Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis

Foram examinados 10 bezerros da raça Holandesa com duas a quatro semanas de idade, distribuídos aleatoriamente em dois grupos, controle e infectado. Os bezerros do grupo-controle foram inoculados por via intrabronquial com 5ml de solução salina fosfato-tamponada Dulbecco (DPBSS). Os do grupo infectado receberam um inóculo contendo 5x10(9) unidades formadoras de colônia-UFC de Mannheimia (Pasteurella) haemolytica, suspensas em 5ml de DPBSS. As amostras de sangue foram colhidas 15 minutos antes e uma, duas, quatro e seis horas após a inoculação. O proteinograma sérico foi obtido por eletroforese em gel de acrilamida. As concentrações séricas das proteínas de pesos moleculares 125.000 D (ceruloplasmina), 60.000 D (a1-antitripsina), 45.000 D (haptoglobina) e 40.000 D (glicoproteína ácida) foram significativamente maiores em bezerros infectados do que nos do grupo-controle. Os resultados indicam que as concentrações das proteínas de fase aguda se elevaram mais rapidamente que o previamente imaginado. O proteinograma sérico pode ser útil no monitoramento da evolução da pneumonia de bezerros causada por M. haemolytica.

A bovine model of a respiratory Parachlamydia acanthamoebae infection.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction.

Molecular cloning restriction enzyme analysis, bovine adenovirus type 2 genome

Adenoviruses are non-enveloped icosahedral-shaped particles which possess a double-stranded DNA genome. Currently, nearly 100 serotypes of adenoviruses have been identified, 48 of which are of human origin. Bovine adenoviruses (BAVs), causing both mild respiratory and/or enteral diseases in cattle, have been reported in many countries all over the world. Currently, nine serotypes of SAVs have been isolated which have been placed into two subgroups based on a number of characteristics which include complement fixation tests as well as the ability to replicate in various cell lines. Bovine adenovirus type 2 (BAV2), belonging to subgroup I, is able to cause pneumonia as well as pneumonic-like symptoms in calves. In this study, the genome of BAV2 (strain No. 19) was subcloned into the plasmid vector pUC19. In total, 16 plasmids were constructed; three carry internal San fragments (spanning 3.1 to 65.2% ), and 10 carry internal Pstl fragments (spanning 4.9 to 97.4%), of the viral genome. Each of these plasmids was analyzed using twelve restriction endonucleases; BamHI, CiaI, EcoRl, HiOOlll, Kpnl, Noll, NS(N, Ps~, Pvul, Saj, Xbal, and Xhol. Terminal end fragments were also cloned and analyzed, sUbsequent to the removal of the 5' terminal protein, in the form of 2 BamHI B fragments, cloned in opposite orientations (spanning 0 to 18.1°k), and one Pstll fragment (spanning 97.4 to 1000/0). These cloned fragments, along with two other plasmids previously constructed carrying internal EcoRI fragments (spanning 20.6 to 90.5%), were then used to construct a detailed physical restriction map using the twelve restriction endonucleases, as well as to estimate the size of the genome for BAV2(32.5 Kbp). The DNA sequences of the early region 1 (E1) and hexon-associated gene (protein IX) have also been determined. The amino acid sequences of four open reading frames (ORFs) have been compared to those of the E1 proteins and protein IX from other Ads.

Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, there is little information on strains infecting herds in Canada. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%. Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by real-time PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). Among the selected M. hyopneumoniae-positive lungs (n = 25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida. MLVA and PCR-RFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains.

Diferencias microbiológicas en pacientes con exacerbación severa de EPOC con o sin consolidación neumónica

