Incorporating animal forensics in routine meat inspection in the Philippines PDF

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Forensic Science International: Animals and Environments 1 (2021) 100020 Contents lists available at ScienceDirect Forensic Science International: Animals and Environments journal homepage: www.sciencedirect.com/journal/forensic- science-international-animals-and-environments Review Article Incorpor...

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Forensic Science International: Animals and Environments 1 (2021) 100020

Contents lists available at ScienceDirect

Forensic Science International: Animals and Environments journal homepage: www.sciencedirect.com/journal/forensicscience-international-animals-and-environments

Review Article

Incorporating animal forensics in routine meat inspection in the Philippines Enrykie B. Fortajada a, b, Ian Kendrich C. Fontanilla a, c, Maria Corazon A. De Ungria a, d, * a

Biodiversity, Ethnicity and Forensics Program, Philippine Genome Center, University of the Philippines, Philippines Science Education Institute, Department of Science and Technology, Taguig City, Philippines c DNA Barcoding Laboratory, Institute of Biology, University of the Philippines Diliman, Philippines d DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Philippines b

A R T I C L E I N F O

A B S T R A C T

Keywords: Meat adulteration Animal forensics Meat inspection Meat species identification DNA barcoding DNA mini-barcoding

Robust species identification of unprocessed and processed meat is essential to ensure the safety and quality of food products. Meat adulteration results from the wrong identification of animal sources, contamination of different meats during processing, or intentional meat substitution using those from other species and non-meat products of lower economic value. This review discusses the potential applications of DNA barcoding in routine meat inspections in the Philippines. Developing mini-barcode primer sets to enhance the utility of conventional techniques is critical in adopting DNA barcoding technology as a robust tool for routine inspections of meat sold commercially, including those intended for the halal meat industry. Increasing the ability of the Philippine National Meat Inspection Service to document the number, types, and scope of meat fraud is a step forward in finally using animal forensic science as a valuable component of its regulatory functions for the protection of the meat-consuming public.

Introduction Humans have been hunting animals as sources of meat, e.g., the flesh of mammalian animals and fowl, since the prehistoric period. Human civilization’s turning point from having a nomadic lifestyle of huntinggathering to having permanent settlement allowed the domestication of animals such as chicken, pig, sheep, and cattle. Community life eventually led to large-scale meat production from various meat sources using animal farms and slaughterhouses. Slaughtered meat is minced, ground, and seasoned under multiple conditions such as low pH, high temperature, and high pressure to produce meat products. These processes change the physical, chemical, and molecular properties of meat, thus opening the possibility of substitution using less expensive counterparts or any other lower-value non-meat product. The problem of meat adulteration has been reported worldwide [1, 2]. Different techniques such as physical, anatomical, histological, chemical, biological, and molecular techniques have been used to assess the authenticity of meat and meat products [3]. Of these, DNA-based methods used for the identification of biological species are most reliable because these confer accurate species confirmation for the source of meat and meat products throughout the entire supply chain, including those that have already undergone several food processing steps [2,4,5].

Most molecular protocols target species-specific DNA sequences followed by DNA amplification using multi-locus and mono-locus primers in multiplex, nested, and semi-nested PCR [3]. One technique, DNA barcoding, a molecular method that utilizes DNA sequences or ’barcodes’, is used to identify organisms rapidly and accurately [6–8]. Hebert et al. [7] recommended using a 650-bp fragment of the cytochrome c oxidase subunit I (COI) in the mitochondrial genome, the so-called ‘barcode of life’ as the standard gene for barcoding animals. The sequences from DNA samples for identification are compared against a DNA database of similar codes called the Barcode of Life Data System (BOLD) (www.barcodinglife.org) [59]. One major strength of the DNA Barcoding network is the global data sharing of information and results, strengthening the robustness of DNA tests for numerous species across different continents [9]. However, conventional DNA barcoding could not distinguish meat in processed food products with degraded DNA [10] and those with more than one animal source. In the Philippines, traces of pork in seafood products have been reported [11–13]. To date, the National Meat Inspection Service has not used a DNA-based method for routine screening of meat and meat products sold in Philippine markets. This article discusses the potential contribution of DNA barcoding, including DNA mini-barcode methods, in assessing the authenticity of meat and meat

