AU2017202135B2

AU2017202135B2 - Method of reducing the virulence of the infectious bursal disease virus

Info

Publication number: AU2017202135B2
Application number: AU2017202135A
Authority: AU
Inventors: John Bingham; Geoff Knight; Stewart Macpherson
Original assignee: Gobblers Inc Pty Ltd
Current assignee: Gobblers Inc Pty Ltd
Priority date: 2016-04-15
Filing date: 2017-03-31
Publication date: 2019-02-28
Anticipated expiration: 2037-03-31
Also published as: AU2017202135A1; AU2017245455A1

Abstract

Abstract A process for reducing the virulence of infectious bursal disease virus (IBDV) in uncooked poultry meat using high hydrostatic pressure.

Description

METHOD OF REDUCING THE VIRULENCE OF THE INFECTIOUS BURSAL

DISEASE VIRUS

Technical Field [0001] The invention relates to the field of commercial food processing. In particular, the invention relates to the processing of poultry meat products to reduce food-borne pathogens, such as infectious bursal disease virus (IBDV).

Background of the Invention [0002] Infectious food-borne pathogens have the potential to cause significant harm to consumers of products of the meat processing industry. The potential for individual and commercial damage is significant, from spoilage that leads to production losses to acute outbreaks that cause morbidity and mortality.

[0003] Pathogens in poultry meat, for example, can cause serious disease in humans. For example, H5NI influenza viruses in chicken meat can be transferred to humans with resultant health risks. To limit the losses caused by such pathogens, rigorous biocontainment is necessary to prevent their spread.

[0004] Further, one of the principal risks in the importation of chicken products into Australia is the introduction of avian pathogens that are currently exotic to Australia.

[0005] Spread of virus can occur in poultry tissue supporting viral replication or through contamination via poultry tissue for which the virus has a predilection. It is therefore possible to spread the virus through inadvertent consumption of those products. One avenue of containment is the heat inactivation of pathogens within uncooked poultry carcasses and products.

[0006] Biosecurity Australia (BA) currently require that imported chicken meat be cooked at a core temperature of 74°C for 165 minutes or 80°C for 125 minutes. This treatment is designed to achieve a 6xlogio reduction of infectious bursal disease virus (IBDV) and was based on research undertaken by Dennis Alexander (CVL 1997) on very virulent infectious bursal disease virus (IBDV) strain CS88 in a bursa, meat and fat homogenate. Insufficient data points exist to support an extrapolation to a 6logio reduction. Due to a considerable reduction in effectiveness of the inactivation of the virus at temperatures lower than 74°C, cooking at temperatures less than 74°C is not approved. Treatments beyond 80°C are also not approved.

2017202135 31 Mar 2017 [0007] A 6xlog io reduction in IBDV virus was determined by Biosecurity Australia to be an appropriate benchmark when assessing heat treated chicken meat for importation into Australia. In the absence of further scientific data, Biosecurity Australia stipulated that any alternative to heat treatment must be shown to be at least equivalent to the currently recommended heat treatment. That is, it must be shown to inactivate at least 6logioof the infectious bursal disease virus initially present.

[0008] However, a major drawback with such processes is that the ‘cooking’ of the meat renders it far less useful for a variety of further processes and products requiring an ‘uncooked’ poultry meat to meet organoleptic and textural requirements.

[0009] Accordingly, it is an object of the present invention to provide an alternative process that can reduce the risk of the effects of these viruses through uncooked poultry meat and that ameliorates at least some of the problems associated with the prior art.

Summary of the Invention [0010] According to a first aspect of the invention, there is provided a process for reducing the virulence of infectious bursal disease virus (IBDV) and/or other viruses in uncooked poultry meat, the process including the steps of: applying 600MPa of high pressure to the poultry meat at ambient temperature for a period of time between about 15 sec and 2 minutes. Preferably, the amount of any live virus present in the poultry meat following application of the process is not capable of causing detectable infections in chickens.

[0011] This process has been demonstrated to reduce the infectivity of strains of IBDV, even where the virus has not been destroyed, or its numbers not reduced by a factor of more than 6xlogio. This result allows the inventive process to form the basis of a treatment of uncooked poultry meat that provides commercial advantages, such as allowing some uncooked poultry meat to meet the biosecurity requirements for import into certain countries.

