1. T. Allen 1, D. O’Toole 1, R. Hunter 2, L. Corbeil 3 1. University of Wyoming, Department of Veterinary Science 2. 778 Sybille Creek Road, Wheatland, WY 82072 3. Department of Pathology, University of California

2. Gram-negative pleomorphic rod Bacterial pathogen in Bovine Respiratory Disease Complex Alone or concurrent with P. multocida and M. haemolytica Diseases associated with H. somni Thrombotic meningoencephalitis Laryngitis-tracheitis Abortion Other; synovitis; otitis; ophthalmitis; Myocarditis

3. Not traditionally recognized in United States Canadian feedlots document the disease in sudden death and myocarditis Inconsistent isolation from affected tissue Antibiotic use Vaccination use Moderately fastidious organism Chronic infections Autolysis/overgrowth Low BVD association Experimental reproduction unsuccessful

4. 1. Document histophilosis-associated myocarditis in Wyoming over a 3 month period (Nov ‘08-Jan ‘09) 1. Historically, this is when disease peaks 2. Obtain and characterize isolates from hearts and establish bank of isolates 3. Develop hypothesis for basis of cardiac localization 4. Determine other, concurrent, causes of death

5. Referring veterinarian Two ranches Whole Heart Un-incised Lung Aseptic collection Urine * Serum * * Urine collected from earlier samples, Serum(pre or post-mortem) from later samples

6. Bacteriology Aseptic swabs or tissue samples from heart and lung Histopathology 10% neutral buffered formalin Heart Interventricular septum Left ventricular papillary muscle Right ventricular papillary muscle Atria Lung

7. Bacterial isolation Pinpoint colonies @ 24 hours Brown colonies @ 48 hours Yellow coloration when swabbed Growth Conditions Columbia Blood Agar 37⁰C 10% CO 2 “Cold Feet” Does not survive moderate freeze Banking Isolates Grown overnight on chocolate slants, covered in BHI Frozen in acetone/dry ice bath Stored at -70⁰C

8. Sectioned at 5µm thickness H. somni membrane insoluble fraction specific immunoglobulin Hematoxylin counterstain Transmission electron microscopy

9. Work done by Dr. L. Corbeil, UC-San Diego Bacterial Isolates Acute/Convalescent Serum Cardiac Strain Compare to lung, brain, fetal isolates Establish presence of IgbpA, DR1, and DR2

10. Acute myocarditis Clinical signs 2 days Left ventricular papillary Note: Cranial PM less commonly affected

11. Chronic Myocarditis Suppurative Clinical signs 8 days

12. Chronic Myocarditis Fibrotic Clinical signs 15 days

13. Hemorrhagic valvular endocarditis Ruptured chordae tendineae Common Findings

14. Lung Purple Spongy No obvious pneumonia Edematous

15. H. somni isolated from 7 of 10 positive cases More often from acute myocarditis 3 cases: concurrent isolation from lung

16. IHC confirmed 7 bacterial cultures and three more Chronic myocarditis confirmed 3 lung samples also IHC positive

17. Bacterial emboli plug capillaries and veins Bacteria adherent to vessel walls

18. Cardiac isolates positive for immunoglobulin binding protein A, direct repeats 1 & 2 1 isolate with hemolytic properties 1 isolate still pending Similar to virulence patterns seen in isolates from pneumonia, encephalitis and abortion

20. 1. H. somni was an important cause of loss on two Wyoming Ranches. 2. Diagnosis was established by clinical signs, gross lesions, bacterial isolation, immunohistochemistry, and electron microscopy 3. Aerobic isolation was less sensitive than immunohistochemistry to confirm presence of H. somni 4. Lesions involved left ventricular myocardium predominately posterior papillary muscle

21. 5. Western blots established that virulence factors of H. somni were present—there is no cardiac specific strain of H. somni 6. There was limited evidence of antecedent pneumonia to account for septicemia—the portal of entry of H. somni was not determined 7. H. somni enters cardiac parenchyma by direct destruction of endothelium—intracellular infection of endothelium was not identified