Walia 2018

Accepted Manuscript
Qualitative risk assessment of cricket powder to be used to treat undernutrition in

infants and children in Cambodia
K. Walia, A. Kapoor, J.M. Farber
PII: S0956-7135(18)30210-X
DOI: 10.1016/j.foodcont.2018.04.047
Reference: JFCO 6109
To appear in: Food Control
Received Date: 12 February 2018

Revised Date: 12 April 2018
Accepted Date: 21 April 2018
Please cite this article as: Walia K., Kapoor A. & Farber J.M., Qualitative risk assessment of cricket
powder to be used to treat undernutrition in infants and children in Cambodia, Food Control (2018), doi:
10.1016/j.foodcont.2018.04.047.
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1 Qualitative risk assessment of cricket powder to be used to treat undernutrition in

2 infants and children in Cambodia
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4 Walia, K.a,*, Kapoor, A. a, Farber, J.M. a
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6 Canadian Research Institute for Food Safety, Department of Food Science, University
7 of Guelph, Guelph, Ontario, Canada.
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9 * Corresponding Author: Canadian Research Institute for Food Safety, Department of
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10 Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Email:
11 kwalia@uoguelph.ca
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12 ABSTRACT
13 This qualitative risk assessment (QRA) aims to estimate the microbiological risk
14 associated with the consumption of locally sourced cricket powder by infants and
15 children aged 6-23 months to prevent or treat undernutrition in Siem Reap, Cambodia. It
16 follows the Codex principles and guidelines for risk assessment and takes into account all
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17 the potential microbial and microbial related toxic hazards that are associated with the
18 consumption of cricket powder. A comprehensive literature search was carried out for
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19 foodborne pathogens isolated from crickets and edible insects along with other dried
20 foods that are similar to cricket powder such as flour, powdered infant formula, and dried
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21 herbs and spices. From this literature search, the following pathogens were identified and
22 considered for this microbiological QRA: Bacillus cereus, Escherichia coli,
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23 Campylobacter spp., Clostridium perfringens, Cronobacter sakazakii, Listeria
24 monocytogenes, Salmonella spp. and Staphylococcus aureus. Overall, the QRA suggests
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25 that cricket powder presents a potential risk to undernourished infants and children. If
26 porridge fortified with cricket powder is boiled (rolling boil for 5 min) prior to
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27 consumption, the food safety risk to undernourished infants and children is low for S.
28 aureus; low to moderate for B. cereus, C. perfringens Type A, C. sakazakii,
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29 enterohemorrhagic E. coli, L. monocytogenes and Salmonella spp.; and a moderate to

30 serious risk for C. perfringens Type C. However, if the fortified porridge is not boiled,
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31 the food safety risk is increased. This risk can be mitigated if crickets are reared in a
32 controlled, strict containment system that limit microbial hazards, and processed in a
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33 hygienic manner and making every effort to ensure that contamination and/or cross-
34 contamination is mitigated by following hazard analysis critical control point (HACCP)
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35 procedures and using a heat treatment to further reduce the hazard.

36
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37 Keywords: Edible insects; Microbial hazards; Food safety; Foodborne pathogens;

38 Complementary feeding
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39 1 INTRODUCTION
40 1.1 Malnutrition
41 Malnutrition in children is a chronic problem in low- and middle-income
42 countries. The World Health Organization (WHO) defines malnutrition as “deficiencies,
43 excesses or imbalances in a person’s intake of energy and/or nutrients” (WHO, 2017a).
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44 It consists of two conditions: (1) undernutrition, which refers to a vitamin and mineral
45 (micronutrient) deficiency or insufficiency along with stunting (i.e., low height for age),
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46 wasting (i.e., low weight for height), and underweight (i.e., low weight for age); and (2)
47 overweight, obesity and diet-related non-communicable diseases (WHO, 2017a).
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48 Globally, 45% of all under 5 child deaths are attributed to undernutrition, and the
49 proportion of total deaths due to stunting, wasting, severe wasting and underweight is
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50 14.7%, 12.6%, 7.4% and 14.4%, respectively (Black et al., 2013; WHO, 2017a, b).
51 Moreover, in children less than 5 years of age, 155 million are stunted, 52 million are
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52 wasted, 17 million are severely wasted and 41 million are overweight and obese
53 (UNICEF, 2017; WHO, 2017a).
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54 Malnutrition in children directly contributes to diseases, disorders, and in severe

