• View in gallery
    Figure 1—

    Mean + SD percentages (determined on the basis of the total number of cells) of CD4+ (A) and CD8+ (B) T lymphocytes and the ratio of CD4+ to CD8+ T lymphocytes (C) in lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in calves before (day −1) and at intervals after exposure to BRSV-infected (n = 7 [dark gray bars]) or BRSV-free tissue culture medium (mock exposure; 6 [light gray bars]) via aerosolization on day 0. The BRSV-infected and mock-infected calves were administered aerosolized ovalbumin on days 1 through 6 and day 15. *Group value on this day differs significantly (P < 0.05) from the group value on day −1. † On a given day, value for BRSV-infected calves differs significantly (P < 0.05) from the value for mock-infected calves.

  • View in gallery
    Figure 2—

    Mean ± SD total clinical disease scores assigned on days 0 through 11 to the 7 BRSV-infected and 6 mock-infected calves (administered aerosolized ovalbumin on days 1 through 6 and day 15) in Figure 1. Total clinical disease scores were calculated on the basis of assessments of various physical variables; a score ≤ 30 was indicative of a clinically normal calf, whereas a score ≥ 31 was indicative of a calf with clinical disease. Clinical disease scores were assigned before exposure to BRSV-infected or BRSV-free tissue culture medium on day 0.*Signifcant (P < 0.05) differences in mean total daily clinical disease scores between BRSV-infected (black squares) and mock-infected calves (black triangles) were detected on days 6 through 11. No significant differences in mean total clinical disease scores between BRSV-infected and mock-infected calves were detected on days 12 through 16 (data not shown).

  • View in gallery
    Figure 3—

    Mean ± SD daily changes from baseline (day −1) in expressions of selected cytokine genes by Th2 cells from lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in BRSV-infected (black bars) and mock-infected calves (gray bars) exposed to aerosolized ovalbumin on days 1 through 6 and day 15. A quantitative RT-PCR assay was used to quantify the expression of cytokine genes. Relative arbitrary gene expression units were assigned for each cytokine and known standards of the GAPDH housekeeping gene. All samples were normalized to the quantity of the expressed GAPDH gene by use of the following equation: normalized value = cytokine gene CT value/GADPH CT value. All cell samples were assayed in duplicate; results for duplicate samples with an SD > 30% were reassayed. No significant differences in cytokine gene expressions were detected between BRSV-infected and mock-infected calves throughout the study.

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Assessment of IgE response and cytokine gene expression in pulmonary efferent lymph collected after ovalbumin inhalation during experimental infection of calves with bovine respiratory syncytial virus

Laurel J. GershwinDepartment of Pathology, Microbiology, & Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Mark L. AndersonDepartment of Pathology, Microbiology, & Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Chunbo WangDepartment of Pathology, Microbiology, & Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Londa J. BerghausDepartment of Pathology, Microbiology, & Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Thomas P. KennyDepartment of Internal Medicine, School of Medicine, University of California-Davis, Davis, CA 95616.

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Robert A. GuntherDepartment of Surgery, School of Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

Objective—To assess IgE response and cytokine gene expressions in pulmonary lymph collected from bovine respiratory syncytial virus (BRSV)-infected calves after ovalbumin inhalation.

Animals—Thirteen 7- to 8-week-old calves.

Procedures—The efferent lymphatic duct of the caudal mediastinal lymph node of each calf was cannulated 3 or 4 days before experiment commencement. Calves were inoculated (day 0) with BRSV (n = 7) or BRSV-free tissue culture medium (mock exposure; 6) via aerosolization and exposed to aerosolized ovalbumin on days 1 through 6 and day 15. An efferent lymph sample was collected daily from each calf on days −1 through 16; CD4+ and CD8+ T lymphocyte subsets in lymph samples were enumerated with a fluorescence-activated cell scanner. Expressions of several cytokines by efferent lymphocytes and lymph ovalbumin-specific IgE concentration were measured. Each calf was euthanized on day 16 and then necropsied for evaluation of lungs.

Results—Mean fold increase in ovalbumin-specific IgE concentration was greater in BRSV-infected calves than in mock-infected calves. At various time points from days 4 through 10, percentages of T lymphocyte subsets and CD4+:CD8+ T lymphocyte ratios differed between BRSV-infected calves and day −1 values or from values in mock-infected calves. On days 3 through 5, IL-4 and IL-13 gene expressions in BRSV-infected calves were increased, compared with expressions in mock-infected calves. Lung lesions were consistent with antigen exposure.

Conclusions and Clinical Relevance—In response to the inhalation of aerosolized ovalbumin, BRSV infection in calves appeared to facilitate induction of a T helper 2 cell response and ovalbumin-specific IgE production.

Abstract

Objective—To assess IgE response and cytokine gene expressions in pulmonary lymph collected from bovine respiratory syncytial virus (BRSV)-infected calves after ovalbumin inhalation.

Animals—Thirteen 7- to 8-week-old calves.

Procedures—The efferent lymphatic duct of the caudal mediastinal lymph node of each calf was cannulated 3 or 4 days before experiment commencement. Calves were inoculated (day 0) with BRSV (n = 7) or BRSV-free tissue culture medium (mock exposure; 6) via aerosolization and exposed to aerosolized ovalbumin on days 1 through 6 and day 15. An efferent lymph sample was collected daily from each calf on days −1 through 16; CD4+ and CD8+ T lymphocyte subsets in lymph samples were enumerated with a fluorescence-activated cell scanner. Expressions of several cytokines by efferent lymphocytes and lymph ovalbumin-specific IgE concentration were measured. Each calf was euthanized on day 16 and then necropsied for evaluation of lungs.

Results—Mean fold increase in ovalbumin-specific IgE concentration was greater in BRSV-infected calves than in mock-infected calves. At various time points from days 4 through 10, percentages of T lymphocyte subsets and CD4+:CD8+ T lymphocyte ratios differed between BRSV-infected calves and day −1 values or from values in mock-infected calves. On days 3 through 5, IL-4 and IL-13 gene expressions in BRSV-infected calves were increased, compared with expressions in mock-infected calves. Lung lesions were consistent with antigen exposure.

Conclusions and Clinical Relevance—In response to the inhalation of aerosolized ovalbumin, BRSV infection in calves appeared to facilitate induction of a T helper 2 cell response and ovalbumin-specific IgE production.

