Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-18Bitstream added on 2014-06-13T20:19:23Z : No. of bitstreams: 1 camossi_lg_me_botfmvz.pdf: 3533496 bytes, checksum: 60945d70b81603efb8f19a414a05e3a3 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A toxoplasmose em ovinos é uma doença parasitária de grande importância médica veterinária, zootécnica e de saúde pública, uma vez que acarreta prejuízos na criação animal, gerado pelas perdas reprodutivas e econômicas, além de sua implicação na saúde humana, já que o consumo de carne e leite contaminados podem facilitar a transmissão zoonótica. Este estudo tem como propósito maior investigar a resposta imunológica das infecções naturais por Toxoplasma gondii em ovelhas, pela análise seqüencial de imunoglobulinas, objetivando a diferenciação entre os estágios agudo e crônico da doença, bem como a pesquisa do DNA do agente no leite pela técnica de PCR. Para realização do estudo utilizaram-se ovelhas em lactação, naturalmente infectadas por T. gondii, divididas em dois grupos: G1, com sorologia positiva, e G2, sorologicamente negativo, composto por 20 ovelhas cada um. A diferenciação dos estágios da infecção toxoplásmica, agudo ou crônico foi realizada pela técnica de aglutinação direta modificada (MAT) com antígeno fixado com formalina (MAT-AF) e metanol (MAT-AM) e pela Reação de Imunofluorescência Indireta para pesquisa de imunoglobulinas IgM e IgG. Os resultados sorológicos evidenciaram que as ovelhas demonstravam perfil sorológico de cronicidade da infecção. A detecção do parasito no leite foi realizada pela reação em cadeia pela polimerase (PCR), detectando-se o DNA do T. gondii em sete amostras de leite, provenientes de cinco ovelhas soropositivas, sendo que em duas ovelhas o DNA foi detectado no leite por duas vezes. A identidade molecular dos produtos amplificados foi confirmada pelo sequenciamento., obtendo-se de 97 a 100% de identidade com T. gondii, constituindo-se em resultado relevante quanto aos aspectos de saúde pública. / Toxoplasmosis is a major parasitary disease in sheep, due its importance in veterinary medicine and animal science and in public health, causing reproductive and economic losses to the herd, and also the prejudice to human health for the consumption of contaminated meat and milk, which can facilitate the zoonotic transmission. This study aims to investigate the immune response of natural infections by Toxoplasma gondii in sheep by sequence analysis of immunoglobulins, aiming to compare the acute and chronic stages of disease as well as DNA research agent in milk by the technique PCR. To accomplish the goal of this study, naturally infected by T. gondii lactating ewes were used, divided in two groups: G1, serologically positive, and G2, serologically negative, each group were composed by 20 ewes. The differentiation between the acute and chronic stages of the illness was made by the modified direct agglutination test (MAT) with antigens fixed with formaline (MAT-AF) and methanol (MAT-AM) and by the indirect fluorescent antibody test searching for IgM and IgG immunoglobulins. The serological results showed that the sheep showed serological profile of chronic infection. The detection of the parasite in milk was performed by polymerase chain reaction (PCR), detecting the DNA of T. gondii in seven milk samples from five seropositive sheep, and in two sheep DNA was detected in milk twice. The molecular identity of the amplified products was confirmed by sequencing, getting up for 97 to 100% identity with T. gondii, constituting an important result regarding the public health aspects

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Apesar do empenho do MinistÃrio da SaÃde para a eliminaÃÃo da hansenÃase, o Brasil à o segundo paÃs em nÃmero de casos no mundo, precedido pela Ãndia, e responsÃvel por 80% dos casos no continente americano, sendo o Nordeste do paÃs e, em especial, o Estado do Cearà considerado uma regiÃo de alta endemicidade. De acordo com a classificaÃÃo de Ridley e Jopling, as formas clÃnicas dividem-se em virchowiana, dimorfa-virchowiana, dimorfa-dimorfa, dimorfa-tuberculÃide e tuberculÃide. O trabalho foi realizado com 87 pacientes com hansenÃase, sendo 51,72% do sexo feminino e 48,3% do sexo masculino; destes 87 pacientes, 77,01% vacinados em algum perÃodo da vida entre a infÃncia a adolescÃncia. Os pacientes incluÃdos no estudo encontravam-se nÃo tratados (n=23) ou em tratamento (n=64), apresentando a maioria dos pacientes a hansenÃase pela primeira vez e alguns apresentavam recidiva (n=11). A sorologia de IgG sÃrica anti-PGL1 foi realizada em 83 pacientes, tendo sido as concentraÃÃes de maiores diferenÃas ocorridas entre os grupos com a forma tuberculÃide e dimorfa-tuberculÃide e o grupo com a forma dimorfa-virchowiana. As 87 amostras analisadas foram amplificadas quanto à seqÃÃncia de 171 pb de uma regiÃo altamente conservada dos aminoÃcidos 671-692 do C-terminal