Bovine respiratory syncytial virus is an important pathogen of the respiratory tract in cattle, with an infection and disease pattern that remarkably parallels that of the closely related RSV in humans.1 There is epidemiologic evidence that infection with RSV influences the development of allergic sensitization and, potentially, asthma in humans.2,3 Although cattle do not develop the clinical syndrome referred to as asthma, chronic lung disease is a common sequel to calfhood respiratory tract disease. Moreover, exposure of cattle to environmental antigenic inhalants is an unavoidable consequence of housing conditions. In other studies4,5 conducted by our laboratory group, there was an increase in disease severity in calves infected with BRSV and exposed by aerosol to the thermophilic actinomycete Saccharopolyspora rectivirgula (formerly known as Micropolyspora faeni). This organism is often found in moldy hay dust and has been associated with Farmer's lung in humans and cattle.
Similar to RSV in humans, BRSV causes an acute and often severe infection of the lungs in young cattle and less severe infection of the nasal cavities and trachea in adult cattle. Pathogenic processes that are made more severe by the immune response of the host have been ascribed to a preponderance of a Th2-mediated mechanism.6,7 Analysis of results of an aforementioned study4 conducted by our laboratory group suggests that BRSV infection alters the persistence of antigen in the lungs, thereby providing increased access to antigen by antigen-presenting cells in the lungs. To examine this hypothesis, we used scintigraphic evaluation of clearance of a radioactively labeled aerosol from the lungs of calves before and after experimentally induced infection with BRSV. In the study, 99mTc-DTPA, a small molecule commonly used to evaluate the integrity of the pulmonary epithelium, was administered to calves by nebulization, and clearance was evaluated by use of a G camera. Next, clearance of a larger molecule (ie, OA [45 kd]), also labeled with 99mTc, was evaluated. In another study8 conducted by our laboratory group, we used OA, a common prototype allergen, to evaluate immune responses to an aerosolized antigen in calves.
Evaluation of pulmonary clearance of 99mTc-labeled molecules is a useful clinical tool as well as a method by which hypotheses on alterations in pathophysiologic processes of the lungs can be addressed. Studies9–13 have been performed to evaluate clearance of 99mTc-DTPA in sheep. In 1 study,14 sheep with chronic lymphatic fistulae in the lungs were used to evaluate clearance of 99mTc-DTPA from air spaces into pulmonary lymph. In another study,15 appearance of 99mTc-albumin in pulmonary lymph was examined in sheep whose lungs were injured by IV infusion of oleic acid. Clearance of 99mTc-DTPA has also been evaluated in anesthetized dogs. Mucociliary clearance of a 99mTc-labeled sulfur colloid complex was evaluated in a study16 in calves, but investigators in that study measured clearance that focused on the larynx. To our knowledge, the effect of viral infection of the respiratory tract on clearance of 99mTc-DTPA or protein antigen from the lungs of calves has not been addressed.
In the study reported here, calves were exposed to aerosolized 99mTc-DTPA or 99mTc-OA to establish the amount of time required to clear the material from the lungs. Calves were then inoculated with BRSV to induce experimental infection, and clearance was examined on several days after inoculation to evaluate the effect of infection on clearance of the aerosolized protein from the lungs. In a subset of calves, the temporal appearance of 99mTc-OA in plasma (8 calves) and pulmonary lymph (1 calf) was also examined to evaluate the effect of viral infection of the respiratory tract on movement of 99mTc-OA from the lungs into blood and lymph.
Materials and Methods
Animals—Eleven conventionally raised 6- to 8-week-old Holstein bull calves were used in the study; each calf served as its own control animal. Calves were assessed at the beginning of the study by use of an indirect immunofluorescence assay performed by personnel at the Center for Animal Health and Food Safety at the University of California, Davis; all calves were seronegative for BRSV. Calves were housed at the Center for Laboratory Animal Medicine at the University of California, Davis. Calves were fed alfalfa hay, and salt and water were available ad libitum. The study was conducted in accordance with a protocol approved by an institutional animal care committee.
