Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vii, 43 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 31-41).

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes. / Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Transmission of the disease occurs through direct contact or aerosols. The bacteria possess many virulence factors, namely capsular polysaccharides, lipopolysaccharides, four Apx toxins and iron acquisition mechanisms. To this day, an efficient cross-serotype vaccine has yet to be developed. In order to investigate regulation mechanisms in A. pleuropneumoniae and to identify new potential targets for the synthesis of subunit vaccines, the transcriptomic profile of the bacteria under conditions that simulate the infection and following a natural acute infection in vivo were studied. The experiences relied on first and second generation microarrays (AppChip1 and AppChip2) designed using 2025 ORFs of the draft version of the A. pleuropneumoniae serotype 5b strain L20 genome and 2033 ORFs of the final and annotated version of the same genome respectively. First, experiments were conducted under iron-restricted conditions and 210 genes were deemed differentially expressed, 92 of which were up-regulated. Some new putative iron acquisition mechanisms were identified, including genes homologous to those of the Yersinia pestis YfeABCD chelated-iron acquisition system, as well as other genes homologous to components of the HmbR iron uptake from hemoglobin system of Neisseria meningitidis. When cultured in conditions promoting biofilm production, genes tadC and tadD from a putative tad (« tight adherence locus ») operon, genes pgaABC involved in the biosynthesis of a polysaccharide of the biofilm matrix as well as two ORFs encoding a putative autotransporter adhesins similar to the Haemophilus influenzae Hsf adhesin were all significantly overexpressed. Many of these genes were also overexpressed when lung epithelial cells were infected with A. pleuropneumoniae. While 170 genes were differentially expressed after planktonic growth in the culture medium above the cells, another 131 were identified following direct adhesion to the cells. Genes tadB and rcpA of the tad locus, as well as genes pgaBC and an ORF coding for the Hsf homolog where all found among overexpressed genes. When A. pleuropneumoniae was cultured in contact with broncho-alveolar lavage fluids (BALF), 156 genes were significantly differentially expressed and gene apxIVA, which was up-regulated, was detected for the first time during in in vitro growth conditions. Finally, experiments were conducted in vivo in animals naturally infected with A. pleuropneumoniae in the late stage of the acute phase in order to identify genes that are expressed during the infection of the natural host. While 150 genes were deemed differentially expressed, genes apxIVA as well as two genes from an operon coding for a putative type IV fimbriae were up-regulated. Out of those 72 genes that were overexpressed, 3 encode proteins or lipoproteins of the outer membrane which are conserved among all serotypes of the bacteria. Overall, we were able to identify several new potential virulence factors for A. pleuropneumoniae in the course of our experiments, as well as several new potential targets for the elaboration of an efficient cross-serotype vaccine.

Dans ce travail de thèse, l'efficacité de la voie intrapulmonaire et les paramètres biopharmaceutiques influençant la diffusion pulmonaire après nébulisation d'antibiotiques ont été évalués. La présence et l'impact de certaines pompes d'efflux dans un modèle in vitro de cellules primaires épithéliales pulmonaires de rat ont été testés. Trois fluoroquinolones et la colistine ont été utilisées comme molécules de référence. La combinaison des molécules testées a permis d'obtenir une vue d'ensemble des caractéristiques de diffusion intrapulmonaire des antibiotiques. L'étude in vivo avec les fluoroquinolones a démontré que les concentrations pulmonaires de ces molécules sont plus importantes que dans le plasma, probablement dû à la présence des transporteurs comme la glycoprotéine-P. La présence de ces transporteurs a été confirmée dans le modèle de cellules pulmonaires de rats. L'étude in vivo avec la colistine a montré qu'une lente diffusion pourrait conférer un avantage à la nébulisation par rapport à l'administration intraveineuse. En conclusion, l'administration par nébulisation des molécules, qui traversent les tissus lentement (colistine), pourrait être avantageuse, alors que pour d'autres, qui traversent vite la barrière (fluoroquinolones), la voie nébulisée pourrait ne pas présenter des avantages par rapport à la voie intraveineuse. De plus, les résultats ont démontré qu'une faible perméabilité à travers le poumon (colistine) pourrait donner un avantage à la nébulisation des antibiotiques, tandis qu'une affinité pour des transporteurs (fluoroquinolones) semble présenter un intérêt aussi bien dans le cadre d'une nébulisation que d'une administration intraveineuse. / The aim of this study was to investigate the efficiency of intrapulmonary administration and the biopharmaceutical parameters regulating the pulmonary diffusion following nebulization. We examined whether certain efflux pumps were present in an in vitro model of rat lung cells and whether these efflux pumps could be beneficial by increasing lung concentrations in vivo. Fluoroquinolones and colistin were the molecules used as reference. These different molecules allowed an overview of the intrapulmonary diffusion characteristics of antibiotics. The in vivo study with fluoroquinolones showed that their lung concentrations are higher than in plasma, probably due to glycoprotein-P. The presence of this efflux pump was confirmed in the model with rat lung cells. The in vivo study with colistin showed that a slow diffusion may confer an advantage for nebulization over intravenous administration. In conclusion, the nebulization molecules passing slowly (colistin) across the tissues may be advantageous, whereas for others, with a fast passage across the barrier (fluoroquinolones), the pulmonary route may not provide an advantage over the intravenous administration. Moreover, the results showed that a slow permeability across the lung (colistin) may confer an advantage for the antibiotic nebulization, while affinity by transporters (fluoroquinolones) is beneficial for both nebulization and intravenous administration.

