo: No final da década de 1990 observou-se um aumento significativo de uma nova modalidade de afecção, o prolapso vaginal parcial ou total recorrente não associado à gestação, principalmente em vacas zebuínas. Em razão do alto valor comercial dos animais acometidos, inicialmente, indicou-se as técnicas convencionais como as de Caslick, Bühner ou Flessa, porém sem o sucesso esperado devido à recorrência da alteração. Considerando esta dificuldade, este trabalho objetivou descrever duas novas técnicas cirúrgicas na correção do prolapso vaginal, denominadas vaginectomia parcial e vaginopexia dorsal em vacas. A condução do estudo foi a campo, utilizando-se 812 vacas zebuínas em idade reprodutiva, alojadas em diversas propriedades de diferentes estados brasileiros. Foram selecionados animais que apresentavam a afecção e que mantiveram os parâmetros clínicos de frequência cardíaca, frequência respiratória e temperatura retal, dentro da normalidade. O diagnóstico do prolapso vaginal foi realizado por meio de anamnese e dos sinais clínicos como exposição da vagina pela rima vulvar, tenesmo, inquietação, lesões da porção evertida, retenção urinária e vaginite. A avaliação dos animais permitiu definir o estágio do prolapso, com a finalidade de eleger a técnica cirúrgica mais adequada. Os protocolos anestésicos foram cumpridos, considerando-se a técnica cirúrgica eleita. A vaginectomia parcial foi utilizada para o tratamento do prolapso vaginal de grau 1 e a vaginopexia dorsal para os de grau 2 e 3. Os resultados pós-cirúrgicos das duas técnicas indicaram alta porcentagem de recuperação (93.44% para vaginectomia parcial e 96,14% para vaginopexia dorsal) e baixo número de recidivas (6,25% e 3,66%, para vaginectomia parcial e vaginopexia dorsal, respectivamente, e baixa mortalidade (entre 0,20 a 0,31%), podendo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: At the end of the decade of 1990, a significant increase of a new affection was observed in the field. This occurrence, recurrent partial or total vaginal prolapse is not associated with gestation, is seen primarily in zebu cows where, in extreme cases, resulted in a total prolapse of the vagina, which included exteriorization of the cervix. Given the high commercial value of elite animals presenting this condition, the increase in cases observed led to attempts to solve the condition with conventional techniques such as those of Caslick, Bühner and Flessa. However, reoccurrence of the prolapse was commonly observed soon after the use of these techniques. Considering these difficulties, the aim of the present work was to develop two new surgical techniques employed in the correction of vaginal prolapse, named partial vaginectomy and dorsal vaginopexy in cows. The research was conducted in the field, using 812 zebu cows of reproductive age, maintained in several properties in different Brazilian states. The animals selected had the vaginal alteration while maintaining all clinical parameters of heart rate, respiratory rate and body temperature, all within normal range. The diagnosis of a vaginal prolapse was performed by anamnesis and analysis of the clinical signs, such as visualization of the vagina through the vulva, tenesmus, agitation, lesions in the everted portin and vaginitis. Evaluation of the animals allowed for the definition of stages of the disease, aiding the choice of an adequate surgical technique. Anesthetic protocols were performed taking into consideration the selected surgery. Partial vaginectomy was employed for the treatment of vaginal prolapses of grade 1, whereas dorsal vaginopexy was used for the grade 2 and 3 prolapses. Post-surgical results for both techniques indicated a high percentage of recovery... (Complete abstract click electronic access below) / Doutor