Bovine respiratory syncytial virus is a single-stranded negative-sense RNA virus in the family Paramyxoviridae. In young calves, BRSV infection causes moderate to severe disease characterized by bronchitis, bronchiolitis, and interstitial pneumonia. Disease associated with BRSV infection in cattle is remarkably similar to disease associated with RSV infection in humans; RSV infection is a frequent cause of pneumonia and bronchiolitis in infants and young children. In both cattle and humans, the most severe disease forms are observed in the young individuals; adults have milder clinical signs, and reinfection is common. In contrast to many viruses that primarily elicit IgG and cellular immune responses, RSV has the ability to skew the immune response toward production of Th2 cytokines, particularly in humans that have a genetic predisposition for atopy.1,2 The parallelism of disease characteristics between RSV infection in children and BRSV infection in calves also includes the development of IgE antibodies against the virus during infection and after vaccination with formalin-inactivated vaccines.3–6

Bovids do not naturally develop asthma. However, natural or experimental exposure to antigens can induce development of IgE antibodies with subsequent impairment of respiratory function after reexposure to the inciting antigen. To examine the dynamics and persistence of inhaled antigens in lungs of cattle, we previously have studied the effect of BRSV infection on pulmonary clearance of inhaled antigens.7 Clearance of an inhaled prototype allergen (ovalbumin) in calves was prolonged because of infection with BRSV.7 The link between RSV infection early in life and allergic sensitization has been substantiated in previous studies.7–9 Among 140 children, RSV-associated bronchiolitis was found to be the most important factor for subsequent development of allergic sensitization and development of asthma.8,9 In another study,10 33% of children that had RSV-associated bronchiolitis were positive for IgE antibodies, compared with only 2.3% of children in a birth cohort that did not have RSV infection.

The effect of RSV infection on allergic sensitization in mice has also been investigated. Effects of concurrent RSV infection and aerosol exposure with ovalbumin on cytokine and IgE production vary depending on the experimental protocol; however, most RSV-infected mice developed enhanced sensitization and production of Th2 cytokines that were attributable to RSV infection.11 A study12 was also performed in mice to determine whether enhancement of allergic sensitization was unique to RSV infection or a general effect observed with other viral lung infections in mice. The allergic sensitization effect was found to be unique to paramyxoviruses, RSV, and pneumonia virus of mice.12 Although ovalbumin is the most commonly used prototype allergen for experimentally induced allergy models, the effect of RSV on allergic sensitization in mice has also been detected in experiments involving ragweed, cockroach allergen, and house dust mite allergens.13–15

Cannulation of the efferent caudal mediastinal lymphatic duct can be used to obtain samples of lymph draining from the lungs because such samples are otherwise impossible to obtain. Cells and antibodies exiting the lung through the efferent lymphatic duct ultimately reach the systemic circulation but only after dilution. Thus, serum composition is less reflective of the pulmonary environment than efferent lymph composition. Lung lavage techniques access airway lumens and provide cell and protein samples that are from a different compartment than that of an efferent lung lymphatic duct. Lymphatic duct cannulation in a calf allows for the observation of temporal changes in cell and antibody populations that exit the lungs via the lymphatic duct in response to infection or allergen challenge.

The economic impact of bovine respiratory tract disease not only includes loss because of death but also diminished weight gain in calves with chronic cough; furthermore, these calves do not thrive and may be culled long after signs of acute respiratory tract infection have resolved. The association of RSV infection in children with subsequent chronic allergic respiratory tract disease, coupled with the consistent parallel between RSV infection of children and BRSV infection of calves, provides a basis for the hypothesis that, in BRSV-infected calves, development of IgE antibodies against antigens inhaled during infection may be enhanced. Acceptance of this hypothesis supports a contention that chronic coughing in some cattle can be the result of allergic bronchitis that develops in response to inhaled environmental antigens. The purpose of the study reported here was to evaluate the effect of BRSV infection on ovalbumin-specific IgE production and cytokine responses in pulmonary lymph collected from the caudal mediastinal lymph node of calves exposed to inhaled ovalbumin and to monitor changes in variables of immune function during the course of infection. These experiments were intended to support or refute the hypothesis that infection with BRSV could enhance the sensitization of calves to environmental allergens inhaled during the early phase of BRSV infection and could thereby predispose these calves to the development of IgE-mediated respiratory tract disease.

Materials and Methods

Animals—Over a 2-year period, approximately 25 conventionally raised Holstein bull calves located on a dairy farm in Tracy, Calif, were screened for the presence of BRSV-specific antibodies. Thirteen calves without circulating anti-BRSV antibodies or with low titers of maternal anti-BRSV antibodies, which are not considered protective, were selected for use in the study. When these calves were 7 to 8 weeks old, they were transported to animal facilities at the University of California-Davis, maintained individually in stalls in a screened-in barn, fed alfalfa hay, and provided water and salt ad libitum. Each calf was maintained free of ectoparasites and endoparasites during the study. This study was approved by the Institutional Animal Care Committee at the University of California-Davis.

Schedule of experimental procedures—After arrival at the animal facility, all calves were acclimated for approximately 7 days. Each calf underwent cannulation of the efferent lymphatic duct of the caudal mediastinal lymph node 3 to 4 days before experimental exposure to BRSV (n = 7 calves) or BRSV-free tissue culture medium (6) via aerosolization on day 0. All calves were administered aerosolized ovalbumin once daily on days 1 through 6 and day 15. Calves were monitored daily for clinical signs of illness throughout the 16-day study period. A nasal swab was collected from each calf once daily on days 0 through 10 for assessment of the shedding of BRSV. Blood samples were collected on days −1, 0, 7, 10, and 16 for detection of serum anti-BRSV antibodies. A lymph sample was collected once daily on days −1 through 16 for evaluation of ovalbumin-specific IgE response, lymphocyte subsets, and cytokine RNA. Calves were euthanized on day 16, and lungs were examined for gross and histologic lesions consistent with antigen exposure. In addition, lung tissues were submitted for bacterial culture and immunohistochemical staining for virus detection.

Cannulation of the efferent lymphatic duct of the caudal mediastinal lymph node—The cannulation procedure used was a modification of a previously de-scribed16 method for cannulation of the lymphatic duct of sheep. In each calf, anesthesia was induced on day −3 or −4 via IV administration of 15 mg of thiamylal sodium/kg and maintained with isoflurane vaporized in 100% oxygen. Each anesthetized calf was positioned in left lateral recumbency and aseptically prepared for surgery. A thoracotomy was performed in the right dorsolateral portion of the thorax. The caudal mediastinal lymph node was located, and a silastic cannula was inserted into the efferent lymphatic duct of the node. The cannula exited through the muscles of the seventh intercostal space and was secured to a plastic tube holder that was sutured to the skin. After recovery from anesthesia, each calf was observed 3 to 4 times daily for changes in appetite, attitude, and patency of the cannula. Lymph flowed continuously, and when not being collected, lymph dripped freely to the ground. Erythrocyte contamination of the lymph samples was evaluated via cytologic examination of samples collected for 4 days after cannulation on day −3 or −4.

