Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: evidence for the involvement of complement and mast cell mediators

Indicators of immune-mediated disease were studied in calves with severe natural bovine respiratory syncytial virus infection. Although antigen and antibody were detected concurrently in most calves, immune complexes were not detected by use of immunofluorescence, ELISA, and binding of the 1q component of complement. Complement component C3, however, was observed by immunofluorescence in the cranioventral, virus-infected portion of the lungs of 19 of 25 calves. Reductions in the amount of histamine and in the numbers of mast cells and mast cell granules in the virus-positive cranioventral and virus-negative caudodorsal portions of the lungs, indicated activation of mast cells and liberation of their granule contents. On the basis of these and previous findings, a model for the pathogenesis of bovine respiratory syncytial virus-induced disease was proposed.     

Cytotoxic T cell responses in lambs experimentally infected with bovine respiratory syncytial virus

The role of cell-mediated immune response in the immunopathogenesis of bovine respiratory syncytial virus (BRSV) infection is not well established. In the present study, cytotoxic T cell responses of BRSV-infected lambs were examined using the chromium release assay. Lambs experimentally infected with BRSV developed cytotoxic lymphocytes in the peripheral blood and the spleen, which lysed BRSV-infected but not uninfected cells. Peak cytotoxic activity occurred 10-14 days after infection. Pretreatment of mononuclear cells with anti-CD8 monoclonal antibodies and rabbit complement significantly reduced cytotoxic activity (P less than 0.05). It appears, therefore, that lambs experimentally infected with BRSV develop virus-specific, predominantly CD8+, cytotoxic lymphocytes in the peripheral blood and spleen.     

Laboratory diagnosis methods for bovine respiratory syncytial virus

Laboratory diagnosis of bovine respiratory syncytial (BRS) virus no longer poses a problem. Clinical diagnosis, based on signs of pulmonary emphysema manifest in autumn, should be confirmed by laboratory techniques. Direct isolation of the BRS virus from field samples in cell cultures is often unsuccessful, whereas detection of the viral antigens by staining ultra-thin tissue sections with fluorescein isothiocyanate antibody conjugates is highly effective. Complement fixation and especially indirect immunofluorescence tests are still very useful for the detection of BRS specific antibodies in serum and nasal mucus.

---

Kurzfassung Die Erkrankungen der Atemwege die durch den Sinzizialatmungsvirus der Rinder hervorgerufen werden, können zur Zeit mit Leichtigkeit diagnostiziert werden. Die klinische Diagnostik, die auf die Anzeichen eines Lungenemphysems beruhen und im Herbst auftreten, muss durch eine Labordiagnose bestätigt werden. Die Sichtbarmachung der viralen Antigenen mittels Färbung ultradünner Schnitte mit einem durch Fluoreszeinisothiozianat markierten Serum erweist sich wirksam und zuverlässig. Die Isolierung des Virus in den Zellkulturen ist oft sehr schwierig. Bei der Aufstellung der Diagnose ist die Suche nach Antikörpern in den gekoppelten Seren, mit der Komplementbindungsmethode und besonders mit der indirekten Immuno-Fluoreszenz, von grosser Wichtigkeit.

---

Resume En cas de troubles respiratoires dus au virus Respiratoire Syncytial Bovin (RSB) le diagnostic peut être posé actuellement sans grande difficulté. Le diagnostic clinique, basé sur les signes d'emphysème pulmonaire, apparaissant en automne, doit être confirmé par un diagnostic de laboratoire. L'isolement de l'agent viral sur culture cellulaire est souvent difficile. La mise en évidence des antigènes viraux par coloration de coupes ultra-fines à l'aide d'un sérum marqué à l'isothiocyanate de fluorescéine est efficace et fiable. La recherche d'anticorps dans des sérums couplés, par les méthodes de fixation du complément et principalement d'immunofluorescence indirecte, est de grande utilité pour l'établissement du diagnostic.

---

Riassunto Attualmente la diagnosi sulle turbe respiratorie causate dal virus respiratorio sinciziale bovino (RSB) non presenta difficoltà di rilievo. La diagnosi clinica, basata sui sintomi di enfisema polmonare, che si manifestano in autunno, deve essere confermata mediante una diagnosi di laboratorio. L'isolamento dell'agente virale su coltura cellulare risulta spesso difficile. La messa in evidenza degli antigeni virali mediante colorazione di tagli ultrafini con un siero marcato all'isotiocianato di fluorescina è efficace e ed affidabile. Per stabilire la diagnosi è di grande utilità la ricerca di anticorpi nei sieri combinati, con i metodi del la fissazione del complemento e in particolare con l'immunofluorescenza indiretta.