Schedule of procedures—Clearance of 99mTc–DPTA was examined in 2 calves (Nos. 1 and 2) before inoculation with BRSV (day of inoculation was designated as day 0) and on 5 days during the course of the disease. Clearance was evaluated in calves 1 and 2 on days –3, 2, 4, 7, 9, and 16. Clearance of 99mTc-OA was evaluated in 6 calves (Nos. 3 through 8) before inoculation with BRSV (day –3) and at various time points during the course of the disease (calves 3 and 4, day 7; calves 5 and 6, days 7, 11, and 16; and calves 7 and 8, days 4, 7, 9, and 11). Additionally, a lymphatic cannula was surgically implanted17 in calf 8 on day –4 for subsequent collection of efferent lung lymph. To examine the effect of repeated administration of OA aerosol on subsequent OA clearance from the lungs, 2 additional calves (Nos. 9 and 10) were exposed via aerosol to 99mTc-OA; clearance was measured before inoculation (days –19, –12, –9, –7, and –5) and during infection (days 5, 7, 9, and 14). Clearance of 99mTc-OA in a control calf (No. 11) was measured before (days –19, –12, –9, –7, and –5) and after (days 5, 7, 9, and 14) mock inoculation. Baseline clearance was designated as values obtained on day –3 for calves 1 through 8 and day –5 for calves 9 through 11. Finally, 99mTc-OA concentrations were measured in plasma obtained from calves 7 through 11 and in lymph collected from calf 8.
Clearance of 99mTc-DTPA and 99mTc-OA—Food was withheld from all calves beginning at noon the day before imaging procedures. The 99mTc-DTPAa was purchased commercially, and OAb was labeled with 99mTc by use of a technique described elsewhere.18 Appropriate precautions for handling of Tc (as dictated by the campus environmental health and safety officer) were adhered to, including use of a dosimeter for all personnel that handled an animal or were present in the room during 99mTc aerosol exposures. Each of the 2 calves received a 100 mCi dose of 99mTc-DTPA. The other calves received 0.4 mg of OA labeled with 60 mCi of Tc in a volume of 3.5 mL.
All aerosols were administered through a bronchoscope. Each unsedated calf was positioned in a stanchion in front of a G camerac while the 99mTc-OA or 99mTc-DTPA was nebulized. The nebulizerd unit contained an aerosol delivery system and was connected to an oxygen source. It delivered an aerosol that had an aerodynamic diameter of approximately 1 Mm, which was administered through an endotracheal tube. The cuff on the endotracheal tube was inflated to prevent escape of Tc into the room, thus making it a closed circuit. Air exhaled by each calf was passed through a breathing circuit filter before being returned to the room. The nebulizer and filter were contained in a lead-lined box. Each calf remained attached to the breathing circuit for 5 minutes after nebulization to allow the filter unit to collect any 99mTc-OA or 99mTc-DTPA that had not been taken up in the lungs. Lateral images were obtained of the right lung field with a large field-of-view G camerac fitted with a parallel-hole low-energy collimator linked to a dedicated computer system. Data were stored on magnetic tape for subsequent analysis with software.e
A 99mTc sample was taped to the camera face and shielded with lead; images were then continuously acquired for the first 15 minutes after the beginning of aerosol administration. One-minute images were acquired for 30 minutes after nebulization and at 30-minute intervals until counts were noticeably less than half of the initial counts (after the observed half-life). Data were expressed as the number of minutes it required for half of the nebulized 99mTc-OA or 99mTc-DTPA to leave the lungs (ie, half-life).
Measurement of 99mTc-OA concentrations in plasma and lymph after aerosol exposure—For calves 7 through 11, plasma samples were obtained at 1-hour intervals until 4 hours after aerosol exposure. For calves 9 and 10, additional samples of plasma were obtained at 5- to 10-minute intervals for the first hour. Lymph samples were collected from calf 8 at 30-minute intervals until 7 hours after aerosol exposure. Plasma and lymph samples were evaluated for 99mTc-OA concentrations. Blood samples were collected into heparinized tubes, which were then centrifuged. Plasma was harvested, and an aliquot (0.5 mL) was placed into a counter tube with 0.5 mL of PBS solution. To evaluate 99mTc-OA without interference from unbound 99mTc, differential centrifugation was performed. A direct count was made from the aforementioned sample (bound 99mTc + unbound 99mTc). An ultrafiltration devicef with a molecular exclusion limit of 10 kd was used to separate bound 99mTc from unbound (filtrate) 99mTc. Then, 1.0 mL of the concentrate was counted to determine the amount of bound 99mTc. The identical procedure was performed on lymph samples. Results for plasma and lymph were reported as adjusted data (percentage of the 99mTc-OA counts in the lungs measured 15 minutes after beginning of aerosol exposure).