Lymph collection and processing—Beginning on day −1, approximately 20 to 50 mL of lymph was collected once daily from each calf into a 50-mL centrifuge tube containing 0.5 mL of heparin sodium (5,000 U/mL) on days −1 through 16. A Giemsa-stained smear was prepared from each sample for confirmation of cell morphologies before the sample was centrifuged at 1,600 × g to separate the cellular fraction of the lymph. The cellular fraction was immediately processed for analysis by FACS and frozen at −80°C for preservation of RNA within the cells. The remaining fluid fraction was frozen at −20°C for later analysis with an anti-ovalbumin IgE assay.

FACS analysis—Cells were collected from lymph samples on days −1 through 16 or until the cannula was no longer patent as described. A Hank's balanced salt solution was used to wash 4 × 107 cells that were previously separated from each lymph sample. After washing, cells were centrifuged at 1,600 × g for 5 minutes. A lysis buffer was added to the pelleted cells to lyse RBCs, and the cells were washed again. Cells were resuspended in Hank's balanced salt solution. A 1- to 2-mL aliquot of the suspended cells was then transferred, counted, and diluted to 107 cells in 0.1 mL of PBS and processed in preparation for FACS analysis. Antibodies used to phenotype the cells included a 1:10 dilution of a mouse anti-bovine CD8:FITC conjugated MCAB37Fa antibody and a 1:10 dilution of a mouse anti-bovine CD4:FITC conjugated MCA1654Fb antibody. The conjugate used was donkey anti-mouse IgG (heavy and light γ chain specific) R-phycoerythrin conjugated with 715–116,150c in a 1:20 dilution. Cell populations stained with these antibodies were examined by use of a FACS analysis system.d Briefly, the gated cell population was evaluated for forward and side scatter; after evaluation, staining of the cell populations with each antibody was determined, and the percentage and absolute number of cells in each stained population were determined by use of a software program.e

Exposure of calves to BRSV or tissue culture medium—With the exception of 1 calf, calves were studied in pairs and were randomly allocated to undergo exposure to BRSV (BRSV-infected group; n = 7 calves) or BRSV-free tissue culture medium (mock-infected group; 6) via aerosolization on day 0. The lymph cannula of the seventh calf in the mock-infected group developed a blockage by day 0 and, therefore, was removed from the study. Cell culture was used to propagate BRSV in primary bovine turbinate cells. Calves in the BRSV-infected group were administered 5 mL of cell culture medium that contained 3 × 104 TCID50 to 4 × 104 TCID50 of clinical isolate CA-1 via aerosolization with a nebulizer systemf attached to a face mask, as previously described.17,18 Calves in the mock-infected group were administered sterile bovine turbinate tissue culture medium via aerosolization with the same type of nebulizer system.e

Administration of ovalbumin—The same nebulizer system used to administer BRSV tissue culture medium via aerosolization was used to administer ovalbumin to the study calves. On days 1 through 6 and day 15, the mask was secured over the nose and mouth of each calf and the apparatus was used to administer 15 mL of a 1% solution of ovalbuming in PBS solution. In addition, approximately 200 mL of lactated Ringer's solution was administered SC once daily during ovalbumin administration for the maintenance of hydration.

Calf monitoring and clinical disease scoring— From days −1 to 16, each calf was evaluated once daily for the development of clinical signs associated with respiratory tract disease. A clinical disease scoring sys-tem19 modified for use in this study (Appendix 1) was applied by a veterinarian (LJG) to assess clinical signs of disease in each calf. A score was assigned to each of several physical variables on the basis of objective and subjective assessments. Scores for each variable then were manipulated mathematically, and the sum of the results of calculations for all variables (total clinical disease score) was recorded for further analysis.

Detection of BRSV shedding—A swab specimen was obtained daily from deep within the nasal cavity of each calf beginning on day 0 and ending on day 10. Specimens were collected before the administration of aerosolized BRSV or tissue culture medium on day 0 and before the administration of ovalbumin daily. Each swab was immediately inserted into 1 mL of Dulbecco minimal essential medium, briefly swirled to loosen cells from the swab, and stored on ice until processed. A cytospin slide was prepared in duplicate from each sample by placing 200 μL of fluid from the nasal swab tube into the concentrating funnel of a cytospin apparatus.h Slides with the deposited cell samples were fixed in acetone and stained with rabbit anti-RSV-FITC antibody. Slides were washed in PBS solution and then briefly rinsed in distilled water. The portion of the slide with the collected cell fraction was covered with several drops of a PBS-glycerol solution and covered with a coverslip. Slides were evaluated under a microscope equipped with epi-illumination, and the number of cells with fluorescence of the cytoplasm was counted. Shedding of BRSV from the nasal cavity was indicated by the detection of the fluorescence of the cytoplasm.

Serum anti-BRSV antibody titers—Before exposure to aerosolized BRSV or tissue culture medium on day 0 and at necropsy on day 16, a 10-mL blood sample was collected via venipuncture of a jugular vein into an evacuated tube that contained no additive from each calf for the detection of serum anti-BRSV antibodies by use of an indirect immunofluorescence assay. Serum samples were separated from clotted blood by centrifugation at 1,600 × g. All assays were performed by the California Animal Health and Food Safety Laboratory in Davis, Calif.

Measurement of ovalbumin-specific IgE response—Lymph obtained on days 0 through 16 were analyzed for ovalbumin-specific IgE by use of an ELISA. Ovalbumin diluted in a carbonate-bicarbonate buffer (pH, 9.6) was used to coat 96-well ELISA plates (1 μg of ovalbumin/well). Coated plates were then incubated for 18 hours at 4°C. Plates were then blocked with a 1% solution of rabbit serum albumin in carbonate-bicarbonate buffer for 1 hour at 37°C. After blocking of the plates, serum samples were thawed and treated with a 27.5% ammonium sulfate solution to remove most of the IgG antibodies that could compete for antigen binding with ovalbumin-specific IgE.20 Plates were washed 6 times and allowed to soak once for 10 minutes in a 0.1% solution of PBS-Tween 20. Serum with a high ovalbumin-specific IgE content was collected from a calf that was previously immunized with ovalbumin-alum and used as a positive control sample in the assay. Fetal bovine serum and PBS-Tween 20 solution were used as negative control samples. All serum samples and control samples were incubated for 1 hour at 37°C. A previously developed21 anti-bovine IgE monoclonal antibody (E5–490) was used as the primary antibody in the assay. A goat anti-mouse IgG horseradish peroxidase conjugatej that recognizes both γ heavy and light chains was diluted 1:2,500 in PBS solution and used as the secondary antibody. The plates were incubated a final time for 1 hour at 37°C and then washed with the PBS-Tween 20 solution. Next, 200 μL of an o-phenylenediamine substrate was added to each well, and the absorbance (optical densities at 450 and 650 nm) was read on an ELISA plate readerk after 15 minutes. Absorbance values were corrected for interplate variation by use of the positive control samples, and daily responses of each calf were determined. On day 16, the final IgE response result was tabulated as a fold change from the IgE response value on day 0 from each calf. Mean IgE responses were calculated for the BRSV-infected and mock-infected calves. The IgE response of each calf was determined daily. All samples and control samples were assayed in duplicate.

