Performing Department
Veterinary Sciences
Non Technical Summary
The hypothesis driving this study is that Histophilus somni is an important and underestimated cause of respiratory disease in western cattle. H. somni is one recognized cause of bovine pneumonia, most often when large groups of weaned calves are stressed. It is a leading cause of feedlot disease in Canada on the basis of multiple studies. By contrast, its importance in the United States is more opaque. One major US view is that H. somni is largely a secondary, opportunistic pathogen in the bovine respiratory disease (BRD) complex. Our impression from routine diagnostic accessions is that H. somni is commonly present in BRD in Wyoming and that other facets of histophilosis, such cardiac disease, are missed unless they are specifically sought. The proposed 3-year study characterizes and quantifies the impact of H. somni on BRD in selected commercial operations in Wyoming. Large cohorts of cattle entering feedlots will be tested at feedlot entry, and 30 and 60 days after placement, for evidence of infection with H. somni using two recently developed serological assays. Lungs from animals dying of pneumonia between feedlot entry (day 0) and 60 days after placement will be assessed to establish how many contain H. somni, either as sole pathogen or along with other BRD agents (M. haemolytica; P. multocida; M. bovis; BoHV-1; BRSV; PI-3; BVDV). Better control of BRD by vaccination and/or mass medication is premised on understanding the role of H. somni in cattle, as well as when animals become infected, and on increased awareness by producers and clinical veterinarians of its impact.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
(N/A)
Developmental
25%
Goals / Objectives
i: Seroprevalence: Determine seroprevalence (= proportion of positive cattle) of H. somni in calves as they enter feedlots, and during the major risk period for bacterial pneumonia (0 - 60 days after placement). This involves using two recently developed serological assays specific for H somni. Testing will be done in replicate and in cooperation with Wyoming producers with endemic H. somni on their properties (years 2 and 3).ii: H. somni in fatal pneumonia: Establish the relative importance of H. somni as a contributor to fatal pneumonia in cattle by frequency of detection (by laboratory culture, PCR and IHC) relative to other infectious causes of pneumonia (years 2 and 3).iii: H. somni and Mycoplasma bovis concurrence in fatal pneumonia: Define concurrence of H. somni with M. bovis in pneumonia (years 2 and 3).
Project Methods
#1. Application of multiplex PCR and IHC to at least 30 cases of BRD submitted from across Wyoming, to establish the frequency with which H. somni is detected, and the concurrence of other pathogens.#2. Immune response to H. somni during first 60 days on feed. We predict a high proportion of cattle will be seronegative on entry, become infected by H. somni, and seroconvert during the first 60 days. To assess this, 250 cattle coming into each of two commercial properties will be bled at 0, 30 and 60 days on feed (= 3,000 serum samples over two years). The enzyme linked immunosorbent assay (ELISA) detects antibodies to H. somni biofilm exopolysaccharide (ELISA #1). Several lines of evidence suggest EPS is formed by H. somni in response to stress, such as inflammation.31 When a cut off value of 0.268 is used, the sensitivity of the assay for infected calves is 90.5%, and specificity for healthy calves is 92.5%. The second ELISA is directed against surface immunoglobulin binding proteins (IgBPs) which are a critical virulence factor encoded by ibpA along with an upstream gene, ibpB. IbpA encodes the IgBP protein (named IbpA). As with the ELISA #1, the IbpA assay (ELISA #2) has been used to assess exposure to infection with H. somni. The proposed study will be the first time the assays having been compared side by side using cattle in commercial settings. We predict 50% seroprevalence at 60 days. For this the sample size calculation is 424, allowing for 10% attrition.#3. Role of H. somni in fatal pneumonia. Calves that die will be examined post-mortem by premise employees or a clinical veterinarian. Lungs of calves with gross evidence of pneumonia will be submitted overnight by United Parcel Service (UPS) to the Wyoming State Veterinary Laboratory (WSVL). They will be examined by a pathologist (DO'T, with KS as backup) along with the undergraduate or graduate student. Gross lesions will be quantified, photographed and sampled. Samples will be taken for multiplex PCR detection of BVDV, PI-3, BRSV and BoHV-1 (currently in use in virology section), and PCR detection of H. somni, M. haemolytica, P. multocida, M. bovis and T. pyogenes (bacteriology). Gross examination will follow standard WSVL protocols, with the addition that lungs will be scored on a +1 to +4 scale for severity of lesions. Comparison of the proportion of lungs with bacterial lesions will be evaluated by ANOVA (comparing three time periods) and by Chi-square. Aerobic culture will be performed on samples of pneumonic lungs and, when present, from lesioned heart. The following will be harvested and placed in 10% neutral buffered formalin; three 2 × 2 cm samples of lung, including consolidated areas, all obvious lesions, and grossly normal areas; a 1- to 2-cm sample from right ventricular myocardium and intraventricular septum of heart; and two 2 x 2 samples from the wall of the left ventricle of the heart (including papillary muscles, where H. somni commonly localizes). Any cardiac tissue with obvious abscesses or inflammation will be sampled. Tissues will be embedded in paraffin, dehydrated, and sectioned for staining with hematoxylin and eosin. Immunohistochemical staining will be done using published methods. Two sections from each block will tested for antigens of the following 4 agents: H. somni, M. hemolytica, M. bovis, and P multocida; additional testing (e.g., for BVDV) will be done as required. The primary antibody reagents used in the stains will be: M. bovis-polyclonal rabbit antiserum at dilutions of 1/1000 or 1/2000 (source: VMRD); M. hemolytica and P. multocida monoclonal antibodies ascites fluid diluted 1/2000 and 1/4000 (source: Veterinary Infectious Diseases Organization, Saskatoon, Saskatchewan); H. somnus rabbit polyclonal antiserum diluted 1/1000 and 1/2000 (source: Dr. L. Corbeil). Appropriate positive and negative control sections from isolation-confirmed cases will be tested concurrently with test tissues. Tissues will be scored for bacteria, without knowledge of gross or microbiological findings, as either positive or negative. The results will be recorded in a spreadsheet and in WSVL's data management system (VADDS).#4. H. somni and Mycoplasma bovis concurrence in fatal pneumonia. These two agents frequently occur together in lungs with chronic caseonecrotic lesions, most commonly in foci of necrosis and in distal airways. Others made similar observations in the past. At present the assumption is that M. bovis is the major factor causing necrosis and thereby responsible for antibiotic failure. We will assess the frequency of co-localization by the two agents by a combination of immunohistochemical scoring and PCR. The correlation of H. somni and M. bovis will be calculated using the kappa statistic.