[0012] More preferably, multiple applications of 600 MPa for between 10 and 15 seconds are used.

[0013] According to a second aspect of the invention, there is provided the use of a process according to any preceding claim to inactivate avian viruses such as avian influenza virus (AIV), Newcastle disease virus (NDV), and to reduce the virulence of

2017202135 31 Mar 2017 infectious bursal disease virus (IBDV) in commercial grade poultry meat, according to that described above.

[0014] According to a third aspect of the invention, there is provided edible poultry meat that has been subjected to a treatment according to that described above and which meets the biosecurity requirements discussed above.

[0015] Now will be described, by way of specific, non-limiting examples, preferred embodiments of the invention.

Detailed Description of the invention [0016] High pressure processing (HPP) of foods is gaining popularity in the food industry, in part, because of its ability to inactivate microorganisms and enzymes at room temperature, whilst valuable, low molecular weight food constituents, such as vitamins, volatiles and colour pigments, remain largely unaffected.

[0017] Over previous decades, equipment development has progressed rapidly and HPP food products, including meat and fish products, are now available in many countries. HPP is known to effectively eliminate pathogens and prolong the shelf life of raw chicken meat. However, the pressure labile nature of some meat protein systems such as myosin or myoglobin often limits the range of attractive commercial applications.

[0018] In commercial settings, pressure pasteurization of foodstuffs typically involves application of static pressure of between 350 MPa and 600 MPa for a few seconds or up to several minutes, in order to destroy food spoilage bacteria.

[0019] The sensitivity of viruses to pressure, and thus extent of their inactivation, is dependent on several environmental (e.g. pH, water activity) and processing factors, for example pressure holding time.

[0020] It is primarily desired to reduce the threat of IBDV infection in animals from imported poultry meats. This virus is well known and usually susceptible to heat treatments, but it is desired to use HPP to manage this threat without the concomitant denaturing of the meat that is associated with conventional heat treatment.

[0021] It was previously known that HPP had a deleterious effect on the viability on some strains of IBDV, at pressure of up to 240 MPa for periods of up to 200 minutes.

However, the possibility of strain differences and the effect of treatment at higher

2017202135 31 Mar 2017 pressures for commercially practical times (i.e. a few minutes only) were heretofore largely unknown.

[0022] The balance between the level of virus inactivation required to guarantee consumer safety, including the likelihood of significant variation in susceptibility to pressure between strains, and a shorter, more commercially viable treatment regime was needed in the poultry processing and importation industry.

[0023] The present invention is embodied in a high-pressure process that has been developed for the inactivation of IBDV viruses in uncooked poultry meat to render it commercially viable. It will be appreciated that this embodiment is by way of example only and the inventive process could be used to treat a wide range of other raw poultry products, and poultry products generally.

[0024] Food safety authorities in some jurisdictions mandate a specific ‘x log 1 o’ factor reduction in particular types of viruses and other spoilage organisms that must be achieved for the food to be considered commercially sterile and safe to be imported into the country. As discussed, Biosecurity Australia require a treatment process to achieve a logio reduction of 6x (i.e. a 1,000,000-fold reduction) in certain viruses before uncooked poultry meat can be imported into Australia. IBDV is used as a benchmark by Biosecurity Australia as it is the most thermally and pressure resistive of the avian viruses.

[0025] In HPP, there are two key variables that define the treatment process: time spent under pressure; and the magnitude of pressure applied. Every different food type requires testing to ascertain at what point the process has been effective in inactivating the target pathogens.

[0026] Studies were conducted to examine the efficacy of HPP in inactivating avian pathogens in chicken products. On the recommendation of Biosecurity Australia, the study included six strains of IBDV (2 classical (Aphis & 52/70), 2 very virulent (CS88 and Tasik94) and 2 variant strains (GLS-5 and Variant E)), avian influenza virus and Newcastle disease virus.

[0027] The samples were prepared and treated at 600 MPa at room temperature at a range of treatment times. For each pathogen, the virus titre in sub-samples of divided meat-virus homogenate was determined.