Necropsy—Calves were euthanized by an IV overdose of pentobarbital sodium on day 16. A complete necropsy of each calf, which included gross evaluation of all organ systems and histologic examination of any lesions observed, was performed by a board-certified veterinary pathologist (MLA). The respiratory tract was isolated, and a description of gross pathological lung lesions was recorded. Fixed tissue samples were prepared from the left lung by perfusion of those lobes with a solution of neutral-buffered 10% formalin. Fresh and frozen tissue samples were prepared from the accessory, caudal, cranial, and middle lobes of the right lung. Bacterial culture for respiratory pathogens and immunohistochemical staining for respiratory viruses were performed and evaluated.

Assessment of viral respiratory infections—Quantitation of anti-BVDV antibody titers in serum samples was performed. In addition, immunohistochemical analysis was performed on lung tissue sections for the presence of BRSV, BVDV, infectious bovine rhinotracheitis virus, and parainfluenza virus that commonly infect the bovine respiratory system.

Quantitation of cytokine gene expression by use of a quantitative RT-PCR assay—Frozen cell lysates stored at −80°C were thawed, and total RNA was isolated and extracted by use of a kit.1 Next, 1.0 to 1.5 μg of RNA was primed with an oligonucleotide (deoxyribonucleotide triphosphate) and reverse transcribed by use of a kit1 in a 20-μL RT reaction according to the manufacturer's recommendations. Quantitative PCR assays for the detection of gene sequences of the cytokines IL-4, IL-13, IFN-γ, and RANTES were performed by use of a thermocyclerm equipped with a 384-well format. The forward and reverse primers included in the PCR reactions were summarized (Appendix 2). The cDNA samples were diluted 1:10 with nuclease-free water. Each well contained a 10-μL PCR reaction that was prepared by the addition of 5.0 μL of PCR master mix,n 0.4 μL each of the forward and reverse primers, 2.2 μL of nuclease-free water, and 2 μL of cDNA. For each gene, a standard curve was prepared by use of a pooled cDNA stock solution sample. Negative control samples were prepared as reaction mixtures in which the volume of the diluted cDNA sample was substituted with nuclease-free water or the product of a sham RT reaction (ie, a reaction containing all RT reaction components except for cDNA). The PCR assay conditions were performed in a thermocyclerm with temperature cycle conditions as follows: an initial cycle at 95°C for 10 minutes and 40 cycles at 94°C for 15 seconds, then 60°C for 1 minute. A software programm was used to generate standard curves and relative expression values for the samples. The r2 value of all standard curves was > 0.980. For analysis of samples, relative arbitrary gene expression units were assigned for each cytokine and known standards of the GAPDH housekeeping gene. All samples were normalized to the expressed GAPDH gene by use of the following equation:

article image

All cell samples were assayed in duplicate, and results for duplicate samples with an SD > 30% were reassayed.

Statistical analysis—Statistical analysis was performed by use of a statistical software program.o Mean values for disease scores, T-cell subpopulations, ovalbumin-specific IgE response, and cytokine gene expression in the BRSV-infected and mock-infected calves were compared by use of a Wilcoxon-Mann-Whitney rank sum test. A 1-way ANOVA procedure with a Dunnett multiple comparison was used to evaluate changes in variables over time. A value of P < 0.05 was used to indicate significance in all analyses.

Results

Rate of lymph flow from the cannula and concentration of cells collected—Although a cannula became nonpatent on day 4 in 1 calf in the mock-infected group, on day 13 in 2 calves in the BRSV-infected group, and on day 14 in 1 calf in the BRSV-infected group, the remaining cannulas were patent and lymph flowed for the duration of the experiment. Data recorded from the calf in the mock-infected group with the nonpatent cannula on day 4 were not included in the statistical analyses. The mean flow rate of lymph from all cannulas was 15 mL/h. The concentration of cells in lymph varied from 1 × 107 cells/mL to 4 × 107 cells/mL. As determined via cytologic examination of lymph samples collected after cannulation of the efferent lymphatic duct of the caudal mediastinal lymph node, erythrocyte contamination of lymph decreased and the flow rate of lymph from the cannula stabilized by day −1.

FACS analysis—Subsets of T cells were detected with anti-CD4+ and anti-CD8+ T lymphocyte antibodies and reported as the percentage of total cells collected from lymph samples (Figure 1). Mean percentages of CD4+ T lymphocytes were significantly increased on day 4 (P = 0.050) and 6 (P = 0.008) for BRSV-infected calves, compared with mean percentages in BRSV-infected calves on day −1. However, no significant differences were detected for mean percentages of CD4+ T lymphocytes in mock-infected calves on days 0 through 16, compared with mean percentages of CD4+ T lymphocytes in mock-infected calves on day −1. Mean percentages of CD8+ T lymphocytes in BRSV-infected calves were significantly increased on days 8 (P < 0.001) and 10 (P = 0.024), compared with mean percentages in mock-infected calves. A significant (P < 0.05) difference in mean percentages of CD8+ T lymphocytes was detected as early as day −1 between BRSV-infected and mock-infected calves. Mean percentages of CD4+ and CD8+ T lymphocytes varied between 45% and 65% and between 18% and 30% of the total lymphocytes, respectively, throughout the study. The ratio of CD4+ to CD8+ T lymphocytes was significantly (P = 0.006) greater on day 6 than on day −1 for the BRSV-infected calves. The CD4+:CD8+ T lymphocyte ratio was significantly increased in BRSV-infected calves on days 8 (P = 0.038) and 10 (P = 0.024), compared with this ratio in mock-infected calves on day −1. Throughout the study, the ratio of CD4+ to CD8+ T lymphocytes in mock-infected calves was not significantly different from the ratio on day −1.

Figure 1—
Figure 1—

Mean + SD percentages (determined on the basis of the total number of cells) of CD4+ (A) and CD8+ (B) T lymphocytes and the ratio of CD4+ to CD8+ T lymphocytes (C) in lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in calves before (day −1) and at intervals after exposure to BRSV-infected (n = 7 [dark gray bars]) or BRSV-free tissue culture medium (mock exposure; 6 [light gray bars]) via aerosolization on day 0. The BRSV-infected and mock-infected calves were administered aerosolized ovalbumin on days 1 through 6 and day 15. *Group value on this day differs significantly (P < 0.05) from the group value on day −1. † On a given day, value for BRSV-infected calves differs significantly (P < 0.05) from the value for mock-infected calves.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.134

Clinical disease scores in BRSV-infected and mock-infected calves—Although mock-infected calves did not develop clinical disease (total clinical disease scores, = 30), BRSV-infected calves developed clinical disease (total clinical disease scores, = 31). Increases in clinical disease scores for BRSV-infected calves were recorded for adventitial lung sounds (eg, rales and wheezing), anorexia, coughing, fever, and signs of depression; there was no change in clinical disease scores for mock-infected calves. An increase in mean total clinical disease scores in BRSV-infected calves was observed on days 5 through 11; a peak mean score was recorded on day 9 (Figure 2). Significant differences (P < 0.05) in mean clinical disease scores between the BRSV-infected and mock-infected calves occurred on days 6 through 11.