Experimental infection with BRSV— Calves were inoculated with a virulent field isolate of BRSV (CA-1) grown on bovine turbinate cells and prepared as described elsewhere.19,20 A representative sample was assayed to determine the titer of the virus preparation; titer of the virus used was 4 × 105 TCID50/mL to 5 × 105 TCID50/mL. Calves received 5 mL of virus suspension by aerosol via a face mask, as described.4,19,20 The mock-infected calf received aerosol of spent tissue culture medium without virus.
Evaluation of clinical signs—A score for clinical signs was determined each day for each calf. A physical examination was performed by an investigator (LJG), and scoring of clinical signs was modified from a method reported elsewhere.19,21 Clinical scores were determined by assignment of points based on variables such as rectal temperature, coughing, nasal exudate, results of lung auscultation, dyspnea, wheezing, anorexia, and lethargy. Scores were modified to decrease the emphasis of rectal temperature (decreased from multiplication by a factor of 200 to multiplication by a factor of 100) on overall disease expression.
Blood gas analysis—On days 0 and 7, a sample of arterial blood was collected from the auricular artery of each BRSV-inoculated calf. Samples were analyzed by use of a blood gas analyzer within minutes after collection.
Shedding of BRSV—Virus shedding was monitored by evaluation of nasopharyngeal swab specimens. Samples were inoculated onto bovine turbinate cells grown on slides.g Slides were then fixed by incubation with acetone for 1 minute, and viral antigen was detected by staining of slides with fluorescein isothiocyanate–conjugated rabbit anti-RSV.h Positive (infected) and negative (uninfected) control samples were evaluated on the same slides.
BRSV titers—Antibody titers against BRSV were determined in serum samples obtained before and 2 to 3 weeks after inoculation. Titers were determined by use of an indirect immunofluorescence assay by personnel at the California Animal Health and Food Safety Laboratory in Davis, Calif.
Statistical analysis—Clearance of 99mTc-OA was evaluated by use of a paired t test to detect significant differences over time. Repeated-measures ANOVA was used to evaluate clinical signs over time, and the Student-Newman-Keuls multiple comparisons test was used to compare scores of clinical signs on various days. The PaO2 and PaCO2 values on days 0 and 7 were compared by use of paired t tests. All statistical evaluations were performed by use of a commercially available program.i
Results
Clearance of 99mTc-DTPA from the lungs—Clearance of 99mTc-DTPA was examined only in calves 1 and 2. Enhanced clearance of 99mTc-DTPA from the lungs was evident at 15 minutes after aerosol exposure on day 7 (Figure 1). The number of subjects exposed to 99mTc-DTPA was insufficient for statistical analysis; however, mean values for clearance half-time on days –3 and 7 were calculated (Figure 2). There was increased clearance of 99mTc-DTPA in both calves on day 7, compared with baseline clearance measured before inoculation.