Mise en contexte: Le syndrome d'aspiration méconiale (SAM) sévère est une détresse respiratoire causée par l'inhalation de liquide amniotique teinté de méconium. Les nouveau-nés atteints d'un SAM sévère nécessitent souvent un support ventilatoire invasif qui mène à un haut taux de morbidité et de mortalité. La ventilation liquidienne totale (VLT) est considérée comme une avenue thérapeutique prometteuse de lavage bronchoalvéolaire (LBA) effectué avec un perfluorocarbone dans le cas d'un SAM sévère. L'équipe Inolivent a développé récemment un 4 e prototype de ventilateur liquidien total (Inolivent 4) conçu pour des applications pédiatriques qui fait l'objet d'études expérimentales. Objectif: Les objectifs de cette maîtrise étaient de valider le ventilateur liquidien, d'optimiser les paramètres ventilatoires de la VLT, de mettre au point un modèle néonatal ovin de SAM ainsi que de caractériser l'efficacité et la tolérance d'un VLT-LBA en comparaison avec un lavage bronchoalvéolaire contenant une dilution de surfactant exogène (S-LBA) dans ce même modèle. Notre hypothèse est que le VLT-LBA sera mieux toléré et plus efficace qu'un S-LBA dans le cas d'un SAM sévère. Matériel et méthode: La validation du prototype 4 a été effectuée avec cinq agneaux à poumons sains et plusieurs paramètres ont été testés de façon à optimiser la ventilation. Ensuite, sept agneaux ont servis à développer le modèle pathologique de SAM. Étude animale randomisée contrôlée approuvée par notre comité d'éthique institutionnel. Une fois anesthésiés et paralysés, 20 agneaux nouveau-nés ont subis une chirurgie visant le monitorage des gaz du sang (GDS) et les paramètres hémodynamiques par thermodilution (Pulsion medical system, Ger). Un SAM sévère fut alors induit à l'aide de l'instillation de 2 aliquotes de 1mL/kg d'une dilution à 25% de méconium humain dans le salin. Les agneaux ont ensuite été randomisés dans deux groupes : VLT-LBA (n=10) à une ventilation minute de 160 mL/kg/min avec le PFDEC (Perfluorodecalin; F2Chemicals, UK) et S-LBA (n=10) ventilé conventionnellement en administrant deux LBA de 15 mL/kg contenant 5mg/mL de surfactant (BLES biochemicals Inc., Canada). Les deux groupes furent ventilés pour un total de 4 heures suivant la randomisation. Résultats: Le VLT-LBA permet une PaO[indice inférieur 2] significativement supérieure au S-LBA tout au long de l'expérimentation. Les valeurs de PaCO[indice inférieur 2], de pH ainsi que les paramètres hémodynamiques sont comparables pour les deux groupes à l'exception d'une augmentation de la pression artérielle pulmonaire moyenne (PaPm) durant la VLT. Le VLT-LBA permet le lavage de 43.14% du méconium instillé alors qu'avec le S-LBA on en retire 28.10% (p < 0,022). L'analyse histologique des coupes de poumons n'a montré aucune différence entre les scores totaux. Conclusion: Le VLT-LBA est bien toléré et est plus efficace que le S-LBA dans ce model expérimental ovin de SAM sévère. Les niveaux surélevés de PaPm durant la VLT nécessite d'être investigués. Cette expérimentation ouvre la voie à une éventuelle étude clinique avec le VLT-LBA comme traitement d'un SAM sévère.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A hemorragia pulmonar induzida por esforço (HPIE) ocorre com freqüência em cavalos atletas acarretando prejuízo econômico já que estes deverão ficar temporariamente afastados dos treinamentos e provas, além de receber tratamento adequado. O Puro-Sangue Inglês (PSI) utilizado em provas de corrida é o animal mais afetado devido ao grande esforço ao qual é submetido, porém animais de outras raças utilizados em outras modalidades de esportes hípicos também podem apresentar HPIE. O presente estudo teve como objetivo quantificar a ocorrência de casos de HPIE em animais utilizados em provas de salto, através do exame endoscópico e da análise citológica de amostras do lavado traqueobrônquico (LTB). Para a realização do presente experimento foram utilizados 50 cavalos adultos