Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Large Animal Clinical Sciences
Non Technical Summary
Bovine respiratory disease (BRD) refers to a clinical syndrome of bronchopneumonia that is caused by a combination of multiple environmental, host and pathogen risk factors. Mannheimia haemoltyica is the most commonly isolated bacterial pathogen from BRD cases and is associated with high levels of morbidity and mortality. Among the multiple etiologic agents of BRD, M. haemolytica is known to cause the greatest amount of pulmonary inflammation and tissue damage. It is estimated that BRD costs the cattle industry approximately $1 billion dollars in animal death, morbidity, production losses, and treatment costs. No true gold standard for the diagnosis of BRD exists. Currently diagnosis is based solely on clinical examination findings and subjective assessment of cattle behavior. Prompt and accurate diagnosis is critical for successful treatment. All mammals produce four surfactant proteins: surfactant protein A, B, C, and D. Elevated surfactant protein D (SP-D) in bronchoalveolar lavage fluid and serum has been implicated as a sensitive and specific marker of early alveolar inflammation and basal membrane damage in humans and other species. This protein has yet to be evaluated in bovine bronchopneumonia. We expect that bronchopneumonia induces enough pulmonary inflammation in cattle to cause the release of SP-D into the blood. If elevated levels of SP-D can be consistently detected in the blood of cattle suffering from bronchopneumonia, then it can serve as a better diagnostic test than is currently available. We have developed a bovine model for the induction of bronchopneumonia using bronchoselective inoculation of Mannheimia haemolytica into the right apical lung lobes. In this study, we can use this model to determine the changes in SP-D in serum and bronchoalveolar fluid and ascertain if SP-D is in fact sensitive and specific for pulmonary inflammation in cattle and could be further investigated as a potential diagnostic marker for BRD.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The specific aims of this study are to develop an assay for the detection of bovine surfactant protein D in bronchoalveolar lavage fluid and serum of cattle with experimentally-induced bronchopneumonia.
Project Methods
This project will use 12 feedlot aged calves (approximately 500 lb body weight). Prior to enrollment, calves will be procured and transferred to the University of Tennessee livestock research center. Upon arrival, calves will be clinically evaluated for concurrent illness. Ear notch samples will be collected for bovine viral diarrhea virus antigen capture ELISA testing. Calves found to be suffering from illness or persistently infected with BVDV will not be enrolled in the study. Each calf will be monitored for signs of illness for 7 days prior to the initiation of the study. Trial Design This trial is designed as a prospective controlled study to determine the changes in SP-D in BAL fluid and serum in calves induced with M. haemolytica. Calves will be randomly assigned to 1 of 2 treatment groups: A) Negative control group (healthy calves, sham inoculated, n=4) and B) Principal group (induced pneumonia, n=8). Calves will be induced with bronchopneumonia 7 days after arrival at the research facility. Serum and BAL fluid samples will be collected prior to and after induction of bronchopneumonia at pre-determined time points. Calves will be monitored for 7 days post-infection. All calves will beeuthanized and necropsy examination performed on day 7. This end point is based on our previous work with this model. Trial Timeline Study Day Procedure -7 Arrival, weight, examination -7 to 0 Acclimation to environment 0 Blood collection, Bronchoaveolar lavage, induction of pneumonia 1 Blood collection, Bronchoaveolar lavage 2 Blood collection 3 Blood collection, Bronchoveolar lavage 4 Blood collection 5 Blood collection, Bronchoaveolar lavage 6 Blood collection 7 Blood collection, Bronchoaveloar lavage Conclude live study, necropsy Bronchopneumonia induction Bacteria Lyophilized M. haemolytica serotype 1, originally isolated from a case of pneumonic pasteurellosis in a feedlot calf (OSU Strain) will be reconstituted in sterile water and grown on brain heart infusion (BHI) agar supplemented with 5% bovine blood in a CO2 incubator at 37°C for 18-22 hr. Colonies will then be selected and suspended in BHI broth and incubated on a rotary shaker (180 rpm) for 5.5 hr at 37°C. Bacteria will be pelleted by