Thesis (M.S.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

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Made available in DSpace on 2018-08-01T22:56:47Z (GMT). No. of bitstreams: 1 tese_6708_TALITA PAOLI20140326-101818.pdf: 1646891 bytes, checksum: de14d9da58942614842c7040aa3e1f9d (MD5) Previous issue date: 2013-08-20 / A tuberculose bovina é uma doença bacteriana crônica, causada pelo Mycobacterium bovis, que atinge animais e humanos. Além de uma importante zoonose, também causa perdas econômicas na produção de carne e leite. Até o momento a prevalência e distribuição regional da tuberculose não estão bem caracterizadas para o território brasileiro e este conhecimento é fundamental para o direcionamento das ações de controle dessa doença. Pelo exposto, buscou-se para o Estado do Espírito Santo estimar a prevalência e determinar o risco dos bovinos nas diferentes regiões do Estado de desenvolverem tuberculose com base nos achados de lesões tuberculosas em bovinos abatidos em matadouros estaduais no período de janeiro de 2009 a dezembro de 2012. A análise estatística dos dados revelou prevalência de 0.23% para tuberculose bovina no Estado. Os bovinos da região D, que abrange os municípios do extremo sul capixaba, apresentam risco relativo de desenvolver tuberculose 11.36, 7.07 e 1.7 vezes superior às regiões A, B e C respectivamente. O município de Muqui apresentou a maior prevalência (1%) e risco relativo para tuberculose bovina até 19.6 vezes superior quando comparado aos demais municípios da região D. Estes resultados indicam a necessidade de elaboração e adoção de medidas que visem o controle da tuberculose no Estado, sobretudo na região de maior prevalência.

Ph.D. / Bovine babesiosis, or redwater, is at present known to be caused by Babesia bigemina and Babesia bovis in the Republic of South Africa. Until recently, however, the only information on the natural transmission of these parasites in the country was based on observations made during the early part of this century and information on the developmental cycle of the parasites in their vectors was superficial or nonexistent ...

Bovine tuberculosis (BTB) is a chronic infectious disease caused by the bacterium Mycobacterium bovis. It infects animals and can be a source of zoonosis. In the last five years, Canada has faced two outbreaks of bovine tuberculosis in the years 2018 and 2016. BTB is mainly diagnosed using the Tuberculin skin test, a test that detects the cellular immune response to administered purified protein derivative (PPD). A drawback of this test is the high level of false-positive test results caused by the immune response to PPD proteins that are conserved in non-tuberculous mycobacteria. Current serodiagnostic tests can detect the disease especially in the advanced state with, low sensitivity ranging between 9-45%. It is hypothesized that the profiling of the humoral immune responses to selected proteins will lead to the discovery of novel immunogenic protein antigens for improved BTB diagnostic tests. Bioinformatic tools used in this research for the prediction of extracellular or outermembrane proteins from M. bovis genome sequences identified 96 protein candidates. Also, a proteomic study conducted to identify proteins secreted by M. bovis, together with the review of previous proteomic studies of the PPD, led to the identification of an additional 92 protein candidates. A high throughput system was developed to efficiently express and analyze these antigens involving the PCR amplification, in vivo cloning, and in vitro expression of open reading frames (ORFs) coding for selected protein candidates. This was followed by a high throughput recombinant protein purification and a microarray analysis of these purified recombinant proteins with sera from M. bovis-infected animals. The system successfully amplified, cloned, and expressed 159 recombinant proteins. From the microarray screening, 13 antigens exhibited immunological reactions and were able to differentiate the sera of the infected animals from the controls. Out of those 13 antigens, 4 novel antigens Mb2740c, Mb0598c, Mb3469c, and Mb3453c, in addition to two well-known antigens MPB70 and MPB83, gave the highest reactivity; they were selected for further evaluation for diagnostic applications using ELISA and dot blot assays. Two antigens Mb3469c and Mb3453c had a significant ability to differentiate between infected and control cattle as tested in ELISA and dot blot assays, respectively and demonstrated by ROC curve analysis. These two novel antigens could be added to the panel of serodiagnostic antigens for improving BTB serodiagnosis and could be beneficial in the detection of outbreaks caused by certain M. bovis strains.

