- Email: [email protected]
Investigation of avian influenza viral ribonucleic acid destruction in poultry co-products under rendering conditions1
©2012 Poultry Science Association, Inc.
Investigation of avian influenza viral ribonucleic acid destruction in poultry co-products under rendering conditions1 A. B. Leaphart,*† T. R. Scott,*‡ S. D. Chambers,* W. C. Bridges Jr.,*§ and A. K. Greene*‡2
Primary Audience: Flock Supervisors, Animal Feed Sales Personnel, Quality Assurance Personnel, Researchers, Veterinarians, Epidemiologists, Regulatory Officers, Poultry Co-product Renderers, Import/Export Agents, Poultry Processors SUMMARY This study was conducted to determine the time and temperature requirements needed to destroy avian influenza viral RNA in high-fat poultry tissues and to determine whether those conditions are met by rendering cooking processes. Because rendered poultry products are used worldwide for feed ingredients, it is imperative to validate the destruction of avian influenza viral RNA to ensure the safety of rendering co-products and avoid cyclic reinfection and disease in poultry, livestock, and potentially humans. Typical high-fat poultry offal was spiked with a known quantity of chemically inactivated, intact H5N9 low-pathogenicity avian influenza viral RNA. After subjecting the material to a variety of thermal doses, RNA was extracted from the material and assayed for the presence of the virus by using real-time reverse-transcription PCR. With thermal treatment at 100°C for 30 s or longer or at 110°C and above for 15 s or longer, the RNA of low-pathogenicity avian influenza virus A/Turkey/Wisconsin/68 H5N9, equivalent to 6 log10 of viable virus, was destroyed within the poultry rendering materials. These thermal treatment conditions are well below the range of temperatures and times used for rendering poultry carcasses and offal in the United States and Canada, where rendered materials are subjected to a heat treatment for a minimum of 30 min with a cooker exit temperature of not less than 118°C. Key words: animal co-product, avian influenza viral ribonucleic acid, rendering, thermal destruction, validation 2012 J. Appl. Poult. Res. 21:719–725 http://dx.doi.org/10.3382/japr.2011-00345
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Technical Contribution No. 5719 of the Clemson University Experiment Station (Clemson, SC). Corresponding author: [email protected]
Downloaded from http://japr.oxfordjournals.org/ at North Dakota State University on June 7, 2015
*Animal Co-Products Research and Education Center, †Livestock-Poultry Health, ‡Department of Animal and Veterinary Sciences, and §Department of Applied Economics and Statistics, Clemson University, Clemson, SC 29634
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DESCRIPTION OF PROBLEM
MATERIALS AND METHODS Virus Inoculum A β-propiolactone-inactivated strain of lowpathogenicity avian influenza viral RNA A/ Turkey/Wisconsin/68 H5N9 [4] was used as a nonviable strain of low-pathogenicity avian influenza viral RNA with an intact viral capsid for use under biosafety level 2 conditions. Because of biosecurity restrictions on the receipt and use of live avian influenza, this experiment was designed within allowed working parameters using the genomic RNA of the inactivated virus. Experimental Design Samples of high-fat-content poultry rendering materials were collected from the exit screws of 3 continuous cookers at rendering plants in local southeastern states (South Carolina, Georgia, and North Carolina). The materials were transported to the laboratory and stored under refrigeration until needed. Materials were blended in a commercial food processor [5] for 5 min to reduce particle size. Samples were analyzed for chemical composition, and results indicated that fat content ranged from 47.8 to 65.0% as sampled (Table 1). One gram of rendered poultry material was added to sterile stainless steel tubes. The tubes were manufactured by a local metal fabrication company by boring 304 stainless steel rods (8.5 cm length, 1.6 cm outer diameter, 1.3 cm inner diameter). Samples were allowed to warm Table 1. Chemical analyses (%) of rendered poultry samples from plants A, B, and C (as sampled) Item CP Fat Moisture DM
Plant A
Plant B
Plant C
21.6 65.0 1.8 98.2
31.0 47.8 1.5 98.5
26.5 60.5 1.4 98.6
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Offal generated from poultry production and poultry mortalities can harbor a variety of bacteria and viruses. These tissues are thermally treated in a process known as rendering. Rendering has a 3-fold purpose: 1) to remove moisture, 2) to separate fat from protein materials, and 3) to inactivate microorganisms. Raw rendering materials are primarily composed of fat, protein, bone, and moisture. High-fat matrices have been shown to offer a protective advantage against thermal inactivation of pathogens because of decreased water activity [1]. American and Canadian rendering cookers process materials at atmospheric conditions by immersion in molten fat with concurrent agitation, which converts moisture in the tissues to steam for removal as vapor. As a result of the immersion in molten fat, rendering materials within the cooker often contain high levels of fat. In the United States and Canada, rendered materials are subjected to a heat treatment for a minimum of 30 min, with a cooker exit temperature of not less than 118°C and often at temperatures up to 145°C for 40 to 90 min, depending on the system and the types of materials being processed [2, 3]. Leading up to discovery or after confirmation of an avian influenza disease outbreak, avian influenza viruscontaminated rendering materials derived from either offal or depopulated whole birds may be presented to a rendering facility. Because finished rendered animal products are used for a variety of applications, including as ingredients in animal feeds such as pet food, poultry feeds, and livestock feeds, it is imperative to validate thermal destruction of avian influenza viral RNA to ensure that poultry meal will not be a source of transmission of avian influenza viral RNA to animals and humans. Currently, only anecdotal evidence is available to suggest that rendering destroys avian influenza viral RNA. However, rendering companies, feed companies, and regulatory agencies need validation that the thermal conditions used for rendering destroy avian influenza viral RNA. The objectives of this study were to determine the temperature and time parameters necessary to inactivate avian influenza virus, by using a model system of destruction of avian influenza viral RNA, in rendered animal co-products and
to compare those conditions with the rendering industry cooking conditions used in the United States and Canada for the purpose of determining whether the current rendering methods are sufficient to destroy avian influenza virus and produce safe products.
