Experimental infection of calves with respiratory syncytial virus.

Three bovine isolates and one human isolate of RS virus were given intranasally to gnotobiotic, colostrum-deprived and conventional calves. All isolates produced a biphasic pyrexia associated with a serous nasal discharge. Virus was recovered from nasal secretions 4-10 days after inoculation from nasal, tracheal and bronchial mucosae and lung of animals killed 7-13 days after inoculation. Infection did not produce any macroscopic lesions, but histologically there was a focal degenerative rhinitis and a catarrhal bronchiolitis with the occasional formation of syncytia in bronchioles and alveoli.     

Respiratory syncytial virus pneumonia in young calves: clinical and pathologic findings

Nine young calves given respiratory syncytial virus by a combined intranasal and intratracheal route developed a severe respiratory tract disease in which coughing, tachypnea, and hyperpnea were prominent clinical features. Calves were euthanatized on postinoculation (initial) days (PID) 1 to 13. At necropsy, large areas of consolidation were present in the cranial, middle, accessory, and cranial parts of the caudal lung lobes of calves killed between PID 4 and 13. Histopathologic examination revealed widespread and severe lesions in small bronchi, bronchioli, and alveoli. Multinucleate epithelial syncytia on bronchiolar and alveolar walls, many containing eosinophilic intracytoplasmic inclusion bodies, were present in the lungs of calves killed on PID 4, 5, and 6. Necrosis and epithelial loss, hyperplasia, and metaplasia were also observed in the epithelium of small bronchi and bronchioli. The lumina of these airways were occluded to varying degrees with exudate. Exudate was present within alveoli, and interalveolar septa were markedly thickened. Collapse of the thickened septa produced large areas where alveolar air spaces were totally obliterated. Repair was evident in the lungs of calves killed at PID 10 and 13 with reepithelialization of damaged bronchiolar mucosa, organization of bronchiolar exudate leading to bronchiolitis obliterans, and peribronchial and peribronchiolar fibrosis. Inoculation of 3 calves by an intranasal route alone produced a less severe clinical disease with only minimal lesions present at necropsy.     

Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus

Mycoplasma bovis was inoculated alone or in combination with respiratory syncytial virus into the respiratory tracts of 12 gnotobiotic calves. Clinical signs ranged from transient pyrexia to protracted fever accompanied by severe lower respiratory signs and in one case, arthritis. Pulmonary lesions included foci of coagulative necrosis surrounded by mononuclear cells and suppurative bronchiolitis with varying degrees of lympho-reticular hyperplasia. No enhancement of lesions occurred in the combined infections of M. bovis and respiratory syncytial virus. M. bovis was identified by immunoperoxidase labelling in lesions of necrosis, especially at interfaces between the lesion and mononuclear cells and in bronchiolar exudates. Organisms were also located in necrotic lesions of joint capsules, in tonsillar crypts, and in liver.     

Flunixin meglumine in calves with natural bovine respiratory syncytial virus infection

Twenty-three calves (three to eight months of age) with serological evidence of bovine respiratory syncytial virus infection were used in this study. The calves originated from four herds with respiratory tract disease. In a double blind trial the calves were injected intravenously with either flunixin meglumine (2 mg/kg body weight) or with a placebo. The effect on the course of disease was measured using the PO2 in capillary blood samples from the ears of the calves and by the effect on body temperature and respiratory rate. Mean body temperature fell significantly in the flunixin meglumine treated group. Statistically significant differences were not found between the treated and control group during the seven-day examination period.     

Clenbuterol hydrochloride in calves with a natural bovine respiratory syncytial virus infection

The effect of clenbuterol hydrochloride on the course of disease in calves with a natural bovine respiratory syncytial virus infection was examined. Six calves (three to nine months of age) originating from four herds with respiratory tract disease and serological evidence of a bovine respiratory syncytial virus infection were used in this study. The calves were injected intravenously with clenbuterol hydrochloride. The effect of clenbuterol on the course of disease was measured using the PO2 in blood taken from an indwelling canula inserted in the caudal auricular artery and by clinical signs. Clenbuterol did not improve clinical signs. After clenbuterol administration arterial PO2 values decreased significantly in five out of six patients. Six to eight hours after medication the mean arterial PO2 values were higher than initial values. The moderate positive effect of clenbuterol after six to eight hours may be caused by enhancing ciliary activity and by the secretolytic activity of clenbuterol.     