no domÃnio intracelular do receptor Toll-like 2 e foi aplicada a tÃcnica de AnÃlise do Polimorfismo Conformacional de Fita Ãnica (SSCP). O perfil eletroforÃtico das amostras testadas encontradas foram de duas e trÃs bandas, mostrando-se necessÃrio o sequenciamento,este apresentou heterozigose marcado pela presenÃa das bases C e T na posiÃÃo C2029T, e alÃm de dois outros perfis de heterozigose nas posiÃÃes C2006T (encontrado em todas as amostras sequenciadas) e T2008G (amostra 82). O trabalho sugere que houve heterozigose na regiÃo do Ãxon 3 do gene do receptor toll-like tipo 2, diferentemente do encontrado por Kang e Chae (2001) o que pode significar que o perfil de suscetibilidade em nossa populaÃÃo à distinta daqueles encontrados na Ãndia e na CorÃia. / Although several efforts from Ministry of Health have been made in order to eliminate leprosy, Brazil is still the second country with the highest number of cases in world after India and is responsible for 80% of the cases in the American continent, the Ceara state situated in the Norheastern region is considered to have high incidence rates of leprosy cases. According to Ridley and Jopling classification, the clinical forms of leprosy can be divided in lepromatous leprosy, borderline lepromatous leprosy, borderline borderline leprosy, borderline tuberculoid leprosy, and tuberculoid leprosy. This work was done with 87 patients with leprosy, being 51.72 % of the female gender and 48.3% of the male gender; from the total of patients, 77.0% had been vaccinated with BCG once in life. All the patients enrolled in the study were not treated (n=23) or in treatment (n=64). Most of the patients had suffered from leprosy for the first time and some (n=11) had suffered from leprosy recidive. The anti-PGL1 serum IgG serology has been performed in 83 patients, and the most significant differences were found comparing the tuberculoid leprosy and borderline tuberculoid leprosy groups with the borderline lepromatous leprosy group. All DNA samples (n=87) were amplified in respect to the 171 bp highly conserved sequence of the aminoacids 671-692 from the C-terminal intra-cellular domain of Toll-like 2 receptor, and they were submitted to a Single Strain Conformation Polymorphism Technique (SSCP). The eletrophoretic profile found of the samples showed two and three bands, it is essential to the sequencing, this showed heterozygosis at position C2029T, and in addition two other sections of heterozygosity at positions C2006T (found in all four sequenced samples) and 2008 (sample 82). The work showed that the heterozygosity found in the gene exon 3 of the tool-like receptor type 2, unlike the one found by Kang and Chae (2001) which may mean that the susceptibility profile from our population is distinct from those found in India and Korea.

Master of Science in Biomedical Sciences / Department of Clinical Sciences / Elizabeth G. Davis / To protect horses from disease, equine practitioners typically prescribe a protocol of an initial primary vaccination followed by a booster vaccination 3-4 weeks later. Subsequent boosters are given every 6-12 months depending on the pathogen of concern. Each vaccination incurs an additional cost and increased chance for adverse reactions. Despite wide-spread protocol acceptance, duration of effectiveness of vaccines in protecting horses from disease is not well documented. It was hypothesized that horses vaccinated annually since birth have increased antibody production that remains consistent and sufficient for long-term protection from common diseases. This work resulted in the development of a novel, multiplex-magnetic bead-based indirect immunoassay to screen sera from vaccinated adult horses to measure antibody levels in response to vaccine administration. Antigens tested included West Nile Virus, Eastern Equine Encephalitis, Western Equine Encephalitis, Equine Influenza Virus, Equine Herpes Virus 1 and 4, Tetanus, and 7 different Rabies antigens (3 lab and 4 wild strains). The developed assay was a 7-plex capture antibody, which quantified equine IgG (Immunoglobulin G) that binds viral antigens derived from different rabies virus strains along with pure vaccine samples of the 7 different antigens. A 7-point standard curve was developed to quantify the viral-antigen reactive IgG concentration in vaccinated horse serum. Vaccinated horses increased serum antibody concentration for each antigen post-vaccination with the percent increase ranging between 34.0% for Equine Herpes Virus 4 and 257.3% for Equine Influenza Virus. Use of the novel assay will provide equine veterinarians with an economical method to measure immune activation toward common pathogens of concern. This methodology will provide foundation level information regarding antigen specific IgG concentrations that ultimately may be extrapolated to establish protective levels of immunity resulting in establishment of vaccine protocols.