Leaphart et al.: RENDERING VS. AVIAN INFLUENZA VIRAL RNA
RNA Extraction and rRT-PCR Ribonucleic acid was extracted with an RNeasy Kit [10] with a modified protocol for rendered material. Briefly, the rendered material was removed from the test tube using Dacrontipped swabs and resuspended in 500 µL of the kit-supplied buffer RLT. The slurry was allowed to remain at room temperature for 10 min. To this volume, 500 µL each of brain heart infusion broth and 70% ethanol was added and the combination was inverted several times. This mixture was then centrifuged at 5,000 × g in a microcentrifuge [11] for 5 min at 20 to 25°C to separate the solid and liquid phases. After centrifugation, an upper oil/fat layer was observed, followed by an aqueous layer and then a solid material layer. Trizol extraction of the oil/fat and solid layers in a non-heat-treated sample indicated no viral RNA was present (data not shown). The aqueous layer was carefully removed and then applied to the spin column, and the remainder of the protocol was performed according to the manu-
facturer’s instructions. Real-time reverse-transcription PCR was conducted using the primer and probe sequences specific for the influenza A matrix gene M1 as described by Spackman et al. [12]. Conditions for reverse transcription were defined as 30 min at 50°C followed by 15 min at 94°C. Thermal cycling conditions were established as a 2-step cycling procedure for 55 cycles of 94°C for 10 s followed by an annealing step at 60°C for 20 s. Fluorescence data were acquired during the annealing step. A Qiagen One-Step RT-PCR Kit [13] was used to make a 17-µL reaction mixture containing 10 pmol of each primer and the hydrolysis probe, 400 µM deoxynucleoside triphosphate mix, 2.5 mM MgCl2, and 6.5 U of ribonuclease inhibitor [14]. Reactions were carried out using a SmartCycler II real-time PCR system [15]. Data were recorded as threshold cycle (Ct) values, where the Ct value denotes how many PCR cycles are required for sample fluorescence to reach the threshold level, which is a fluorescent signal significantly above background fluorescence. At the Ct, a detectable amount of amplified target nucleic acid has been generated during the early exponential phase of the reaction. The Ct is thus inversely proportional to the relative level of sample presence of the gene of interest. Stated otherwise, the more target nucleic acid present in a sample, the lower the Ct value will be. Using the rRT-PCR method described by Spackman et al. [12], a cutoff Ct value of 37 was used to determine the presence or absence of viral RNA based on the established limit of detection of the assay. That is, any Ct value above 37 was deemed unreliable for predicting the presence of avian influenza virus RNA, and was therefore regarded as negative for containing viral RNA. Virus Inoculum Copy Number Determination The sample copy number for the inactivated viral preparation was estimated using the influenza A copy number standard supplied in a TaqManAIV-M Reagent Kit [16] and the rRT-PCR assay used above. A standard curve correlating the matrix gene copy number to Ct value was developed using serial dilutions of the influenza copy number standard. Ribonucleic acid was extracted from the inactivated avian influenza virus preparation mixed with rendered material
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to room temperature over a 3-h period. Subsequently, 250 µL of inactivated, intact avian influenza virus preparation was added to the 1-g quantities of the rendering materials and mixed vigorously by vortex for approximately 1 min to ensure homogeneity. These samples were prepared in duplicate for each time point from each of the 3 rendering plant samples. Samples were thermally treated in temperature and time combinations of 110, 120, 130, and 140°C for 0, 15, 30, 60, and 120 s. Controls included nondosed samples and samples dosed for 0 s. Sample tubes were heated in an analog dry block heater [6]. Additional thermal trials were conducted in rendering materials at 80 and 100°C for 10, 20, 30, and 60 s. Temperature and time combinations were selected to allow determination of the minimum thermal conditions required to destroy the viral inoculum in the rendering materials. Additional control tubes were included in each thermal test round for monitoring temperature using thermocouples [7] connected to a data logger [8] with computer software control [9]. After thermal treatment, the tubes were plunged into ice to cool. Samples were frozen until processing via real-time reverse-transcription PCR (rRT-PCR).
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Statistical Analysis The means were compared among times and temperatures using ANOVA followed by Fisher’s LSD test. Any means with similar letters in Figures 1 and 2 are not significantly different (P > 0.05). All calculations were performed using SAS software [17].
RESULTS AND DISCUSSION During this study, an ELISA system was initially developed and tested for use in detecting viral components. However, because of the high fat content of the rendering materials, the ELISA was unsuccessful for use in this thermal trial. Therefore, RNA extraction and rRT-PCR were used for all subsequent measurements of the β-propiolactone-inactivated strain of avian influenza virus (A/Turkey/Wisconsin/68 H5N9) in this study. β-Propiolactone inactivation of strains of high-pathogenicity avian influenza viruses is commonly used to create vaccines [18]. In this case, the inactivated virus retains sufficient viral characteristics to initiate an immunological response to protect the vaccinated animal against future exposure to the live virus. The inoculum used in this study was selected as an inactivated strain of a low-pathogenicity avian influenza virus with an intact viral capsid for use under biosafety level 2 conditions. Jonges et al. [19] reported that the residual virus genomic RNA is protected by viral nucleoprotein. Jonges et al.