Figure 2—
Figure 2—

Mean ± SD total clinical disease scores assigned on days 0 through 11 to the 7 BRSV-infected and 6 mock-infected calves (administered aerosolized ovalbumin on days 1 through 6 and day 15) in Figure 1. Total clinical disease scores were calculated on the basis of assessments of various physical variables; a score ≤ 30 was indicative of a clinically normal calf, whereas a score ≥ 31 was indicative of a calf with clinical disease. Clinical disease scores were assigned before exposure to BRSV-infected or BRSV-free tissue culture medium on day 0.*Signifcant (P < 0.05) differences in mean total daily clinical disease scores between BRSV-infected (black squares) and mock-infected calves (black triangles) were detected on days 6 through 11. No significant differences in mean total clinical disease scores between BRSV-infected and mock-infected calves were detected on days 12 through 16 (data not shown).

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.134

BRSV shedding, serum anti-BRSV antibody titers, and BVDV infection—Mock-infected calves did not shed BRSV during the study. Duration of BRSV shedding in BRSV-infected calves was variable on days 3 and 10. A mild increase in serum anti-BRSV antibody titers occurred in 2 BRSV-infected calves (titers of 80 and 40); however, anti-BRSV antibody titers > 320 were detected in the remaining BRSV-infected calves. Samples of the ileum obtained at necropsy from both calves that had minimal increases in anti-BRSV antibody titers (titers of 80 and 40) were stained with an immunoperoxidase and determined to be positive for BVDV infection.

Pathological lung changes and results of bacterial isolation—Evidence of pathological lung consolidation (percentage of lung affected, < 1% to 50%) was observed in all BRSV-infected calves. However, 7% and 25% of the lungs in 2 mock-infected calves had evidence of consolidation. The BRSV-infected calves had pathological lung lesions that were typical of BRSV infection, including evidence of bronchiolitis, bronchitis, and interstitial pneumonia. Bacteria were not cultured from lung tissues from 4 of the 7 BRSV-infected calves and from 4 of the 6 mock-infected calves. Pasteurella multocida was isolated from all 3 remaining BRSV-infected calves and from 1 of the 2 remaining mock-infected calves; in addition, Mycoplasma bovis was isolated from lung tissue samples from both of those mock-infected calves. Pathological lung lesions that were consistent with antigenic stimulation were observed in BRSV-infected and mock-infected calves; the lesions consisted of lymphoplasmacytic infiltrates observed in the bronchial mucosa, peribronchial lymphoid infiltrates, and lymphocytic nodules. A suppurative secondary bronchopneumonia was observed in all 5 calves in which bacteria were isolated.

Ovalbumin-specific IgE response in lymph—Ovalbumin-specific IgE responses were detected in lymph samples collected from the efferent lymphatic duct of the caudal mediastinal lymph node of BRSV-infected and mock-infected calves. For each sample, the difference in corrected absorbance from the day 0 value was calculated and expressed as the fold change from the day 0 value. The mean ± SD fold increase in ovalbumin-specific IgE concentration for the BRSV-infected calves was 2.694 ± 1.410, compared with 1.562 ± 0.678 for the mock-infected calves. The increase in ovalbumin-specific IgE concentration in efferent lung lymph was not significantly different between groups, although the P value (P = 0.083) was close to our cutoff for significance (P < 0.05).

Remarkable variability in the calf IgE response to ovalbumin administration was observed. One BRSV-infected calf had a gradual increase in IgE response over time; on day 16, the absorbance was 5.2 times as high as that of the absorbance on day 0. In contrast, there was a 2-fold increase in the absorbance in a second BRSV-infected calf on day 7, compared with the absorbance on day 0; thereafter, the absorbance decreased over time. A third BRSV-infected calf had an initial progressive increase in IgE response, and the absorbance on day 10 was 3.2 times as high as the absorbance on day 0; the absorbance declined thereafter and, on day 16, was almost unchanged from the absorbance on day 0. A fourth BRSV-infected calf failed to show evidence of ovalbumin-specific IgE production throughout the 16 days of the experiment. A fifth BRSV-infected calf had a steady increase in the IgE response; on day 15, the absorbance was 4 times as high as the absorbance on day 0. The IgE response for a sixth BRSV-infected calf peaked on day 7, and the peak absorbance was slightly > 2 times as high as the absorbance on day 0; this peak absorbance remained unchanged until day 15. The seventh BRSV-infected calf had a minimal ovalbumin-specific IgE response throughout the experimental period; this calf was considered not to have an ovalbumin-specific IgE response to ovalbumin administration. Three mock-infected calves did not have an ovalbumin-specific IgE response after ovalbumin administration. A fourth mock-infected calf had a gradually increasing ovalbumin-specific IgE response until day 16, when the absorbance was 2.2 times as high as the absorbance on day 0. A fifth mock-infected calf had a minimal ovalbumin-specific IgE response until day 10, when the absorbance was 1.6 times as high as the absorbance on day 0; however, an absorbance was not reported on day 16 for this calf because of complications related to the flow of lymph from the cannula. The IgE response for a sixth mock-infected calf was not determined because of complications related to the flow of lymph from the cannula on day 4.

Cytokine gene expression in cells from efferent lymph—Mean ± SD cytokine gene expressions in BRSV-infected and mock-infected calves on days −1 through 16 were analyzed after normalizing data by use of the GAPDH housekeeping gene expression values (Table 1). No significant differences in cytokine gene expressions were detected between BRSV-infected and mock-infected calves throughout the study. However, some patterns of change were observed in BRSV-infected calves. Mean changes in gene expressions of IL-4 and IL-13 were increased on days 3 through 5, compared with mean changes in mock-infected calves (Figure 3). The expression of IL-4 and IL-13 was greatest in BRSV-infected calves on day 4; thereafter, both IL-13 and IL-4 expression decreased until day 9. Lower expression of IL-4 and IL-13 in mock-infected calves was observed until day 15 when increases in IL-4 and IL-13 gene expressions were detected in both BRSV-infected and mock-infected calves. On days 5, 7, and 8, BRSV-infected calves had an increase in IFN-γ expression; on day 9, gene expression levels returned to the levels observed on day 6. The expression of RANTES was increased in BRSV-infected calves on days 4 and 5, compared with the expression of RANTES in mock-infected calves.