Gamma camera image of 99mTc-DTPA (A and B) and 99mTc-OA (C and D) in the lung of representative calves 15 minutes after aerosol exposure on day –3 (A and C) and on day 7 (B and D). The images were obtained as a lateral view on standing calves. The 99mTc–DTPA and 99mTc –OA are evident in the lungs as an increase in opacity. The lung volume containing 99mTc–DTPA has diminished on day 7 after inoculation with BRSV, compared with results for day –3; this indicates that more of the 99mTc–DTPA has been cleared from the lung. In contrast, the opacity in the lung on day 7 indicates that clearance of 99mTc–OA has not been altered, compared with clearance on day –3. Day 0 = Day of BRSV inoculation.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Gamma camera image of 99mTc-DTPA (A and B) and 99mTc-OA (C and D) in the lung of representative calves 15 minutes after aerosol exposure on day –3 (A and C) and on day 7 (B and D). The images were obtained as a lateral view on standing calves. The 99mTc–DTPA and 99mTc –OA are evident in the lungs as an increase in opacity. The lung volume containing 99mTc–DTPA has diminished on day 7 after inoculation with BRSV, compared with results for day –3; this indicates that more of the 99mTc–DTPA has been cleared from the lung. In contrast, the opacity in the lung on day 7 indicates that clearance of 99mTc–OA has not been altered, compared with clearance on day –3. Day 0 = Day of BRSV inoculation.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Gamma camera image of 99mTc-DTPA (A and B) and 99mTc-OA (C and D) in the lung of representative calves 15 minutes after aerosol exposure on day –3 (A and C) and on day 7 (B and D). The images were obtained as a lateral view on standing calves. The 99mTc–DTPA and 99mTc –OA are evident in the lungs as an increase in opacity. The lung volume containing 99mTc–DTPA has diminished on day 7 after inoculation with BRSV, compared with results for day –3; this indicates that more of the 99mTc–DTPA has been cleared from the lung. In contrast, the opacity in the lung on day 7 indicates that clearance of 99mTc–OA has not been altered, compared with clearance on day –3. Day 0 = Day of BRSV inoculation.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Mean ± SD clearance half-time of 99mTc-DPTA (2 calves; A) and 99mTc-OA (8 calves; B) before (baseline) and on day 7 after inoculation with BRSV. Day of inoculation was designated as day 0. Because of the small number of calves, no statistical analysis was performed for the 99mTc-DPTA data. For part B, lower and upper corners of each box represent the 25th and 75th percentiles, respectively; the horizontal bar within each box represents the mean, and the bars represent the SD. *Value differs significantly (P = 0.043) from the baseline value.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Mean ± SD clearance half-time of 99mTc-DPTA (2 calves; A) and 99mTc-OA (8 calves; B) before (baseline) and on day 7 after inoculation with BRSV. Day of inoculation was designated as day 0. Because of the small number of calves, no statistical analysis was performed for the 99mTc-DPTA data. For part B, lower and upper corners of each box represent the 25th and 75th percentiles, respectively; the horizontal bar within each box represents the mean, and the bars represent the SD. *Value differs significantly (P = 0.043) from the baseline value.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Mean ± SD clearance half-time of 99mTc-DPTA (2 calves; A) and 99mTc-OA (8 calves; B) before (baseline) and on day 7 after inoculation with BRSV. Day of inoculation was designated as day 0. Because of the small number of calves, no statistical analysis was performed for the 99mTc-DPTA data. For part B, lower and upper corners of each box represent the 25th and 75th percentiles, respectively; the horizontal bar within each box represents the mean, and the bars represent the SD. *Value differs significantly (P = 0.043) from the baseline value.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Clearance of 99mTc-OA from the lungs—Clearance of OA, a larger molecule (45 kd) than DTPA, was examined in 8 calves. This larger molecule induced a clearance pattern that differed from the pattern for DTPA, as indicated by evaluation of images obtained on days –3 and 7 (Figure 1). In calves 3 to 8, the time required to clear 99mTc-OA from the lungs was greatest on day 7 after inoculation. Mean clearance half-time for 99mTc-OA at baseline was 675.95 minutes, which was significantly (P = 0.043) less than the value of 1,044.37 minutes on day 7.
To evaluate the possibility that repeated exposure to 99mTc-OA aerosol would influence clearance independent of BRSV infection, 2 additional inoculated calves (Nos. 9 and 10) and the mock-infected calf (No. 11) were tested for clearance of 99mTc-OA aerosol on multiple days before and after inoculation. Those calves failed to have a significant increase in clearance time on any day during the preinoculation testing, yet calves 9 and 10 both had an increase in clearance time after inoculation, similar to results for calves that received only the single baseline exposure before inoculation. The mock-infected calf did not have an increase in clearance time after mock inoculation (Figure 3). Mean 99mTc-OA clearance time for all 8 calves exposed to 99mTc-OA aerosol before and on day 7 after inoculation revealed a significant (P = 0.011) enhancement in persistence of OA in the lungs (Figure 2).
