Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Elizabeth Davis / The immune status of an individual host or among a population is affected by important variables including the source and route of potential natural exposure and for vaccination consist of vaccine type, potency, and virus strain; vaccination route and schedule; and individual host factors. Although, perhaps, often overlooked, it is essential to have a basic understanding of the laboratory methods used to measure and assess the host’s immune status. The precision, accuracy, sensitivity, and specificity of a method must be well defined. Moreover, an “adequate,” acceptable, or diagnostic value for each method must be clearly defined so that a particular test result for a patient can be meaningfully interpreted in relation to the patient’s history and clinical management. The reasons for performing rabies serology can range from diagnosis of infection to investigation of epitope specificity of an anti-rabies virus glycoprotein monoclonal antibody. Characterization of an antibody’s affinity, specificity, quantity, and neutralizing function, and class/subclass are achieved by various methods. Many serological techniques developed over the past five decades differ not only in their ability to detect the function, affinity and specificity of rabies virus antibodies, but also in the ease and practicality with which they are performed. To select an appropriate method and appropriately interpret test results, it is essential to understand the specific strengths, weaknesses, and limitations of available methods. The decision to use a specific assay should start with the purpose of testing and the intended application of results. Other factors to consider are the assay complexity, degree of precision and/or accuracy, specificity and range of detection. Given the importance of RVNA levels in the prevention of human and animal rabies, guidelines for adequate vaccination should be stated in terms that are readily understood by individuals-at-risk and health care providers, both veterinary and medical, who will use the recommendations for clinical management of humans or animals. Across the globe, the standardization of rabies serologic assays has a direct effect on the clinical use of human and animal products, including direct assessment of, and assessment of host responses to, rabies vaccines for the prevention of rabies.

Digitized by Kansas Correctional Industries

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

Creation of a reliable and easy to use serologic test would greatly improve ante mortem diagnosis of Mycobacterium avium subsp. avium and aid in the control of avian mycobacteriosis, particularly in captive birds. In order to determine whether serodiagnostics could be of value in testing ring-neck doves (Streptopelia risoria) for M. a. avium infection, Western blot analysis was used to assess the humoral response of ring-neck doves exposed to M. a. avium, and to evaluate whether an association could be made between humoral response and necropsy findings, histopathology, culture, and PCR testing. Western blot results were examined for reactivity patterns associating the humoral response with infection status, severity and type of lesions (diffuse vs multifocal granulomatous inflammation) and phenotype (white vs non-white). A sensitivity of 88.24% and a specificity of 100% were achieved utilizing Western blot analysis to detect M. a. avium infection in ring-neck doves, offering a negative predictive value of 93% and a positive predictive value of 100%. While Western blot analysis results did not reflect lesion severity, lesion type did partially correspond with the humoral response. The findings of the present study indicate that serologic testing can be used as a valuable ante mortem screening tool for identifying ring-neck doves infected with M. a. avium.

No description available.