residentes nos clubes hípicos de São Paulo, que foram divididos em dois grupos, A e B, conforme as diferentes alturas dos obstáculos utilizados nas provas em que participaram. No grupo A foram inseridos os cavalos que saltam obstáculos de 1,00m a 1,20m de altura, e no grupo B, os cavalos que saltam obstáculos de 1,30m a 1,50m de altura. Foram realizados exames físicos destes animais, antes e depois da participação em uma prova. Após 1 hora do final da prova, os mesmos foram submetidos a um exame endoscópico em que eram observadas as possíveis alterações do trato respiratório anterior do animal sendo a principal delas a hemorragia pulmonar induzida por esforço. Foi realizada citologia do lavado traqueobônquico (LTB) por microscopia após citocentrifugação das amostras e posterior confecção das lâminas que foram então coradas por Rosenfeld. A principal observação durante a leitura das lâminas foi à presença de hemossiderófagos. No trabalho realizado são apresentados além da citologia do trato respiratório dados como os valores médios de freqüência cardíaca, freqüência respiratória e temperatura corpórea dos animais após a participação em uma prova. Estes dados foram relacionados às alturas dos obstáculos existentes em cada prova realizada pelos animais. Em relação à ocorrência de HPIE observamos que no grupo A 50% dos animais apresentaram HPIE e no grupo B a ocorrência foi de 66,67%. Foram observados todos os graus de HPIE, porém somente no grupo B houve ocorrência de graus IV e V, demonstrando assim a correlação entre o nível de esforço realizado pelos cavalos de salto e o grau de HPIE apresentado por eles. / Effort induced pulmonary hemorrhage (EIPH) frequently occurs in athlete horses causing economic losses not only due to temporary removal from training and competition, but also due to the cost of adequate treatment. Thoroughbreds used in racing are the most frequently affected breed due to the great effort to which they are submitted; however other breeds and crossbred sport horses can also have EIPH. The present study´s objective is to quantify the occurrence of cases of EIPH in animals used in show jumping, through the endoscopic examination and cytological analysis of tracheobronchial lavage (TBL) samples. For the present experiment fifty adult horses, housed in maneges in the city of São Paulo, were used. Horses were divided into two groups, named A and B according to the different heights of the obstacles. Group A included horses that jump heights of 1.0 to 1.2 m and group B, obstacles of 1.3 to 1.5 m. Physical examinations of these animals were carried through, before and after participation in each test. One hour after the end of each series, the same ones had been submitted to an endoscopic examination where the possible alterations of previous the respiratory treatment of the animal were observed being main of them the induced pulmonary hemorrhage for effort. Cytology of tracheobronchial lavage (TBL) was performed in cytocentrifuge slides prepared on the same day, slides were stained using Rosenfeld Romanov and observed examined under light microscopy. The major focus during the observation of the slides was the presence of hemosiderophages and grading of EIPH intensity (I through V) Besides the cytological evaluation of the respiratory tract, this study includes clinical data such as the average values of cardiac frequency, respiratory frequency and body temperature of the animals after the each competition. These data were correlated with the heights of the existing obstacles jumped by each athlete horse. In relation to the EIPH occurrence we observed that in the group A 50% of the animals presented EIPH and in group B the occurrence was of 66,67%. All grades of EIPH were observed when both groups were considered, however only animals in group B showed intense hemorrhage levels (IV or V) thus demonstrating and the correlation between the levels of effort the intensity of EIPH in jumpers.