---

---

This article was originally written in French. Copies of the French version may be obtained free of charge by writing to: Mr J. Rodesch, Commission of the European Communities, DG XIII, Bâtiment Jean Monnet, Rue Alcide de Gasperi, Kirchberg, Luxembourg.     

Acute respiratory distress syndrome.

Several combination therapies have been described throughout this article: in all likelihood, it is combination therapy that will allow improved survival of ARDS patients. As medicine moves into the future, clinical trials evaluating the efficacy of therapies for ARDS will be performed. In human critical care medicine, a large forward step was taken when ALI and ARDS were clearly defined. Unfortunately. as good as the definition is, ALI and ARDS occur secondary to many different underlying pathologic processes,perhaps obscuring the benefits of certain therapies for ARDS based on the underlying condition, for example, trauma versus sepsis. Selection of patients entering any ARDS trial is crucial: not only must those patients meet the strict definition of ARDS but the underlying disease process should be clearly identified. Identification of patients suffering from different disease processes before the onset of ARDS will allow for stratification of outcomes according to the intervention and the underlying pathology--comparing apples to apples and not to oranges. We are in a unique position in veterinary medicine. Although frequently financially limited by our clients, we have the opportunity to achieve several goals. First, we need to clearly define what constitutes ALI and ARDS in veterinary medicine. Do we want to rely on the human definitions? Probably not; however, as a group, we need to determine what we will accept as definitions. For example, we may not be able perform right heart catheterizations on all our patients to meet the wedge pressure requirement of human beings of less than 18 mm Hg. Do we agree that a PAOP of less than 18 mmHg is appropriate for animals, and is it appropriate for all animals? Will we accept another measure, for example, pulmonary artery diameter increases with echocardiographic evidence of acceptable left heart function? What is acceptable left heart function? As veterinarians, what do we consider to be hypoxemia? Is it the same in all species that we work with? What do we define as acute onset? Most human ARDS cases occur while patients are in hospital being treated for other problems, whereas many of our patients present already in respiratory distress. If we are unable to ventilate patients for economic or practical reasons, what do we use as the equivalent of the Pao2/Flo, ratio'? Reliance on the pathologist is not reasonable, because many disease processes can look similar to ARDS under the microscope. If anything, ALI and ARDS are clinical diagnoses. It is time for veterinarians to reach a consensus on the definition for ALI and ARDS in our patients.Only when we have a consensus of definition can rational prospective clinical trials of therapies be designed.     

Experimental reproduction of respiratory tract disease with bovine respiratory syncytial virus

An experiment was conducted to reproduce respiratory tract disease with bovine respiratory syncytial virus (BRSV) in one-month-old, colostrum-fed calves. The hypothesized role of viral hypersensitivity and persistent infection in the pathogenesis of BRSV pneumonia was also investigated. For BRSV inoculation a field isolate of BRSV, at the fifth passage level in cell culture, was administered by a combined respiratory tract route (intranasal and intratracheal) for four consecutive days. Four groups of calves were utilized as follows: Group I, 6 calves sham inoculated with uninfected tissue culture fluid and necropsied 21 days after the last inoculation; Group II, 6 calves inoculated with BRSV and necropsied at the time of maximal clinical response (4-6 days after the last inoculation); Group III, 6 calves inoculated with BRSV and necropsied at 21 days after the last inoculation; Group IV, 6 calves inoculated with BRSV, rechallenged with BRSV 10 days after initial exposure, and necropsied at 21 days after the initial inoculation. Clinical response was evaluated by daily monitoring of body temperature, heart rate, respiratory rate, arterial blood gas tensions, hematocrit, total protein, white blood cell count, and fibrinogen. Calves were necropsied and pulmonary surface lesions were quantitated by computer digitization. Viral pneumonia was reporduced in each principal group. Lesions were most extensive in Group II. Disease was not apparent in Group I (controls). Significant differences (p less than 0.05) in body temperature, heart rate, respiratory rate, arterial oxygen tension, and pneumonic surface area were demonstrated between control and infected calves. Results indicate that severe disease and lesions can be induced by BRSV in one-month-old calves that were colostrum-fed and seropositive to BRSV. BRSV rechallenge had minimal effect on disease progression. Based on clinical and pathological response, results did not support viral hypersensitivity or persistent infection as pathogenetic mechanisms of BRSV pneumonia.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24–96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p<0.05).     

Lesions in lambs experimentally infected with bovine respiratory syncytial virus

Respiratory syncytial virus was inoculated intratracheally into five 1-week-old lambs. Three of the lambs responded clinically with fever, hyperpnea, and listlessness. Pulmonary lesions consisted of multifocal areas of consolidation, with necrosis of individual epithelial cells of the airways and accumulation of necrotic debris, macrophages, and few neutrophils in terminal airways and alveoli. Pulmonary septa in affected areas were infiltrated with numerous macrophages and lymphocytes. Viral particles were seen as buds on epithelial cells and free in bronchioles and alveoli.     