[19] further indicated that β-propiolactone did not have a major effect on the hemagglutinin and neuraminidase characteristics in avian influenza Type A (H7N3). However, they did report that β-propiolactone inactivation reduced the hemagglutination titer and the neuraminidase activity in a human type A (H3N2) influenza virus, and they hypothesized that this observation was due to a reduction in glycoprotein functionality from creation of the RNA-/DNA-protein complexes. Wainberg et al. [20] reported that β-propiolactone treatment had no effect on the viral envelope of the Sendai virus. Franchini et al. [21] reported that inactivation of the herpes simplex virus by β-propiolactone did not affect the capsid, as verified using electron microscopy. No published data were located for the effect of β-propiolactone inactivation on the integrity of the strain of avian influenza virus used in this experiment (A/Turkey/Wisconsin/68 H5N9). It is not known whether the β-propiolactone inactivation process had any effect on the thermal resistance of the inactivated virus in comparison with the non-inactivated virus. However, because of biosecurity and shipping restrictions, it was not possible to obtain a live virus to determine if there was a difference in this study. This factor needs to be tested in further studies by laboratories capable of working with live strains of high-pathogenicity influenza. For the standard curve developed, results of the thermal dose treatments are shown in Figures 1 and 2 as mean log M1 matrix gene copy number. Using the assumption that 1 copy of the M1 matrix gene is present per viral genome, these data roughly estimated the number of viral particles present before and after thermal trials. In the study, an average of approximately 1.97 × 106 copies of the M1 matrix gene was added per gram of rendering materials (n = 4). On the basis of analysis of the unheated, inoculated controls, approximately 1.83 × 106 copies of the matrix gene were recovered by extracting the rendering sample (n = 4). No background M1 matrix gene was detected in the uninoculated rendering material. Because of the nature of the test procedure, the PCR assay used in this experiment was designed to determine if any type A H5N9 influenza RNA was destroyed in the samples. Complete destruction of the RNA strand was assumed,
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(unheated), and rRT-PCR was conducted to determine the Ct value. Using the standard curve developed with the best fit line, a mean of approximately 1.97 × 106 copies of the M1 matrix gene was added per gram of rendering materials (n = 4). Upon extraction recovery from the rendering materials, the copy number was extrapolated to 1.83 × 106 per gram of inoculated rendered material based on an average value over 4 unheated, inoculated samples, so minor losses in the copy number were observed because of passage through the rendering materials. With the assumption that 1 copy of the M1 matrix gene is present per viral genome, this number can be roughly equated to the number of viral particles added.
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based on the lack of observable viral RNA. With this assay, the viral RNA was totally destroyed after treatment for 30 s or longer at 100°C or for 15 s or longer at 110°C or above (Figures 1 and 2), resulting in greater than a 6-log reduction in copy number of the M1 matrix gene. At a thermal treatment of 80°C (Figure 2), viral RNA survival was noted at 10, 20, 30, and 60 s. It was noted (Figure 1) that the thermal dose applied simply during the time (approximately 2 min) to reach temperatures of 110, 120, 130, or 140°C (reported as 0 s) caused a 3-log or greater reduction in observable copy numbers of the M1 matrix gene. Commercial rendering has been used to destroy viral diseases such as pseudorabies in animal tissues [22]. This project was designed to determine the thermal treatment and time com-
binations that would destroy avian influenza virus RNA in rendered products. Rendering would be effective in destroying viral RNA in rendering materials containing virus particles, based on the results of this study. Swayne [23] indicated that avian influenza virus was killed at 70°C within a few seconds in edible chicken thigh and breast meat products. According to Article 10.4.25 [24] of the OIE (World Organisation for Animal Health) Terrestrial Animal Health Code, avian influenza is inactivated in eggs and egg products at 60°C for 188 s for whole eggs, at 60°C for 188 s or at 61.1°C for 94 s for whole egg blends, at 55.6°C for 870 s or at 56.7°C for 232 s for liquid egg whites, and at 62.2°C for 138 s for 10% salted yolks. Treating these products at these temperature and time parameters results in a 7-log de-
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Figure 1. Mean log avian influenza virus matrix gene copy number of β-propiolactone-inactivated avian influenza virus A/Turkey/Wisconsin/68 H5N9 in replicate samples from plants A, B, and C (3 plants × 2 replications each = 6 measurements/time point) that were unheated or heated at 110, 120, 130, and 140°C at treatment times of 0, 15, 30, 60, and 120 s. Time and temperature combinations with similar letters (A–E) are not significantly different. Significance tests were based on ANOVA followed by Fisher’s LSD methods, with α set at 0.05. Zero seconds was the first time point measured after reaching the specified treatment temperature. The mean M1 matrix gene copy number from RNA extracted (as purchased) was 1.97 × 106 (n = 4). The mean M1 matrix gene copy number from unheated, inoculated, rendered material and subsequently extracted was 1.83 × 106 (n = 4). The mean M1 matrix gene copy number in uninoculated rendered material was 0. The SE was ±0.26 log gene copy number.
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struction of avian influenza. Article 10.4.26 [24] of the Terrestrial Animal Health Code describes procedures for inactivation of avian influenza in poultry meat and states that a core temperature of 65.0°C for 42 s, 70.0°C for 3.5 s, or 73.9°C for 0.51 s is sufficient to cause a 7-log destruction of avian influenza. Rendered materials, although prepared for consumption by animals, are termed “inedible materials” because these are not for human consumption. The overall composition of the inedible rendered material was considered a more complex matrix than edible breast meat alone (i.e., much less uniform and containing considerably higher fat content than edible chicken thigh and breast meat). More rigorous thermal conditions were required to destroy the H5N9 viral RNA in the higher fat inedible rendering matrix
than to destroy the live virus in edible poultry meat and eggs. The data obtained in this study on inedible rendered animal co-products in no way contradicts or challenges the data for thermal destruction of avian influenza in edible eggs and meat. Upon obtaining results in this study, the authors hypothesized that the higher fat and increased complexity of the inedible rendered material matrix influenced thermal destruction as compared with meat and eggs. Alternatively, the β-propiolactone viral inactivation process used on the inoculum in this experiment may have enhanced the thermal resistance of the viral RNA. A third hypothesis is that inactivation of the live avian influenza virus may not require as much thermal rigor as destruction of the RNA as measured in this experiment. However, the design of this experiment did not allow for test-
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Figure 2. Mean log avian influenza virus matrix gene copy number of β-propiolactone-inactivated avian influenza virus A/Turkey/Wisconsin/68 H5N9 in replicate samples from plants A, B, and C (3 plants × 2 replications each = 6 measurements/time point) that were unheated or heated at 80 and 100°C at treatment times of 0, 10, 20, 30, and 60 s. Time and temperature combinations with similar letters (A–E) are not significantly different. Significance tests were based on ANOVA followed by Fisher’s LSD methods, with α set at 0.05. Zero seconds was the first time point measured after reaching the specified treatment temperature. The mean M1 matrix gene copy number from RNA extracted (as purchased) was 1.97 × 106 (n = 4). The mean M1 matrix gene copy number from unheated, inoculated, rendered material and subsequently extracted was 1.83 × 106 (n = 4). The mean M1 matrix gene copy number in uninoculated rendered material was 0. The SE was ±0.49 log gene copy number.