* Corresponding author at: DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Philippines. E-mail addresses: [email protected] (E.B. Fortajada), [email protected] (I.K.C. Fontanilla), [email protected] (M.C.A. De Ungria). https://doi.org/10.1016/j.fsiae.2021.100020 Received 10 March 2021; Received in revised form 10 May 2021; Accepted 18 June 2021 Available online 1 July 2021 2666-9374/© 2021 The Author(s). Published by Elsevier B.V. This is an (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Forensic Science International: Animals and Environments 1 (2021) 100020

products. This protocol could also be used to screen halal foods and detect ‘haram’, if present, in these products. For Muslim Filipinos and the halal meat industry, the detection of all contaminants in halal meat sold is of utmost significance.

Welfare Act of 1998” [23] except for those used as part of indigenous rituals. Imported meat, particularly those originating from China, Europe, and the US that are significant trading partners of the Philippines, must also be inspected. For example, a widespread inspection of meat covering many areas in Europe revealed that 61 % of food products labeled as 100 % beef contained horse meat [24]. The consumption of horse meat poses a health risk to humans because it can contain phenylbutazone residues. This chemical used to treat lameness in horses is hazardous in humans [25]. In addition, preliminary screening of meat products for horse and rat DNA in the Philippines reported negative testing results [17] but the work was limited to one province. Also, another increasing concern is the amount of meat that is regularly being imported from China, wherein fox, mink, and rat had been used as beef and mutton [26]. Another concern in the Philippines is the exploitation of wildlife for bushmeat. Bushmeat, or the meat of wild animals, is used in trade, diet, and culture in many parts of the world [27]. Illegally traded species in the Philippines such as Philippine warty pig (Sus philippinensis), Visayan spotted deer (Rusa alfredi), land snail locally known as “bayuku” (Ryssota otaheitana), Philippine crocodile (Crocodylus mindorensis), and Philippine duck (Anas luzonica), among others, are sold in local markets. The Philippine pangolin (Manis culionensis), considered critically endangered and the most trafficked animal, is sold for its valued scales and meat used as traditional Chinese medicine [28]. Although the capture, sale, and transport of these threatened wildlife species is unlawful in the Philippines [29], enforcement of this law is difficult once the meat is butchered and is no longer visually recognizable. Hence, rapid and accurate identification of animals from seized meat samples, including those that had been butchered and processed, can be achieved through DNA barcoding. As a start, the Philippines has established hygiene protocols for fresh meat and meat preparations, including laboratory tests to diagnose disease conditions and monitor zoonoses [30]. The protocol prescribes an accredited inspector regulating the type of animal for minced raw meat or used in meat preparations. However, the protocol did not include meat species identification at the post-processing stage when adulteration of meat likely happens. Meat source identification is critical in the preparation and sale of halal food. Halal (’lawful, permissible’) food refers to food that is permitted for consumption and prepared according to the halal dietary laws [31]. Halal meat can be obtained from domesticated animals such as cattle, sheep, camel, buffalo, goat, chicken, duck, turkey, etc. as well as non-predatory wild animals such as deer, antelope, wild cattle, etc. and non-predatory birds such as pigeon, sparrow, quail, ostrich, etc. [32]. ’Haram’ or forbidden animals that should not be consumed include the meat of pig, boar, and swine, carnivorous animals, e.g., lion, tiger, cheetah, cat, dog, wolf, and birds of prey such as eagle, falcon, osprey, vulture, and goshawk [32]. The halal meat industry is significant in the Philippines because the number of Muslims in the archipelago has reached 6 million as of 2015 (psa.gov.ph/) [65]. Several cases that involved the adulteration of halal foods with haram ingredients had already been reported in Malaysia [33]; hence the Philippines must avoid similar cases from happening locally. A National Standard on Code of Halal Slaughtering Practices for Ruminants is in place in the Philippines [34]. However, this code is limited only to ruminants, and an overall national standard for halal food is required to cover all types of halal meat.