Abstract Pteropid bats are species of zoonotic significance and are known to be reservoir hosts of many viral diseases recently emerged in Asia and Australia. The transfer of these organisms from bats into terrestrial hosts resulted in lethal illnesses in humans and animals. Implementing attentive strategies for the detection and monitoring of these organisms is essential to the protection of humans, animals and the balance of the global economy. Unfortunately the paucity of information on these mammals’ immune system and the absence of diagnostic tools for the detection and for the studying of the humoral response in these animals disengage us from responding promptly when such outbreaks occur and prevent us from describing the possible underling causes that may be responsible for the absence of symptoms in bats infected with such organisms. It was assumed that flying foxes have immunoglobulin like molecules that provide humoral response and are involved in mediating secondary effector functions such as complement fixation and activation of other components of the cellular response in a similar manner as it occurs in humans and other mammals. The aims of this study which include immunoglobulin isotype identification, purification and characterisation as well as generation of reagents that are immunoglobulin class specific, provide the primary platform that should enable us to begin examining these questions. IgG was purified from the black flying fox’s serum by affinity chromatography using Protein G and Protein A. Protein L was ineffective in purifying any antibody from the bat serum. The heavy chain of IgG was also purified by gel electroelution. IgG was digested with papain to yield Fab and Fc fragments. The identity of the bat IgG was confirmed by N-terminal sequencing of the heavy chain and light chain of immunoglobulins separated by SDS-PAGE and by N-terminal sequencing of Fc and Fc' fragments. IgM has also been purified using methods that have not been previously explored to our knowledge. These methods consisted of the purification of Fab specific antibodies from antisera generated against Fab, and using these antibodies to capture other immunoglobulin classes in samples that had been previously enriched by classical fractionation methods. Antisera against the whole IgG molecule, Fc, Fab, IgG heavy chain and IgM heavy chain have been produced in rabbits and tested by Western blot and ELISA. The antisera against the whole IgG molecule and against the Fc were also utilized to detect antibodies to Nipah virus in bats that were found positive to Serum Neutralisation Test. Failure to identify the bat IgA in the bat serum poses questions on the presence, abundance and functional significance of such molecule in bats. The tools that were generated in this study recognise immunoglobulin isotypes, which enabled us to detect and measure antibodies and will allow the study of the humoral response in infected bats to a large extent. Tedious approaches routinely adopted for the purification of antibodies involve a series of pre-fractionation steps or affinity chromatography which rely on the use of expensive immobilised novel or partially characterised ligands, with no guarantee of affinity for the immunoglobulin isotype of interest. The method adopted for immunoglubulin isotype purification described in this study proved to be an effective, reasonably quick and economic solution for immunoglubulin isotype purification from any mammalian species.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-12Bitstream added on 2014-06-13T19:03:34Z : No. of bitstreams: 1 augustofilho_o_dr_botfmvz.pdf: 3263131 bytes, checksum: 3dff38635500878cf1ed7a081766be74 (MD5) / Universidade Estadual Paulista (UNESP) / Bezerros nascidos de vacas revacinadas contra raiva no terço final de gestação foram vacinados aos dois, quatro e seis meses de idade e revacinados 30 dias após. Os títulos de anticorpos (Ac) foram mensurados nas mães e em seus descendentes 48 horas após o parto. Os bezerros foram separados em quatro lotes com dez animais, sendo representados por bezerros não vacinados, vacinados com dois meses, quatro meses e seis meses de idade. O título de Ac antirrábicos nos bezerros dos diferentes grupos foi avaliado mensalmente e 15 dias pós vacinação e revacinação até completarem 12 meses de vida. Todas as vacas apresentaram titulo de anticorpos antirrábicos soroneutralizantes (AcSN) superiores a 0,5UI/mL 48 horas após o parto. Anticorpos transferidos pelo colostro com títulos semelhantes ao materno foram observados em todos os bezerros estudados 48 horas após o nascimento. Títulos de AcSN superiores a 0,5UI/mL se mantiveram até o 5º mês nos bezerros vacinados aos dois meses sendo que nos demais lotes houve queda nos títulos a partir do 3º mês de vida apresentando um período de desproteção decorrente de queda de anticorpos colostrais superior ao grupo vacinado aos dois meses de idade. Nenhum dos grupos de bezerros estudados apresentou resposta sorológica satisfatória a primo vacinação, porém, todos apresentaram resposta adequada após a revacinação. O estudo demonstrou que é recomendável a vacinação antirrábica em bezerros aos dois meses de idade independentemente do estado vacinal materno seguida de revacinação 30 dias após, e ao completarem 12 meses de vida / Calves born from cows revaccinated against rabies in the final third of pregnancy were vaccinated at two, four and six months of age and revaccinated 30 days later. Seraneutralizing antibody titers (AcSN) against rabies were measured in mothers and their calves 48 hours after delivery. The calves were separated into four groups of 10 animals, represented by non-vaccinated and vaccinated calves with two months, four months and six months of age. The AcSN titers was evaluated monthly and 15 days after vaccination and revaccination until 12 months of life in each group. All cows had AcSN greater than 0,5 UI/mL 48 hours after delivery. Antibodies trasnferred by colostrum with similar titers were observed in all calves studied 48 hours after birth. AcSN titers higher than 0,5UI/mL remained to the 5th month in calves vaccinated at two month of age in contrast to the others groups that presented decrease of AcSN begining at 3th month of age until 30 days after first vaccination representing a longer period of lack of protection comparatively to the group vaccinated at two month of age. Absence of satisfatory antibody response was observed in all groups after the first vaccination, however, all calves had an adequate response after revaccination. The study demonstrated that antirabies vaccination can be recommended in calves at two months of age regardless of vaccination status of mothers being mandatory the revaccination 30 days after and at 12 months of life