Figure 3—
Figure 3—

Mean ± SD daily changes from baseline (day −1) in expressions of selected cytokine genes by Th2 cells from lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in BRSV-infected (black bars) and mock-infected calves (gray bars) exposed to aerosolized ovalbumin on days 1 through 6 and day 15. A quantitative RT-PCR assay was used to quantify the expression of cytokine genes. Relative arbitrary gene expression units were assigned for each cytokine and known standards of the GAPDH housekeeping gene. All samples were normalized to the quantity of the expressed GAPDH gene by use of the following equation: normalized value = cytokine gene CT value/GADPH CT value. All cell samples were assayed in duplicate; results for duplicate samples with an SD > 30% were reassayed. No significant differences in cytokine gene expressions were detected between BRSV-infected and mock-infected calves throughout the study.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.134

Table 1—

Expressions of selected cytokine genes* by cells from lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in calves before (day −1) and at intervals after exposure to BRSV-infected (n = 7) or BRSV-free tissue culture medium (mock exposure; 6) via aerosolization on day 0 and administration of aerosolized ovalbumin on days 1 through 6 and day 15.

 IL-4INF-γIL-13RANTES
DayBRSVMockBRSVMockBRSVMockBRSVMock
−14.94±7.604.60±4.550.82±0.271.97±2.338.87±17.384.71±5.625.71±6.944.56±3.20
04.32±10.503.51±2.340.72±0.521.30±0.500.15±0.344.33±3.579.73±13.564.19±1.82
13.34±3.334.50±5.260.64±0.231.11±1.033.42±3.455.09±6.253.93±2.465.19±3.10
24.98±6.889.89±11.750.99±0.560.64±0.385.01±8.0514.12±17.045.57±5.997.71±6.16
38.66±13.043.01±3.741.47±1.930.52±0.1911.65±17.955.17±8.157.21±8.313.69±3.99
415.37±17.264.99±5.411.14±0.870.63±2.1916.65±21.006.28±7.1410.15±9.945.07±5.21
54.89±4.591.63±1.7010.51±20.382.14±2.794.85±4.631.35±1.368.00±8.892.22±0.46
61.71±2.592.46±2.234.40±3.611.33±1.422.18±3.283.89±4.132.12±2.063.66±1.66
71.85±3.832.38±2.276.68±7.851.32±0.862.63±4.922.44±2.382.10±2.133.26±1.44
81.59±2.4511.03±18.637.37±10.972.01±1.902.10±2.9316.77±29.042.56±1.4310.68±13.74
97.15±13.612.99±3.264.43±7.572.67±1.267.72±14.764.09±5.328.00±9.614.82±4.91
102.27±2.412.30±1.692.82±2.462.87±3.023.31±4.272.81±2.595.32±4.524.41±2.82
112.65±2.741.881.69±2.031.98±2.559.46±10.151.51
123.48±3.991.38±0.800.95±0.541.02±1.442.81±3.211.65±0.775.83±3.815.20±3.56
1310.69±10.273.85±4.671.71±0.961.73±2.4413.98±14.014.53±6.4011.65±11.464.39±2.40
143.50±5.030.72±0.705.98±8.500.74±0.474.05±6.560.51±0.7320.97±30.521.65±0.19
155.33±4.446.56±9.161.90±0.771.17±1.385.94±5.073.87±5.196.40±3.737.87±5.10
162.12±2.090.88±0.821.18±1.031.58±0.892.01±1.791.01±1.373.92±0.825.59±4.39

Data are reported as mean 6 SD.

A quantitative RT-PCR assay was used to quantify the expression of cytokine genes. Relative arbitrary gene expression units were assigned for each cytokine and known standards of the GAPDH housekeeping gene. All samples were normalized to the quantity of the expressed GAPDH gene by use of the following equation: normalized value = cytokine gene CT value/GAPDH CT value.

An SD was not determined because a sample was obtained for only 1 animal.

— = Not determined because samples were unavailable.

Discussion

To our knowledge, reports describing an IgE response to aerosolized antigens in cattle are limited. However, we have previously reported information regarding the immune response of 4- to 5-month-old calves exposed to aerosolized ovalbumin during a 9-week period22; only 1 of 3 calves had an ovalbumin-specific IgE response. This finding is not surprising because among humans and in inbred mouse strains, there are individuals that have high (atopic) and low IgE responses after antigen exposure.23 Indeed, the interaction of genetic background with environmental factors is critical for development of an allergic phenotype.24 Moreover, we have previously reported that BRSV infection alters the IgE response to 2 environmentally encountered microbes, Micropolyspora faeni (formerly Saccharopolyspora retivirgula) and Aspergillus fumigatus.25,26

Infection of calves with BRSV caused an alteration in the clearance of inhaled ovalbumin from the lungs.6 Clearance of inhaled technetium-labeled ovalbumin in calves was decreased most when the severity of clinical signs was greatest after experimental infection with BRSV on day 8. This decreased clearance rate persisted until ≥ 15 days after infection with BRSV. Persistence of antigens in the lungs allows antigen-presenting cells to process antigen and lymphocytes to respond to antigens found within the lung environment for a longer period. On the basis of these data and the fact that BRSV infection can increase the serologic response to an antigen in sensitized calves, the study reported here was designed to examine the local pulmonary immune response to aerosolized ovalbumin that was administered daily for 6 days during the early phase of BRSV or mock infection in calves that were not previously exposed to ovalbumin. Analysis of efferent lymph collected from the caudal mediastinal lymph node daily for assessment of cytokine gene expressions and ovalbumin-specific IgE content provided data reflective of the temporal fluctuations of these immune responses in the lungs of calves. Previous studies25,26 by our group to investigate BRSV infection and aerosol exposure to allergens in calves have been limited to the evaluation of IgE responses in serum.

Cannulation of the efferent lymphatic duct of the caudal mediastinal lymph node for the collection of lymph from the lungs of calves has been evaluated in mock-infected27 and BRSV-infected calves.17 The findings of those studies provide historical control data for comparison with results of subsequent investigations. Historical control ratios of CD4+ to CD8+ T lymphocytes in calves on day 10 after mock exposure vary from 2.82 to 1.91; on the day after BRSV exposure, the historical control ratio varies from 2.02 to 1.87. In the present study, ratios ≥ 3 were detected in mock-infected calves on days 4 through 10 and in BRSV-infected calves on days 4 through 8. These differences between the findings of the present study and the historical control data reflect an increase in the number of CD4+ T lymphocytes in the lungs of calves in the present study. The increase in the number of CD4+ T lymphocytes was most likely because of the administration of aerosolized ovalbumin. The percentages of CD4+ T lymphocytes in BRSV-infected calves on days 4 and 6 were significantly different, compared with the percentage on day −1, but a similar significant difference was not detected in mock-infected calves; this suggests that there was a greater effect of the virus than that of the aerosolized antigen on CD4+ T lymphocytes. As would be expected in calves with a viral infection, BRSV-infected calves had a moderate, gradual, and significant increase in the percentage of CD8+ T lymphocytes beginning on day 8. The percentage of CD8+ T lymphocytes changed significantly from the percentage on day −1 only in BRSV-infected calves. The CD4+:CD8+ T lymphocyte ratios in BRSV-infected and mock-infected calves differed significantly only on day 8; at that time point, the ratio in mock-infected calves was greater. This difference was caused by an increase in the number of CD8+ T lymphocytes in BRSV-infected calves.