The meat industry in the Philippines and the need for meat identification Food products in the Philippine market are classified into primary production and post-harvest stages of the food supply chain and processed food [14]. The meat industry in the Philippines consists of large meat factories and numerous small-scale hog and cattle farms that do not pass through government facilities for inspection before going to market [15–17]. Because it is decentralized, food manufacturers themselves need to regularly assess their supply chain from farm to market and maintain the high quality of their meat following trade standards. Interestingly, small-scale or backyard piggeries with less than 100 hogs contribute to 70 % of the total domestic pork supply and 80 % of the national aggregate inventory from more than 100 meat processing establishments nationwide [18]. The entire slaughter volume that the Philippines produced from licensed meat establishments in 2019 amounted to ~1.1 billion kg, in which the top three highest slaughter volumes were from chicken, pork, and beef (Table 1) [19]. These numbers reflect the enormous potential impact of adopting more regulatory requirements for the commercial meat industry. To date, different government agencies regulate a specific category of food and food products. For example, the regulation of primary production and post-harvest stages of the food supply chain falls under the Department of Agriculture (DA) and its regulatory agencies, e.g., the National Meat Inspection Service (NMIS) for meat and meat products. Processed food and food products are under the jurisdiction of the Food and Drug Administration of the Department of Health (DOH) [14]. As part of its function, NMIS is mandated to formulate and implement policies governing the flow of meat (including, but not limited to, pork, beef, and chicken) through various marketing stages and inspection through "The Meat Inspection Code of the Philippines of 2003" [20]. The Meat Inspection Code also requires the routine post-mortem inspection of carcasses and animal parts consumed as food. Illegally slaughtered meat, which includes ’hot meat’, is one of the Philippine meat industry’s significant problems. ’Hot meat’, is the carcass or animal parts that reach the market from unregistered and uninspected establishments [21]. Consumption of illegally slaughtered meat poses health risks to humans; hence animal identification systems are needed at the primary production level [15]. The animal sources for these products should be traced back to the production site during a regulatory investigation [22]. In 2019, NMIS found at least 12 confiscated samples from local markets that were confirmed to be dog meat out of the 455 analyses conducted [19]. The consumption of dog meat is ’dangerous’ because of the risk of trichinellosis, cholera, and rabies (hsi. org/) [63], and was banned in the Philippines through "The Animal Table 1 The volume of slaughtered meat in the Philippines in 2019. Food Animal Chicken Hogs Cattle Carabao Duck Goat Crocodile Horse Sheep Ostrich Total

Amount of Slaughtered Meat Volume (in heads)

Volume (in kg)

651,176,815 4,920,381 172,116 71,978 65,359 30,611 2331 1799 78 88 656,441,556

768,721,558 323,598,987 25,068,913 12,051,238 179,130 207,255 9774 194,250 1781 7059 1,130,039,945

Molecular tests used for meat species identification in the Philippines In 2013, the Meat Inspection Code, was amended to include laboratory and analytical services for the detection of contaminants, pathogens, veterinary drug residues, meat parasites, and identify adulterated meat and meat products in NMIS [21]. These laboratory services include 2

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molecular tests (Table 2). Unfortunately, these services are not routinely requested because their availability is not known to the general public. Also, there is no report available to show if these tests have been tested against different types of halal foods albeit some molecular tests have not detected haram substances due to failed PCR amplification [35].

formation, hairpin formation, and false priming [40]. Some oligonucleotide species-specific primers that amplify smaller PCR products that are treated as DNA mini-barcodes have been used to identify common meat sources (Table 3) and those used as illegal substitutes (Table 4). A rapid multiplex PCR assay was designed to generate different sized amplicons that were used to identify 14 fish species [44]. DNA mini-barcoding showed a 93 % success rate compared to full DNA barcoding with 21 % when tested against 44 processed fish products [50]. The authors believe that combining conventional and mini- DNA barcodes will provide the best molecular testing regimen for routine inspections of meat and meat products. The species-specific primers designed for goat, chicken, cattle, sheep, and pig [45] were successfully tested using multiplex PCR at high temperatures. However, DNA from cooked horse samples failed to amplify due to the 439 bp DNA fragment’s size. These same primers were later reported to anneal to cattle and other meat [51] and hence were not suitable for further development. To show the danger of undetected meat, Kane & Hellberg [52] used real-time PCR (rt-PCR). In their study of 48 samples, only 39 samples were successfully sequenced in both directions using conventional DNA barcodes for beef, chicken, lamb, turkey, pork, and horse. Ten samples were contaminated with meat, not on the product label were detected using rt-PCR. Given these reports, the authors believed that the utility of real-time PCR beyond DNA quantitation with applications in detecting multiple DNA sources should be explored further [53].