A Mannheimia haemolytica é uma importante bactéria relacionada ao Complexo Doença Respiratória dos Bovinos e a essa atribui-se uma evolução para uma forma grave de pneumonia fibrinonecrótica. É considerada um habitante comensal da nasofaringe que em situações de comprometimento da resposta imune adquire um perfil oportunista. O presente estudo buscou observar por meio de modelo de infecção experimental, as possíveis alterações locais e sistêmicas causadas pela M. haemolytica em bezerros experimentalmente inoculados. Dessa forma seria possível de maneira longitudinal, acompanhar a dinâmica dos principais aspectos de defesa das vias aéreas posteriores durante a infecção e após o tratamento com o antimicrobiano norfloxacina associado ou não à flunexina meglumina. Avaliou-se por exame físico acrescido de broncoscopia, alterações funcionais das células de defesa e mediadores inflamatórios, tanto séricos quanto locais e a atividade in vitro da norfloxacina sobre a função dos fagócitos sanguíneos e do lavado broncoalveolar (LBA). Para tal foram utilizados 12 bezerros sadios que foram experimentalmente infectados por M. haemolytica dos quais foram avaliadas as alterações clínicas e, quantitativamente e funcionalmente, as populações leucocitárias no sangue e no LBA, assim como o efeito da norfloxacina sobre a atividade funcional dos fagócitos no sangue e no LBA. Os resultados do presente estudo demonstraram o sucesso da infecção experimental por M. haemolytica pelos achados clínicos, broncoscópicos e citológicos. Ademais, a infecção experimental por M. haemolytica foi associada a alterações nas subpopulações de linfócitos T CD8+ e &#1091;&delta;, na produção intracelular de espécies reativas de oxigênio (ERO), fagocitose e viabilidade pelas células CD14+ sanguíneas e do LBA e granulócitos do sangue e, expressão de L-selectina pelos leucócitos polimorfonucleares do sangue. Nenhuma alteração evidente foi observada na expressão de citocinas IL-1&#946;, IL-8 e TNF-&#945; nas células sanguíneas e do LBA. O tratamento com antimicrobiano associado ou não ao anti-inflamatório foi capaz de curar a infecção e reestabelecer os parâmetros avaliados à sua condição basal. Portanto, não se observou benefícios com a utilização adicional do anti-inflamatório no reestabelecimento do quadro clínico e da resposta imune neste experimento. Contudo, apesar de algumas alterações na resposta sistêmica durante o quadro infeccioso, as alterações locais foram mais perceptíveis. Outro aspecto importante encontrado foi o efeito in vitro da norfloxacina na produção intracelular de ERO, fagocitose bacteriana pelas células CD14+ sanguíneas e do LBA e em leucócitos polimorfonucleares no sangue. Conclui-se que as alterações funcionais dos fagócitos apresentaram papel importante na patogenia da mannheimiose, que foram condizentes com os achados clínicos da mannheimiose e da evolução do tratamento quando realizado no início do processo nosológico. / Mannheimia haemolytica is an important bacterial pathogen associated with Bovine Respiratory Disease Complex (BRDC) and it is believed to be the predominant cause of the disease&rsquo;s evolution into a fibrinonecrotic pneumonia. A commensal inhabitant of the nasopharynx, M. haemolytica acts as an opportunist when host defenses are compromised. This study used an experimental infection model to investigate the possible local and systemic changes caused by M. haemolytica in inoculated calves. It sought to linearly follow the dynamics of the lower respiratory tract defense mechanisms, during the course of infection and after treatment with the antibacterial norfloxacin, which was administrated both with and without the anti-inflammatory flunixin meglumine. With clinical examination followed by bronchoscopy, this study evaluated the physiological modifications in defense cells and mediators of inflammation, and the in vitro influence of norfloxacin on phagocytes from the peripheral blood and Bronchoalveolar Lavage Fluid (BLF). Twelve (12) healthy calves were infected with M. haemolytica and posteriorly physically examined, and had samples of white cells from the peripheral blood and BLF analyzed for changes in count and physiology, further, the norfloxacin effect on phagocytes from the peripheral blood and BLF was also studied. The experimental infection proved itself to be successful based on clinical, bronchoscopic and cytological findings. Furthermore, the M. haemolytica experimental infection was associated with modifications in the subpopulations of lymphocytes CD8+ and &#1091;&delta; T cells, in intracellular production of Reactive Oxygen Species (ROS), viability and phagocytosis activity of CD14+ cells from the peripheral blood and BLF and granulocytes from the peripheral blood. No obvious change was observed in the expression of cytokines IL-1&#946;, IL-8 e TNF-&#945; by cells from the peripheral blood or BLF. The treatment with the antibacterial agent, with or without the anti-inflammatory, was proved to be successful in curing the disease, thus, the addition of an anti-inflammatory was considered unnecessary to revert the clinical infection and in the immune response. Although there was a systemic response during the course of infection, the local response was more noticeable. Another key finding of the present study was the in vitro effect of norfloxacin on the intracellular production of ROS and on phagocytosis activity of CD14+ cells from the peripheral blood and BLF and granulocytes from the peripheral blood. In conclusion, the functional changes in phagocytes play an important role in the pathogenesis of pulmonary infection caused by M. haemolytica, as they were consistent with the clinical findings of mannheimiosis and with the treatment when it was administrated in the beginning of the infection.