Clearance of 99mTc-OA from the lungs of calves 9 (white diamonds) and 10 (black squares) on multiple days before and after inoculation with BRSV and of calf 11 (gray triangles) before and after mock inoculation (A), and plasma concentrations of 99mTc-OA in calf 10 at multiple time points after aerosol exposure to 99mTc-OA before (black diamonds) and on days 5 (white squares), 7 (black triangles), 9 (black squares with a white cross inside), and 14 (black squares) after inoculation with BRSV (B). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Clearance of 99mTc-OA from the lungs of calves 9 (white diamonds) and 10 (black squares) on multiple days before and after inoculation with BRSV and of calf 11 (gray triangles) before and after mock inoculation (A), and plasma concentrations of 99mTc-OA in calf 10 at multiple time points after aerosol exposure to 99mTc-OA before (black diamonds) and on days 5 (white squares), 7 (black triangles), 9 (black squares with a white cross inside), and 14 (black squares) after inoculation with BRSV (B). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Clearance of 99mTc-OA from the lungs of calves 9 (white diamonds) and 10 (black squares) on multiple days before and after inoculation with BRSV and of calf 11 (gray triangles) before and after mock inoculation (A), and plasma concentrations of 99mTc-OA in calf 10 at multiple time points after aerosol exposure to 99mTc-OA before (black diamonds) and on days 5 (white squares), 7 (black triangles), 9 (black squares with a white cross inside), and 14 (black squares) after inoculation with BRSV (B). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Detection of 99mTc-OA in lymph and plasma— Plasma and lymph data were adjusted for each calf and reported as a percentage of the counts per minute in the lungs of each respective calf 15 minutes after the 99mTc-OA aerosol. For those calves in which the plasma concentration of 99mTc-OA was measured, the peak of 99mTc-OA was detected between 10 and 15 minutes after beginning of aerosol administration, with a gradual decrease during the next 4 hours (Figure 3). Generally on day 7, the amount of 99mTc-OA in plasma had decreased substantially below the amount at baseline, and on days 9, 11, and 14, the amount of 99mTc-OA in plasma was still less than the amount at baseline. The value for 99mTc-OA measured in plasma was greatest for most calves at baseline through 5 hours after aerosol administration. Infection diminished the amount of detectable 99mTc-OA in plasma. The number of calves was insufficient for statistical evaluation.
Samples of lymph were obtained from the cannulated calf (No. 8) at 30-minute intervals for 7 hours on each day that clearance was evaluated. On day –3 before inoculation, the peak concentration of 99mTc-OA was detected in lymph at 1 hour after 99mTc-OA aerosol exposure. On day 4, at all collection time points until 5.5 hours after aerosol exposure, the amount of 99mTc-OA detected in lymph was greater than on any other days. Also on day 4, clearance of 99mTc-OA from the lung of calf 8 was decreased to 554.9 minutes, compared with the baseline value of 850.8 minutes. Subsequent days (7, 9, and 11) revealed lesser amounts of 99mTc-OA measurable in the lymph than at baseline. At all time points on days 9 and 11, adjusted amounts of 99mTc-OA measured in lymph were < 10%. Even at 7 hours after aerosol administration on days –3 and 4, amounts of detectable 99mTc-OA were > 30%, whereas on days 9 and 11, 99mTc-OA was almost undetectable in lymph (Figure 4).
Amount of 99mTc-OA in samples of lymph (A) and plasma (B) obtained from a lymphatic cannulated calf (No. 8) at various time points after 99mTc-OA aerosol exposure before (black squares) and on days 4 (white triangles), 7 (black inverted triangles), 9 (asterisk), and 11 (white circles) after inoculation with BRSV. See Figure 2 for reminder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Amount of 99mTc-OA in samples of lymph (A) and plasma (B) obtained from a lymphatic cannulated calf (No. 8) at various time points after 99mTc-OA aerosol exposure before (black squares) and on days 4 (white triangles), 7 (black inverted triangles), 9 (asterisk), and 11 (white circles) after inoculation with BRSV. See Figure 2 for reminder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Amount of 99mTc-OA in samples of lymph (A) and plasma (B) obtained from a lymphatic cannulated calf (No. 8) at various time points after 99mTc-OA aerosol exposure before (black squares) and on days 4 (white triangles), 7 (black inverted triangles), 9 (asterisk), and 11 (white circles) after inoculation with BRSV. See Figure 2 for reminder of key.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
In calf 8, plasma concentrations of 99mTc-OA were measured, but at 1-hour intervals (Figure 3). The pattern for concentrations in plasma differed from that of concentrations in lymph. Beginning at 1 hour after 99mTc-OA aerosol exposure on day –3, values for 99mTc-OA were greater than values for all days after inoculation until 6 hours after aerosol exposure; on day 4, there was a peak in detectable 99mTc-OA at 6 hours after aerosol exposure. Similar to results for the other calves on days 7, 9, and 11, the adjusted 99mTc-OA concentration detected in plasma failed to reach 15% at any time point.
