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1.
Vet Immunol Immunopathol ; 236: 110251, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33901710

RESUMO

Bacterial lung disease caused by Mannheimia haemolytica inflict significant mortality and morbidity resulting in enormous economic losses to cattle industry. The use of antibiotics is becoming more challenging because of development of anti-microbial resistance. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3), a pattern-recognition receptor is produced at sites of inflammation by many cell types, recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in lungs of normal and M. haemolytica-infected calves and normal and E. coli lipopolysaccharide-treated cattle neutrophils using light and electron microscopic immunochemistry and Western blots. Immunohistology showed the presence of PTX3 in airway epithelial cells, alveolar septa and macrophages in normal and inflamed lungs of calves and the blots showed a significant increase in the expression of PTX3 in lungs from infected calves. Immuno-gold electron microscopy showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages (PIMs). Immunohistochemical staining for PTX3 in peripheral blood neutrophils shows an altered staining pattern in neutrophils stimulated with lipopolysachharide (LPS). However, western blots no significant change in PTX3 amount in LPS-treated neutrophils compared to the controls. These are the first data on the expression of PTX3 in the lungs and the neutrophils of cattle which may add to our understanding of innate immunity in cattle lungs.


Assuntos
Proteína C-Reativa/genética , Expressão Gênica , Pulmão/microbiologia , Mannheimia haemolytica/imunologia , Neutrófilos/imunologia , Pneumonia Enzoótica dos Bezerros/microbiologia , Componente Amiloide P Sérico/genética , Fatores Etários , Animais , Bovinos , Imunidade Inata , Pulmão/citologia , Pulmão/imunologia
2.
J Immunol Methods ; 493: 113037, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33722512

RESUMO

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli-based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens.


Assuntos
Actinobacillus pleuropneumoniae/química , Ensaio de Imunoadsorção Enzimática , Mannheimia haemolytica/química , Neisseria meningitidis/química , Proteína B de Ligação a Transferrina/imunologia , Actinobacillus pleuropneumoniae/imunologia , Avidina/química , Avidina/imunologia , Biotina/química , Biotina/imunologia , Mannheimia haemolytica/imunologia , Neisseria meningitidis/imunologia , Poliestirenos/química , Cloreto de Polivinila/química , Proteína B de Ligação a Transferrina/química
3.
Vet Clin North Am Food Anim Pract ; 36(2): 349-359, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32451029

RESUMO

Calves vary considerably in their pathologic and clinical responses to infection of the lung with bacteria. The reasons may include resistance to infection because of pre-existing immunity, development of effective immune responses, or infection with a minimally virulent bacterial strain. However, studies of natural disease and of experimental infections indicate that some calves develop only mild lung lesions and minimal clinical signs despite substantial numbers of pathogenic bacteria in the lung. This may represent "tolerance" to pulmonary infection because these calves are able to control their inflammatory responses or protect the lung from damage, without necessarily eliminating bacterial infection. Conversely, risk factors might predispose to bovine respiratory disease by triggering a loss of tolerance that results in a harmful inflammatory and tissue-damaging response to infection.


Assuntos
Complexo Respiratório Bovino/imunologia , Complexo Respiratório Bovino/microbiologia , Mannheimia haemolytica/imunologia , Animais , Complexo Respiratório Bovino/patologia , Bovinos , Mannheimia haemolytica/patogenicidade
4.
J Vet Med Sci ; 81(9): 1355-1358, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31316038

RESUMO

To evaluate the preventive effects of combined vaccination for Pasteurella multocida, Mannheimia haemolytica and Histophilus somni on respiratory diseases in Japanese Black calves, 295 calves at one farm were alternately assigned to two groups; 147 calves received the vaccine at 4 and 8 weeks of age (vaccination group), and the other 148 calves did not receive vaccine (control group). The incidences of respiratory diseases were 25.9 and 70.9% in the vaccination and control groups, respectively, and the odds ratio for comparison between the two groups was 0.143 (95% confidence interval: 0.086-0.238). Administration of the multiple vaccine to Japanese black calves might be one of effective factor for prevention of respiratory diseases.