Memoria para optar al Título Profesional de Médico Veterinario / La tuberculosis bovina (TBB) es una enfermedad crónica y zoonótica, producida por Mycobacterium bovis. El costo de la enfermedad se relaciona a la disminución en la productividad de los animales severamente infectados, al control de movimiento animal, pruebas de diagnóstico para la detección y eliminación de animales positivos para evitar la propagación del agente, los decomisos y el menor valor de la leche. Se considera como una enfermedad ocupacional, ya que las personas que tienen mayor riesgo de contagiarse con M. bovis son quienes pasan largos periodos en contacto cercano con el ganado. La TBB en Chile se encuentra presente, y para apoyar el plan para su control y erradicación, se está estudiando el uso de la vacuna con cepa BCG de M. bovis en el ganado bovino. Fue desarrollada por Calmette y Guérin, quienes informaron la inducción de protección en el ganado contra la exposición a la bacteria. Como esta vacuna corresponde a una cepa atenuada de M. bovis, el objetivo de este trabajo es evaluar la interferencia que tiene su aplicación, con el diagnóstico tradicional de la infección en planteles lecheros de la Región Metropolitana. Para ello, 56 vaquillas fueron vacunadas y 48 fueron inyectadas con NaCl 0,9% a modo de controles. A todas se les tomó muestra de sangre tres y seis meses después de la vacunación las que luego fueron estimuladas con distintos antígenos y se dejaron incubar. Una vez finalizado el tiempo de incubación, se cosecharon los plasmas y finalmente se procedió a realizar el ensayo diagnóstico, ELISA IFN-γ con antígenos DIVA añadidos. Los antígenos DIVA corresponden a antígenos que fueron eliminados de la cepa vacunal. Los resultados demostraron un aumento en la cantidad de individuos falsos positivos al diagnóstico tradicional de TBB en los primeros seis meses después de la vacunación. Esto quiere decir, que fueron positivos a la tuberculina bovina, pero negativos a la prueba DIVA (Detecting Infected among Vaccinated Animals), debido a la reacción con la vacuna. Se concluye que se hace necesario utilizar una prueba complementaria para el diagnóstico de la tuberculosis en el ganado, en este caso, la prueba DIVA, durante los primeros meses después de vacunar con la cepa M. bovis BCG / Bovine tuberculosis is a chronic and zoonotic disease, caused by Mycobacterium bovis. The cost of the disease is associated to the decrease in the productivity of severely infected animals, the restrictions for the animal movement, the diagnostic tests for the detection and elimination of positive animals to avoid the spread of the agent, confiscations and the lower price of milk. It is considered an occupational disease, since people who have the highest risk for M. bovis zoonotic infection are those who spend long time in close contact with livestock. Bovine tuberculosis in Chile is an endemic disease, and the plan for the control and eradication of the disease is being supported by the study of the vaccination with the M. bovis BCG strain in cattle. It was developed by Calmette and Guerin, who reported the induction of protection in cattle against exposure with M. bovis. As this vaccine corresponds to a strain of M. bovis, the objective of this work is to evaluate the interference that has its application, with the traditional diagnosis of infection in dairy farms from the Metropolitan Region. For this, 56 heifers were vaccinated and 48 were injected with NaCl 0,9%. Blood samples were taken at three and six months after the vaccination. Once at the lab, samples were stimulated with different antigens and, after an incubation period, plasmas were harvested and IFN-γ detected by carrying out an ELISA diagnostic assay.-- The results showed an increase in the number of false positive individuals to the traditional diagnosis of bovine tuberculosis in the first six months after vaccination. This means that they were positive to bovine tuberculin, but negative to the DIVA (Detecting Infected among Vaccinated Animals) test, due to the reaction with the vaccine. It is concluded that it is necessary to use a complementary test for the diagnosis of tuberculosis in cattle, in this case, the DIVA test, during the first months after vaccination with the M. bovis BCG strain / Convenio SAG-FAVET