Orientador: Virgínia Bodelão Richini-Pereira / Resumo: O Zika virus (ZIKV) é um flavivírus transmitido por mosquito que causa Febre Zika e está associado às Malformações Congênitas e Síndrome de Guillain-Barré integrando a lista de agentes etiológicos que são um problema de saúde pública internacional. Apresentamos uma investigação sorológica e molecular do ZIKV em amostras humanas na região de Bauru do estado de São Paulo, Brasil a fim de contribuir na determinação dos primeiros casos de infecção de ZIKV nos locais do estudo. Foi realizada a investigação sorológica por técnica de imunocromatografia em 2000 amostras Dengue virus negativas referentes aos anos de 2014-2016, sendo 159 (7,9%) positivas para ZIKV, evidenciando que a circularização do ZIKV na região de estudo teve início em 2014. Foi investigado pela técnica de Reação em Cadeia da Polimerase quantitativa por transcrição reversa a presença de ácido ribonucleico (RNA) do ZIKV nas 159 amostras positivas a nível sorológico. Porém o RNA viral não foi detectável. Esse resultado negativo quanto a molecular a partir de soro reforça a possibilidade de aderência do ZIKV em eritrócitos. Esses achados levantam o questionamento se outros membros do gênero Flavivirus possuem o mesmo potencial. Para complementar os dados realizamos uma análise filogenética da espécie ZIKV. Com o propósito de geração de hipóteses referentes a sua dinâmica de distribuição no Brasil. Para tal utilizamos os bancos de dados, algoritmos e parâmetros presentes nos softwares Virus Pathogen Resource e MEGA7. ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Zika virus (ZIKV) is a mosquito-borne flavivirus that causes Zika Fever and is associated with Congenital Malformations and Guillain-Barré Syndrome, which has been declared an international public health problem by the World Health Organization. We conduct a serological and molecular investigation of ZIKV in human samples in the Bauru region of the state of São Paulo, Brazil, in order to contribute to the determination of the first cases of ZIKV infection in the study sites. Serological investigation was made by immunochromatography technique performed in 2000 Dengue virus negative samples referring to the years 2014-2016, 159 (7.9%) was positive for ZIKV, evidencing that the circularization of ZIKV in the region of study began in 2014. The presence of ZIKV ribonucleic acid (RNA) in the 159 serologically positive samples was investigated by the Quantitative Reverse Polymerase Chain Reaction technique. All did not show detectable RNA. This molecular negative result from serum enhances the possibility of ZIKV adhesion in erythrocytes. These findings raise the question of whether other flaviviruses have the same potential. We also performed a phylogenetic analysis of the ZIKV species. With the purpose of generating hypotheses referring to its distribution dynamics. For this we use the databases, algorithms and parameters present in the software Virus Pathogen Resource and MEGA7. Our evidence allows us to infer that the ZIKV presents a possible adaptation in the Northeast Region ... (Complete abstract click electronic access below) / Mestre

Thesis (DPhil (Biomedical Science))--Cape Peninsula University of Technology, 2019. / Knowledge of population vulnerabilities to infectious diseases is key in managing many public health problems and for mapping appropriate strategies for prevention or intervention. A number of genes associated with resistance to HIV infection, such as the double deletion of 32 base pairs in the CCR5 gene , have been described and potentially account for lower HIV infections in some populations. The magnitude of the HIV pandemic in Sub-Saharan Africa warrants an investigation of the peculiar genetic factors that may have exacerbated its spread. An understanding of the genetic factors that are involved may aid in the development of specific strategies for prevention such as vaccine development, genetic counselling as well as gene therapy. The aim of this project was therefore to study the relationship between blood groups and HIV-infection in Botswana. HIV infection in Africa has not been linked to particular blood groups. The project was undertaken in two phases from December 2012 to December 2017. In the first phase, 346 subjects of known HIV status (negative or positive) were phenotyped for 23 erythrocyte antigens via standard scientific procedures. A Chi-square analysis was used to determine those antigens associated with increased or reduced risk of HIV infection. In the second phase, 120 samples were phenotyped for the protective blood group (RhC) and the risk-associated groups (Lub and P1). The samples were also characterized according to their laboratory results for viral load, lymphocyte sub-populations, complete blood count and blood chemistry, including total cholesterol. Some of