Assuntos
Doenças dos Bovinos/prevenção & controle , Mannheimia haemolytica/imunologia , Pasteurella multocida/imunologia , Pasteurellaceae/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Doenças Respiratórias/imunologia , Doenças Respiratórias/prevenção & controle , Vacinação/veterinária
5.
Arch Razi Inst ; 74(2): 111-118, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31232560

RESUMO

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-β-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Expressão Gênica , Mannheimia haemolytica/genética , Mannheimia haemolytica/imunologia , Formação de Anticorpos , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
6.
J Anim Sci ; 97(7): 2739-2749, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31069378

RESUMO

The goal of this study was to compare the cell-mediated immune responses of highly commingled, sale-barn origin calves (STR; n = 10) to those of single source calves that had been weaned for 60 d (UNS; n = 10). Peripheral blood mononuclear cells and neutrophils (PMNs) were isolated from jugular venous blood of each calf. Peripheral blood mononuclear cells were stimulated with Concanavalin A (ConA), BVDV-1, BVDV-2, BHV-1, Mannheimia haemolytica, and Pasteurella multocida and evaluated for clonal proliferation and secretion of IL-8 into cell culture supernatants. The native functional capacities of PMNs were evaluated in response to stimulation with heat-killed Escherichia coli and Staphylococcus aureus. Complete blood counts and serum biochemical profiles were performed for each animal at the time of sample collection. Compared with STR calves, UNS calves had greater lymphocyte proliferative responses following stimulation BVDV1 (P = 0.041), BVDV2 (P = 0.002), BHV-1 (P = 0.001), M. haemolytica (P = 0.016), and P. multocida (P = 0.049). In addition, PMNs isolated from UNS calves had a greater ability to phagocytose E. coli (P = 0.001) and S. aureus (P = 0.003) when compared with STR calves. Serum nonesterified fatty acids were higher in STR calves (P < 0.001). Serum ß-hydroxybutyrate was lower in STR calves (P < 0.003). These data suggest that immunologic and physiologic differences exist between STR and UNS calves. Although the underlying mechanisms for these differences are not clear, it is possible that combinations of energy imbalances, stress-induced immunosuppression, and general immune naiveté may predispose STR calves to an increased risk of morbidity and mortality due to bovine respiratory disease.


Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Bovinos/imunologia , Imunidade Celular , Vacinas Virais/imunologia , Animais , Bovinos , Doenças dos Bovinos/prevenção & controle , Concanavalina A/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Escherichia coli/imunologia , Herpesvirus Bovino 1/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Mannheimia haemolytica/imunologia , Neutrófilos/imunologia , Pasteurella multocida/imunologia , Distribuição Aleatória , Staphylococcus aureus/imunologia , Estresse Fisiológico , Vacinas Atenuadas/imunologia , Desmame
7.
J Anim Sci ; 97(3): 1171-1184, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30597005

RESUMO

Objectives were to determine whether live yeast (LY) supplementation would affect daily dry matter feed intake, body weight (BW), immune, and febrile responses to a viral-bacterial (VB) respiratory challenge. Crossbred heifers (N = 38, BW = 230 ± 16.4 kg) were allocated into a 2 × 2 factorial treatment arrangement: Factor 1 = roughage-based diet with or without LY (Saccharomyces cerevisiae boulardii CNCM I-1079, 62.5 g/hd/d), Factor 2 = VB, intranasal administration of bovine herpesvirus-1 (BHV-1, 2 ×108, PFU) on day 0 and endobronchial inoculation with Mannheimia haemolytica (5.4 × 1010, CFU) on day 3, or intranasal saline administration followed by inoculation with phosphate buffer solution (PBS). Heifers were fed their respective diets for 27 d prior to VB challenge on day 0. Heifers were housed by treatment and group-fed using electronic feedbunks. Thermo-boluses (Medria; Châteaubourg, FR) measured rumen temperature (RUT) at 5-min intervals and rectal temperature and whole blood samples were collected on days 0, 3 to 8, 10, 13, and 15. Data were analyzed using repeated measures in the mixed procedure of SAS with fixed effects of day, diet, inoculation, and their interactions. Animals fed LY exhibited a 16% increase (P = 0.02) in neutrophils relative to CON. Diet × inoculation × day interactions were detected for monocytes and haptoglobin. The VB-LY had the greatest (P < 0.05) concentration of monocytes on day 4, followed by VB-CON which was greater (P < 0.05) than PBS treatments. Haptoglobin concentration was greatest (P < 0.02) for VB-CON on day 5, followed by VB-LY which was greater (P < 0.05) than PBS. Heifers supplemented with LY had less (P < 0.05) haptoglobin production than CON. The VB challenge produced nasal lesions that increased (P < 0.01) with day, reaching a zenith on day 6 with 70% of the nares covered with plaques, and increased (P < 0.05) neutrophils on days 3 to 5. The VB challenge increased RUT (P < 0.05) days 2 to 7 and rectal temperature (P < 0.05) on days 0 and 3 to 6. The increased rectal temperature on day 0 was likely due to increased ambient temperature at time of challenge, as VB heifers were processed after the PBS heifers to avoid contamination. The VB challenge was effective at stimulating immune responses, and RUT was effective for measuring febrile responses. These results indicate that prior LY supplementation altered the leukogram in response to VB challenge, suggestive of increased innate immune response.