An ovalbumin-specific IgE response was detected in efferent lymph collected from the caudal mediastinal lymph node in the calves of the present study. Comparison of the mean absorbance of the final lymph samples obtained on day 16 with that of the mean absorbance of samples obtained on day 0 for each group revealed that the BRSV-infected group had a greater concentration of ovalbumin-specific IgE antibodies than did the mock-infected group. Considerable variability in the ovalbumin-specific IgE response, temporal changes in peak responses, and the relatively small sample size contributed to a lack of statistical significance for differences between BRSV-infected and mock-infected calves in the study reported here. Nonetheless, because of the patterns of change in individual calves and the fact that the P value (P = 0.083) was close to our cutoff for significance, our findings could support the hypothesis that BRSV infection enhances an ovalbumin-specific IgE response following inhalation of ovalbumin during the first 6 days after BRSV infection. The high degree of variability in IgE responsiveness among calves receiving similar treatment in the same environment was apparent and similar to the findings of other studies.5,6,17 This variability supports the premise that 1 or more genes interact with environmental factors to modulate IgE responsiveness in calves.

Measurement of cytokine gene expressions in lymphocytes collected from lymph provided information regarding temporal changes in the immune environment of the lungs of calves. Lymph samples collected on days −1 and 0 were used to provide baseline values for cytokine gene expressions for each calf; this enabled us to evaluate the relationship between the cytokine response to BRSV or mock infection and the administration of aerosolized ovalbumin (Figure 3). Results for cytokine gene expressions (IL-4, IL-2, and IFN-γ) in BRSV-infected and mock-infected calves that were not exposed to aerosolized ovalbumin have also been re-ported17; furthermore, similarities between the findings of the present study and results of those previous studies are apparent. In the other study,17 expression of the IL-4 gene was greater in all BRSV-infected calves and in 2 of the 5 mock-infected calves on days −1 through 4; however, the IL-4 gene was not expressed in mock-infected calves after day 4. The BRSV-infected calves had a more consistent level of IL-4 gene expression; furthermore, expression of the IL-4 gene was detected in 4 of the 8 BRSV-infected calves from day −1 through day 7. In both groups, expression of the IL-2 gene was detected on all 10 days this was assayed. The expression of the IFN-γ gene was detectable in both groups but was most consistent in the BRSV-infected group in which 9 of 9 calves expressed the IFN-γ gene on days 5 through 7. In the study reported here, expression of the IL-4 gene increased from day −1 to a peak in expression on day 4 in the BRSV-infected calves that were administered aerosolized ovalbumin. As expected, IL-13 gene expression was similar to the change in the expression of the IL-4 gene in the BRSV-infected calves. On days 5 to 10 of the present study in the BRSV-infected group, the IFN-γ gene was expressed to a greater extent than were all other cytokine genes. Mean cytokine gene expression values were lower in the mock-infected calves that were administered aerosolized ovalbumin but similar to previously reported results for mock-infected calves. In addition, in the previous study,17 expression of the IFN-γ gene was detected in both groups but was more consistently expressed in the BRSV-infected group in which 100% (9/9) of calves expressed the IFN-γ gene on days 5 through 7.

Cytokines that regulate humoral versus cellular immune responses include IL-4 and IL-13 for Th2 and IFN-γ for Th1. Both IL-4 and IL-13 facilitate IgE production, whereas IFN-γ downregulates IgE production.28 Expression of cytokine genes associated with Th2 cell responses and expression of IFN-γ were determined on days 0 through 16 or until lymph could not be collected from the cannula because of flow stoppage (Table 1). There was no distinct pattern of cytokine gene expressions in relation to ovalbumin-specific IgE response at each time point. However, expression of the IL-13 gene was greater in BRSV-infected calves, compared with expression in mock-infected calves. For example, the 2 BRSV-infected calves that had peak levels of IL-13 gene expression in the absence of IFN-γ had ovalbumin-specific IgE antibodies detectable in lymph samples at the time of the peak in IL-13 gene expression. In contrast, 1 BRSV-infected calf that did not have an ovalbumin-specific IgE response had almost no expression of the IL-13 gene but had consistent expressions of both the IL-4 and IFN-γ genes. Expression of the IFN-γ gene in mock-infected calves, when evident, occurred in conjunction with expression of the IL-4 gene. Expression of the IL-4 and IFN-γ genes in the BRSV-infected calves occurred independently and concurrently with each other. The concurrent production of Th2 and Th1 cytokines and IFN-γ would be expected to exert a modulatory effect on IgE production, as demonstrated in calves that failed to have a strong IgE response to aerosolized ovalbumin. Results of cytokine gene expressions indicated that BRSV-infected calves had greater increases in expressions of both Th2 cytokines, compared with expressions in mock-infected calves. In addition, BRSV-infected calves had comparatively greater IgE responses to aerosolized ovalbumin.

To our knowledge, expression of the RANTES gene in efferent pulmonary lymph obtained from calves after BRSV or mock infection has not been reported. Expression of RANTES, which is a chemokine of the a family that is also designated as CCL5, by bronchial epithelial cells after RSV or BRSV infection and by lymphocytes during an allergic response has been detected.29,30 Therefore, increased expression of the RANTES gene was expected in both groups of calves administered aerosolized ovalbumin in the present study. Although expression of the RANTES gene in BRSV-infected and mock-infected calves increased throughout the experiment, no significant difference between groups was detected. This lack of significance in RANTES gene expression in lymph samples between calf groups was likely a consequence of the high individual variability for cytokine production and the relatively low number of animals in each study. However, expression did increase during the first 5 days (days 0 through 4) of BRSV infection when the expression of Th2 cytokines peaked. These results support the contention that induction of the expression of RANTES by lymphocytes is facilitated more by BRSV infection than by administration of aerosolized ovalbumin. The accumulation of inflammatory cells in the lungs of BRSV-infected and mock-infected calves is not commonly observed in calves infected with BRSV that are not administered an aerosolized antigen. Within these lymphoplasmacytic accumulations, T lymphocytes were presumably stimulated by local antigen-presenting cells to express cytokines that modulate the influx of inflammatory cells.