55 cases, death. It also impairs cognitive development resulting in poor performance at
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56 school and during later stages when working (UNICEF, 2017). As such, malnutrition
57 poses a great socio-economic burden on a nation.
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58 Key factors that contribute to micronutrient deficiencies (i.e., undernutrition due

59 to a lack of essential vitamins and minerals) in children are a lack of diversity in diet and
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60 poor breastfeeding practices. Cambodia, the target country for this risk assessment, has a
61 high prevalence of undernutrition in children less than 5 years of age (Kosal et al.,
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62 2015). For example, in rural Cambodia, 33.8%, 9.9%, 2.4% and 25.4% of children under
63 5 years are stunted, wasted, severely wasted, and underweight, respectively. In
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64 comparison, in urban Cambodia, the percentages are lower, but still substantial with
65 23.7%, 7.5%, 2.0%, and 14.8% of children under 5 years being stunted, wasted, severely
66 wasted and underweight, respectively (Kosal et al., 2015).
67 The majority of the rural population in Cambodia (80.5% versus 19.5% in urban
68 areas) depend on rice as a staple food with limited availability of farmed vegetables, fruit
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69 and animal-based foods (Kosal et al., 2015). This contributes to high rates of chronic
70 malnutrition and micronutrient deficiencies (Müller & Krawinkel, 2005; WHO, 2017a).
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72 1.2 Treating Undernutrition with Local Foods
73 The United Nations (UN) General Assembly has identified 2016 to 2025 as the
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74 UN Decade of Action on nutrition, the aim of which is to ensure that all people have
75 access to nutritious and sustainable foods, which can help to combat the two conditions
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76 of malnutrition – undernutrition and overweight, obesity and diet-related non-
77 communicable diseases (WHO, 2016). For the purposes of this risk assessment, the
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78 former (i.e., undernutrition) will be the main focus; however, brief mention to the latter
79 will be made when necessary.
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80 One approach to treating undernutrition is to use locally available nutrient-dense
81 foods, which can address micronutrient deficiencies. As described by the Food and
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82 Agriculture Organization of the United Nations (FAO) and International Life Science
83 Institute (ILSI), these diverse food-based strategies are a preventative, comprehensive
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84 and sustainable way to meet nutrient requirements. In addition, they are cost-effective,
85 meet traditional and local food consumption practices, and can benefit large populations
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86 (FAO & ILSI, 1997; WHO, 2012).

87 The integration of locally available nutrient-dense cricket powder to the diet is
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88 one example of a potential solution to address the issue of undernutrition amongst

89 infants and children in Cambodia (Hanboonsong, Jamjanya, & Durst, 2013).
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91 1.3 Edible Insects
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92 Entomophagy, the eating of insects, is a traditional practice in many nations. To-