Assuntos
Complexo Respiratório Bovino/imunologia , Doenças dos Bovinos/imunologia , Suplementos Nutricionais , Herpesvirus Bovino 1/imunologia , Mannheimia haemolytica/imunologia , Saccharomyces cerevisiae , Ração Animal/análise , Animais , Complexo Respiratório Bovino/microbiologia , Bovinos , Doenças dos Bovinos/microbiologia , Dieta/veterinária , Ingestão de Alimentos , Feminino , Haptoglobinas/análise , Imunidade Inata , Rúmen/fisiologia , Regulação para Cima
8.
J Anim Sci ; 97(2): 596-609, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496421

RESUMO

Objectives of this experiment were to examine the effects of live yeast (LY) supplementation on immunological, physiological, and behavioral responses in steers experimentally challenged with Mannheimia haemolytica (MH). Thirty-six crossbred Angus steers (BW = 352 ± 23 kg) seronegative for MH were allocated within a 2 × 2 factorial arrangement: Factor 1 = roughage-based diet with LY (Saccharomyces cerevisiae boulardii CNCM I-1079, 25 g·per steer daily) or negative control (CON). Factor 2 = bronchoselective endoscopic inoculation with MH or phosphate buffer solution (PBS). Steers were fed their respective diets for 28 d prior to MH challenge on day 0. Reticulo-rumen temperature (RUT; ThermoBolus, Medria) was measured continuously at 5-min intervals and rectal temperature on days -4, 0, 1, 2, 3, 5, 7, 10, and 14 relative to MH inoculation. Compared with PBS-treated steers, the steers inoculated with MH had increased (P < 0.05) RUT from 2 to 24 h, reaching a zenith (>41 °C) 9 to 11 h post-MH challenge, whereas rectal temperature was increased (P < 0.04) in MH-inoculated steers on day 1 post-MH challenge. Supplementation with LY increased (P < 0.05) rectal temperature on days 0, 7, and 10, relative to CON steers. There were inoculation x day interactions (P < 0.01) for lymphocyte, neutrophil, leukocyte, and haptoglobin concentrations. Steers challenged with MH had increased (P < 0.05) neutrophil concentration from days 1 to 3, leukocyte concentration on days 1 and 2 and haptoglobin concentration on days 1 to 5 post-MH challenge compared with PBS-treated steers. Steers supplemented with LY exhibited increased (P < 0.02) cortisol throughout the study compared with the CON treatment. Following inoculation, MH-challenged steers exhibited reduced (P < 0.05) DMI, eating rate, frequency, and duration of bunk visit (BV) events compared with PBS-treated steers. Results from this study demonstrate that the experimental challenge model effectively stimulated acute-immune responses and behavioral changes that are synonymous with naturally occurring bovine respiratory disease (BRD). However, supplementation with LY minimally altered the impact of the MH challenge on physiological and behavioral responses in this study. Continuously measured RUT was more sensitive at detecting febrile responses to MH challenge than rectal temperature. These results serve to guide future research on behavioral and physiological alterations exhibited during a BRD infection.


Assuntos
Bovinos/fisiologia , Suplementos Nutricionais , Comportamento Alimentar , Mannheimia haemolytica/imunologia , Saccharomyces cerevisiae , Ração Animal/análise , Animais , Bovinos/imunologia , Bovinos/microbiologia , Dieta/veterinária , Ingestão de Alimentos/efeitos dos fármacos , Haptoglobinas/análise , Masculino , Rúmen/fisiologia , Regulação para Cima
9.
Vet Immunol Immunopathol ; 196: 53-59, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29695326

RESUMO

The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ±â€¯42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14+ macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.


Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Macrófagos/metabolismo , Mannheimia haemolytica/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Fagocitose , Ficoeritrina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Animais , Bovinos , Citometria de Fluxo/veterinária , Macrófagos/química , Macrófagos/imunologia , Macrófagos Alveolares/química , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Mannheimia haemolytica/imunologia , Microscopia Confocal/veterinária , Monócitos/química , Monócitos/imunologia , Neutrófilos/química , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/análise
10.
Anim Health Res Rev ; 19(2): 79-99, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30683173

RESUMO

Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/normas , Mannheimia haemolytica , Pasteurelose Pneumônica/prevenção & controle , Animais , Proteínas da Membrana Bacteriana Externa , Bovinos , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/imunologia , Pasteurelose Pneumônica/microbiologia , Fatores de Virulência
11.
Vet Immunol Immunopathol ; 195: 19-24, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29249313

RESUMO

This study examined the use of comparative genomic analysis for vaccine design against Mannheimia haemolytica, a respiratory pathogen of ruminants. A total of 2,341genes were identified in at least half of the 23 genomes. Of these, a total of 240 were identified to code for N-terminal signal peptides with diverse sub-cellular localizations (78 periplasmic, 52 outer membrane, 15 extracellular, 13 cytoplasmic membrane and 82 unknown) and were examined in an ELISA assay using a coupled-cell free transcription/translation system for protein expressionwith antisera from cattle challenged with serovars 1, 2 or 6 of M. haemolytica. In total, 186 proteins were immunoreactive to at least one sera type and of these, 105 were immunoreactive to all sera screened. The top ten antigens based on immunoreactivity were serine protease Ssa-1 (AC570_10970), an ABC dipeptid transporter substrate-binding protein (AC570_04010), a ribonucleotide reductase (AC570_10780), competence protein ComE (AC570_11510), a filamentous hemagglutinin (AC570_01600), a molybdenum ABC transporter solute-binding protein (AC570_10275), a conserved hypothetical protein (AC570_07570), a porin protein (AC569_05045), an outer membrane assembly protein YeaT (AC570_03060), and an ABC transporter maltose binding protein MalE (AC570_00140). The framework generated from this research can be further applied towards rapid vaccine design against other pathogens involved in complex respiratory infections in cattle.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Mannheimia haemolytica/imunologia , Animais , Proteínas de Bactérias/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Sistema Livre de Células/microbiologia , Simulação por Computador , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaios de Triagem em Larga Escala/veterinária , Infecções por Pasteurellaceae/prevenção & controle , Infecções por Pasteurellaceae/veterinária
12.
Vaccine ; 35(12): 1630-1636, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28228321

RESUMO

Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible. A vaccine that does not require booster doses, therefore, is ideal for vaccination of BHS. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation which obviates the need for booster administration. The objective of this study was to evaluate the potential of bovine herpesvirus 1 (BHV-1) as a vector encoding M. haemolytica immunogens. As the first step towards this goal, the permissiveness of BHS for BHV-1 infection was determined. BHS inoculated with wild-type BHV-1 shed the virus following infection. The lytic phase of infection was superseded by latency, and treatment of latently-infected BHS with dexamethasone reactivated the virus. A recombinant BHV-1-vectored vaccine encoding a leukotoxin-neutralizing epitope and an immuno-dominant epitope of the outer membrane protein PlpE was developed by replacing the viral glycoprotein C gene with a leukotoxin-plpE chimeric gene. Four of six BHS vaccinated with the recombinant virus developed significant leukotoxin-neutralizing antibodies at day 21 post-vaccination, while two of six BHS developed significant surface antigen antibodies at day 17 post-vaccination. These antibodies, however, were inadequate for protection of BHS against M. haemolytica challenge. These data indicate that BHV-1 is a suitable vector for immunization of BHS, but additional experimentation with the chimeric insert is necessary for development of a more efficacious vaccine.