Objetivou-se caracterizar clinicamente a Doença Inflamatória de Vias Aéreas (DIVA) assim como verificar o comportamento da atividade dos macrófagos alveolares destes animais. Utilizaram-se 17 eqüinos adultos machos e fêmeas, com idade variando entre 11 e 24 anos, sendo oito do Regimento Escola de Cavalaria Andrade Neves (grupo Controle) e nove da Polícia Militar do Rio de Janeiro (grupo Doente, com DIVA). Estes animais não apresentavam história pregressa recente (dois meses) compatível com doença respiratória, nesse período não foram submetidos a nenhum tipo de tratamento e apresentavam resultados de leucogramas e determinações de fibrinogênio plasmático dentro da normalidade. Realizaram-se exames físicos, endoscópicos, mensurações da diferença de pressão intrapleural máxima (Ventigrafia), e citologia broncoalveolar. Para o grupo Controle os animais foram selecionados levando-se em conta os resultados de normalidade nos exames executados. O grupo Doente foi selecionado considerando-se achados de anormalidade compatíveis com alterações inflamatórias pulmonares no mínimo em dois exames, sendo obrigatórios os exames endoscópicos e de citologia broncoalveolar. Para a avaliação da atividade macrofágica, macrófagos alveolares obtidos através de lavagem broncoalveolar foram cultivados a 37ºC em atmosfera de 7% CO2, aderidos em lamínulas de vidro estéril, de 13 mm de diâmetro em placas de 24 poços contendo 300 µL de RPMI 1640 enriquecido com 10% de Soro Fetal Bovino inativado pelo calor. Realizaram-se testes de fagocitose de Zymosan (por uma hora) e de interação com Promastigotas de Leishmania braziliensis cepa 3456 (por uma até 48 horas pós infecção). A análise das funções vitais e das mensurações da diferença de pressão intrapleural máxima revelou, ainda que dentro de valores fisiológicos, diferenças significativas apenas para a temperatura corpórea e freqüência cardíaca. Para os achados do exame do exame físico, não foi detectada diferença estatística entre os grupos. Com relação ao lavado broncoalveolar, o grupo Doente apresentou aumento na contagem celular total, moderado aumento na porcentagem de neutrófilos, discreta redução na porcentagem de macrófagos, porém com aumento no número de macrófagos espumosos e de células epiteliais além de discreto aumento na porcentagem de eosinófilos. Não observou-se diferença, entre os grupos, na capacidade fagocítica com relação ao Zymosan. Porém, uma diferença significativa na capacidade fagocítica após uma hora de interação e redução do índice de sobrevivência de L. braziliensis após 48 horas de cultivo no grupo Doente foi observada. Os resultados demonstraram que a DIVA nos animais de policiamento apresentou caráter assintomático e que os macrófagos alveolares provenientes destes animais, quando comparados aos do grupo Controle, não apresentaram alteração em sua capacidade fagocítica para Zymosan, mas sim para L. braziliensis e um maior estado de ativação, caracterizado pela maior redução no índice de sobrevivência deste parasita após 48 horas de cultivo. / The objectives were to characterize clinically the inflammatory airway disease (IAD), as well as to verify the behavior of the alveolar macrophages activity in the horses. Seventeen adult horses, from both sexes, from 11 to 24 years old, were used, eight from the Regimento Escola de Cavalaria Andrade Neves (Control group) and nine from the Polícia Militar do Rio de Janeiro (Diseased group, with IAD). These animals did not present recente previous history (two months) compatible with respiratory disease. In this period the horses did not undergo any kind of treatment and presented leucogram and plasmatic fibrinogen determination results under normality. Physical and endoscopic examination, measurement of the maximal intrapleural pressure (Ventigraphy) and bronchoalveolar cytology were done. For the Control group, the animals were selected considering the normal results of the exams. The Diseased group was selected considering abnormal findings compatible with inflammatory pulmonary alterations at least in two exams, obligatory the endoscopic examination and bronchoalveolar cytology. To evaluate the macrophagic activity, alveolar macrophages obtained through bronchoalveolar lavage were cultured under 37oC in a 7% CO2 atmosphere, adhered to 13 mm diameter sterile glass cover slips , in 24 wells polystyrene plates containing 300µL of RPMI 1640 enriched with 10% Foetal Calf Serum inactivated by heat. Assays for phagocytosis of Zymosan (for 1h) and for promastigotes