Clinical signs following experimental lungworm infection and natural bovine respiratory syncytial virus infection in calves

Similar clinical signs have been reported in calves infected either by Dictyocaulus viviparus or bovine respiratory syncytial virus. Three experiments were carried out to establish the clinical picture and the course of the disease in animals with these infections. The clinical signs of calves infected with lungworm included coughing, nasal discharge, tachypnoea, abdominal breathing and pyrexia, and auscultation of their lungs revealed increased bronchial sounds. Similar signs were also observed after infection with bovine respiratory syncytial virus, but the signs were more acute and resolved more rapidly than in animals infected with lungworm larvae. Calves infected with lungworm had more serious clinical signs after infection with bovine respiratory syncytial virus than calves, which were not infected with lungworm.     

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.     

Mismatching of ventilation and perfusion in calves with natural bovine respiratory syncytial virus infection

The cause of arterial hypoxia during natural infection with bovine respiratory syncytial virus was studied in seven calves (three to nine months of age) originating from five herds with respiratory tract disease and serological evidence of infection with the virus. Blood gas values were measured during ambient air breathing and during 100 per cent oxygen breathing. The percentages of contribution to the arterial hypoxia from alveolar hypoventilation, mismatching of ventilation and perfusion, and right-to-left shunting were calculated from the measured parameters. Calculated percentages of total venous admixture varied from 14 per cent of cardiac output in relatively mild cases to 48 per cent in the worst affected animal. This venous admixture had been caused mainly by right-to-left shunting of blood, while mismatching of ventilation and perfusion became important in the more severely affected animals. Alveolar hypoventilation was only important in the worst affected animal.     

Experimental infection of calves with bovine respiratory syncytial virus (Quebec strain).

An experiment was designed to evaluate the clinical, haematological, viral and serological aspects of bovine respiratory syncytial virus infection in calves. Eleven calves were inoculated intranasally with bovine respiratory syncytial virus (Quebec strain) in aerosol. Clinical, haemotological and serological responses of the calves and virus shedding were monitored. The experimentally infected animals manifested moderate to severe signs of respiratory disease. The parameters used to evaluate the severity of the disease included ocular discharge, conjunctivitis, lung sounds, nasal discharge, pyrexia and leukopenia. The animals were scored accordingly (scale infected 70.8-148.5, control 22-29.3). Highest disease scores were observed between day 6-9 after infection. Virological and serological assessment demonstrated that the observed clinical picture was due to bovine respiratory syncytial virus infection.     

Immunofluorescence in the serological diagnosis of parainfluenza type 3 and respiratory syncytial virus infection in calves

The fluorescent antibody (FA) test is compared with the haemagglutination inhibition (HI) test for parainfluenza virus type 3 (PI-3) and virus neutralisation (VN) test for respiratory syncytial (RS) virus for detection and titration of virus-specific antibodies. In experimentally inoculated calves PI-3 and RS virus FA tests detected seroconversion at the same time as HI and VN tests, however, in serially diluted sera, the FA test was positive to higher dilution. In studies with paired samples from calves from four farms with respiratory problems, the FA test gave similar results to PI-3 HI and RS virus VN tests. Large increases in antibody titre to RS virus detected by FA and VN tests indicated this was the problem on two of the farms. Individual animals showed large rises to PI-3 by FA and HI test on three farms. It is concluded that the FA test provides a rapid and sensitive alternative to the more conventional serological tests for respiratory viruses.     

Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: morphologic and serologic findings

Lesions in 32 calves that died or were euthanatized during the course of severe natural infection with bovine respiratory syncytial virus (BRSV) are described. All calves had been dyspneic for 1 to 2 days. At necropsy, lesions that could be related to dyspnea included congested and cyanotic mucosae and widespread petechiae. The lungs had various lesions in the cranioventral (CV) and caudodorsal (CD) portions. The CV portion of the lungs was consolidated, firm, and edematous. Histologically, the main characteristic was degenerative, necrotic bronchiolitis, with few syncytial cells. Signs of repair, such as epithelial hyperplasia, fibrosis, and bronchiolitis obliterans, often were observed. The CD portion of the lungs was markedly distended, owing to severe edema and emphysema. Bronchiolar lesions were lacking in the CD portion. In 14 calves, hyaline membranes were seen in the CV and CD portions. Results of immunofluorescence for BRSV were positive in 24 calves, but only in the CV portion of the lungs. The calves had variable concentrations of BRSV-specific IgG1 and IgM in serum, lung lavage fluid, or both. The BRSV-specific IgA, on the contrary, was seldom detected. Thus, 2 discrepancies existed. Although the clinical picture appeared to be acute, bronchiolar lesions and serotest results suggested infection of longer duration. Also, although virus and viral cytopathologic features were detected only in the CV portion of the lungs, the CD portion had extensive lesions that consisted of emphysema and edema.     

Bovine respiratory syncytial virus infection

Bovine respiratory syncytial virus (BRSV) belongs to the pneumovirus genus within the family Paramyxoviridae and is a major cause of respiratory disease in young calves. BRSV is enveloped and contains a negative sense, single-stranded RNA genome encoding 11 proteins. The virus replicates predominantly in ciliated respiratory epithelial cells but also in type II pneumocytes. It appears to cause little or no cytopathology in ciliated epithelial cell cultures in vitro, suggesting that much of the pathology is due to the host's response to virus infection. RSV infection induces an array of pro-inflammatory chemokines and cytokines that recruit neutrophils, macrophages and lymphocytes to the respiratory tract resulting in respiratory disease. Although the mechanisms responsible for induction of these chemokines and cytokines are unclear, studies on the closely related human (H)RSV suggest that activation of NF-kappaB via TLR4 and TLR3 signalling pathways is involved. An understanding of the mechanisms by which BRSV is able to establish infection and induce an inflammatory response has been facilitated by advances in reverse genetics, which have enabled manipulation of the virus genome. These studies have demonstrated an important role for the non-structural proteins in anti-interferon activity, a role for a virokinin, released during proteolytic cleavage of the fusion protein, in the inflammatory response and a role for the SH and the secreted form of the G protein in establishing pulmonary infection. Knowledge gained from these studies has also provided the opportunity to develop safe, stable, live attenuated virus vaccine candidates.     

Observations on outbreaks of respiratory disease in calves associated with parainfluenza type 3 virus and respiratory syncytial virus infection.

In four outbreaks of indoor calf pneumonia, dyspnoea was a prominent clinical finding. At necropsy it was associated with pneumonia involving the cranial lobes of the lung and severe pulmonary emphysema. Histological examination of lung tissue revealed bronchiolitis and alveolitis with alveolar epithelial cell hyperplasia and multinucleate syncytium formation. Intraalveolar haemorrhage, intra-alveolar oedema and hyaline membrane formation were also noted. In all cases parainfluenza type 3 (PI3) virus was isolated from the lungs. In each of the four outbreaks there was evidence of PI3 virus and respiratory syncitial virus (RSV) infection.     

Effect of infection with bovine respiratory syncytial virus on pulmonary clearance of an inhaled antigen in calves

OBJECTIVE: To evaluate the effect of infection with bovine respiratory syncytial virus (BRSV) on clearance of inhaled antigens from the lungs of calves. ANIMALS: Eleven 6- to 8-week-old Holstein bull calves. PROCEDURES: Aerosolized (99m)technetium ((99m)Tc)-labeled diethylene triamine pentacetate (DTPA; 3 calves), commonly used to measure integrity of the pulmonary epithelium, and (99m)Tc-labeled ovalbumin (OA; 8 calves), commonly used as a prototype allergen, were used to evaluate pulmonary clearance before, during, and after experimentally induced infection with BRSV or sham inoculation with BRSV. Uptake in plasma (6 calves) and lung-efferent lymph (1 calf) was examined. RESULTS: Clearance of (99m)Tc-DTPA was significantly increased during BRSV infection; clearance of (99m)Tc-OA was decreased on day 7 after inoculation. Clearance time was correlated with severity of clinical disease, and amounts of (99m)Tc-OA in plasma and lymph were inversely correlated with clearance time. Minimum amounts of (99m)Tc-OA were detected at time points when pulmonary clearance of (99m)Tc-OA was most delayed. CONCLUSIONS AND CLINICAL RELEVANCE: BRSV caused infection of the respiratory tract with peak signs of clinical disease at 7 or 8 days after inoculation. Concurrently, there was a diminished ability to move inhaled protein antigen out of the lungs. Prolonged exposure to inhaled antigens during BRSV infection may enhance antigen presentation with consequent allergic sensitization and development of chronic inflammatory lung disease. IMPACT FOR HUMAN MEDICINE: Infection of humans with respiratory syncytial virus early after birth is associated with subsequent development of allergic asthma. Results for BRSV infection in these calves suggested a supportive mechanism for this scenario.     