Assuntos
Vacinas Bacterianas/imunologia , Portadores de Fármacos , Herpesvirus Bovino 1/genética , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/prevenção & controle , Doenças dos Ovinos/prevenção & controle , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Bovinos , Vetores Genéticos , Herpesvirus Bovino 1/fisiologia , Ovinos , Carneiro da Montanha , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Ativação Viral , Latência Viral
13.
Res Vet Sci ; 109: 21-28, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27892870

RESUMO

Bovine respiratory disease complex (BRDc) is a multi-factorial disease, involving both viral and bacterial pathogens, that negatively impacts the cattle feedlot industry. A nitric oxide releasing solution (NORS) has been developed and shown to have potential in the prevention of BRDc. This study investigated the underlying immunological mechanisms through which the nitroslyating agent NORS provides protection against the development of BRDc in susceptible cattle. An in vitro BRDc experimental model was designed using bovine peripheral blood mononuclear cells (PBMCs) which were infected with bovine herpesvirus 1 (BHV-1) and subsequently cultured with lipopolysaccharides (LPS) extracted from Mannheimia haemolytica bacteria. The cells were treated with NORS following viral infection to reflect the timing of administering the NORS treatment in feedlots during initial processing. An expression and protein analysis of key genes involved in the innate immune response was carried out. The BRDc model produced significant increases in gene expression (p<0.01) and protein release (p<0.05) of the proinflammatory cytokines IL-1ß and TNF. Treatment with NORS reduced the protein levels of IL-1ß (0.39-fold↓) (p<0.05) and TNF (0.48-fold↓) (p<0.01) in the BRDc experimental group when compared against the non-treatment BRDc controls. TLR4 expression, having been significantly reduced under the BRDc experimental conditions (0.33-fold↓) (p<0.05), increased significantly (0.76-fold↑) (p<0.05) following NORS treatment. This study provides evidence suggesting that NO may protect against the development of BRDc by limiting deleterious inflammation while simultaneously increasing TLR4 expression and enhancing the ability of the host to detect and respond to bacterial pathogens.


Assuntos
Complexo Respiratório Bovino/imunologia , Herpesvirus Bovino 1/imunologia , Leucócitos Mononucleares/fisiologia , Lipopolissacarídeos/toxicidade , Mannheimia haemolytica/metabolismo , Óxido Nítrico/metabolismo , Animais , Bovinos , Células Cultivadas , Citocinas/genética , Regulação da Expressão Gênica/imunologia , Lipopolissacarídeos/imunologia , Mannheimia haemolytica/imunologia
14.
J Anim Sci ; 94(11): 4799-4808, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27898956

RESUMO

Bovine respiratory disease (BRD) is the most common and economically detrimental disease of beef cattle during the postweaning period, causing the majority of morbidity and mortality in feedlots. The pathogenesis of this disease often includes an initial viral infection, which can predispose cattle to a secondary bacterial infection. The objective of this experiment was to determine the effects of timing of an intratracheal (MH) challenge relative to 72 h of natural exposure to bovine viral diarrhea virus (BVDV) type 1b persistently infected (PI) calves on performance, serum antibody production, total and differential white blood cell (WBC) count, rectal temperature, clinical severity score (CS), and haptoglobin (Hp). Steers ( = 24; 276 ± 31 kg initial BW) were randomly allocated to 1 of 3 treatments (8 steers/treatment) in a randomized complete block design. Treatments were steers not exposed to calves PI with BVDV 1b and not challenged with MH (CON), steers intratracheally challenged with MH 84 h after being exposed to calves PI with BVDV 1b for 72 h (LateCh), and steers intratracheally challenged with MH 12 h after being exposed to calves PI with BVDV 1b for 72 h (EarlyCh). Performance (ADG, DMI, and G:F) was decreased ( < 0.001) for both EarlyCh and LateCh from d 0 to 4. From d 5 to 17, LateCh appeared to compensate for this lost performance and demonstrated increased ADG ( = 0.01) and G:F ( = 0.01) compared with EarlyCh. Both EarlyCh and LateCh had decreased platelet counts ( < 0.001) compared with CON. Antibody concentrations of BVDV and MH were higher ( < 0.05) for both EarlyCh and LateCh compared with CON. Rectal temperature, CS, and Hp increased ( < 0.001) across time from h 4 to 48, h 4 to 36, and h 8 to 168, respectively. Within 24 h of MH challenge, WBC and neutrophil concentrations within the blood increased whereas lymphocyte concentrations decreased. The timing of BVDV exposure relative to a MH challenge appears to influence the CS and acute phase response associated with BRD. As typical beef cattle marketing channels allow for variation in the timing of respiratory pathogen exposure, understanding the physiological changes in morbid cattle will lead to improved management of BRD.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Bovinos/fisiologia , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Mannheimia haemolytica/imunologia , Infecções por Pasteurellaceae/veterinária , Animais , Temperatura Corporal , Doença das Mucosas por Vírus da Diarreia Viral Bovina/complicações , Doença das Mucosas por Vírus da Diarreia Viral Bovina/metabolismo , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos/imunologia , Haptoglobinas/análise , Haptoglobinas/metabolismo , Masculino , Infecções por Pasteurellaceae/complicações , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/microbiologia , Distribuição Aleatória , Carne Vermelha , Fatores de Tempo
15.
Vet Res ; 47(1): 93, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27599994