Clinical signs and blood gas concentrations—All BRSV-inoculated calves developed clinical signs of disease. Significant differences were apparent in mean clinical scores from days 6 through 9. Differences were most evident when comparing scores for days 8 and 9 with scores for days 0 through 4. A correlation of mean clinical score versus 99mTc-OA clearance half-time revealed a significant positive correlation (R2 = 0.614). The temporal relationship between severity of clinical disease and clearance half-time revealed an increase in both beginning at day 5 and peaking on days 7 and 8 (Figure 5). Analysis of arterial blood gas data revealed a significant difference between baseline values and values on day 7. Mean PaO2 was 87.9 mm Hg at baseline, and it decreased significantly (P = 0.032) to 68.4 mm Hg on day 7. Mean PaCO2 increased significantly (P = 0.029) from 33.18 mm Hg at baseline to 39.7 mm Hg on day 7.
Temporal relationship between mean ± SD clearance of aerosolized 99mTc-OA (squares) and clinical scores (diamonds) in 8 calves on various days after inoculation with BRSV. There was a significant correlation (R2 = 0.614; P < 0.05) between clearance and clinical scores.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Temporal relationship between mean ± SD clearance of aerosolized 99mTc-OA (squares) and clinical scores (diamonds) in 8 calves on various days after inoculation with BRSV. There was a significant correlation (R2 = 0.614; P < 0.05) between clearance and clinical scores.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Temporal relationship between mean ± SD clearance of aerosolized 99mTc-OA (squares) and clinical scores (diamonds) in 8 calves on various days after inoculation with BRSV. There was a significant correlation (R2 = 0.614; P < 0.05) between clearance and clinical scores.
Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.416
Shedding of BRSV—All calves shed virus at some time point during the 10 days after inoculation. Generally, virus was detectable on days 5 and 6, but some calves shed virus as early as day 3, and several were still shedding virus on day 8 (data not shown).
Antibody titers against BRSV— Calves were chosen on the basis of a very low (seronegative or < 4 on the indirect immunofluorescence assay) maternal-derived titer against BRSV. By day 21 (or sooner), all calves seroconverted to BRSV, with most calves having a titer > 360 (data not shown).
Discussion
In other studies19,20 conducted by our laboratory group in which we used the same method to induce BRSV infection, we determined that clinical signs begin on day 4 and gradually increase to day 7 or 8, after which most calves continue to get better until day 10 when the disease is mostly resolved. Calves in the study reported here had this expected course of disease. Clinical signs and blood gas concentrations during the peak days of illness indicated moderate to severe disease in all infected calves. Other indicators of successful infection included seroconversion and shedding of BRSV.
The hypothesis that BRSV infection alters the clearance of inhaled antigen was substantiated. Size of the nebulized molecules had a noticeable effect on whether clearance time from the lungs was increased or decreased. Altered permeability of bronchial epithelium, perhaps by viral effects on tight junctions, increased the movement of the small molecule DPTA from the lungs, whereas the larger molecule OA was delayed in passage from the lungs at similar time points. On the basis of other studies19,20 conducted by our laboratory group in which we used an identical experimental protocol and virus isolate, we know that there is some loss of virusinfected epithelium into small airways as well as the development of interstitial pneumonia. Indeed, the decrease in clearance of 99mTc-OA was most pronounced when the clinical signs were greatest. It is possible that aerosolized OA molecules were trapped in the lungs by an intense inflammatory response.