La EPOC es una causa importante de morbilidad y mortalidad en el mundo y su prevalencia en Bogotá alcanza hasta 8,5%. Las exacerbaciones están asociadas a deterioro funcional y de la calidad de vida por lo que se consideran un factor cardinal de la enfermedad. En la literatura se ha descrito que las infecciones por bacterias y/o virus son las responsables del 78% de las exacerbaciones. Estos datos han sido descritos en poblaciones diferentes y no hay datos en la literatura que muestren cual es la epidemiología local de las exacerbaciones de EPOC y menos aún de aquellas que se asocian a consolidaciones neumónicas. Objetivo: Comparar la microbiología de las exacerbaciones severas de la EPOC que requieren ingreso a UCI con y sin infiltrados alveolares. Materiales y métodos: Estudio de corte transversal en el que se estudiaron pacientes con EPOC que ingresaron a la UCI Médica de la FCI-IC por exacerbación severa, asociada o no a infiltrados alveolares. Se tomaron muestras de microbiología, serológicas y radiografía de tórax para evaluar la etiología de la exacerbación, si se asocia a coinfección viral y a consolidación neumónica o no. Resultados: No se encontró una diferencia estadísticamente significativa en la microbiología de los diferentes grupos evaluados. Se encontró un resistencia global del 24% y llama la atención que hay una alta prevalencia de Serratia Marcescens AMPc entre los 2 grupos, germen que no está descrito como patógeno común en la literatura. Se encontraron diferencias en cuanto a factores de riesgo para presentar neumonía asociada como lo son un mayor índice de paquetes/año (55.1.6 vs. 36.3 paq/año, sig.=0.021). Así mismo se demostró que los pacientes con neumonía asociada presentan mayor necesidad de IOT (48.9 vs. 23.9, sig.=0.013). No hay diferencia significativa en desenlaces como mortalidad (20.5 vs. 13.0, sig.=0.346). Conclusiones: A pesar de no haber diferencia microbiológica entre los 2 grupos se encontraron variables como factores de riesgo y variables clínicas que pueden ayudar a proponer planes de manejo en los dos escenarios. El hecho de encontrar un paciente con neumonía asociada al cuadro de exacerbación no debe afectar en la toma de decisiones en relación al tratamiento antibiótico.

Estudo clínico e laboratorial da pneumonia de bezerros induzida pela inoculação intrabronquial de Mannheimia haemolytica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mannheimiose pulmonar experimental em bezerros: swab nasal e nasofaringeano como auxílio diagnóstico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lavado traqueobrônquico por via nasotraqueal como metodologia de colheita de células do trato respiratório de ovinos sadios e portadores de afecções pulmonares

Os estudos das secreções traqueobrônquicas são amplamente utilizados nas pesquisas de doenças pulmonares nas diversas espécies animais, inclusive no homem. Os objetivos desta pesquisa foram a viabilização da técnica de colheita de lavado traqueobrônquico na espécie ovina e o estudo da relação clínico-citológica do lavado de ovinos portadores de broncopneumonia e sadios. Foram utilizados 33 ovinos, 18 sadios e 15 portadores de enfermidade respiratória com sinais clínicos de envolvimento das vias aéreas, divididos nos respectivos grupos, GS e GD. Após o exame físico foi realizado o lavado traqueobrônquico por via nasotraqueal. A colheita do lavado foi feita com a inoculação e aspiração de solução fisiológica estéril. As amostras foram processadas citologicamente através de citocentrifugação e coradas pelos métodos Wright-Giemsa e Shorr. Tanto a contagem total de células epiteliais quanto o número de hemácias por mililitro foi maior no grupo de animais com broncopneumonia. Nos animais sadios notou-se predomínio de macrófagos, seguido por células epiteliais cilíndricas, neutrófilos e linfócitos. No grupo de animais doentes havia menor número de macrófagos, e predomínio da população de neutrófilos. Por ser de fácil realização, pouco dispendiosa e pela obtenção representativa de material, a técnica estudada mostrou-se eficaz na obtenção de fluidos traqueobrônquicos e, portanto um bom método de colheita de células para uso nas pesquisas de vias aéreas.

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

Objective-To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils.Sample Population-Blood collected from 5 healthy adult Holstein cows.Procedure-Isolated neutrophils or whole brood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured.Results-incubation with TNF-alpha, IL-8; PAF, and IFS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils.Conclusions and Clinical Relevance-Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and setectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.

Effect of porcine reproductive and respiratory syndrome virus on subsequent Pasteurella multocida challenge in pigs