[0028] From each batch, one sub-sample was left untreated and the remainder were pressure treated for varying times. IBDV suspensions were prepared in infected SPF

2017202135 31 Mar 2017 chickens. The 3 weeks-old chickens were infected by instillation of viral inoculum by the mucosal route (onto the eyes and in the mouth), and they were euthanased 48 hours later and the bursae of Fabricius removed. Bursae were ground in a sterile pestle and mortar, and mixed with sterile phosphate-buffered saline (PBS-A, pH 7.2, Oxoid, Cambridge, UK) in a ratio of 1:5 to make a 20% suspension. This was centrifuged to separate ground tissue and the supernatant was stored in aliquots at -80°C.

[0029] Chicken breast meat from non-vaccinated SPF chickens was ground in an electric blender (Ultima FP35-AA, Black & Decker, Mooroolbark, Victoria. Australia). Aliquots of 9g were mixed with 1 mL virus suspension and further ground with a pestle and mortar until the fluid and meat were fully homogenised. The homogenate was split into up to 5 plastic sachets, which were then double sealed with a heat sealer (Impulse Sealer ME-300HI, Mercier Corporation, Taipei Hsien, Taiwan). They were stored temporarily on ice and pressure treated within 2 hours of preparation.

[0030] High pressure treatments were carried out using a lab-scale 2-litre high pressure system (2.0 litre Food Processing Unit, Flow International Corporation, Kent, WA, USA) using water at 22°C as the compression fluid. The sachets with the meat-virus homogenate were subjected to 600 MPa pressure for the appropriate times. The treatment time was measured beginning immediately as the target pressure was reached. The compression rate was standardised at 50MPa/sec and decompression was immediate. The temperature of the water was measured at the end of the compression cycle and varied from 19 to 23°C [0031] After treatment, the sample was mixed with 10 mL of PBS-A, homogenised with a sterile pestle and mortar, and centrifuged to pellet the muscle tissue. The supernatant extract was harvested for titration, which was performed on the same day. An untreated meat-virus homogenate sample, which was prepared, handled and tested in a manner identical to, and simultaneously with, the treated samples, was included with each batch.

[0032] The very virulent strains of IBDV (such as Tasik94) propagate best in the allantoic cavity of chicken embryos, while other strains of IBDV grow inefficiently in eggs and therefore are best propagated and tested in chickens. Preparations containing IBDV Variant E and GLS-5 were tested in chickens, while preparations of all other viruses were tested in eggs (Table 1). For each procedure, an untreated sample of the same meatvirus homogenate was titrated in parallel.

2017202135 31 Mar 2017 [0033] Supernatants of treated and untreated IBDV were titrated by chorioallantoic membrane (CAM) inoculation of 9-11 day old embryonated chicken eggs (from SPF and unvaccinated parent stock) (OIE, 2008b). Each dilution was inoculated into 3-5 eggs. The eggs were incubated for 7 days; those that died in the first 48 hours after inoculation were discarded.

[0034] CAMs were harvested from all eggs that died after 48 hours and all those alive at 7 days. Infection was detected by an IBDV antigen-capture ELISA on CAM homogenates, described below. Allantoic fluids from all inoculated eggs dead after the first day, and all that survived 5 days were tested by haemagglutination (OIE, 2008a). Virus titres were calculated by the Spearman-Karber method (Hawkes, 1979).

[0035] Aliquots of treated and untreated mixtures of IBDV were titrated by inoculation of serial dilutions in groups of 3-week-old SPF chickens (OIE, 2008b). A total volume of 0.2ml of each dilution was inoculated into each of five chickens by the mucosal route (instillation onto the eyes, into the nostrils and mouth). The chickens were euthanased 23 days after challenge and their bursae were harvested for histopathology and immunohistochemistry.

[0036] The chorioallantoic membrane (CAM) was harvested from eggs and placed into a 10 mL tube with PBS-A. After being frozen and thawed three times, the CAM supernatants were tested by an antigen capture enzyme linked immunosorbent assay (AC-ELISA) (OIE, 2008b).

[0037] The procedure for the ELISA is briefly as follows: the capture antiserum (100μΙ per well of 1/1000 dilution) was incubated in flat bottom, polystyrene 96-well microtitre plates (Nunc Maxosorb, Nunc, Roskilde, DK) and incubated for 60 min at 37°C (this and all subsequent steps were, unless indicated, done at this temperature on a shaker). After this, and between all subsequent steps, the plates were washed three times in PBS-A containing 0.05% Tween® 20 (Sigma-Aldrich, Castle Hill, NSW, Australia). 10OpL of each sample (or control) suspension was placed into two wells and incubated for 30 min. 10OpL of anti-IBDV monoclonal antibody was added to the wells and incubated for a further 30min. 100 pL of anti-mouse horseradish peroxidase conjugate was added for 30 min. Tetramethylbenzidine substrate was added at room temperature and 5min later the reaction was stopped by adding 50μΙ of 1M H2SO4.