the samples were also assessed for erythrocyte-associated viral RNA. Generally, the prevalence of the blood groups in the general population in Botswana did not differ with the known prevalence for Africans broadly. Three novel findings were established. First, the blood group Rh(C) was associated with a 40% risk reduction for HIV infection. Immunologically, carriage of the C antigen was associated with a more robust cell-mediated immunity as evidenced by enhanced cytotoxic T cell counts. Moreover, this antigen occurred with a frequency lower than 30% in all countries where HIV prevalence was high. There was therefore an inverse relationship between Rh(C) frequency and HIV prevalence. An examination of reports from previous studies revealed that the pattern was consistent in Africa, Europe, Asia, South America and Caribbean countries. It appears that the population frequency of this antigen explains, at least in part, a genetic factor that puts some African populations at higher risk for HIV infection. These results are novel in that Rh antigens have not been previously associated with immunity in any reports. Novel findings regarding the P1 blood group was its association with a double risk for HIV infection. While the plasma viral load did not differ between P1-positive and P1-negative subjects, P1-positive erythrocyte lysate yielded more viral RNA than P1-negative cells, implying more intracellular HIV RNA. Intra-erythrocytic viral RNA was detected even in patients with an undetectable plasma viral load. Glycosphingolipids, of which P1 is an example, have been documented to promote viral fusion to cells independent of CD4 receptors or other ligands. In at least one report, the presence of sphingolipids in lipid rafts was considered to be sufficient for viral fusion. The presence of viral RNA even in erythrocyte lysates corroborates this phenomenon and potentially explains the double risk of HIV infection observed. The occurrence of HIV RNA in erythrocyte lysate is a novel finding that suggests a new viral reservoir. Apparently, P1 has a high frequency among Africans and low in other races.

Orientador: Fabiano Antonio Cadioli / Coorientador: Luiz Carlos Marques / Banca: Marcos Rogério André / Banca: Letícia Andreza Yonezawa / Resumo: Infecções pelo hemoprotozoário T. vivax causam grandes prejuízos à pecuária da África e Américas Central e do Sul. Surtos devido a este protozoário têm ocorrido com frequência cada vez maior no Brasil. O conhecimento das alterações que ocorrem durante a evolução desta enfermidade podem ser de grande valia. Para tanto foram estudados os sinais clínicos, parasitemia, alterações hematológicas, bioquímicas, sorológicas e anatomopatológicas de três bovinos naturalmente infectados (grupo N) e outros três experimentalmente infectados (grupo E) pelo T. vivax. com média de peso de 500 kg e 548 kg, respectivamente. O período pré-patente foi de dois a três dias (grupo E). Animais do grupo N apresentaram perda de peso intensa (140 Kg). No grupo E observou-se constante diminuição da contagem global de eritrócitos, teor de hemoglobina e volume globular (VG). O leucograma revelou alterações mais evidentes para o grupo E, com leucopenia por neutropenia e linfopenia durante a fase aguda da enfermidade. O grupo N apresentou um quadro de leucocitose por neutrofilia. Foram observados diminuição do colesterol total, colesterol ligado ao HDL, albumina, aspartato aminotransferase (AST), lactato desidrogenase (LDH) e aumento da glicose, globulinas, proteínas e fosfatase alcalina (FA) para o grupo E. Os animais do grupo E mostraram-se positivos tanto para Reação de Imunofluorescência Indireta (RIFI) quanto para o ensaio de imunoabsorção enzimático (ELISA), fato que não ocorreu no grupo N, sendo este positivo para RIFI e alternando entre períodos positivos e negativos frente ao ELISA. No proteinograma, os bovinos do grupo E apresentaram inicialmente um incremento de ceruloplasmina, com posterior redução; valores elevados de transferrina praticamente por todo o período experimental; e um aumento inicial de haptoglobina com posterior redução. O grupo N apresentou valores baixos de ceruloplasmina, transferrina e ... / Abstract: Infections by T. vivax cause great losses to livestock in Africa and Central and South America. Outbreaks due to this parasite have been occurred with increasing frequency in Brazil. Knowledge of changes that occur during the development of disease can be of great value. In order to evaluate clinical signs, parasitemia, hematologic, biochemical, serological and pathological changes caused by T. vivax, three naturally (group N) and three experimentally (group E) infected bovines by T. vivax, with an average weight of 500 and 548 kg, respectively, were studied. The observed prepatent period was two to three days (group E). Animals of group N showed severe weight