This trial was conducted to evaluate the effect of Porcine reproductive and respiratory syndrome virus (PRRSv) on a subsequent challenge with Pasteurella multocida in pigs. Sixteen, 3-4 week-old piglets, from a PRRSv and Aujeszky disease virus (ADV) free herd were used. Animals were equally and randomly allocated in four groups which were treated according the following schedule: Group I: negative controls; Group II: inoculation with only PRRSV; Group III: inoculation with PRRSV and P. multocida; Group IV: inoculation with ADV and multocida (positive controls), PRRSV and ADV were inoculated intranasally, at the doses of 10(4.6) and 10(4.5) TCID50/ml, respectively. Five days later, pigs from groups III and IV were inoculated intranasally, with two ml of a 10(9) CFU/mL suspension of equal parts of P. multocida, strains A52 and A24. No lesions were observed in piglets of group I. Microscopically, interstitial pneumonia was identified in all piglets of groups II and III and 3/4 piglets from group IV. Bronchopneumonia was detected in 3/4 of the piglets from group III and in all animals of group TV which, additionally, showed meningo-encephalitis and purulent rhinitis. Macroscopically, only piglets of groups III and IV had lung consolidation. However, much lower pneumonic scores (2.3%) were observed in group III, where 3 of 4 piglets were affected. on the other hand, all piglets of group IV showed some degree of pulmonary consolidation, with a mean score of 13.7%. Based on these results, it appears that the role of PRRSV as a initiator of secondary diseases is still undefined, but is probably mild, There was no clear interaction between PRRSV and Pasteurella multocida under the conditions and strains tested here. (C) 1997 Elsevier B.V. B.V.

Mannheimiose pneumônica experimentalmente induzida em bezerros pela Mannheimia (Pasteurella) haemolytica A1-cepa D153: achados do exame físico, hemograma e swabs nasal e nasofaringeano

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8+ T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366–374 (DbNP366). The numbers of CD8+ DbNP366+ cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8+ DbNP366+ cells tended to be more “activated” than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8+ DbNP366+ set was always much higher than the percentage of CD8+ T cells that could be induced to synthesize interferon γ after short-term, in vitro stimulation with the NP366–374 peptide, whereas these values were generally comparable for virus-specific CD8+ T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8+ T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8+ T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

Measuring the diaspora for virus-specific CD8+ T cells

The CD8+ T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of DbNP366- and DbPA224-specific CD8+ T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8+ tetramer+ populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the “whole mouse” virus-specific CD8+ T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8+DbNP366+ and CD8+DbPA224+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 “activation marker” were detected consistently on virus-specific CD8+ T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69hi T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of “resting” CD8+ memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M.bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.

Setúbal e a Primeira Guerra Mundial (1914-1918)

A presente dissertação é circunscrita no espaço e no tempo, tendo como objecto de estudo fundamental o concelho de Setúbal durante a I Guerra Mundial (1914-1918). Enquadrando historicamente os impactos do conflito no contexto nacional, Setúbal é revisitada entre os finais de oitocentos e os primórdios do século XX a fim de retratar política, económica, social e culturalmente um dos maiores centros urbanos portugueses dos finais da Monarquia Constitucional e do início da I República. Numa lógica de impactos políticos e económico-sociais, esta investigação promove o redescobrir das vivências quotidianas de Setúbal durante um dos períodos mais negros da História da Humanidade. Através do estudo dos mecanismos de acção e intervenção municipais, bem como dos resultados eleitorais e dinâmicas políticopartidárias locais, transmite-se como foram vividos acontecimentos marcantes da vida pública de Portugal, nomeadamente o 14 de Maio de 1915 e o coup d’état sidonista, realçando como dimensões operárias anárquicas e sindicalistas conviviam com grupos conservadores, católicos e monárquicos. Num plano de economia de guerra, o mundo agrícola é abordado em diferentes sectores, nomeadamente na laranja, na cortiça e nos vinhos, dando maior atenção às questões relacionadas com a decadência da fruta e à abertura de novos mercados exportadores, como o Brasil, pela Casa José Maria da Fonseca. A indústria de conservas de peixe, que viveu um boom neste período como resultado da intensa procura externa dos países aliados que tinham nas latas de conserva o alimento das tropas nas trincheiras, representa outro foco de análise deste trabalho. Consequentemente aborda-se o papel da estrutura portuária local na resposta às necessidades de consumo britânicas, francesas e italianas. Por fim, os impactos sociais do conflito em Setúbal constituem um dos enunciados desta investigação, compreendendo como o estudo de uma imprensa dividida na questão intervencionismo versus anti-intervencionismo reflectiu um concelho que viveu como poucos a crise de abastecimentos e a carestia de vida. A proliferação de greves, manifestações contra a guerra, as deserções militares e os assaltos colectivos representam o culminar de um vulcão que entrou em erupção e que ainda sofreu as agruras da epidemia da gripe pneumónica até ao final do ano de 1918.