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-09Bitstream added on 2014-06-13T20:33:24Z : No. of bitstreams: 1 nardo_cdd_me_araca.pdf: 584996 bytes, checksum: d064cf941273079c61aca57c35d57979 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A leishmaniose visceral é uma zoonose causada por um protozoário do gênero Leishmania, cujo agente etiológico no Brasil é a Leishmania chagasi. O primeiro caso canino autóctone no Estado de São Paulo, Brasil, foi identificado em 1998 no município de Araçatuba. Desde então a doença vem se espalhando por todo o Estado. O objetivo do presente estudo foi determinar a freqüência de ocorrência de anticorpos anti-Leishmania chagasi em amostras de soro de 584 cães de São José do Rio Preto, São Paulo, área não endêmica para a doença e que dista 160 Km de Araçatuba. Asoroprevalência foi de 0,86% por ELISA e 0,17% por imunocromatografia. Areação de imunofluorescência indireta foi utilizada em 138 amostras eidentificou 1,45% de soropositividade. O ELISA identificou cinco cães positivos,a RIFI dois e um cão foi identificado pela imunocromatografia. Um dos cãespositivo pela RIFI havia sido vacinado para leishmaniose visceral. Dois cãesque foram considerados positivos pelo ELISA indireto foram testados por PCRpara detecção da presença de Leishmania chagasi, mas os resultados foram negativos, não confirmando a doença. Somente um cão foi soropositivo em todos os métodos. Ele apresentava sinais clínicos inespecíficos e foi adquirido pelo proprietário em uma área endêmica para a doença. O diagnóstico não pôde ser confirmado por exame parasitológico direto ou PCR porque o cão morreu antes dos resultados dos exames sorológicos. Os resultados obtidos na população do presente estudo permitem concluir que São José do Rio Preto deve ser, ainda, área não endêmica para leishmaniose visceral canina. / Visceral leishmaniasis is a zoonosis caused by a protozoa of the genus Leishmania, whose etiological agent in Brazil is Leishmania chagasi. The first canine autochthonous case of the disease in the São Paulo State, Brazil, was identified in 1998 in Araçatuba. Since then, the disease has spread to great part of the State. The aim of the present study was to determine the frequency of occurrence of anti-Leishmania chagasi antibodies in serum samples of 584 dogs from São José do Rio Preto, São Paulo, a non endemic area for the disease, 160km far from Araçatuba. Seroprevalence was 0,86% by ELISA and 0,17% by imunochromatography. IFAT, used to test 138 samples, identified 1,45% of seropositivity. ELISA identified five positive dogs, IFAT two, and immunochromatography one. One of the dogs that were considered positive by IFAT has been vaccinated for visceral leishmaniasis. Two dogs that were considered positive by indirect ELISA were tested by PCR for the presence of leishmania chagasi, but the results were negative, not confirming the disease. Only one dog was seropositive by all methods. He presented inespecific clinical signs and was acquired by his owner in an endemic area for the disease. The diagnoses could not be confirmed by direct parasitological test or PCR, because the dog has died before serologic results. The population results achieved in this study allow us to conclude that São José do Rio Preto city still must be a canine visceral leishmaniasis non-endemic area.