Leaphart et al.: RENDERING VS. AVIAN INFLUENZA VIRAL RNA ing of these possible explanations, and further research will be needed. The US and Canadian rendering cookers process materials for a minimum of 30 min at 118°C and often process them at even more rigorous conditions of up to 145°C for 40 to 90 min, depending on the system and the types of materials being processed. The minimum rendering processing conditions are much more rigorous than the time and temperature combinations tested in this study.
1. With thermal treatment for 30 s or longer at 100°C or for 15 s or longer at 110°C or above, RNA of low-pathogenicity avian influenza virus A/Turkey/Wisconsin/68 H5N9, equivalent to 6 log10 of viable virus, was destroyed within the poultry rendering materials. These conditions are well below the range of temperatures and times used for rendering poultry carcasses in the United States and Canada, indicating that the current rendering protocols should be sufficient to destroy the avian influenza virus. 2. Future research is needed by laboratories equipped to handle live avian influenza virus to compare the thermal degradation rate of the β-propiolactone-inactivated strain of avian influenza virus A/Turkey/ Wisconsin/68 H5N9 with the live strain as well as to study the thermal death time of high-pathogenicity live virus. Because of biosafety constraints, this study relied on the use of an inactivated strain and measurement of the destruction of virus RNA to levels at which it could no longer be detected by rRT-PCR as a proxy to indicate that the virus would be destroyed.
REFERENCES AND NOTES 1. Brown, M. H. 2000. Processed Meat Products. Pages 389–391 in The Microbiological Safety and Quality of Food. Vol. 1. B. M. Lund, T. C. Baird-Parker, and G. W. Gould, ed. Aspen Publishers, Gaithersburg, MD. 2. Pearl, G.G. 2004. Rendering 101. Render Mag. Aug.:30–38. 3. Meeker, D. L., and C. R. Hamilton. 2006. An overview of the rendering industry. Pages 1–16 in Essential Ren-
dering. D. L. Meeker, ed. Natl. Renderers Assoc., Alexandria, VA. 4. Catalog Number 204-ADV, US Department of Agriculture National Veterinary Services Laboratory, Ames, IA. 5. Model R2N Ultra, Robot Coupe, Ridgeland, MS. 6. Model 12621-108 equipped with Model 13259-162 heating blocks, both from VWR International, Suwanee, GA. 7. Ecklund-Harrison CNS thermocouples, EcklundHarrison Technologies Inc., Fort Myers, FL. 8. CALPLex Data Logger, TechniCAL Inc., Kenner, LA. 9. CALSoft Software, TechniCAL Inc., Kenner, LA. 10. RNeasy Kit, Qiagen, Valencia, CA. 11. Model 5415D, Eppendorf, Westbury, NY. 12. Spackman, E., D. A. Senne, T. J. Myers, L. L. Bulaga, L. P. Garber, M. L. Perdue, K. Lohman, L. T. Daum, and D. L. Suarez. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol. 40:3256–3260. 13. One Step RT-PCR kit, Qiagen, Valencia, CA. 14. RNase inhibitor, Promega, Madison, WI. 15. SmartCycler II, Cepheid, Sunnyvale, CA. 16. Taq-ManAIV-M Reagent Kit, Applied Biosystems, Foster City, CA. 17. SAS Institute Inc., Cary, NC. 18. Lee, C.-W., D. A. Senne, and D. L. Suarez. 2006. Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin. Vaccine Immunol. 13:395–402. 19. Jonges, M., W. M. Liu, E. van der Vries, R. Jacobi, I. Pronk, C. Boog, M. Koopmans, A. Meijer, and E. Soethout. 2010. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J. Clin. Microbiol. 48:928–940. 20. Wainberg, M. A., R. N. Hjorth, and C. Howe. 1971. Effect of β-propiolactone on Sendai virus. Appl. Microbiol. 22:618–621. 21. Franchini, M., C. Abril, C. Schwerdel, C. Ruedl, M. Ackermann, and M. Suter. 2001. Protective T-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus. J. Virol. 75:83–89. 22. Pirtle, E. C. 1990. Stability of the pseudorabies virus (PRV) in meat and bone meal and intermediate rendering products. Directors Digest 203. Fats and Proteins Research Foundation, Bloomington, IL. 23. Swayne, D. E. 2006. Microassay for measuring thermal inactivation of H5N1 high pathogenicity avian influenza virus in naturally infected chicken meat. Int. J. Food Microbiol. 108:268–271. OIE (World Organisation for Animal Health). 24. 2012. Avian influenza. Chapter 10.4 in Terrestrial Animal Health Code. Accessed April 2012. http://www.oie. int/fileadmin/Home/eng/Health_standards/tahc/2010/en_ chapitre_1.10.4.htm.
Acknowledgments
This work was supported with funds provided by various sponsors of the Fats and Proteins Research Foundation (Bloomington, IL).