loss (140 kg). Reduction of erythrocyte numbers, hemoglobin concentration and PCV were observed in group E. WBC showed pronounced changes for group E, such as severe neutropenia and lymphopenia during the acute phase of the illness. Group N showed leukocytosis by neutrophilia. Decrease in total cholesterol, HDL cholesterol, albumin, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and increased in glucose, globulin, protein, and alkaline phosphatase (ALP) were observed in group E. Animals from group E were positive for both Immunofluorescence Assay (IFA) and for the Enzyme Linked Immunosorbent Assay (ELISA), which did not occur in group N that was positive in IFAT and alternating between positive and negative periods in ELISA. Proteinogram of group E showed initially an increase of ceruloplasmin, which was later reduced, high values of transferrin almost the entire experimental period. On the hand, haptoglobin showed an initial increase with subsequent reduction in group E. In group N, low levels of ceruloplasmin, transferrin and haptoglobin were observed. Histopathological examination showed alterations mainly in were liver, spleen, heart, lung, lymph node, kidney and central nervous system. Changes mainly consisted in mononuclear inflammatory infiltrate in liver ... / Mestre

Orientador: Marcos Rogério André / Banca: Heitor Miraglia Herrera / Banca: Rosangela Zacarias Machado / Banca: Lucia Helena O'dwyer de Oliveira / Banca: Estevam Guilherme Lux Hoppe / Resumo: As enfermidades transmitidas por artrópodes vêm sendo recentemente estudadas em animais selvagens brasileiros, os quais podem atuar como hospedeiros tanto para os vetores quanto para os patógenos, muitos dos quais apresentam potencial zoonótico. O presente estudo tem como objetivo investigar a ocorrência de agentes transmitidos por artrópodes (agentes Anaplasmataceae, Bartonella spp., mycoplasmas hemotróficos, Rickettsia spp., Hepatozoon spp. e piroplasmideos) em animais selvagens, cães domésticos e seus respectivos ectoparasitos, amostrados na região do Pantanal sul matogrossense, por meio de métodos sorológicos e moleculares. Para tal, 31 Nasua nasua, 78 Cerdocyon thous, sete L. pardalis, 42 cães, 110 roedores e 30 marsupiais foram capturados. Os carrapatos recolhidos (1582) dos animais pertenciam às espécies Amblyomma sculptum, Amblyomma parvum, Amblyomma ovale, Amblyomma tigrinum, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato e Amblyomma auricularium. Adicionalmente, 80 pulgas Polygenis (Polygenis) bohlsi bohlsi foram recolhidas. Quatorze (17,9%) C. thous, sete (16,6%) cães e um (3,2%) N. nasua mostraram-se soropositivos (títulos≥80) para Ehrlichia canis, com títulos de anticorpos variando de 80 a 1280. Nenhum animal mostrou-se soropositivo Anaplasma phagocytophilum. Nove cães, dois C. thous, um N. nasua, oito roedores, cinco marsupiais e um pool de pulgas P. (P.) b. bohlsi mostram-se positivos para Ehrlichia spp. Todos os cães e o pool de pulgas P. (P.) b.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The diseases transmitted by arthropods have been recently studied in Brazilian wildlife, which can act as hosts for vectors and pathogens, many of which have zoonotic potential. The present work aimed to investigate the occurrence of tick-borne agents (Anaplasmataceae agents, Bartonella spp., hemotropic mycoplasmas, Rickettsia spp., Hepatozoon spp. and piroplasms) in wild animals, domestic dogs and their respective ectoparasites, in southern Pantanal region, central-western Brazil, by serological and molecular assays. For this reason, 31 Nasua nasua, 78 Cerdocyon thous, seven L. pardalis, 42 dogs, 110 rodents and 30 marsupials were captured. The ticks collected (1582) from animals belonged to the species Amblyomma sculptum, Amblyomma parvum, Amblyomma ovale, Amblyomma tigrinum, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato and Amblyomma auricularium. Additionally, 80 Polygenis (Polygenis) bohlsi bohlsi fleas were collected. Overall, 14 (17.9%) C. thous, seven (16.6%) dogs and one (3.2%) N. nasua were seroreactive (titer≥80) to Ehrlichia canis, with titers ranging from 80 to 1280. No animal showed to be seroreactive for A. phagocytophilum antigen. Nine dogs, two C. thous, one N. nasua, eight rodents, five marsupials and one P. (P.) b. bohlsi pool were positive for Ehrlichia spp. All positive dogs and the only P. (P.) b. bohlsi pool positive for Ehrlichia spp. were also positive in specific E. canis-qPCR based on dsb gene. Seven N. nasua, two dogs, one C. thous, ... (Complete abstract click electronic access below) / Doutor

