Immunohistochemical Characterization of Current and Emerging Infectious Diseases of Livestock and Commercial Birds

NON-TECHNICAL SUMMARY: The complexity of infectious diseases of livestock requires specific and sensitive methods for diagnosis. Immunohistochemistry is a sensitive and specific methodology to detect virus, bacteria, and fungi in tissue sections. Immunohistochemistry is the gold standard for surveillance of such livestock diseases of livestock as bovine virus, diarrhea virus, scrapie, chronic wasting disease and West Nile virus. During the previous 5 years more than 30,000 samples have been tested by Immunohistochemistry to detect livestock infectious diseases. <P>APPROACH: Tissue samples will be fixed in formaldehyde and processed using a paraffin embedding tissue processor. Once paraffin blocks are prepared, they will be cut with a rotary microtome and put in positive charged glass slides. Immunohistochemical staining will consist of the following stages: Step 1 includes all pre-immunologic procedures done before incubation with the primary bindings, blocking endogenous activities (e.g., peroxidase, phosphatase, biotin), and antigen retrieval. Step 2 includes all the immunologic reactions between the primary antibody and tissue antigens, the primary antibody and secondary antibody and some additional chemical reactions necessary to bind the reporter molecule (label) such as peroxidase or alkaline phosphatase to the performed immune complex. Step 3 includes all the procedures necessary to visualize the antigen-antibody binding. In immunohistochemistry, this is achieved with a chemical reaction in which the label reacts with its substrate and a chromogen to produce a colored reaction product. Then, immunolabeled tissue sections are counterstained and coverslipped. Step 4 includes interpretation and report of the immunohistochemical results. <P>PROGRESS: 2006/10 TO 2007/09<BR>
OUTPUTS: During this period we tested tissues (approximately 650 cases) by immunohistochemistry for one or more of the following infectious agents: adenovirus, Aspergillus, bovine and porcine coronavirus, bovine herpesvirus, bovine respiratory syncytial virus, equine herpesvirus, infleunza A, Leptospira, Neospora caninum, porcine circovirus 2, Sarcocystis neurona, and Toxoplasma. We followed standardized protocols for each test. In addition, we have tested over 8,000 samples of ovine and deer tissues for surveillance of transmissible spongiform encephalopathies (TSEs[crapie in sheep and chronic wasting disease in cervids]).<BR> PARTICIPANTS: Animal Disease Diagnostic Laboratory staff <BR> TARGET AUDIENCES: Livestock producers and large animal veterinarians
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IMPACT: 2006/10 TO 2007/09<BR>
These results follow studies done during the past year. These tests have improved the quality of service provided to livestock owners. We are actively implementing new tests to be used in diagnostic and research settings. As in past years, this laboratory has been heavily involved in surveillance of TSEs with immunohistochemistry following protocols dictated by USDA.
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PROGRESS: 2005/10/01 TO 2006/09/30<BR>
During this period we have collected samples to standardize the following immunohistochemical tests for the following infectious agents: Caprine arthritis encephalitis virus, Cryptosporidium, avian influenza, Sarcocystis neurona, and Toxoplasma. Standardization of immunohistochemical tests was performed for each antigen to determine optimal assay conditions. These conditions included antigen retrieval methods (e.g., proteinase K, steamer), different dilutions of the primary antibody. To rule out possible cross-reactivities, we tested tissues lacking the antigen investigated (negative tissue controls). Results were compared based on the intensity of the reaction and the number of infected cells. Positive results were those in which the reaction was present in the expected location (intracellular, membrane or intranuclear) and absent in the negative control. We have started to use immunohistochemical tests to confirm the presence of the above-mentioned infectious agents in diagnostic specimens received at the Animal Disease Diagnostic Laboratory. In addition, we have tested over 6,000 samples of ovine and deer tissues for surveillance of transmissible spongiform encephalopathies (scrapie in sheep and chronic wasting disease in cervids). Immunohistochemical testing for porcine circovirus 2 was done in over 150 cases.
<P>
IMPACT: 2005/10/01 TO 2006/09/30<BR>
These results follow studies done during the past year. These tests have improved the quality of service provided to livestock owners. We are actively implementing new tests to be used in diagnostic and research settings. This year we also have been heavily involved in surveillance of TSEs with immunohistochemistry following protocols dictated by USDA.
<P>
PROGRESS: 2004/10/01 TO 2005/09/30<BR>
During this period we have collected samples to standardize the following immunohistochemical tests for the following infectious agents: Aspergillus, bovine respiratory syncytial virus, bovine coronavirus, and bovine herpesvirus 1. Standardization of immunohistochemical tests was performed for each antigen to determine optimal assay conditions. These conditions included antigen retrieval methods (e.g., proteinase K, steamer), different dilutions of the primary antibody. To rule out possible cross-reactivities, we tested tissues lacking the antigen investigated (negative tissue controls). Results were compared based on the intensity of the reaction and the number of infected cells. Positive results were those in which the reaction was present in the expected location (intracellular, membrane or intranuclear) and absent in the negative control. We have started to use immunohistochemical tests to confirm the presence of the above-mentioned infectious agents in diagnostic specimens received at the Animal Disease Diagnostic Laboratory. In addition, we have tested over 6,000 samples of ovine and deer tissues for surveillance of transmissible spongiform encephalopathies (scrapie in sheep and chronic wasting disease in cervids).
<P>
IMPACT: 2004/10/01 TO 2005/09/30<BR>
These results follow studies done during the past year. These tests have improved the quality of service provided to livestock owners. We are actively implementing new tests to be used in diagnostic and research settings. This year we also have been heavily involved in surveillance of TSEs with immunohistochemistry following protocols dictated by USDA.

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PROGRESS: 2003/10/01 TO 2004/09/29<BR>
During this period we have collected samples to standardize the following immunohistochemical tests for the following infectious agents: porcine circovirus 2, porcine adenovirus, Lawsonia intracellularis, porcine coronavirus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and Neospora caninum. Standardization of immunohistochemical tests was performed for each antigen to determine optimal assay conditions. These conditions included antigen retrieval methods (e.g., proteinase K, steamer), different dilutions of the primary antibody. To rule out possible cross-reactivities, we tested tissues lacking the antigen investigated (negative tissue controls). Results were compared based on the intensity of the reaction and the number of infected cells. Positive results were those in which the reaction was present in the expected location (intracellular, membrane or intranuclear) and absent in the negative control. We have started to use immunohistochemical tests to confirm the presence of the above-mentioned infectious agents in diagnostic specimens received at the Animal Disease Diagnostic Laboratory.
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IMPACT: 2003/10/01 TO 2004/09/29<BR>
These preliminary results permitted us to start using immunohistochemistry in the diagnosis of infectious diseases of food animals. Although the number of samples tested is limited, we believe that using these tests has improved the quality of service provided to livestock owners. We are actively implementing new tests to be used in diagnostic and research settings.