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CONCLUSIONS AND APPLICATIONS
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Investigation of avian influenza viral ribonucleic acid destruction in poultry co-products under rendering conditions1 A. B. Leaphart,*† T. R. Scott,*‡ S. D. Chambers,* W. C. Bridges Jr.,*§ and A. K. Greene*‡2
Primary Audience: Flock Supervisors, Animal Feed Sales Personnel, Quality Assurance Personnel, Researchers, Veterinarians, Epidemiologists, Regulatory Officers, Poultry Co-product Renderers, Import/Export Agents, Poultry Processors SUMMARY This study was conducted to determine the time and temperature requirements needed to destroy avian influenza viral RNA in high-fat poultry tissues and to determine whether those conditions are met by rendering cooking processes. Because rendered poultry products are used worldwide for feed ingredients, it is imperative to validate the destruction of avian influenza viral RNA to ensure the safety of rendering co-products and avoid cyclic reinfection and disease in poultry, livestock, and potentially humans. Typical high-fat poultry offal was spiked with a known quantity of chemically inactivated, intact H5N9 low-pathogenicity avian influenza viral RNA. After subjecting the material to a variety of thermal doses, RNA was extracted from the material and assayed for the presence of the virus by using real-time reverse-transcription PCR. With thermal treatment at 100°C for 30 s or longer or at 110°C and above for 15 s or longer, the RNA of low-pathogenicity avian influenza virus A/Turkey/Wisconsin/68 H5N9, equivalent to 6 log10 of viable virus, was destroyed within the poultry rendering materials. These thermal treatment conditions are well below the range of temperatures and times used for rendering poultry carcasses and offal in the United States and Canada, where rendered materials are subjected to a heat treatment for a minimum of 30 min with a cooker exit temperature of not less than 118°C. Key words: animal co-product, avian influenza viral ribonucleic acid, rendering, thermal destruction, validation 2012 J. Appl. Poult. Res. 21:719–725 http://dx.doi.org/10.3382/japr.2011-00345
1 2
Technical Contribution No. 5719 of the Clemson University Experiment Station (Clemson, SC). Corresponding author: [email protected]
Downloaded from http://japr.oxfordjournals.org/ at North Dakota State University on June 7, 2015
*Animal Co-Products Research and Education Center, †Livestock-Poultry Health, ‡Department of Animal and Veterinary Sciences, and §Department of Applied Economics and Statistics, Clemson University, Clemson, SC 29634
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DESCRIPTION OF PROBLEM
MATERIALS AND METHODS Virus Inoculum A β-propiolactone-inactivated strain of lowpathogenicity avian influenza viral RNA A/ Turkey/Wisconsin/68 H5N9 [4] was used as a nonviable strain of low-pathogenicity avian influenza viral RNA with an intact viral capsid for use under biosafety level 2 conditions. Because of biosecurity restrictions on the receipt and use of live avian influenza, this experiment was designed within allowed working parameters using the genomic RNA of the inactivated virus. Experimental Design Samples of high-fat-content poultry rendering materials were collected from the exit screws of 3 continuous cookers at rendering plants in local southeastern states (South Carolina, Georgia, and North Carolina). The materials were transported to the laboratory and stored under refrigeration until needed. Materials were blended in a commercial food processor [5] for 5 min to reduce particle size. Samples were analyzed for chemical composition, and results indicated that fat content ranged from 47.8 to 65.0% as sampled (Table 1). One gram of rendered poultry material was added to sterile stainless steel tubes. The tubes were manufactured by a local metal fabrication company by boring 304 stainless steel rods (8.5 cm length, 1.6 cm outer diameter, 1.3 cm inner diameter). Samples were allowed to warm Table 1. Chemical analyses (%) of rendered poultry samples from plants A, B, and C (as sampled) Item CP Fat Moisture DM
Plant A
Plant B
Plant C
21.6 65.0 1.8 98.2
31.0 47.8 1.5 98.5
26.5 60.5 1.4 98.6
Downloaded from http://japr.oxfordjournals.org/ at North Dakota State University on June 7, 2015
Offal generated from poultry production and poultry mortalities can harbor a variety of bacteria and viruses. These tissues are thermally treated in a process known as rendering. Rendering has a 3-fold purpose: 1) to remove moisture, 2) to separate fat from protein materials, and 3) to inactivate microorganisms. Raw rendering materials are primarily composed of fat, protein, bone, and moisture. High-fat matrices have been shown to offer a protective advantage against thermal inactivation of pathogens because of decreased water activity [1]. American and Canadian rendering cookers process materials at atmospheric conditions by immersion in molten fat with concurrent agitation, which converts moisture in the tissues to steam for removal as vapor. As a result of the immersion in molten fat, rendering materials within the cooker often contain high levels of fat. In the United States and Canada, rendered materials are subjected to a heat treatment for a minimum of 30 min, with a cooker exit temperature of not less than 118°C and often at temperatures up to 145°C for 40 to 90 min, depending on the system and the types of materials being processed [2, 3]. Leading up to discovery or after confirmation of an avian influenza disease outbreak, avian influenza viruscontaminated rendering materials derived from either offal or depopulated whole birds may be presented to a rendering facility. Because finished rendered animal products are used for a variety of applications, including as ingredients in animal feeds such as pet food, poultry feeds, and livestock feeds, it is imperative to validate thermal destruction of avian influenza viral RNA to ensure that poultry meal will not be a source of transmission of avian influenza viral RNA to animals and humans. Currently, only anecdotal evidence is available to suggest that rendering destroys avian influenza viral RNA. However, rendering companies, feed companies, and regulatory agencies need validation that the thermal conditions used for rendering destroy avian influenza viral RNA. The objectives of this study were to determine the temperature and time parameters necessary to inactivate avian influenza virus, by using a model system of destruction of avian influenza viral RNA, in rendered animal co-products and
to compare those conditions with the rendering industry cooking conditions used in the United States and Canada for the purpose of determining whether the current rendering methods are sufficient to destroy avian influenza virus and produce safe products.