DNA barcoding: conventional and mini-barcoding strategies DNA barcoding has been used for species identification of various organisms. It uses a 650-bp fragment of cytochrome c oxidase subunit I (COI) in the mitochondrial genome as the standard gene for barcoding animals [7]. This DNA region exhibits a relatively low divergence level within species and a high divergence level between species [36]. A species is identified if its sequence matches one in the barcode library. If the sequence is unique, it could be a novel DNA barcode (new haplotype or geographical variant) for a given species, or it belongs to an unknown species [37]. This technology initially used for taxonomic and phylogenetic studies is now finding applications for the authentication of animals used as meat sources for human consumption [8,38]. Species-specific primer pairs enabled direct identification of meat from three species of animals (bovines, pigs, and ducks) that were already mixed in food [39]. However, there are drawbacks in obtaining a full-length barcode from processed meat products particularly those that have undergone extreme processing conditions such as low pH, high temperature, and high pressure resulting in negative results [36]. To address the issue of non-amplification using conventional primers, some DNA mini-barcodes with smaller PCR product sizes increased the likelihood of successful DNA amplification [10,40]. Meusnier et al. [10] designed a universal primer set targeting a short DNA region of 100 and 250 bp on the COI gene which achieved 90 % and 95 % success rates, respectively. Identifying the animal sources of processed meat products with multiple species also posed some challenges in using a conventional DNA barcode approach. To discriminate closely related buffalo species, Ramadan [41] used a 422 bp fragment of mitochondrial 16S rRNA D-loop gene as a DNA mini-barcode marker. Hellberg et al. [36] compared the ability of full-length COI DNA barcoding (658 bp) and mini-barcoding (127 bp) in identifying meat species in processed products. Mini-barcoding out-performed conventional DNA barcoding to identify canned products (23.8 % vs. 19.0 % success), including turkey and duck products.However, the primer set poorly performed when tested against chicken, beef, and buffalo products. Because mini-barcodes are shorter than the typical barcode sequence, some taxonomically important genetic information may be missed if the target sequences are not adequately tested or validated [42]. The selection of target sites for DNA mini-barcodes and DNA quality also affect the identification efficiency of the DNA mini-barcoding method [43]. Some unsuccessful identifications involved the amplification of non-specific DNA sequences generally observed in samples with lower DNA quality [36]. Also, novel primer pairs based on 100–200 bp sequences must be designed that avoid dimer

DNA barcoding in meat inspection in the Philippines The application of DNA barcoding in meat species identification of raw meat and meat-derived products is not well explored in the Philippines. Ramos et al. [17] were the first to use DNA barcoding to confirm the absence of horse and rat DNA in 30 meat and poultry products tested. Sarmiento et al. [13] analyzed popular street food such as fish balls, fish nuggets, squid balls, and shrimp balls. They reported that only 63 % of samples tested produced by recognized commercial establishments were correctly labeled. More importantly, 100 % of the samples from unknown companies sold by street vendors and commercial stalls were positive for pig and chicken DNA. DNA barcoding technology could not determine if the DNA is from actual meat or animal fat used to prepare these products. Out of all the food products analyzed, 50 % contained meat sources other than seafood. The undeclared substances could mislead consumers into believing that these are ‘safe street foods’, including those who do not consume pork. DNA barcoding has been used to identify confiscated samples suspected to originate from illegally traded wildlife species. Luczon et al. [54] used DNA barcoding on several incidences to determine the taxonomy of pangolins that were confiscated in the Philippines. Out of 85 unidentified pangolin carcasses, 73 were identified as the Philippine pangolin (Manis culionensis), endemic in Palawan, Philippines, while the other 12 individuals were identified as Malayan pangolin (M. javanica). This information provided critical leads to Philippine law enforcement that co...