of Leishmania braziliensis strain 3456 binding (for one until 48 hours post-infection) were done. The vital functions and the measurement of the maximal intrapleural pressure measurement analyses showed, even under physiological values, significant differences for body temperature and heart rate. There was not statistical difference between the groups for the physical examination findings. In relation to the bronchoalveolar lavage, the Diseased group presented an increase in total cell count, a moderate increase in neutrophil percentage, a discrete reduction in macrophage percentage, but with a higher number of foamy macrophages and epithelial cells, and also a discrete increase of eosinophil percentage. It was not observed any difference between groups in the phagocytic capability in relation to Zymosan. However, a significant difference in the phagocytic capability after one hour interaction and a reduction of the survival index of L. braziliensis after 48 hours of culture in the Diseased group was observed. The results demonstrated that IAD in the police horses presented an asymptomatic characterization and that the alveolar macrophages from these horses, when compared to the Control group, did not present alteration in their phagocytic capacity for Zymosan, but did for L. braziliensis, and a greater activation status, characterized by a greater reduction of the survival index of L. braziliensis after 48 hours of culture.

As influências etárias do sistema imune de bezerros são descritas na primeira fase de vida desses animais e a hipótese de também ocorrerem ariações nos principais mecanismos de resposta inata do pulmão pode identificar períodos de maior suscetibilidade às principais doenças respiratórias que acometem os bezerros nesse período. Com a finalidade de minimizar os prejuízos econômicos associados às doenças respiratórias em bezerros, este estudo objetivou avaliar a dinâmica imunológica inata do sistema respiratório de bezerros sadios nos três primeiros meses de vida, no qual nove bezerros sadios foram acompanhados por três meses e submetidos a oito avaliações imunológicas. O material recuperado do lavado broncoalveolar colhido por broncoscopia foi submetido à avaliação funcional dos macrófagos alveolares utilizando as provas de fagocitose (SaPI e E.coli), burst oxidativo, quantificação de imunoglobulinas e expressão de CD14. Os dados foram avaliados pelo teste ANOVA oneway (unstacked) (paramétricos) e pelo teste Mann-Whitney (não paramétricos). Verificaramse alterações funcionais de fagócitos CD14+, que apesar de se manterem constantes em seus valores relativos durante todo o período, apresentou intensidade de fagocitose elevada pontual na terceira semana de vida e um aumento da fagocitose por mononucleares CD14+ aos 45 dias de idade com diminuição da intensidade da fagocitose por essas mesmas células a partir dessa idade. Conclui-se que a partir de 45 dias de vida os animais começam a montar uma resposta imune própria, porém pontual e que até os 90 dias não atingem a estabilidade necessária para atestar a conclusão do processo de maturação da resposta inata local. / The influences of age in calves\' immune system are described in their first phase of life. The hypothesis that variations occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. With the purpose of minimizing the economic losses associated with respiratory disease in calves, this study aimed to evaluate the innate immune dynamics of the respiratory system of healthy calves in the first three months of life. Nine healthy calves were monitored for three months and eight immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. Then, the alveolar macrophages in samples were identified by protein expression of CD14 and undergone functional evaluation of phagocytosis (SAPI and E.coli) and oxidative burst. Immunoglobulin were also quantified in samples. Data was assessed by one-way ANOVA (unstacked) (parametric) and the Mann-Whitney test nonparametric). Functional alterations in phagocytes CD14 + were observed, and although their relative values were kept throughout the period, higher intensity of phagocytosis in the third week and increased phagocytosis by macrophages CD14 + at 45 days of life was observed. Decreased intensity of phagocytosis was observed after this age. It is concluded that from 45 days of life on, calves began to maintain their immune response, but until 90 days of life they did not achieve the stability to conclude the maturation of local innate response.