RESUMO

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 µM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lactoferrina/metabolismo , Mannheimia haemolytica/metabolismo , Animais , Apoproteínas/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Bovinos , Eletroforese em Gel Bidimensional , Imunidade Inata , Lactoferrina/imunologia , Mannheimia haemolytica/imunologia , Simulação de Acoplamento Molecular , Pasteurelose Pneumônica/imunologia , Pasteurelose Pneumônica/metabolismo
16.
Vet Immunol Immunopathol ; 175: 36-41, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27269790

RESUMO

Mannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Exotoxinas/imunologia , Mannheimia haemolytica/imunologia , Mannheimia haemolytica/patogenicidade , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Carneiro da Montanha/imunologia , Carneiro da Montanha/microbiologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Neutralizantes/biossíntese , Especificidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Exotoxinas/química , Exotoxinas/genética , Feminino , Vetores Genéticos , Herpesviridae/genética , Mannheimia haemolytica/genética , Camundongos , Camundongos Endogâmicos BALB C , Pasteurelose Pneumônica/imunologia , Pasteurelose Pneumônica/microbiologia , Pasteurelose Pneumônica/prevenção & controle , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ovinos , Doenças dos Ovinos/prevenção & controle , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
17.
PLoS One ; 11(3): e0151083, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26942409

RESUMO

Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and γδ T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing γδ T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and γδ T cells in the pathogenesis of BRDC.


Assuntos
Coinfecção/imunologia , Interleucina-17/biossíntese , Mannheimia haemolytica/imunologia , Infecções por Pasteurellaceae/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Bovino/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Bovinos , Células Cultivadas , Coinfecção/microbiologia , Coinfecção/virologia , Modelos Animais de Doenças , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Infecções por Pasteurellaceae/sangue , Infecções por Pasteurellaceae/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Vírus Respiratório Sincicial/sangue , Infecções por Vírus Respiratório Sincicial/virologia , Células Th17/imunologia , Vacinação , Vacinas Atenuadas/imunologia
18.
J Microbiol Methods ; 116: 53-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26149626

RESUMO

Advancements in high-throughput "omics" technologies have revolutionized the way vaccine candidates are identified. Now every surface expressed protein that an organism produces can be identified in silico and possibly made available for the rapid development of recombinant/subunit vaccines. However, evaluating the antigenicity of a large number of candidate proteins is an immense challenge, typically requiring cloning of several hundred candidates followed by immunogenicity screening. Here we report the development of a rapid, high-throughput method for screening candidate proteins for vaccines. This method involves utilizing a coupled, cell-free transcription-translation system to screen tagged proteins that are captured at the C-termini using appropriate ligand coated wells in 96 well ELISA plates. The template DNA for the cell-free expression is generated by two sequential PCRs and includes gene coding sequences, promoter, terminator, other necessary cis-acting elements and appropriate tag sequences. The process generates expressible candidate proteins containing two different peptide tags at the N- and the C-termini of the protein molecules. Proteins are screened in parallel for their quantity and immunoreactivity with N-terminal tag antibodies and antisera raised against the pathogen of interest, respectively. Normalization against the total detectable bound protein in the control wells allows for the identification of highly immunoreactive candidates. For this study we selected 30 representatives of >300 potential candidate proteins from Mannheimia haemolytica, a bacterial agent of pneumonia in feedlot cattle for expression with N-terminal Strep-II and C-terminal His(x6)-tag and evaluated their relative immunoreactivities using Strep-tactin-HRP and rabbit antisera generated against M. haemolytica. Using this system we were able to swiftly and quantitatively analyze and rank the suitability of proteins to identify potentially viable vaccine candidates, with the majority of the high ranking candidates being associated with virulence and pathogenicity. The system is adaptable to any bacterial target and presents an alternative to conventional laborious cloning, expression and screening procedures.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Vacinas Bacterianas , Ensaios de Triagem em Larga Escala/métodos , Mannheimia haemolytica/química , Mannheimia haemolytica/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática , Mannheimia haemolytica/patogenicidade , Reação em Cadeia da Polimerase/métodos , Proteômica , Coelhos , Virulência
19.
Vet Microbiol ; 177(3-4): 386-93, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25843945