[0038] The optical density was read at 450 nm. The cut-off was calculated as double the OD values of the negative CAM control values. The capture antibody used was the convalescent antiserum of a chicken infected with IBDV (strain 002/73). The detection

2017202135 31 Mar 2017 antibody was a monoclonal antibody, 9-6, prepared and characterised as described in a separate study (Fahey, et al., 1991).

[0039] Bursae were processed by routine histological methods. Sections of bursa were stained with haematoxylin and eosin for detection of the characteristic lesions of IBDV: lymphocytic, necrosis and depletion. Histological sections were also stained in an immunohistochemistry test for detection of IBDV antigen. For the immunoperoxidase test a recombinant chicken, antibody, Crab 20, with an E-tag label was used as the primary antibody. Briefly, histologically-prepared sections were autoclaved (Pascal pressure cooker, DakoCytomation, Groskup, DK) at 121 °C for 15 min in citrate-citric acid buffer (0.1 M, pH 6.0). They were incubated for 60 min with the labelled Crab20 antibody; the sections were incubated with anti-E-tag conjugated to horseradish peroxidise (GE Healthcare, Buckinghamshire, UK) for 45 min. Antigen staining was developed with aminoethylcarbazol (AEC) substrate chromogen (DAKO Envision) twice for 5min each. The sections were counterstained with haematoxylin, and mounted under aqueous mountant for viewing under standard light microscopy.

[0040] Table 1 shows the reduction in titre of various avian viruses in chicken meat homogenate after pressure treatment at 600 MPa and room temperature. Application of 600 MPa for 1 min and 2 min inactivated more than 6xlogio EID5o/O.2ml of AIV (H7N7) and 7xlogioEID5o/0.2ml of NDV (Herts) in chicken meat homogenate, respectively. Pressure treatments at 600 MPa for only 15 seconds resulted in more than 2xlogiolDso/0.2

2017202135 31 Mar 2017 ml inactivation of selected IBDV strains in chicken meat homogenate.

Table 1: Reduction in titre of various avian viruses in chicken meat homogenate after treatment at 600 MPa and room temperature

Virus	Pressure holding time (sec)	Titration method	Start titres (log₁₀EID	End titres '5o/O.2 ml)
AIV (H7N7 Bendigo)	60	AS^a	6.5	0
NDV (Herts)	120	AS	8.3	0.9
IBDV (Tasik94)	120	CAM^b	5.7	1.7
IBDV (CS88)	120	CAM	4.5	0
IBDV (Aphis)	120	CAM	4.3	0
IBDV (52/70)	120	CAM	3.2	0
IBDV (variant E)	15	Chick	2.1	0
IBDV (variant E)	120	Chick⁰	<2.7	0
IBDV (GLS-5)	15	Chick	2.5	0
IBDV (GLS-5)	120	Chick	<2.8	0

^a AS - allantoic sac inoculation followed by haemagglutinating effect of allantoic fluid after embryo death or 5 days incubation ^b CAM — chorioallantoic membrane inoculation followed by antigen capture ELISA for IBDV on CAM after death of embryo or 7 days after inoculation ^c Chick — inoculation onto the mucous membranes of 3 week-old chicks and histological analysis of bursa at 3 days following inoculation.

[0041] Pressure treatments at 600 MPa for only 15 sec resulted in more than 2 logiolDso/0.2 ml inactivation of selected IBDV strains in chicken meat homogenate. Most strains of IBDV showed no residual infectivity after 2 min treatment at 600 MPa. However, 1.7 logic EID5o/O.2ml of IBDV-Tasik94 remained after the treatment. This indicates a relatively high pressure resistance of this strain.

2017202135 31 Mar 2017 [0042] Inactivation of the strain IBDV-Tasik94 in chicken meat homogenate showed high initial titre reductions within 15 sec. of treatment at 600MPa followed by significant slowing of inactivation and thus persistence after longer treatment times. It appears that economically practical treatment times (e.g. 150 sec) did not completely eliminate the residual live virus.