Leaphart et al.: RENDERING VS. AVIAN INFLUENZA VIRAL RNA
RNA Extraction and rRT-PCR Ribonucleic acid was extracted with an RNeasy Kit [10] with a modified protocol for rendered material. Briefly, the rendered material was removed from the test tube using Dacrontipped swabs and resuspended in 500 µL of the kit-supplied buffer RLT. The slurry was allowed to remain at room temperature for 10 min. To this volume, 500 µL each of brain heart infusion broth and 70% ethanol was added and the combination was inverted several times. This mixture was then centrifuged at 5,000 × g in a microcentrifuge [11] for 5 min at 20 to 25°C to separate the solid and liquid phases. After centrifugation, an upper oil/fat layer was observed, followed by an aqueous layer and then a solid material layer. Trizol extraction of the oil/fat and solid layers in a non-heat-treated sample indicated no viral RNA was present (data not shown). The aqueous layer was carefully removed and then applied to the spin column, and the remainder of the protocol was performed according to the manu-
facturer’s instructions. Real-time reverse-transcription PCR was conducted using the primer and probe sequences specific for the influenza A matrix gene M1 as described by Spackman et al. [12]. Conditions for reverse transcription were defined as 30 min at 50°C followed by 15 min at 94°C. Thermal cycling conditions were established as a 2-step cycling procedure for 55 cycles of 94°C for 10 s followed by an annealing step at 60°C for 20 s. Fluorescence data were acquired during the annealing step. A Qiagen One-Step RT-PCR Kit [13] was used to make a 17-µL reaction mixture containing 10 pmol of each primer and the hydrolysis probe, 400 µM deoxynucleoside triphosphate mix, 2.5 mM MgCl2, and 6.5 U of ribonuclease inhibitor [14]. Reactions were carried out using a SmartCycler II real-time PCR system [15]. Data were recorded as threshold cycle (Ct) values, where the Ct value denotes how many PCR cycles are required for sample fluorescence to reach the threshold level, which is a fluorescent signal significantly above background fluorescence. At the Ct, a detectable amount of amplified target nucleic acid has been generated during the early exponential phase of the reaction. The Ct is thus inversely proportional to the relative level of sample presence of the gene of interest. Stated otherwise, the more target nucleic acid present in a sample, the lower the Ct value will be. Using the rRT-PCR method described by Spackman et al. [12], a cutoff Ct value of 37 was used to determine the presence or absence of viral RNA based on the established limit of detection of the assay. That is, any Ct value above 37 was deemed unreliable for predicting the presence of avian influenza virus RNA, and was therefore regarded as negative for containing viral RNA. Virus Inoculum Copy Number Determination The sample copy number for the inactivated viral preparation was estimated using the influenza A copy number standard supplied in a TaqManAIV-M Reagent Kit [16] and the rRT-PCR assay used above. A standard curve correlating the matrix gene copy number to Ct value was developed using serial dilutions of the influenza copy number standard. Ribonucleic acid was extracted from the inactivated avian influenza virus preparation mixed with rendered material
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to room temperature over a 3-h period. Subsequently, 250 µL of inactivated, intact avian influenza virus preparation was added to the 1-g quantities of the rendering materials and mixed vigorously by vortex for approximately 1 min to ensure homogeneity. These samples were prepared in duplicate for each time point from each of the 3 rendering plant samples. Samples were thermally treated in temperature and time combinations of 110, 120, 130, and 140°C for 0, 15, 30, 60, and 120 s. Controls included nondosed samples and samples dosed for 0 s. Sample tubes were heated in an analog dry block heater [6]. Additional thermal trials were conducted in rendering materials at 80 and 100°C for 10, 20, 30, and 60 s. Temperature and time combinations were selected to allow determination of the minimum thermal conditions required to destroy the viral inoculum in the rendering materials. Additional control tubes were included in each thermal test round for monitoring temperature using thermocouples [7] connected to a data logger [8] with computer software control [9]. After thermal treatment, the tubes were plunged into ice to cool. Samples were frozen until processing via real-time reverse-transcription PCR (rRT-PCR).
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Statistical Analysis The means were compared among times and temperatures using ANOVA followed by Fisher’s LSD test. Any means with similar letters in Figures 1 and 2 are not significantly different (P > 0.05). All calculations were performed using SAS software [17].
RESULTS AND DISCUSSION During this study, an ELISA system was initially developed and tested for use in detecting viral components. However, because of the high fat content of the rendering materials, the ELISA was unsuccessful for use in this thermal trial. Therefore, RNA extraction and rRT-PCR were used for all subsequent measurements of the β-propiolactone-inactivated strain of avian influenza virus (A/Turkey/Wisconsin/68 H5N9) in this study. β-Propiolactone inactivation of strains of high-pathogenicity avian influenza viruses is commonly used to create vaccines [18]. In this case, the inactivated virus retains sufficient viral characteristics to initiate an immunological response to protect the vaccinated animal against future exposure to the live virus. The inoculum used in this study was selected as an inactivated strain of a low-pathogenicity avian influenza virus with an intact viral capsid for use under biosafety level 2 conditions. Jonges et al. [19] reported that the residual virus genomic RNA is protected by viral nucleoprotein. Jonges et al.