A hemorragia pulmonar induzida por esforço (HPIE) ocorre com freqüência em cavalos atletas acarretando prejuízo econômico já que estes deverão ficar temporariamente afastados dos treinamentos e provas, além de receber tratamento adequado. O Puro-Sangue Inglês (PSI) utilizado em provas de corrida é o animal mais afetado devido ao grande esforço ao qual é submetido, porém animais de outras raças utilizados em outras modalidades de esportes hípicos também podem apresentar HPIE. O presente estudo teve como objetivo quantificar a ocorrência de casos de HPIE em animais utilizados em provas de salto, através do exame endoscópico e da análise citológica de amostras do lavado traqueobrônquico (LTB). Para a realização do presente experimento foram utilizados 50 cavalos adultos residentes nos clubes hípicos de São Paulo, que foram divididos em dois grupos, A e B, conforme as diferentes alturas dos obstáculos utilizados nas provas em que participaram. No grupo A foram inseridos os cavalos que saltam obstáculos de 1,00m a 1,20m de altura, e no grupo B, os cavalos que saltam obstáculos de 1,30m a 1,50m de altura. Foram realizados exames físicos destes animais, antes e depois da participação em uma prova. Após 1 hora do final da prova, os mesmos foram submetidos a um exame endoscópico em que eram observadas as possíveis alterações do trato respiratório anterior do animal sendo a principal delas a hemorragia pulmonar induzida por esforço. Foi realizada citologia do lavado traqueobônquico (LTB) por microscopia após citocentrifugação das amostras e posterior confecção das lâminas que foram então coradas por Rosenfeld. A principal observação durante a leitura das lâminas foi à presença de hemossiderófagos. No trabalho realizado são apresentados além da citologia do trato respiratório dados como os valores médios de freqüência cardíaca, freqüência respiratória e temperatura corpórea dos animais após a participação em uma prova. Estes dados foram relacionados às alturas dos obstáculos existentes em cada prova realizada pelos animais. Em relação à ocorrência de HPIE observamos que no grupo A 50% dos animais apresentaram HPIE e no grupo B a ocorrência foi de 66,67%. Foram observados todos os graus de HPIE, porém somente no grupo B houve ocorrência de graus IV e V, demonstrando assim a correlação entre o nível de esforço realizado pelos cavalos de salto e o grau de HPIE apresentado por eles. / Effort induced pulmonary hemorrhage (EIPH) frequently occurs in athlete horses causing economic losses not only due to temporary removal from training and competition, but also due to the cost of adequate treatment. Thoroughbreds used in racing are the most frequently affected breed due to the great effort to which they are submitted; however other breeds and crossbred sport horses can also have EIPH. The present study´s objective is to quantify the occurrence of cases of EIPH in animals used in show jumping, through the endoscopic examination and cytological analysis of tracheobronchial lavage (TBL) samples. For the present experiment fifty adult horses, housed in maneges in the city of São Paulo, were used. Horses were divided into two groups, named A and B according to the different heights of the obstacles. Group A included horses that jump heights of 1.0 to 1.2 m and group B, obstacles of 1.3 to 1.5 m. Physical examinations of these animals were carried through, before and after participation in each test. One hour after the end of each series, the same ones had been submitted to an endoscopic examination where the possible alterations of previous the respiratory treatment of the animal were observed being main of them the induced pulmonary hemorrhage for effort. Cytology of tracheobronchial lavage (TBL) was performed in cytocentrifuge slides prepared on the same day, slides were stained using Rosenfeld Romanov and observed examined under light microscopy. The major focus during the observation of the slides was the presence of hemosiderophages and grading of EIPH intensity (I through V) Besides the cytological evaluation of the respiratory tract, this study includes clinical data such as the average values of cardiac frequency, respiratory frequency and body temperature of the animals after the each competition. These data were correlated with the heights of the existing obstacles jumped by each athlete horse. In relation to the EIPH occurrence we observed that in the group A 50% of the animals presented EIPH and in group B the occurrence was of 66,67%. All grades of EIPH were observed when both groups were considered, however only animals in group B showed intense hemorrhage levels (IV or V) thus demonstrating and the correlation between the levels of effort the intensity of EIPH in jumpers.