Ribavirin inhibits proliferation of bovine respiratory syncytial virus in vitro

Bovine respiratory syncytial virus (BRSV) is an important pathogen in bovine respiratory diseases in the United States. Proliferation of the disease can reach epidemic proportions with mortality reaching as high as 20%. In vitro work shown here suggests that the antiviral compound Ribavirin will be effective in the treatment of infected animals. Treatment at three dose levels with Ribavirin have shown significant inhibition of BRSV proliferation. Bovine turbinate cells were host cells for this study. Tissue culture specimens, infected and noninfected, were carried for 10 days. Presence of BRSV was verified with the use of monoclonal antibody. In addition, the infection with BRSV and consequential treatment with Ribavirin of calves demonstrated a noticeable reduction in viral symptom but no apparent systemic reaction to drug therapy.     

Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs

Twenty-four 6-8-week-old conventionally reared lambs were inoculated intranasally and intratracheally with bovine respiratory syncytial virus. Infected lambs showed mild clinical signs characterized by slight serous nasal discharge, coughing, lachrymation and bronchovascular sounds on the middle part of the lung 5-9 days post-inoculation (PI). Virus was isolated in nasal swabs from 9 of 24 lambs between 3 and 7 days PI. However, virus was recovered from tracheal and lung tissue of all lambs killed between 3 and 11 days PI. Virus-specific antibodies appeared as early 6 days PI but high titres were attained 14-21 days PI. Lungs of lambs killed on different days PI had multifocal areas of consolidation. There was an increase of lymphocytes with a T-suppressor cell marker and a decrease in those with a T-helper marker in lung lavages obtained 5 days PI.     

Serologic studies of bovine respiratory syncytial virus in Minnesota cattle

Serum antibody titers to bovine respiratory syncytial virus were determined for 559 cattle. Serum samples were obtained through the Minnesota State-Federal Brucellosis Laboratory and were collected over a 1-year period. Results of this study revealed an antibody prevalence of 65.5% to bovine respiratory syncytial virus. The distribution of antibody titers is presented, as well as analysis of titers based on breed, sex, and age of the cattle.     

Cell-mediated immune response in cattle to bovine respiratory syncytial virus

Cattle inoculated with bovine respiratory syncytial virus (BRSV) were evaluated for the development of a cell-mediated immune response. Results of the leukocyte migration-inhibition test under agarose and the delayed hypersensitivity test indicated that a cell-mediated immune response was elicited after intranasal inoculation of calves with BRSV. Migration inhibition in the leukocyte migration-inhibition test was detected by postinoculation day (PID) 5 and reached maximum inhibition on PID 21. Inhibition of leukocyte migration was still evident by PID 42 when values were still appreciably greater than preinoculation values. All of the calves inoculated with BRSV developed a delayed hypersensitivity skin response when challenge exposed intradermally with BRSV antigen.     

Experimental bovine respiratory syncytial virus infection in conventional calves: ultrastructural respiratory lesions

The morphogenesis and repair of airway and alveolar injury induced by bovine respiratory syncytial virus (BRSV) was studied ultrastructurally in conventional calves to characterize pulmonary cell types susceptible to viral infection and cytopathologic changes associated with infection. Viral nucleocapsids and budding virions were present in tracheal and bronchial ciliated and nonciliated epithelial cells and mucous cells 3, 5, and 7 days after inoculation and in bronchiolar ciliated and nonciliated epithelial cells 5 days after inoculation. Mild interstitial pneumonia was observed 5 days after inoculation and was characterized by swelling of type 1 and type 2 alveolar epithelial cells, interstitial edema, and infiltration by lymphocytes and macrophages. Viral assembly and release in tracheal and bronchial epithelial cells was associated with loss of cilia from ciliated cells, formation of syncytial epithelial cells, swelling of mitochondria and endoplasmic reticulum, and cell necrosis. Neutrophils, lymphocytes, and macrophages were present in close association with the viral-infected and damaged epithelial cells. There was intercurrent hyperplasia of basal epithelial cells that, in association with other epithelial lesions, resulted in the loss of normal ciliated epithelium in these airways 5 and 7 days after inoculation. Regeneration of airway epithelium was largely completed by 10 days after inoculation, except in 1 of 4 calves that had failure of epithelial repair and that developed secondary bacterial pneumonia. Pulmonary ultrastructure in BRSV-inoculated calves 30 days after inoculation was indistinguishable from that in controls. The results demonstrated that BRSV can induce reversible alterations in airway epithelium, which may cause depression of mucociliary clearance and thereby enhance susceptibility to bacterial infection.     

