clinical course clearly associated with the lower respiratory tract (pneumonia), lung tissue should be submitted. Accurate culture results require using sterile techniques. During a necropsy, these should be the first specimens taken, and the tissue should be minimally or not manipulated. This can be achieved by using sterile instruments and/or a swab stick, or by placing a piece of tissue directly into a sterile container. It is important to alert the microbiology laboratory that the specimen was taken at the time of necropsy. Antibiotic therapy can skew or prevent the culture of many bacteria. Any pre‐mortem therapy should be noted in the submission form and on the necropsy report.2 Molecular diagnostics:Fresh or fresh/frozen lung or upper respiratory tissue samples are necessary to diagnose agents contributing to pneumonia (former) or URI. These samples need to be taken early in the postmortem stage, using sterile technique, and from tissue minimally manipulated. DNA or RNA from the infectious agent will degrade at rates dependent on time, environmental factors (temperature, pH) and the organism itself. Ideally, samples from affected organs are fresh or fresh/frozen for molecular analysis. Most diagnostic laboratories or veterinary schools can offer guidance and a list of possible tests, the preferred or potentially useful tissue samples, and the preferred method of shipment.
Formalin‐fixed tissues
Histological samples should be taken in ALL CASES no matter what supplementary diagnostic tests are performed. Histology can provide a definitive diagnosis, identify possible causes, or confirm or refute the results of other diagnostic tests. The following is a general list for sampling an animal with respiratory disease. In nearly all cases, these samples would be sufficient to diagnose or exclude common shelter respiratory pathogens.
5.5.3.1 Tissue Checklist for Respiratory Disease
Tissues should be no thicker than 1 cm. Tissues should be placed immediately in 10% buffered formalin at a ratio of 1 part tissue to 10 parts formalin.
1 Nasal conchae, sinus
2 Trachea, 1‐2 cartilage rings
3 Lung, multiple samples, including cranioventral portions of the cranial lobe(s), the caudal and dorsal regions of the caudal lobes and hilar region including major bronchus
4 Hilar lymph nodes, and/or lymph nodes from the thoracic inlet
5 Heart‐longitudinal sections including atrium, ventricle and valves from both left and right sides.
5.5.3.2 Common Respiratory Diseases in the Shelter
5.5.3.2.1 Canine Distemper Virus (CDV)
Clinical impressions are rarely sufficient to differentiate canine distemper from other causes of infectious canine respiratory disease. Pre‐mortem testing options are limited; serological tests are limited by viral immunosuppression and interference due to maternal or vaccine‐induced antibodies and fluorescent antibody (FA) testing (cells from the conjunctiva, blood, respiratory tract epithelium, or urinary bladder) is very specific but has low sensitivity. Another pre‐mortem test is PCR (on urine sediment, epithelial swabs, bronchoalveolar lavage, buffy coat preps, or CSF); however, PCR may also detect vaccine virus in recently vaccinated animals and for this reason the use quantitative reverse transcriptase PCR (RT‐PCR) is recommended. If an animal dies of suspected distemper, or if the presentation of the disease is unusual and confirmation is necessary, distemper can be identified reliably on necropsy samples and histopathology by a qualified pathologist.
Gross findings: If the lungs are involved, canine distemper virus will be disseminated and affect all lobes. In most cases, oculo‐nasal discharges are thick and mucopurulent. The lungs are generally edematous or consolidated (interstitial pneumonia). Thick, foamy to mucopurulent hemorrhagic exudates may be found in the airways. Secondary (bacterial) infection is common, both because of viral damage to the airways and because of lymphoid depletion. Therefore, a cranioventral distribution of lung consolidation (bronchopneumonia) does NOT rule out distemper. Lymphoreticular tissues are characteristically involved and are the primary site for viral replication. There can be enlargement of the tonsils and/or atrophy of the thymus. Hyperkeratosis (“hardpad disease”) of the nose and/or footpads is sporadically present. There are no gross lesions of the central nervous system even when nervous signs are uniquely present. The heart should always be examined, opened, and sampled when investigating respiratory outbreaks; right heart failure (e.g. as a result of heartworm disease) often poses clinically as respiratory distress.
Histopathology: In addition to the list above for general respiratory disease, histopathologic samples useful in the diagnosis of CDV include the brain and bladder. Samples submitted for histology and paraffin‐embedded are also used for immunohistochemistry, which is one of the definitive methods of identifying CDV induced respiratory and/or neurologic disease.
Molecular diagnostics: PCR can be used to detect virus in lung, CSF, feces, or urine. False positives are possible within one to three weeks of vaccination. The diagnostic laboratory should be consulted regarding testing details.
5.5.3.2.2 Canine Infectious Respiratory Disease (CIRD, “Kennel Cough,” Multiple Agents)
Canine infectious respiratory disease is caused by a combination of both viral and bacterial agents. The range of morbidity and mortality can be attributed to several problems, including husbandry, but in ANY case, knowing the spectrum of infectious agents that are contributing to the disease is helpful in identifying problems, developing isolation procedures and ongoing therapy. Many laboratories offer tests for common agents of CIRD, though new bacterial and viral agents continue to be identified. Confirmation of specific causative agent(s), whether novel or typical, requires a combination of histology, microbiology, and (for the viral component) PCR or virus isolation. Histology (including PCR or immunohistochemistry) is also necessary to rule in/out viruses as contributory or causative in an outbreak of respiratory disease.
Gross findings: The gross lesions of CIRD typically reflect the aerogenous introduction of bacteria into the pulmonary tissues. The lungs are congested and consolidated, most consistently within the cranial and ventral portions of the cranial lobes. There can be pleural mottling that may involve multiple lobes. Depending upon whether a viral component is acute or chronic, the bronchopneumonia could be superimposed on a more diffuse pattern of lung involvement (interstitial pneumonia). The thoracic cavity should be examined for excessive pericardial and thoracic fluid production (normal is less than 5 ml in both cavities), +/− concurrent pericarditis and pleuritis.
Histological, microbiological (lung), and molecular diagnostic (lung) samples should be taken according to the general respiratory disease protocol above.
5.5.3.2.3 Canine Influenza: (CIV)
As with most viral diseases, it can be difficult to determine the role of a virus in a clinical event. Reliable diagnosis of many viral diseases by serology requires both acute and convalescent serum samples. Virus detection by PCR in respiratory secretions from acutely ill or recently exposed animals is possible, but false negatives are not uncommon. The Becton‐Dickinson Flu‐A ELISA test may also be used on nasal secretions from acutely affected animals. While the mortality rate of canine influenza is fairly low (5–8%), if an animal does die or is euthanized with respiratory disease, the most accurate test for the virus is a PCR test of respiratory tissue. This, combined with histological features of viral‐induced pneumonia, would be the “gold‐standard” for the presence, and effect, of the virus. In several identified cases of CIV, there was a concurrent and severe bacterial pneumonia, so samples for culture and antibiotic sensitivity should be concurrently submitted.
Gross