The greatest concentration of 99mTc-OA was detected in plasma prior to inoculation at baseline, and the amount detected in plasma diminished each day as the clearance time increased. In contrast in lymph, there was a large increase in 99mTc-OA concentration on day 4 after inoculation with a subsequent return to concentrations less than those at baseline, which essentially paralleled concentrations detected in plasma by day 7. In contrast, plasma concentrations of 99mTc-OA on day 4 were less than concentrations at baseline until 6 hours after aerosol exposure (Figure 4). For both plasma and lymph, the concentration of 99mTc-OA on days 7, 9, and 11 were greatly reduced (by approx a third) from those at baseline. Although the data for the lymph was from only 1 calf, the pattern in plasma was representative of data from 8 calves in which plasma concentrations of 99mTc-OA were measured.
Analysis of data obtained from calves exposed to 99mTc-OA multiple times before and after inoculation with BRSV supported the conclusion that the effects of viral infection, rather than a developing immune response, caused a delay in clearance of OA from the lungs. It is unlikely that the small amount of OA administered repeatedly by aerosol exposure would have caused antibody production within the time frame of this study. However, the persistence of OA in the lungs may serve to prime an immune response for an increase in responsiveness to inhaled antigen in the days and weeks following acute BRSV infection. This hypothesis is supported by other experiments22 in which mice and guinea pigs were experimentally infected with human RSV and exposed to OA-containing aerosol.
Examination of plasma concentrations of 99mTc-OA in conjunction with the clearance data for 99mTc-OA suggested that as the clearance time decreased, so did the amount of 99mTc-OA detectable in the plasma (Figure 3). This suggests that during the period of most severe pathologic changes in the lungs, the movement of solutes from the airspace into the vascular system may be compromised. In addition, results for the single mock-infected calf further indicated that the BRSV infection, rather than repeated 99mTc-OA aerosol exposure, was responsible for the increase in clearance time from the lungs.
During acute RSV infection in human infants, experimental infection in mice, and BRSV infection in calves, chemokines, cytokines (Th2), and inflammatory cells increase in the lungs, which creates an environment that facilitates antigen presentation and development of immune responses to inhaled allergens.7,22–25 Experimentally induced RSV infection in primed mice can stimulate a Th2 response and increased amounts of IgE in mice exposed by aerosol to OA.24 Our studies4,5,25 in cattle have revealed that infection with BRSV enhances IgE responses to Micropolyspora faeni (recently renamed Saccharopolyspora rectivirgula) and Th2 responses against inhaled Alternaria alternata. These data prompted us to hypothesize that altered antigen clearance may play a role in the increased immune response to aerosolized antigen-allergen during and after BRSV infection. The importance of these data to the health of cattle is implicit in the understanding that calfhood BRSV infection is common and that dairy, calf-rearing, and feedlot facilities are sources of multiple aeroallergens. Moreover, data from our cattle studies as well as mouse RSV experiments indicate that vaccination against BRSV or RSV may fail to protect and instead can induce a Th2 environment.26 Additional studies will be required to define the immunologic ramifications of these results.
ABBREVIATIONS
| RSV | Respiratory syncytial virus |
| BRSV | Bovine respiratory syncytial virus |
| Th | T-helper |
| 99mTc-DTPA | 99mTechnetium-labeled diethylene triamine pentacetate |
| OA | Ovalbumin |
| 99mTc-OA | 99mTechnetium-labeled ovalbumin |
99mTc-DTPA, Amersham Health, Sacramento, Calif.
Ovalbumin fraction V, Sigma Chemical Co, St Louis, Mo.
Technicare Omega 500, QRS Systems, Cleveland, Ohio.
Aero-Tech II nebulizer, CIS-US Inc, Bedford, Mass.
Nuclear Mac, version 5.21 software, Scientific Imaging Inc, Larkspur, Colo.
Centriprep ultrafiltration device, Amicon, Millipore Corp, Bedford, Mass.
Lab-Tek slides, Baxter Scientific, Brisbane, Calif.
FITC-conjugated anti-RSV, American BioResearch Labs, Seymour, Tenn.
GraphPad Instat, version 3 program, GraphPad Prism Software Inc, San Diego, Calif.
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