Orientador: Karina Paes Bürger / Banca: Luis Antonio Mathias / Banca: Anna Monteiro Correia Lima / Resumo: Este trabalho teve como objetivo padronizar e avaliar a técnica "dot blotting" para o diagnóstico sorológico da brucelose bovina, comparar os resultados com os encontrados nos testes de antígeno acidificado tamponado (AAT), 2-mercaptoetanol (2-ME) e fixação de complemento (RFC), além de estimar a sensibilidade e a especificidade relativa do "dot blotting" em comparação com os testes de diagnóstico oficiais. Para tanto, utilizaram-se 50 amostras de soro sanguíneo bovino para padronização do teste, e foram avaliados dois antígenos: Brucella abortus B19 obtida a partir da vacina ANABORTINA® B19 - Merial, após processo de ruptura do microrganismo, e extrato polissacarídico obtido de amostra de Brucella abortus S99 pelo método água quente - fenol quente. Ao final da padronização foi estabelecida como ideal para a técnica a utilização de membrana de nitrocelulose no formato de círculo, o antígeno obtido da Brucella abortus após passar por um processo de ruptura do microrganismo, como suporte a placa de cultivo celular de 24 poços e fundo chato, a diluição do soro de 1:100 e do conjugado de 1:30.000, além de 3 lavagens por etapa. A avaliação e a comparação da sensibilidade e especificidade relativa aos testes de AAT, 2-ME e RFC foram feitas utilizando 1.315 amostras, sendo estes resultados comparados por meio do indicador Kappa. A comparação dos resultados do "dot blotting" com o AAT mostrou índice Kappa de 0,9081 (IC95%: 0,8542 - 0,9621), sensibilidade 88% (IC95%: 83,75% - 92,25%) e especificidade 99,45% (IC95%: 98,8% - 99,75%); com o 2-ME, índice Kappa de 0,9939 (IC95%: 0,939 - 1,0484), sensibilidade 99,48% (IC95%: 97,11% - 99,91%) e especificidade 99,91% (IC95%: 99,49% - 99,989%); com a RFC, índice Kappa de 0,8226 (IC95%: 0,7690 - 0,8761), sensibilidade 100% (IC95%: 97,42% - 100,0%) e especificidade 95,40% (IC95%: 94,03% - 96,47%). Usando a combinação dos resultados do 2-ME e da RFC para ... / Abstract: This study aimed to standardize and evaluate the dot blotting technique for the serologic diagnosis of bovine brucellosis, compare the results with those found in the Bengal test (AAT), 2-mercaptoethanol (2-ME) and complement fixation (CF) and also estimate the relative sensitivity and specificity of dot blotting in comparison with the official diagnostic tests. In order to do this, 50 samples of bovine blood serum were used for test standardization, and two antigens were evaluated: Brucella abortus B19 bacteria obtained from ANABORTINA® B19 vaccine - Merial, after the rupture process of the microorganism, and a polysaccharide extract obtained from a sample of Brucella abortus S99 by the hot water method - hot phenol. At the end of standardization, the use of nitrocellulose membrane in a circle format was established as ideal for the technique, the antigen obtained from B. abortus bacteria after going through the rupture process of the microorganism, as a support a cell culture plate with 24 flat bottom wells was used, a dilution serum of 1:100 and the conjugate of 1:30,000, and washing 3 times per step. The evaluation and comparison of relative sensitivity and specificity with the AAT, 2-ME and CF tests was done using 1,315 samples, and these results were compared using the Kappa indicator. The comparison between the results of dot blotting and the AAT showed a Kappa index of 0.9081 (IC95%: 0.8542 - 0.9621), sensitivity of 88% (IC95%: 83.75% - 92.25%) and specificity of 99.45% (IC95%: 98.8% - 99.75%); 2-ME test, Kappa index of 0.9939 (IC95%: 0.939 - 1.0484), sensitivity of 99.48% (IC95%: 97.11% - 99.91%) and specificity of 99.91% (IC95%: 99.49% - 99.989%); complement fixation test, Kappa index of 0.8226 (IC95%: 0.7690 - 0.8761), sensitivity of 100% (IC95%: 97.42% - 100%) and specificity of 95.40% (IC95%: 94.03% - 96.47%). Using the combination of the results of 2-ME and RFC to establish the true condition of the animal, the ... / Mestre

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Mannheimia haemolytica is an opportunistic pathogen in cattle and the main bacterial agent in bovine respiratory disease. Despite its economic importance, few studies have characterized the genetic diversity of M. haemolytica, particularly from feedlots. Three genotyping techniques (BOX-PCR, (GTG)5-PCR and PFGE) were compared to discriminate M. haemolytica and strains from the family Pasteurellaceae. PFGE was the most discriminating and repeatable, although BOX-PCR was most accurate in clustering isolates together according to species. Mannheimia haemolytica was isolated from nasal swab samples collected from cattle upon entry and exit from two feedlots in southern Alberta. These were characterized by PFGE and antimicrobial susceptibility using a disk-diffusion assay. Select gene determinants were screened for using PCR. PFGE analysis revealed the isolates to be highly diverse. Ten percent of the isolates exhibited resistance. At present, the development and spread of antimicrobial resistance in M. haemolytica observed within the feedlots examined appears to be low. / xi, 116 leaves : ill. ; 29 cm

Glucocorticoids are commonly used to treat cows with clinical ketosis and fatty liver disease. This study investigated the effects of 10 mg/mL of FlucortRTM and PredefRTM 2X on the day of calving on blood metabolites, immune function and milk composition on 30 transitional Holstein cows. Sample of blood and milk were analyzed for energy metabolites (glucose, NEFA, BHB and insulin), mineral metabolites (Ca, P, Na, K, Cl and Mg), energy function parameters (antibody, lymphocyte), milk compositional parameters (protein, fat, lactose, SCC). There were no differences in glucose, Na, Cl, Mg, antibody, lymphocyte and milk fat, were observed among treatments. FlucortRTM treated cows had significantly lower NEFA on D1, higher BHB on D21 and D28, lower insulin on D14, higher Ca on D1 and lower P on D1. PredefRTM 2X treated cows had significantly higher BHB on D21, higher insulin on D7, lower Ca on D1, higher SCC on D1 and higher milk protein on D1. With respect to the significant data in this study, the use of glucocorticoids FlucortRTM and Predef RTM 2X in a single intramuscular injection on d1 for the treatment of ketosis is not warranted.

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.

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