Effect of vaccination on cell populations in lung washes from calves after infection with respiratory syncytial virus

The inflammatory response in the air-passages of the lungs of calves after intranasal inoculation with respiratory syncytial virus (RSV) was compared in RSV-vaccinated and control animals. Total cells recovered from lung washings remained the same; however, the fold by eight days after infection and the type of cells changed from a predominance (85 per cent) of macrophages to equal proportions of macrophages and neutrophils (45 per cent) during the course of infection. The absolute numbers of neutrophils rose by 15-fold. In contrast, when RSV-vaccinated calves were challenged, the total number of cells recovered from lung washings remained the same; however, the numbers of macrophages decreased and the numbers of neutrophils increased by fivefold. Cytological studies of the lung washings revealed no evidence of an exacerbated inflammatory response in RSV-vaccinated calves. Levels of virus replication were significantly reduced in RSV-vaccinated compared with control animals.     

Efficacy of a saponin-adjuvanted inactivated respiratory syncytial virus vaccine in calves

The objective of this study was to determine whether a commercially available, saponin-adjuvanted, inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from experimental infection with virulent BRSV. This was a randomized controlled trial comprising 14, 8- to 9-week-old calves seronegative for BRSV Group 1 calves (n = 8) were not vaccinated and group 2 calves (n = 6) were vaccinated on days 0 and 19 with an inactivated BRSV vaccine. All calves were challenged with virulent BRSV on day 46. Clinical signs, arterial PO2, and immune responses were monitored after challenge. Calves were euthanatized on day 54 (8 d after challenge) and lungs were examined for lesions. Vaccination elicited increases in BRSV-specific immunoglobulin (Ig) G and virus neutralizing antibody titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, but no signs of clinical disease and minimal or no pulmonary lesions in vaccinated calves. Arterial blood oxygen values on day 53 (7 d after challenge) in control calves were significantly lower than those in vaccinated calves, which remained within normal limits. Control calves shed BRSV for several days after challenge, whereas BRSV was not detected on deep nasal swabs from vaccinated calves. In summary, the results indicated that this inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus 27 d after vaccination and significantly decreased the prevalence and severity of pulmonary lesions. Efficacy was similar to that reported for other commercial inactivated and modified-live BRSV vaccines.     

Correlation of a disease scoring system with arterial PO2 values in respiratory syncytial virus infection in calves

The mean arterial PO2 value measured in blood obtained by puncture of the brachial artery of 20 calves with acute clinical signs of a bovine respiratory syncytial virus (BRSV) infection was 8.4 +/- 1.9 kPa. The values differed significantly from arterial PO2 values of eleven healthy calves (mean 14.2 +/- 1.5 kPa). A disease scoring system is presented based on the type of respiration and the findings on auscultation. A high correlation (r = -0.87) was found between disease scores and arterial PO2 values. This indicates that the described disease scoring system can be a useful tool in the evaluation of the severity and course of BRSV infections in calves, and could be used for evaluating the efficacy of BRSV vaccines in the field. The course of disease was studied in 127 calves with clinical signs of serologically proven BRSV infection. Animals with mild respiratory signs during the acute phase of disease remained free of severe respiratory problems until the end of a 35-day examination period. Mean disease scores indicated that animals with severe signs in the acute phase often developed persistent respiratory problems.