Le champignon microscopique Aspergillus fumigatus provoque un nombre important de maladies graves. Parmi elles, l’aspergillose pulmonaire chronique (APC) et l’aspergillose broncho-pulmonaire allergique (ABPA) affectent 3 et 4,8 millions de personnes dans le monde, respectivement.L’APC est très souvent mortelle si elle n’est pas soignée. Elle se développe très souvent après une tuberculose. C’est donc une maladie des pays émergents, où il n’est souvent pas possible de la diagnostiquer à cause du coût trop important des techniques existantes.L’ABPA est une complication très grave de l’asthme et de la mucoviscidose, qui complique fortement ces maladies. Elle est très difficile à diagnostiquer.Notre travail a donc consisté à développer et évaluer deux tests, un test rapide permettant de poser le diagnostic d’APC sans avoir à utiliser de matériel de laboratoire à destination des pays émergents et un western blot qui permet la confirmation du diagnostic d’ABPA. / Aspergillus fumigatus is a microscopic fungus that can cause numerous diseases. Among them, chronic pulmonary aspergillosis (CPA) and allergic broncho-pulmonary aspergilloses (ABPA) affect 3 and 4.8 million people, respectively.CPA is often fatal if left untreated. It is often a complication of tuberculosis and therefore affect low and middle income countries. However, it is difficult to diagnose it in those countries, as the tests are too expansive.ABPA is a severe complication of asthma and cystic fibrosis, worsening those diseases. It’s very hard to diagnose it.Our work was to develop and evaluate two tests, a rapid test for the diagnosis of CPA that does not require laboratory equipment designed for low and middle income countries and a western blot for confirmation of ABPA diagnosis.

En aquesta Tesis, s'ha estudiat per una banda, 1) la dinàmica de infecció de Mycoplasma hyopneumoniae (M. hyopneumoniae) a nivell de granja i la seva relació amb els diferents sistemes de producció i 2) la correlació entre la detecció d'aquest patogen a diferents nivells del tracte respiratori (cavitat nasal, tonsil.la, i bronqui) amb la seroconversió, i presència de lesions macroscópiques i microscópiques suggestives de la Pneumònia Enzoòtica (PE) a nivell de casos clínics i nivell d'animals de camp. Del estudi de la dinàmica de infecció en vam concloure que característiques inherents de cada granja tenien un efecte molt important en el patró de circulació de M.hyopneumoniae. Fins i tot dins d'una mateixa granja, es podien trobar dinàmiques de infecció diferents entre diferents lots d'animals, repercutint en diferents graus i moments d'aparició de les lesions. A més a més, el tipus de producció utilitzat (1-2 llocs versus 3 llocs) també influenciava la dinàmica de infecció del patogen. Els resultats obtinguts en les granges en 3 llocs, clarament donen suport a la característica tardana aparició del complex respiratori porcí en els sistemes de producció en múltiples fases. De fet, la colonització en transició era menor i més tardana en els sistemes en 3 llocs comparats amb en 1-2 llocs. A més a més, en els sistemes de producció en múltiples fases, s'observava un increment molt brusc de la proporció d'animals infectats associat a brot respiratoris tardans.Malgrat que la infecció per M.hyopneumoniae es va detectar en totes les granges, aquest patogen no estava sempre involucrat en els problemes respiratoris observats. De fet, es van observar garnges infectades clínica i subclínicament per M.hyopneumoniae Aquest fet pot ser important en el moment de prendre decisions sobre les estratègies de prevenció i control de malalties. Els seroperfils no van resultar ser discriminatius per determinar el paper del agent en els problemes respiratoris observats. Els bacterioperfils van ser més útils per determinar el paper del patogen, ja que l'aparició de la simptomatologia clínica estava estretament lligada a un increment de la proporció d'animals infectats (basat en la detecció de M.hyopneumoniae en hisops nasals). Tot i no ser discriminatiu, la proporció d'animals seropositius va ser major també en les granges afectades clínicament que en les subclinicament. La seroconversió en front a aquest agent ha estat descrita com lenta, retardada i molt variable. Ens els estudis de camp presentats en aquesta Tesis, la seroconversió també va ser variable. Sembla ser, que la seroconversió estava més relacionada amb la detecció de M.hyopneumoniae en els hisops bronquials que en els nasal o tonsil.lars, i a la vegada amb el percentatge d'animals amb els hisops bronquials positius.Tal i com s'esperava, l'hisop bronquial va ser el millor en predir la presencia de lesions macroscópiques i microscópiques de PE coincidint amb els treball prèviament publicats. Sorprenentment, la proporció relativa de detecció de M.hyopneumoniae en hisops nasals va ser menor en l'estudi de camp que en l'estudi on les mostres de diagnòstic. La major proporció d'hisops positius en els animals de diagnòstic es podria explicar per: un increment de l'excreció de M. hyopneumoniae degut a situacions estressants (tal com el transport) o el increment de la multiplicació de les