RESUMO

Ovine pneumonia is an economic important disease worldwide for the sheep industry. Multiple serotypes (S) of Mannheimia haemolytica are involved in the disease and S2 and S1 are the most frequent isolates associated with lung lesions in sheep. Vaccines based on some M. haemolytica S2 strains have been shown to have poor immunogenicity. The objective of this study was to determine the cross-protection effect of an S1 strain based vaccine, Bovilis MH, in sheep against an experimental challenge with an S2 strain. Lambs (n=12) in the vaccine group were injected subcutaneously with 1 ml of the Bovilis MH vaccine, and revaccinated 4 weeks later, while the control group (n=12) received 1 ml of saline at each occasion. Two weeks after revaccination, all lambs were challenged intratracheally with parainfluenza virus 3, and with an M. haemolytica S2 strain at day 7 post-viral challenge. The proportion of animals having pyrexia in the first 2 days post-bacterial challenge was significantly less in the vaccine group compared to the control group (P<0.05). The animals in the vaccine group had significantly lower dyspnoea scores and lung/bodyweight ratio than those in the control group (P<0.05). The vaccine provided 49.1% overall protection. Prior to the challenge, the vaccinated animals had significantly higher titres of antibodies to S1 and S2 whole cell antigens and to leukotoxins produced by S1 and S2 strains compared to the control animals (P<0.05). The S1 strain vaccine provided considerable cross-protection against the S2 strain challenge.


Assuntos
Vacinas Bacterianas/imunologia , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/prevenção & controle , Doenças dos Ovinos/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Bovinos , Proteção Cruzada/imunologia , Exotoxinas/imunologia , Imunização/veterinária , Imunização Secundária/veterinária , Injeções Subcutâneas/veterinária , Pulmão/patologia , Mannheimia haemolytica/classificação , Mannheimia haemolytica/genética , Pasteurelose Pneumônica/microbiologia , Distribuição Aleatória , Sorogrupo , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia
20.
Am J Vet Res ; 76(3): 239-45, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25710760

RESUMO

OBJECTIVE: To investigate the association of bovine respiratory disease (BRD) or vaccination with serologic response in calves. ANIMALS: 94 Holstein calves. PROCEDURES: To assess the association between BRD and antibody titers, 38 calves < 3 months old that were treated for BRD were matched with 38 untreated calves. To investigate the effect of vaccination on antibody titers, 24 calves were randomly assigned to be vaccinated against bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus types 1 and 2, bovine herpesvirus type 1 (BHV1), and parainfluenza virus type 3 at 2 weeks of age (n = 6), 5 weeks of age (6), and both 2 and 5 weeks of age (6) or were assigned to be unvaccinated controls (6). Blood samples were obtained at I, 2, 5, and 12 weeks for determination of serum neutralization antibody titers against the vaccine viruses, bovine coronavirus, and Mannheimia haemolytica. Antibody rates of decay were calculated. RESULTS: Calves with initial antibody titers against BRSV < 1:64 that were treated for BRD had a slower rate of anti-BRSV antibody decay than did similar calves that were not treated for BRD. Calves with high initial antibody titers against BRSV and BHV1 had lower odds of BRD than did calves with low initial antibody titers against those 2 pathogens. Vaccination at 2 or 5 weeks of age had no effect on the rate of antibody decay. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical BRD and the serologic response of dairy calves were associated with initial antibody titers against BRSV and BHV1. Serologic or clinical responses to viral exposure may differ in calves with low passive immunity.


Assuntos
Doenças dos Bovinos/prevenção & controle , Infecções por Vírus Respiratório Sincicial/veterinária , Vacinas Virais/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Estudos de Casos e Controles , Bovinos , Doenças dos Bovinos/imunologia , Indústria de Laticínios , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Feminino , Herpesvirus Bovino 1/imunologia , Masculino , Mannheimia haemolytica/imunologia , Vírus da Parainfluenza 3 Bovina/imunologia , Infecções por Vírus Respiratório Sincicial/sangue , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Bovino/imunologia , Estudos Retrospectivos , Vacinação/veterinária
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