[0043] Nevertheless, it was found that residual IBDV-Tasik94 particles in chicken meat homogenate treated at 600 MPa for 30 sec did not cause infection in chickens. It would previously have been expected that at least some, if not most, chickens would be infected by such amounts of virus. Without being bound by theory, the fact that this does not happen suggests that the treated virus has been changed in a way that renders the virions non-infectious when administered by the mucosal route to chickens.

[0044] The results of this study indicated that HPP at 600 MPa is highly effective at causing rapid inactivation of AIV, NDV and six strains of IBDV, when mixed into ground chicken meat.

[0045] IBDV-Tasik94 showed relatively high pressure resistance in chicken meat homogenate compared to other avian viral pathogens with around 0.5-2.0xlogio EID50/0.2ml persistent virus particle even after long treatment times. However, residual IBDV-Tasik94 virus after 30 sec treatment at 600 MPa did not cause detectable infection in chickens, which indicates that processing under these commercially viable conditions may be suitable to avoid the risk of infection of avian viral pathogens via contaminated chicken meat.

[0046] It was found that multiple pressure cycles on IBDV have no noticeable advantage in inactivation effect over a single pressure cycle. Furthermore, despite the detection of live IBDV virus in eggs after pressure treatment, infectivity could not be detected in 3-week-old chickens. These findings can be useful for the application of HPP to the limitation of pathogen spread via chicken meat products, specifically homogenate, and can inform risk assessment policy on importation of these products.

[0047] Therefore, HPP treatment at 600 MPa for 15 sec or more can reduce the viability or virulence of IBDV strains, even where the virus count is not reduced by the usual factor of 6xlogio. This can accordingly form the basis of a processing regime that mitigates the risk of infections being borne by non-cooked poultry meat.

[0048] It will be appreciated by those skilled in the art that the above described embodiment is merely one example of how the inventive concept can be implemented. It will be understood that other embodiments may be conceived that, while differing in their detail, nevertheless fall within the same inventive concept and represent the same invention.

Claims

Claims

1. A commercial process for reducing, by a factor of 6xlogio or greater, the infectivity of any one or more strains of infectious bursal disease virus (IBDv) in uncooked poultry meat, said strains selected from the group consisting of: IBDv Tasik94, IBDv Aphis, IBDv CS88, IBDv variant E, IBDv 52/70 and IBDv GLS-5 ; said process including the steps of: applying 600MPa of hydrostatic pressure to said poultry meat for a period of time between about 15 seconds and 2 minutes.
2. A commercial process of reducing, by a factor of 6xlogio or greater, the infectivity of uncooked poultry meat containing the infectious bursal disease virus (IBDv) with reference to any one or more viruses selected from the group consisting of: IBDv Tasik94, IBDv Aphis, IBDv CS88, IBDv variant E, IBDV 52/70 and IBDv GLS-5; said process including the steps of: applying at least 600MPa of hydrostatic pressure to the poultry meat for a period of time between about 15 seconds and 2 minutes.
3. The process according to any preceding claim, wherein said strain is IBDv Tasik94.
4. The process according to any preceding claim, wherein the amount of any live virus present in the poultry meat following application of the pressure is not capable of causing detectable infections in chickens.
5. The process of any preceding claim, wherein multiple applications of 600 MPa of hydrostatic pressure to the poultry meat for between 10 and 15 seconds are used.
6. The use of a process according to any preceding claim to reduce the infectivity of any one or more strains of infectious bursal disease virus selected from the group consisting of: IBDv Tasik94, IBDv Aphis, IBDv CS88, IBDv variant E, IBDv 52/70 and IBDv GLS-5 in commercial poultry meat.
7. Poultry meat that has been subjected to a treatment according to any one of claims 1 to 5.
8. The use of the any one or more strains of infectious bursal disease virus (IBDv) strains selected from the group consisting of: IBDvTasik94, IBDv Aphis, IBDvCS88, IBDv variant E, IBDv 52/70 and IBDv GLS-5 as a marker to determine the effectiveness of a HPP process to render uncooked poultry meat practically non-infective.

2017202135 17 Jan 2019
9. The use of claim 8, wherein the strain is IBDv Tasik94.