bactèries després d'unes hores de la mort de l'animal. Els resultats d'aquesta Tesis, han demostrat que M. hyopneumoniae es pot detectar en tonsil.la, on no hi ha cèl·lules epitelials. Malgrat el mostreig d'hisops tonsil.lar en animals vius es possible però molt laboriós, pot esdevenir un lloc de mostreig més adequat per monitorejar la infecció per M.hyopneumoniae a nivell de granja. Per altre banda, el resultats obtinguts, demostren que quan l'hisop bronchial és positiu, la probabilitat de tenir lesions macroscópiqes i microscópiques compatibles amb PE és alta. Tanmateix, la presencia d'aquestes lesions no sempre implica la detecció de M.hyopneumoniae en bronqui, ja que poden ser causades per altres patògens com pot ser SIV. / The research presented in this Thesis assessed 1) the dynamics of infection of M. hyopneumoniae at a herd level and its relationship with production systems and 2) its detection in three respiratory levels (nasal cavities, tonsil and bronchi), taking into account the presence of seroconversion and macroscopic and microscopic lung lesions suggestive of Enzootic Pneumonia (EP)The studies on infection dynamics showed that the factor with greatest effect on M. hyopneumoniae circulation pattern was the farm. Even within a farm, significant differences in the dynamics of infection between different batches were observed, which were also related with different degrees and timings of M. hyopneumoniae -related lesions appearance.Moreover, the type of production system (1/2-site versus 3-site herds) was another factor that influenced M.hyopneumoniae infection dynamics. Results of our field trial in regards M.hyopneumoniae, involving 12 Spanish herds clearly support the characteristic late appearance of Porcine Respiratory Disease Complex (PRDC) in multisite production systems. M. hyopneumoniae colonization in nurseries was lower and occurred later in time in 3-site versus in 1/2-site production systems. Moreover, an abrupt increase in the proportion of infected animals, associated with late respiratory outbreaks was seen in the multisite production systems. Although M. hyopneumoniae infection was detected in all farms, it was not always involved in the respiratory disease. An important contribution of this work is that, although seroconversion and M. hyopneumoniae infection were detected in all herds, clinical and subclinical infections were described. This is important for decision making on the control strategies to implement at the farm. Serumprofiles were not discriminative to determine the involvement of M. hyopneumoniae in a clinical outbreak. Bacteriumprofiles were more useful in determining M. hyopneumoniae involvement in respiratory disease, since an increase of the proportion of infected pigs (based on detection of M. hyopneumoniae in nasal swabs) was clearly related with appearance of the clinical outbreak. Although not discriminative, the proportion of seropositive pigs was also greater in clinically than in subclinically infected herds. Dynamics of serocoversion against M. hyopneumoniae was another issue addressed in both, transversal and longitudinal field trials. Several authors have described the time of seroconversion under field conditions as slow, delayed and vaery variable. In field studies presented here time of seroconversion was also variable. Seroconversion was more related to detection of M. hyopneumoniae in bronchial swabs rather than in nasal or tonsillar swabs, and to the detection of a higher percentage of pigs with a positive nPCR bronchial swab. As expected, bronchial swabs were the best predictors of presence of microscopic and gross EP lesions coinciding with previously published studies. However, to sample bronchial swabs from live animals is not possible. Surprisingly, the relative proportion of nasal detection was lower in the field study than in the study using pigs received at the diagnostic laboratory. An increase of M.hyopneumoniae shedding in nasal cavities due to stressful situations (such as transport) or due to increase multiplication of bacteria after the animal's death have been proposed as potential explanations for a larger proportion nPCR positive nasal samples obtained in diagnostic samples.Results of this study demonstrated that detection of M.hyopneumoniae in tonsil where no ciliated epithelial cells exists, is possible. Although tonsillar sampling in live animals is more laborious than nasal samplings, it is possibly more adequate to monitor M. hyopneumoniae detection at a farm level. Results obtained in this Thesis and from previous works, showed that when bronchial swabs are positive the probability to show EP-compatible microscopic lesions in the animal is high. However, presence of EP-compatible lesions does not always imply M. hyopneumoniae detection in bronchi, and that may be caused by other pathogens such as SIV.