[19] further indicated that β-propiolactone did not have a major effect on the hemagglutinin and neuraminidase characteristics in avian influenza Type A (H7N3). However, they did report that β-propiolactone inactivation reduced the hemagglutination titer and the neuraminidase activity in a human type A (H3N2) influenza virus, and they hypothesized that this observation was due to a reduction in glycoprotein functionality from creation of the RNA-/DNA-protein complexes. Wainberg et al. [20] reported that β-propiolactone treatment had no effect on the viral envelope of the Sendai virus. Franchini et al. [21] reported that inactivation of the herpes simplex virus by β-propiolactone did not affect the capsid, as verified using electron microscopy. No published data were located for the effect of β-propiolactone inactivation on the integrity of the strain of avian influenza virus used in this experiment (A/Turkey/Wisconsin/68 H5N9). It is not known whether the β-propiolactone inactivation process had any effect on the thermal resistance of the inactivated virus in comparison with the non-inactivated virus. However, because of biosecurity and shipping restrictions, it was not possible to obtain a live virus to determine if there was a difference in this study. This factor needs to be tested in further studies by laboratories capable of working with live strains of high-pathogenicity influenza. For the standard curve developed, results of the thermal dose treatments are shown in Figures 1 and 2 as mean log M1 matrix gene copy number. Using the assumption that 1 copy of the M1 matrix gene is present per viral genome, these data roughly estimated the number of viral particles present before and after thermal trials. In the study, an average of approximately 1.97 × 106 copies of the M1 matrix gene was added per gram of rendering materials (n = 4). On the basis of analysis of the unheated, inoculated controls, approximately 1.83 × 106 copies of the matrix gene were recovered by extracting the rendering sample (n = 4). No background M1 matrix gene was detected in the uninoculated rendering material. Because of the nature of the test procedure, the PCR assay used in this experiment was designed to determine if any type A H5N9 influenza RNA was destroyed in the samples. Complete destruction of the RNA strand was assumed,
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(unheated), and rRT-PCR was conducted to determine the Ct value. Using the standard curve developed with the best fit line, a mean of approximately 1.97 × 106 copies of the M1 matrix gene was added per gram of rendering materials (n = 4). Upon extraction recovery from the rendering materials, the copy number was extrapolated to 1.83 × 106 per gram of inoculated rendered material based on an average value over 4 unheated, inoculated samples, so minor losses in the copy number were observed because of passage through the rendering materials. With the assumption that 1 copy of the M1 matrix gene is present per viral genome, this number can be roughly equated to the number of viral particles added.
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based on the lack of observable viral RNA. With this assay, the viral RNA was totally destroyed after treatment for 30 s or longer at 100°C or for 15 s or longer at 110°C or above (Figures 1 and 2), resulting in greater than a 6-log reduction in copy number of the M1 matrix gene. At a thermal treatment of 80°C (Figure 2), viral RNA survival was noted at 10, 20, 30, and 60 s. It was noted (Figure 1) that the thermal dose applied simply during the time (approximately 2 min) to reach temperatures of 110, 120, 130, or 140°C (reported as 0 s) caused a 3-log or greater reduction in observable copy numbers of the M1 matrix gene. Commercial rendering has been used to destroy viral diseases such as pseudorabies in animal tissues [22]. This project was designed to determine the thermal treatment and time com-
binations that would destroy avian influenza virus RNA in rendered products. Rendering would be effective in destroying viral RNA in rendering materials containing virus particles, based on the results of this study. Swayne [23] indicated that avian influenza virus was killed at 70°C within a few seconds in edible chicken thigh and breast meat products. According to Article 10.4.25 [24] of the OIE (World Organisation for Animal Health) Terrestrial Animal Health Code, avian influenza is inactivated in eggs and egg products at 60°C for 188 s for whole eggs, at 60°C for 188 s or at 61.1°C for 94 s for whole egg blends, at 55.6°C for 870 s or at 56.7°C for 232 s for liquid egg whites, and at 62.2°C for 138 s for 10% salted yolks. Treating these products at these temperature and time parameters results in a 7-log de-
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Figure 1. Mean log avian influenza virus matrix gene copy number of β-propiolactone-inactivated avian influenza virus A/Turkey/Wisconsin/68 H5N9 in replicate samples from plants A, B, and C (3 plants × 2 replications each = 6 measurements/time point) that were unheated or heated at 110, 120, 130, and 140°C at treatment times of 0, 15, 30, 60, and 120 s. Time and temperature combinations with similar letters (A–E) are not significantly different. Significance tests were based on ANOVA followed by Fisher’s LSD methods, with α set at 0.05. Zero seconds was the first time point measured after reaching the specified treatment temperature. The mean M1 matrix gene copy number from RNA extracted (as purchased) was 1.97 × 106 (n = 4). The mean M1 matrix gene copy number from unheated, inoculated, rendered material and subsequently extracted was 1.83 × 106 (n = 4). The mean M1 matrix gene copy number in uninoculated rendered material was 0. The SE was ±0.26 log gene copy number.
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struction of avian influenza. Article 10.4.26 [24] of the Terrestrial Animal Health Code describes procedures for inactivation of avian influenza in poultry meat and states that a core temperature of 65.0°C for 42 s, 70.0°C for 3.5 s, or 73.9°C for 0.51 s is sufficient to cause a 7-log destruction of avian influenza. Rendered materials, although prepared for consumption by animals, are termed “inedible materials” because these are not for human consumption. The overall composition of the inedible rendered material was considered a more complex matrix than edible breast meat alone (i.e., much less uniform and containing considerably higher fat content than edible chicken thigh and breast meat). More rigorous thermal conditions were required to destroy the H5N9 viral RNA in the higher fat inedible rendering matrix
than to destroy the live virus in edible poultry meat and eggs. The data obtained in this study on inedible rendered animal co-products in no way contradicts or challenges the data for thermal destruction of avian influenza in edible eggs and meat. Upon obtaining results in this study, the authors hypothesized that the higher fat and increased complexity of the inedible rendered material matrix influenced thermal destruction as compared with meat and eggs. Alternatively, the β-propiolactone viral inactivation process used on the inoculum in this experiment may have enhanced the thermal resistance of the viral RNA. A third hypothesis is that inactivation of the live avian influenza virus may not require as much thermal rigor as destruction of the RNA as measured in this experiment. However, the design of this experiment did not allow for test-
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Figure 2. Mean log avian influenza virus matrix gene copy number of β-propiolactone-inactivated avian influenza virus A/Turkey/Wisconsin/68 H5N9 in replicate samples from plants A, B, and C (3 plants × 2 replications each = 6 measurements/time point) that were unheated or heated at 80 and 100°C at treatment times of 0, 10, 20, 30, and 60 s. Time and temperature combinations with similar letters (A–E) are not significantly different. Significance tests were based on ANOVA followed by Fisher’s LSD methods, with α set at 0.05. Zero seconds was the first time point measured after reaching the specified treatment temperature. The mean M1 matrix gene copy number from RNA extracted (as purchased) was 1.97 × 106 (n = 4). The mean M1 matrix gene copy number from unheated, inoculated, rendered material and subsequently extracted was 1.83 × 106 (n = 4). The mean M1 matrix gene copy number in uninoculated rendered material was 0. The SE was ±0.49 log gene copy number.