BACKGROUND:Bovine tuberculosis (TB) caused by Mycobacterium bovis is primarily a disease of ruminants, particularly cattle (Bos primigenius) and buffalo (Syncerus caffer), and is endemic in most developing countries. To date, studies done in Uganda have documented the prevalence of M. bovis in cattle, humans and wild life, in addition to non-tuberculous mycobacteria in pigs. Pigs are increasingly becoming an important component of the livestock sector and share the human ecosystem in rural Uganda. It is therefore of public health interest that they are not a source of human infections. As a follow up to previously published findings on mycobacteria in pigs, this study was aimed at investigating the occurrence and molecular characteristics of M. bovis detected in slaughter pigs in Mubende district, Uganda. One hundred fifty mesenteric lymph nodes with lesions suggestive of mycobacterial infections were collected from approximately one thousand slaughtered pigs in Mubende district over a period of five months. The isolation and identification of M. bovis was done using conventional mycobacteriological methods. Mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC) were identified to species level using deletion analysis. Molecular typing was done using Spoligotyping and MIRU-VNTR analysis. Molecular data were analysed and interpreted using MIRU-VNTR plus, SpolDB4.0 and the Mycobacterium bovis spoligo database.RESULTS:Of the examined animals, one boar and two sows from Madudu Sub County were infected with M. bovis which presented as lesions of a deep yellow colour and a grit-like texture in the mesenteric lymph nodes. This represents 2% (3/150) of the lymph nodes where lesions suggestive of mycobacterial infections were detected. Molecular analysis revealed that the isolates from the infected pigs showed identical MIRU-VNTR profile and spoligotype (SB1469).CONCLUSIONS:This is the first study documenting the occurrence of M. bovis in slaughter pigs in Uganda, revealing that one in fifty slaughter pigs with suspected lesions in mesenteric lymph nodes were infected. Molecular analysis revealed that the isolates were identical, showing a spoligotype previously reported from humans and cattle in the north eastern part of the Uganda cattle corridor. This finding is of public health importance, therefore there is a need for close cooperation between medical and veterinary professionals in designing and implementing control and prevention measures that safeguard the public from this potential source of zoonotic TB in Uganda.

O carrapato Rhipicephalus microplus é um ectoparasito hematófago de grande importância para a pecuária por ser responsável por perdas massivas na produção animal, de forma que o seu controle é economicamente relevante. Este carrapato, além dos danos que causa por si só, é também um importante vetor para a transmissão de microorganismos patogênicos, entre eles o hemoprotozoário intraeritrocítico Babesia bovis. O presente trabalho descreve o desenvolvimento de uma linhagem de B. bovis capaz de expressar um antígeno protetor, uma glutationa S-transferase do carrapato Haemaphysalis longicornis (HlGST), e o teste desta linhagem como uma vacina de vetor vivo para o controle do carrapato R. microplus. B. bovis, em cultivo, da linhagem S74-T3B foram eletroporados em presença de plasmídeo contendo o promotor bidirecional de B. bovis Ef-1 aresponsável pela expressão independente de dois genes: o repórter fusionado ao agente para seleção (GFP-BSD) e HlGST fusionada à sequência codificadora do peptídeo sinal de MSA-1 (merozoite surface antigen-1). Após a eletroporação, foi feita a seleção com blasticidina para obtenção da linhagem nomeada HlGST. A linhagem HlGST é composta por parasitos contendo diferentes padrões de inserção dos genes exógenos, tanto dentro quanto fora do locus Ef-1. Uma linhagem clonal denominada HlGST-Cln expressando HlGST e GFP-BSD foi obtida a partir da linhagem HlGST. Dois ensaios, independentes, de imunização de bovinos com os parasitos clonais foram realizados, sendo usado como controle uma linhagem clonal previamente caracterizada denominada GFP-Cln. Todos os animais inoculados desenvolveram uma forma branda de babesiose, indicando que ambas as linhagens clonais são atenuadas, mas apenas os animais imunizados com a linhagem HlGST-Cln foram capazes de produzir anticorpos anti-HlGST. O segundo procedimento de imunização foi seguido por um desafio com larvas de R. microplus. O desenvolvimento dessas larvas no hospedeiro levou a fêmeas adultas de menor peso e fertilidade. Coletivamente, esses dados mostram a possibilidade de uso de linhagens transfectadas de B. bovis como vacinas de vetor vivo. / The tick Rhipicephalus microplus is a notorious blood-feeding ectoparasite of cattle, responsible for massive losses in animal production. It is the main vector of pathogenic microorganisms, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74- T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1 promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD, and HlGST fused to the MSA-1 (merozoite surface antigen-1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1 a locus. A B. bovis clonal line termed HlGST-Cln expressing HlGST and GFP-BSD was then derived from the mixed parasite line HlGST. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1 locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis indicating that both transfected cloned parasite lines are attenuated. All animals immunized with HlGST-Cln produced detectable anti-glutathione-S-transferase antibodies. After immunization with HlGST-Cln, calves were challenged with R. microplus larva. Development of these larva produce fully engorged female tick with reduced weight and fertility. Collectively, these data show that transfected B. bovis parasites can be used as vectors in live vectored vaccines.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 73-91).

Zugl.: Giessen, Universiẗat, Habil-Schr., 2009.