centrifugation, washed 3 times in sterile phosphate-buffered saline (PBS),aliquoted, and resuspended in PBS. Optical density (650 nm) will be used to approximate a concentration of 1 x 109 colony forming units (cfus) per mL based on a standard curve of cfu vs. optical density. Plate counts will be conducted on the bacterial suspensions to confirm the bacterial concentration. Bacteria administration Bronchopneumonia will be induced using a strain of M. haemolytica validated with this model in calves. Cattle will be restrained in a chute, haltered, and the head elevated and stabilized. The external nasal area will be cleaned and a 5.9-mm endoscope with 2-mm biopsy channel will be introduced into the right nasal passage. The endoscope will be passed through the nasopharyngeal region and subsequently through the laryngeal folds into the trachea. The endoscope will be passed into smaller airways until the tracheal bronchus is identified. A 140-cm long, 1.95-mm diameter polyurethane catheter will be passed through the endoscopic biopsy channel, and inoculum containing approximately 3 to 5 x 109 of M. haemolytica suspended in approximately 5 mL PBS will be delivered through the catheter, followed by a 20-mL wash with PBS. The catheter will be slowly withdrawn and the restraint relieved. Sample Collection Blood and BAL samples will be collected at timesoutlined above. Venous blood will be collected via jugular venipuncture. Blood will be centrifuged, and serum will be aliquotted into cryovials. BAL fluid samples will be collected via bronchoscopy. Once the endoscope has been placed as outlined above, 60 mLPBS will be delivered through a sterile catheter. A syringe will be used to aspirate as much fluid from the area as possible. BAL samples will be aliquotted into cryovials. SP-D levels will be analyzed using an ELISA validated for cattle. Monitoring Calves will be observed daily for signs of clinical illness byexperienced personnel. Calf weight and rectal temperature will also be monitored daily. A clinical illness score will be assigned to each calf based on a modified system from Perino and Apley (see below). Any calf meeting the criteria of a CIS = 4 will immediately beeuthanized and a full gross necropsy performed. Clinical Illness Scores (CIS) CIS Description Clinical Appearance 1 Normal No abnormalities noted. 2 Slightly Ill Mild depression, gaunt, +/- cough 3 Moderate Illness Severe depression, labored breathing, ocular/nasal discharge, +/-cough 4 Severe Illness Moribund, near death, little response to human approach. Post-Mortem Analysis All calves will be euthanized and a full necropsy performed 7 days post challenge. Lungs will be removed and evaluated to determine the overall characteristics of the lung and the effectiveness of the challenge administration model. Aerobic microbial cultures will be performed on lung tissue samples collected at the time of gross necropsy to ensure M. haemolytica colonization of affected lung lobes. The percentage of consolidation in each lobe will be visually estimated. Total percentage lung consolidation will be calculated similar to methods reported by Fajt et al. Histopathologic sections of affected and healthy lung tissue will be made to determine the presence and extent of alveolar inflammation induced by the challenge model. Data Analysis Continuous measurements (SP-D, weight and rectal temperature) will be analyzed using generalized linear models to evaluate changes in variable values from before inoculation and post- treatment. Expected Results/Limitations The expected results of this study are an elevation of SP-D in the serum and BAL fluid of calves with induced bronchopneumonia compared to healthy controls. We anticipate the pattern of elevation to reflect the progression of the disease and to correlate with the severity of inflammation occurring at the level of alveolus. Limitations of this design include the use of a challenge model to induce bronchopneumonia in calves rather than evaluating naturally occurring cases. However, using a challenge model provides essential temporal control over the onset and progression of disease and allows sampling points along an established timeline. The number of calves enrolled in each treatment group may be too small to detect subtle differences in SP-D levels. However, the intent of the project is to develop the assay and gather preliminary data that can be used in garnering extramural funding for larger studies in the future. Unforeseen factors may interfere with the detection of SP-D in either sample types.