Examination of fixed lung tissues from BRSV-infected calves revealed lesions that presumably resulted from BRSV infection and, in some calves, secondary bacterial infection. These pathological changes were similar to findings of other studies19,20 in which calves were experimentally infected with BRSV in a similar manner. However, the microscopic detection of lymphoplasmacytic infiltrates in the bronchial mucosa, lymphocytic nodules, and peribronchial lymphoid infiltrates in fixed lung tissues of calves of the study reported here were suggestive of antigenic stimulation with ovalbumin. Furthermore, those findings were common to both BRSV-infected and mock-infected calves and are not so commonly observed in calves infected with BRSV in the absence of aerosolized antigen. Within these lymphoplasmacytic cellular accumulations, T lymphocytes were presumably stimulated by local antigen-presenting cells to make cytokines that modulate the cellular response in the lung. These observations together with the results of quantitative RT-PCR assay results of cytokine gene expression in efferent lymphocytes suggest that these lymphoplasmacytic accumulations are local sites of immune activation. Expression of the RANTES gene by T lymphocytes detected in lymph supports this premise because RANTES is known to attract basophils, dendritic cells, eosinophils, monocytes, and T lymphocytes. Therefore, the presentation of an inhaled antigen by dendritic cells to T lymphocytes would be an expected outcome.

Results of the present study indicated BRSV infection facilitates an ovalbumin-specific IgE response (detectable in pulmonary efferent lymph samples) following ovalbumin challenge in some calves. However, some ovalbumin-exposed BRSV-infected calves did not have an ovalbumin-specific IgE response. Yet overall, BRSV infection had an influence on the immune response to the administration of aerosolized ovalbumin. The role of genetic factors in an ovalbumin-specific IgE response was implicated by intercalf and intracalf variability and would be a likely target for future investigation. Nonetheless, the enhancement effect of BRSV infection coupled with the administration of aerosolized ovalbumin in calves was apparent, and this could contribute to the development of chronic cough in calves after recovery from BRSV infection and those that undergo environmental exposure to inhaled dusts, molds, or other antigens. The findings of the present study in BRSV-infected and allergen-exposed calves paralleled the findings reported2 for infants who are infected with RSV and exposed to inhalant allergens. In calves and humans, early exposure to environmental antigens alters the immune response to subsequent exposures to inhaled environmental antigens.

ABBREVIATIONS

BRSV

Bovine respiratory syncytial virus

BVDV

Bovine viral diarrhea virus

C

Cycle threshold

FACS

Fluorescence-activated cell scanner

FITC

Fluorescein isothiocyanate

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

IFN-Y

Interferon-γ

IL

Interleukin

RANTES

Chemokine (C-C motif) ligand 5

RSV

Respiratory syncytial virus

RT

Reverse transcription

Th

T helper

a.

Abd Serotec, Oxford, England.

b.

Abd Serotec USA, Raleigh, NC.

c.

Donkey anti-mouse IgG (H+L) R-PE conjugated 715–116150 antibody, Zymed Laboratories Inc, South San Francisco, Calif.

d.

FACScan flow cytometer, Becton-Dickinson, San Jose, Calif.

e.

CellQuest, Applied Biosystems, Foster City, Calif.

f.

DeVilbis nebulizer system model No. 571, DeVilbiss Corp, Somerset, Pa.

g.

Grade V, Sigma Chemical Co, St Louis, Mo.

h.

Cytospin 3, Shandon Laboratories, Shandon, Calif.

i.

American Bioresearch Lab, Seivierville, Tenn.

j.

Zymed Laboratories Inc, South San Francisco, Calif.

k.

VersaMax ELISA plate reader, Molecular Devices, Sunnyvale, Calif.

l.

Ambion, Applied Biosystems, Foster City, Calif.

m.

PRISM 7900 sequence detection instrument ABI SDS 2.02, Applied Biosystems, Foster City, Calif.

n.

2X Syber PCR mix, Invitrogen, Carlsbad, Calif.

o.

Prism, version 4, Graphpad Software Inc, San Diego, Calif.

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Appendix 1

Scoring system used to assign clinical disease scores to calves on the basis of objective and subjective assessments of clinical signs of respiratory tract disease.

VariableScoreFormula*
Anorexia0 or 1Value × 100
Signs of depression0 or 1Value × 100
Cough  
   Spontaneous0 or 1Value × 10
   Induced0 or 1Value × 10
Nasal discharge  
   Right1–3(Value right + value left) × 10
   Left1–3 
Adenitis0 or 1Value × 10
Conjunctivitis0 or 1Value × 10
Ocular exudate  
   Right1–3(Value right + value left) × 10
   Left1–3 
Rectal temperature (°C)Value(Value − 39.5) × 100
Respiratory rate (breaths/min)ValueValue × 1
Dyspnea0 or 1Value × 100
Mouth breathing0 or 1Value × 100
Auscultation of the lungs0 or 1Value × 100

Scores for each variable were mathematically manipulated. A sum of the results of calculations for all variables was recorded as the total clinical disease score for further analysis.

Signs of depression included unwillingness to rise from a recumbent position, disinterest in surroundings, and reluctance to move.

Auscultation of the lungs was used to detect adventitial lung sounds (ie, rales and wheezing).

(Adapted from Collie DD. Pulmonary function changes and clinical findings associated with chronic respiratory tract disease in calves. Br Vet J 1992;148:33–40. Reprinted with permission.)

Appendix 2

Primer sequences used in the quantitative PCR assay reactions for the quantification of cytokine gene expression in lymph samples collected from a cannula inserted into the efferent lymphatic duct of the caudal mediastinal lymph node in calves before (day −1) and at intervals after exposure to BRSV-infected (n = 7) or BRSV-free tissue culture medium (mock exposure; 6) via aerosolization on day 0. The BRSV-infected and mock-infected calves were administered aerosolized ovalbumin on days 1 through 6 and day 15.

GeneDirectionPrimer sequence*
GAPDHForward5′-GCATCGTGGAGGGACTTATGA-3′
 Reverse3′-GGGCCATCCACAGTCTTCTG-5′
IL-4Forward5′-GCCACACGTGCTTGAACAAA-3′
 Reverse3′-TGCTTGCCAAGCTGTTGAGA-5′
IL-13Forward5′-AAGGACCAAGAAGATGCTGAATG-3′
 Reverse3′-CGGACGTACTCACTGGAAACC-5′
IFN-γmForward5′-TGGAGGACTTCAAAAAGCTGATT-3′
 Reverse3′-TTTATGGCTTTGCGCTGGAT-5′
RANTESForward5′-CAACTTTGCTTCTCGCTCTTGTC-3′
 Reverse3′-TGGGAGGAGGGCATTGC-5′

Primer sequences are based on GenBank accession Nos. 281181 (GAPDH), 280824 (IL-4), 281247 (IL-13), 2812237 (IFN-γ), and 327712 (RANTES).

Contributor Notes

Ms. Berghaus' present address is the Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

Supported by the USDA National Research Initiative Grant (No. 2000–02061) and the Center for Food Animal Health at the University of California-Davis.

The authors thank Linda Talken for technical assistance.

Address correspondence to Dr. Gershwin (ljgershwin@ucdavis.edu).