Leaphart et al.: RENDERING VS. AVIAN INFLUENZA VIRAL RNA ing of these possible explanations, and further research will be needed. The US and Canadian rendering cookers process materials for a minimum of 30 min at 118°C and often process them at even more rigorous conditions of up to 145°C for 40 to 90 min, depending on the system and the types of materials being processed. The minimum rendering processing conditions are much more rigorous than the time and temperature combinations tested in this study.
1. With thermal treatment for 30 s or longer at 100°C or for 15 s or longer at 110°C or above, RNA of low-pathogenicity avian influenza virus A/Turkey/Wisconsin/68 H5N9, equivalent to 6 log10 of viable virus, was destroyed within the poultry rendering materials. These conditions are well below the range of temperatures and times used for rendering poultry carcasses in the United States and Canada, indicating that the current rendering protocols should be sufficient to destroy the avian influenza virus. 2. Future research is needed by laboratories equipped to handle live avian influenza virus to compare the thermal degradation rate of the β-propiolactone-inactivated strain of avian influenza virus A/Turkey/ Wisconsin/68 H5N9 with the live strain as well as to study the thermal death time of high-pathogenicity live virus. Because of biosafety constraints, this study relied on the use of an inactivated strain and measurement of the destruction of virus RNA to levels at which it could no longer be detected by rRT-PCR as a proxy to indicate that the virus would be destroyed.
REFERENCES AND NOTES 1. Brown, M. H. 2000. Processed Meat Products. Pages 389–391 in The Microbiological Safety and Quality of Food. Vol. 1. B. M. Lund, T. C. Baird-Parker, and G. W. Gould, ed. Aspen Publishers, Gaithersburg, MD. 2. Pearl, G.G. 2004. Rendering 101. Render Mag. Aug.:30–38. 3. Meeker, D. L., and C. R. Hamilton. 2006. An overview of the rendering industry. Pages 1–16 in Essential Ren-
dering. D. L. Meeker, ed. Natl. Renderers Assoc., Alexandria, VA. 4. Catalog Number 204-ADV, US Department of Agriculture National Veterinary Services Laboratory, Ames, IA. 5. Model R2N Ultra, Robot Coupe, Ridgeland, MS. 6. Model 12621-108 equipped with Model 13259-162 heating blocks, both from VWR International, Suwanee, GA. 7. Ecklund-Harrison CNS thermocouples, EcklundHarrison Technologies Inc., Fort Myers, FL. 8. CALPLex Data Logger, TechniCAL Inc., Kenner, LA. 9. CALSoft Software, TechniCAL Inc., Kenner, LA. 10. RNeasy Kit, Qiagen, Valencia, CA. 11. Model 5415D, Eppendorf, Westbury, NY. 12. Spackman, E., D. A. Senne, T. J. Myers, L. L. Bulaga, L. P. Garber, M. L. Perdue, K. Lohman, L. T. Daum, and D. L. Suarez. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol. 40:3256–3260. 13. One Step RT-PCR kit, Qiagen, Valencia, CA. 14. RNase inhibitor, Promega, Madison, WI. 15. SmartCycler II, Cepheid, Sunnyvale, CA. 16. Taq-ManAIV-M Reagent Kit, Applied Biosystems, Foster City, CA. 17. SAS Institute Inc., Cary, NC. 18. Lee, C.-W., D. A. Senne, and D. L. Suarez. 2006. Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin. Vaccine Immunol. 13:395–402. 19. Jonges, M., W. M. Liu, E. van der Vries, R. Jacobi, I. Pronk, C. Boog, M. Koopmans, A. Meijer, and E. Soethout. 2010. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J. Clin. Microbiol. 48:928–940. 20. Wainberg, M. A., R. N. Hjorth, and C. Howe. 1971. Effect of β-propiolactone on Sendai virus. Appl. Microbiol. 22:618–621. 21. Franchini, M., C. Abril, C. Schwerdel, C. Ruedl, M. Ackermann, and M. Suter. 2001. Protective T-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus. J. Virol. 75:83–89. 22. Pirtle, E. C. 1990. Stability of the pseudorabies virus (PRV) in meat and bone meal and intermediate rendering products. Directors Digest 203. Fats and Proteins Research Foundation, Bloomington, IL. 23. Swayne, D. E. 2006. Microassay for measuring thermal inactivation of H5N1 high pathogenicity avian influenza virus in naturally infected chicken meat. Int. J. Food Microbiol. 108:268–271. OIE (World Organisation for Animal Health). 24. 2012. Avian influenza. Chapter 10.4 in Terrestrial Animal Health Code. Accessed April 2012. http://www.oie. int/fileadmin/Home/eng/Health_standards/tahc/2010/en_ chapitre_1.10.4.htm.
Acknowledgments
This work was supported with funds provided by various sponsors of the Fats and Proteins Research Foundation (Bloomington, IL).
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CONCLUSIONS AND APPLICATIONS
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