The epidemiological study of pathogens largely depends on three technologies, serology, microscopy and the polymerase chain reaction (PCR). Serological methods are unable to differentiate between current and past infections. Microscopy has historically been the mainstay of epidemiological study. In recent times the use of microscopy has been in decline, as it has been shown to have an inherent lack of sensitivity and specificity and produces many false negative results. PCR is now the method of choice for screening samples for the presence or absence of pathogens. Although PCR is widely regarded as an extremely sensitive technique, the fact that it assays a very small volume of sample is often overlooked. If the target pathogen is not present in the tiny aliquot of sample from an infected host, then a false negative results will occur. In endemic situations were the pathogen is present at low infection intensities, then the potential for false negatives results of this type is high. This intensity related false negative effect can lead to serious underestimation of diagnosed prevalence and incidence with consequent misinterpretation of the resulting data. This phenomenon has been reported in the literature for a range of pathogens and especially for epidemiological study of schistosomiasis. The extensive occurrence of false negatives during study of schistosomiasis samples was such an obstacle to epidemiological study it prompted the world health organisation to repeatedly call for quantitative methods to be employed to combat the problem. The main objectives of this thesis are to rationalise and simplify the methods of diagnosing African trypanosomes in epidemiological studies and to investigate the consequences of, and methods of dealing with infection intensity related false negative results that occur as a result of widespread sub-patent infections in the study population A new PCR assay was developed that was capable of analysing whole blood placed onto treated filter paper. The PCR assay was capable of differentiating between all the important African trypanosome species, producing a unique size of amplicon for each species of trypanosome. Initial results from repeated screening of human and cattle samples known to be parasitologically positive indicated that many false negative results occur. A more extensive analysis of thirty five bovine blood samples randomly chosen from a collection of field samples revealed that false negative results occurred regularly. The prevalence of infection after a single screening was 14.3% whereas the cumulative prevalence after over 100 repeated screenings rose to 85.7%. This showed that a severe underestimation of prevalence occurs from a single screening of the samples. In order to investigate the consequences of, and develop methods of dealing with this problem, computer based simulations were used to model the dynamics of screening samples with sub-patent infections. In order to construct the model the data obtained from repeat screening of the thirty-five bovine blood samples was fitted to a number of mathematical distributions. A negative binomial distribution best described the distribution of trypanosomes across the hosts. Exploration of the phenomenon with the resulting model showed the extensive underestimation of true prevalence that is possible. The simulations also showed that it is possible for populations with very different patterns of infection and true prevalence to all have the same diagnosed prevalence from a single screening per sample. Statistical comparison of these very different populations by diagnosed prevalence alone would conclude there was no significant difference between the populations. It was therefore concluded that the diagnosed prevalence from a single (or even multiple) screenings is an inadequate and potentially misleading measure of both infected hosts and parasite numbers. In order to deal with these problems new methods were evaluated for use in epidemiological studies. A simple method of producing quantitative measures of infection was advocated. The insensitivity of existing screening methods in detecting significant difference between populations was highlighted and a greatly improved methodology was shown. Finally, a method for inferring the true population prevalence from the data obtained from repeat screening of samples was suggested. Although some of these new methodologies have limitations, they represent a great improvement on the use of a single diagnostic test for each host. The work presented in this thesis highlights a serious potential limitation to our understanding of the epidemiology of pathogens that exist at sub-patent levels, and develops some possible methods of overcoming these limitations.