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-29Bitstream added on 2014-06-13T18:53:57Z : No. of bitstreams: 1 machado_gc_me_botib.pdf: 359425 bytes, checksum: 96a3b6e269405927664fd4f77072b888 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O fungo termodimórfico Paraccocidiodes brasiliensis, em sua fase leveduriforme, é o agente etiológico de uma das mais importantes micoses sistêmicas da América Latina, a paracoccidioidomicose. A doença pode ser fatal em sua forma aguda, além de gerar, na forma crônica, lesões principalmente de ordem dermatológica e pulmonar. O fato de P. brasiliensis ser considerado uma única entidade biológica vem sendo questionado pelos recentes achados de filogenia molecular. Atualmente acredita-se que P. brasiliensis seja um complexo de quatro espécies crípticas, S1, PS2, PS3 e P. lutzii, sendo esta última a mais distante filogeneticamente. É possível que o fato esteja interferindo em aspectos relacionados à doença, como, por exemplo, a positividade do diagnóstico sorológico. Além disso, tal especiação pode significar diferenças na biologia do fungo, em aspectos como sua morfologia, fisiologia e ocupação de nichos ecológicos.A primeira frente deste trabalho visou produzir e caracterizar antígenos a partir de cepas pertencentes às diferentes espécies de P. brasiliensis, e avaliar o desempenho das mesmas em testes de imunodifusão (ID) contra pacientes das diferentes regiões do Brasil. Melhores resultados foram obtidos com o antígeno produzido por filtrado de cultura de um isolado pertencente ao grupo PS3, o qual apresentou altas taxas do antígeno imunodominante, a glicoproteína gp43, detectável por SDS-PAGE, e apresentou boa positividade nas IDs quando cruzados com soros de pacientes da região de Botucatu, reagindo inclusive com alguns soros falso-negativos para o antígeno considerado padrão, produzido pela cepa B-339. Entretanto, este antígeno, bem como outros aqui avaliados, não foi capaz de apresentar desempenho satisfatório frente a soros de pacientes da Região Centro-Oeste, justamente os que apresentam maiores taxas de negatividade, provavelmente por serem... / The thermo-dimorphic fungus Paraccocidiodes brasiliensis is the causative agent of paracoccidioidomycosis, one of the most important systemic mycosis in Latin America. The disease can be fatal in its acute form, besides causing important lesions especially in lungs and skin when occurs in its chronic form. The fact that P. brasiliensis is considered a single biological entity has been challenged by the recent findings in molecular phylogenetics. Currently, it has been observed that P. brasiliensis is a complex of four cryptic species, S1, PS2, PS3 and Pb01-like, which is the most distantly related, being proposed as new formal species, P. lutzii. It is possible that these facts are interfering with the host/pathogen interactions, as well as with the results of serological diagnosis. Furthermore, this speciation could reflect differences in the biology of the fungus, especially on their morphology, physiology and occupation of ecological niches.The first approach of this work was to produce and characterize antigens from strains belonging to different species of P. brasiliensis, and evaluate their performance in immunodiffusion tests (ID) against sera of pacients from different regions of Brazil. The better results were obtained with the antigen produced by culture filtrate of an isolate belonging to the PS3, AgEpm83, which showed high positivity rates in IDs, against sera of patients from Botucatu. This antigen, in addition, presented high levels of the glycoprotein gp43, the immunodominant component, detectable by SDS-PAGE. However, this antigen, as well as other here evaluated, was unable to provide satisfactory performance against the sera of patients from the Brazilian Midwest Region, probably because they are infected with P. lutzii isolates. The in silico analysis of exon2 of the gene that encodes gp43 indicated that this gene is under purifying selection both... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-18Bitstream added on 2014-06-13T19:14:35Z : No. of bitstreams: 1 queiroz_nmgp_me_araca.pdf: 2616211 bytes, checksum: 581da3d4309fd8ecf96545fb137a9c18 (MD5) / A leishmaniose visceral canina (LVC) é causada pelo protozoário Leishmania (L.) chagasi e a presença deste parasita na pele dos cães reforça a importância destes animais no ciclo de vida da L. (L..) chagasi e na infecção humana. O objetivo deste trabalho foi avaliar o teste imunoistoquímico (IMIQ) e a PCR (Reação em Cadeia da Polimerase) em tecidos cutâneos caninos para o diagnóstico da LVC e compará-los com os exames histoquímico (HE) e sorológicos (ELISA e RIFI). Dos 34 cães naturalmente infectados, classificados em assintomáticos, oligossintomáticos e polissintomáticos, foram colhidas amostras de pele sadia ou com lesão para a realização da IMIQ, HE e PCR. Não somente peles lesionadas (56,5%), mas também peles sadias (31,8%) encontravam-se positivas pela IMIQ, confirmadas posteriormente pela PCR em 97,8% das amostras. No grupo assintomático, 87,5% estavam negativos pelos testes sorológicos, mas positivos em 50% dos casos pela IMIQ e 100% pela PCR. Entre os oligossintomáticos, 100%, 85,7% e 28,6% encontravam-se positivos, respectivamente pela PCR, sorologia e IMIQ. 91,7% dos polissintomáticos eram soro positivos e tinham parasitas intactos na pele. A PCR apresentou maior positividade, detectando amastigotas na pele de 100% dos cães independente do quadro clínico, do resultado dos exames sorológicos e do teste HE. A eficiência de cada teste variou de acordo com a evolução da doença, demonstrando a necessidade da associação de técnicas, usando IMIQ para confirmação da sorologia e a PCR apenas nos casos ainda suspeitos após a IMIQ, podendo assim aumentar os níveis de positividade e contribuir para o controle desta zoonose. / Canine visceral leishmaniasis (CVL) is caused by a protozoa of the specie Leishmania (L.) chagasi and the presence of parasite in the skin reinforces the importance of dogs in the L. (L.). chagasi life cycle and for human infection. The purpose of the present study was to evaluate the immunohistichemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for CVL diagnosis and compared the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitalogical test (microscopic direct examination of the parasite stained with haematoxylin and eosin - HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positives by IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positives and had intact parasites in the skin. PCR showed higher positivity, detecting amastigotes in skins of 100% dogs regardless their clinical status or the serological or IMHC or HE test results. The efficiency of each test varied in agreement with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR only in suspicious cases after IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this zoonoses.