Study on Thermal Death Time of AI Published

By Annel K. Greene, PhD, Professor and Center Director
Clemson University Animal Co-Products Research and Education Center


A study on the thermal destruction of avian influenza (AI) in rendering materials conducted by Clemson University researchers Adam Leaphart, Thomas R. Scott, William C. Bridges Jr., Laine Chambers, and Annel K. Greene has been published in the December 2012 quarterly edition of the Journal of Applied Poultry Research.

Raw poultry offal from poultry processing and mortalities can be a source of a variety of bacteria and viruses, including AI. During the rendering process, these animal tissues are heat-processed to kill inherent microbial agents. Since rendered poultry products are used worldwide for animal feeds, it is imperative that AI is destroyed to prevent passing the disease agent to other poultry, livestock, pets, and even humans. Many decades of anecdotal data have always suggested that rendering destroys AI but validation of the thermal conditions required to destroy the virus in the rendering process has been needed for regulatory issues as well as to address potential customer concerns. This study was conducted to validate the time and temperature required for processing poultry offal to destroy AI viral ribonucleic acid (RNA) and to confirm that current rendering cooking processes are sufficient to prevent spread of the disease via rendered poultry products.

Poultry materials were collected post-cooker from three rendering facilities in South Carolina, North Carolina, and Georgia and evaluated for crude protein, fat content, and moisture. Crude protein ranged from 21.6 percent to 31.0 percent, fat ranged from 47.8 to 65.0 percent, and moisture ranged from 1.4 to 1.8 percent. Most rendering materials contain considerably higher fat than typical food products and the Clemson University researchers have previously determined that higher fat content can cause difficulties with standard methodology originally developed for and currently utilized in food microbiology.

Due to biosecurity restrictions on the receipt and use of live AI strains, the researchers had to design the experiment within regulatory allowances but with material that would still represent typical Type A influenza. A β-propiolactone-inactivated strain of A/Turkey/Wisconsin/68 H5N9 was selected as the test material. This nonviable strain of AI viral RNA has an intact viral capsid as well as AI viral RNA. It was allowed for use under Biosafety Level 2 conditions available at the Clemson University Veterinary Diagnostic Laboratories in Columbia, SC.

Using the selected AI strain, the initial step in the study was to verify ability to detect the virus in high fat rendering materials. During the initial months of the study, Scott developed an enzyme-linked immunosorbent assay detection test that is a common and effective method of measuring virus levels. However, Scott determined the unique high fat qualities of rendering materials interfered with use of this type of test procedure. Therefore, it was determined another method of detection would be needed to determine thermal destruction of the viral RNA.

Scott consulted with Leaphart and a wholly different test procedure was then utilized to detect intact viral RNA. This test procedure, known as real-time reverse-transcription polymerase chain reaction (PCR) is a highly sensitive method that utilizes technology to amplify very low copy numbers of RNA to create thousands to millions of copies of the sequence. Techniques for detection of these sequence copies were subsequently used. In this methodology, even if the sequence is present in the sample in only very small levels, amplifying it will allow its detection. After multiple preliminary experiments using the high fat rendering material matrix and the test strain of AI, it was determined the PCR technique was capable of detecting very low levels of the AI viral RNA in rendering materials.

After determining the methodology capable of detecting the viral RNA, the next step in the study was to add the test strain with its viral RNA to poultry rendering materials and heat process the materials across various times and temperatures. Using custom-made stainless steel tubes and poultry rendering materials collected from South Carolina, North Carolina, and Georgia, the study was conducted at temperatures of 80 degrees Celsius © (176 degrees Fahrenheit (F)), 100 degrees C (212 degrees F), 110 degrees C (230 degrees F), 120 degrees C (248 degrees F), 130 degrees C (266 degrees F) and 140 degrees C (284 degrees F) for times ranging up to 120 seconds. Controls for the experiment included heated, non-dosed samples of rendering materials as well as unheated, dosed samples. After completion of the experimental phase, statistician Bridges analyzed the data.

Experimental results indicated that with a thermal treatment of 30 seconds or longer at 100 degrees C (212 degrees F) or 15 seconds or longer at 110 degrees C (230 degrees F), “the RNA of low-pathogenicity avian influenza virus A/Turkey/Wisconsin/68 H5N9, equivalent to six log10 of viable virus, was destroyed within the poultry rendering materials.”1 The thermal treatment conditions reported by the rendering industry in the United States and Canada indicate rendering materials are treated for a minimum of 30 minutes with a cooker exit temperature of at least 118 degrees C (244.4 degrees F).2,3 Therefore, according to the published validation experiment, the thermal treatment applied during the rendering cooking process is more than sufficient to destroy the AI viral RNA.

In addition, due to the test methodology used, the results of the PCR assay for the experiment actually indicated the entire RNA strand was completely destroyed, or in other words, all Type A influenza RNA was destroyed under these time/temperature conditions in poultry rendering materials. The thermal destruction time/temperature values determined were higher for rendering materials than the thermal destruction values previously noted for destroying AI within poultry breast meat and eggs; however, this difference was attributed to the much more complex matrix as well as higher fat content of the rendering materials than the poultry food products.

Researchers Greene, Bridges, and Chambers, along with Dr. Xiuping Jiang and PhD graduate students M. Melissa Hayes and Yubo Zhang, are continuing studies on thermal death time to validate the time/temperature requirements for destruction of recognized animal feed pathogenic strains of Salmonella as well as for destruction of the sporeforming bacteria Clostridium perfringens. As noted in the AI study, the unique chemical composition of rendering materials (i.e., high fat content) has required modification of test procedures to allow accurate enumeration and identification of the pathogens. This work is continuing in order to provide the rendering industry with solid validation data for pathogens of concern.

References:
1. Leaphart, A.B., T.R. Scott, S.D. Chambers, W.C. Bridges Jr., and A.K. Greene. 2012. “Investigation of AI viral ribonucleic acid destruction in poultry co-products under rendering conditions.” Journal of Applied Poultry Research 21 (4):719-725.
2. Pearl, G.G. 2004. “Rendering 101.” Render 33 (4):30-39.
3. Meeker, D.L., and C.R. Hamilton. 2006. “An Overview of the Rendering Industry.” In Essential Rendering, edited by D.L. Meeker. 1-16. Alexandria:National Renderers Association.


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