Detection of IgE antibodies to bovine respiratory syncytial virus

The role of IgE antibodies against respiratory syncytial virus has attracted attention for both human and bovine disease. To detect such antibodies, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for bovine respiratory syncytial virus (BRSV). Firstly, anti-serum strongly positive for BRSV-specific IgE was produced by immunizing a levamisole-treated calf with BRSV. The presence and specificity of BRSV-specific IgE in this animal was confirmed with the Praunitz-Kustner (PK) technique. Potential interference in an ELISA by other BRSV-specific immunoglobulin isotypes was eliminated by preferential precipitation of serum samples with 27.5% saturated ammonium sulfate. The correlation between the PK and the ELISA assay was greater than 93% and the ELISA was found to be more specific than the PK. Indeed, in a pilot experimental infection study, the serum levels of BRSV-specific IgE were found to correlate with the symptom expression following repetitive live virus aerosolization. This may prove to be a useful rapid test to study both herd immunity and the potential pathogenic influence of IgE.     

Immunology of bovine respiratory syncytial virus infection of cattle

Bovine respiratory syncytial virus (BRSV) is a respiratory pathogen of cattle that causes severe disease in calves alone and as one of several viruses and bacteria that cause bovine respiratory disease complex. Like human RSV this virus modulates the immune response to avoid stimulation of a vibrant CD8+ T cytotoxic cell response and instead promotes a Th2 response. The Th2 skew sometimes results in the production of IgE antibodies and depresses production of the Th1 cytokine interferon γ. Innate immune cells have a pivotal role in guiding the adaptive response to BRSV, with selective secretion of cytokines by pulmonary dendritic cells. Here we review some of the pertinent observations on immune responses to BRSV infection and vaccination and illustrate how experimental infection models have been used to elucidate the immunopathogenesis of BRSV infection. Recent experiments using intranasal vaccination and/or immune modulation with DNA based adjuvants show promise for effective vaccination by the stimulation of Th1 T cell responses.     

Activation of complement by bovine respiratory syncytial virus-infected cells

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.     

Reinfection of lambs with bovine respiratory syncytial virus.

Eight lambs which were experimentally infected with bovine respiratory syncytial virus (RSV) when they were six to eight weeks old were challenged with the same virus seven months later. After reinfection, lambs developed mild clinical disease and the virus was isolated from nasal swabs from three lambs and peripheral blood from two lambs. Reinfection resulted in changes in peripheral blood cell populations. There was an early increase in the number of CD8+ T lymphocytes and B (LCA p220+) lymphocytes but the proportions of CD4+ and CD4-CD8- T lymphocytes were significantly reduced. Peripheral blood mononuclear cells obtained from lambs reinfected with bovine RSV showed significantly higher responses to bovine RSV antigen in vitro than those obtained from control lambs but their responses to the mitogen phytohaemagglutinin were significantly lower than in control lambs. RSV-specific IgG, IgM and IgA levels of serum samples obtained 10 days after challenge were significantly higher than those of serum samples obtained before challenge.     

Human and bovine respiratory syncytial virus: immunopathologic mechanisms

Human respiratory syncytial virus (HRSV) is the major respiratory tract pathogen of infants and young children. Bovine respiratory syncytial virus (BRSV) is recognised as an important cause of respiratory tract disease in calves. Both of these viruses and their respective diseases share many similarities. Immunopathologic mechanisms have been proposed to be involved in the pathogenesis of respiratory syncytial virus (RSV) infections. This review examines the current understanding of the role of immunopathologic mechanisms in RSV infections. The role of vaccines in inducing hypersensitivity is also examined. Additionally, non-immunopathogenic mechanisms involved in RSV infections are discussed.     

Neonatal respiratory distress syndrome in the calf

Thirty-five calves were delivered by caesarean section near to term. During the operation amniotic fluid was collected for determination of the lecithin/sphingomyelin (L/S) ratio. Clinical examination of the calves and analysis of blood gas concentration (venous blood) were carried out within the first hour of life. Fifteen out of 35 calves under examination did not show clinical or blood gas disorders in the course of the first hour of life. In these calves, the L/S ratio, which represents a measure for the maturity of the surfactant system, averaged 2.6. The other 20 calves, however, developed a respiratory distress syndrome together with a progressive respiratory and metabolic acidosis within the first hour of life. The L/S ratio in the animals affected with respiratory distress syndrome reached an average value of 1.5 which was significantly below that of the calves not suffering from respiratory distress. Eleven of the 20 calves which developed respiratory distress syndrome died within the first 60 hours of life. The most striking findings in the post mortem examinations of these animals were intracranial haemorrhages and pulmonary lesions (hyaline membranes, interstitial and alveolar oedema). On the basis of the significantly lower L/S ratio and the post mortem findings, it is to be assumed that the respiratory distress syndrome in calves, equally with that in infants, is attributable to a surfactant deficiency.