93 date, more than 2000 insects have been recognized as being edible (Rumpold &
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94 Schlüter, 2013a). While Western countries have begun to show an interest in the
95 consumption of insects, regions such as Africa, Latin America, as well as Asia and the
96 Pacific, have a long history of using insects for food and feed (van Huis et al., 2013).
97 Insects have a higher fecundity, higher feed conversion efficiency, require only small
98 land for production and produce fewer greenhouse gases (Rumpold & Schlüter, 2013a;
99 van Huis, 2016). Moreover, edible insects are expected to meet the growing demand for
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100 animal protein while contributing to food and nutrition security, i.e., the nutritional value
101 of consuming insects (van Huis, 2013; van Huis et al., 2013).
102 Although the nutritional content of insects can vary due to several factors such as
103 species, gender, developmental stage, and rearing conditions such as temperature, most
104 of the species are nutrient dense, i.e., rich in proteins, fats, essential amino acids (AA),
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105 vitamins, minerals and energy (Rumpold & Schlüter, 2013b). They contain
106 approximately 20 to 76% protein; possess all essential AA, meeting WHO requirements;
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107 are rich in zinc, iron, copper, Vitamins B1 and B2, B12, and have a high but variable
108 lipid content of 7 to 77 g/100 g dry weight, depending on the species (Belluco et al.,
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109 2013; van Huis, 2016). The lipids consist of essential fatty acids such as linoleic and/or
110 linolenic acids and a high polyunsaturated fatty acid (PUFA) to saturated fatty acid
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111 (SFA) ratio (Belluco et al., 2013; van Huis, 2016).
112 Edible insects are harvested in one of three ways: wild harvesting, semi-
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113 domestication or farming. Once harvested, they are killed by freeze-drying, sun-drying
114 or boiling and thereafter can be consumed as whole insects, in the form of a paste, a
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115 powder, or as an extract that can be used to fortify nutrient poor food and feed products
116 to increase their nutritional value (van Huis et al., 2013). As insects are a traditional food
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117 in Cambodia, where many suffer from undernutrition, their use has been proposed by the
118 FAO in fortified blended food products (FBFs) (van Huis et al., 2013). Currently, most
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119 of the FBFs and Ready-to-Use Therapeutic Foods (RUTF) used to treat malnutrition
120 contain ingredients such as soy protein, milk powder, corn blend pre-cooked pulses and
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121 legumes. However, such ingredients are not part of the local diet, are not locally
122 available, and are not a sustainable source. Hence, the protein, fat and micronutrient
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123 content of edible insects are an excellent source for FBFs and RUTF to combat
124 malnutrition. Moreover, their availability, cultural relevance and minimal ecological
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125 impact are well suited in the framework of sustainable diets as defined by FAO.
126 One such programme that uses edible insects to address malnutrition is WinFood.
127 It is a Denmark-based initiative that aims to improve traditional foods globally by
128 developing nutritionally improved foods through the use of spiders and termites to
129 prepare food products in Cambodia and Kenya, respectively (van Huis et al., 2013).
130 WinFood Cambodia’s meal is composed of rice, fish and spiders (Haplopelma
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131 albostriatum), and in a recent study, it was found to be equally beneficial as the other
132 FBFs (Skau et al., 2015). The WinFood Cambodia meal was proven to be a cost-
133 effective, locally available, self-sufficient method to treat malnutrition (Skau et al.,
134 2015). In another study, infants aged 6 to18 months in the Democratic Republic of
135 Congo were fed cereal made with caterpillars. It was found that the infants in the cereal
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136 group had a higher hemoglobin concentration and fewer were anaemic as compared with
137 the usual diet of just cereal. However, this fortification did not reduce the prevalence of
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138 stunting (van Huis, 2016).
139 A second programme is the ‘Funky Foods’ project by World Vision, where
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140 cricket powder is planned to be prepared at the household level, in Cambodia, as a
141 potential treatment for malnutrition. This ‘Funky Foods’ project is the reason for the
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142 subsequent risk assessment in later sections. Processing cricket powder in Cambodia is
143 relatively simple and involves harvesting crickets that are reared outside (normally under
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144 the house) in a soil-free environment in new clean paper egg cartons, which are covered
145 with mosquito netting to prevent access by ants and pests. Figure 1 (Supplementary
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146 Material) depicts the steps of producing cricket powder at both the factory and home
147 settings. In general, Figure 1 showed that following harvesting, World Vision personnel
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148 for the pilot project (denoted as “at factory”, which is where the cricket powder was
149 produced) killed the crickets by suffocation, followed by cleaning with running water,
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150 boiling and then drying by laying the crickets on trays and placing them into a solar
151 dehydrator that was closed, limiting contamination from the outside environment and
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152 encouraging a higher heat transfer of ~80ºC (Personal Communication, World Vision
153 Canada, 2017). These dried crickets were then ground indoors with an electric grinder to
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154 form a powder, which was then put into plastic bags and then into a box for transport.
155 Once the plastic bags were distributed to caregivers, a single bag per day was then added
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156 to cooked porridge and fed to infants and children aged 6-23 months. On the other hand,
157 Figure 1 showed that in a home setting, once crickets are harvested, World Vision (based
158 on first-hand knowledge) anticipates that the crickets will be killed by suffocation,
159 boiled and then laid on trays and sundried or placed into a solar dehydrator that is closed,
160 limiting contamination from the outside environment. These dried crickets are ground
161 outdoors with a mortar and pestle to form a powder, which is then added to cooked
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162 porridge and fed to infants and children of the same age group (Personal
163 Communication, World Vision Canada, 2017).
164 Although the use of insects to treat malnutrition is promising, there are various
165 potential health concerns associated with the production, processing and preparation of
166 these products. Even from the description above, it is evident that ingresses for
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167 contamination in the processing of cricket powder is highly likely and, as such, were
168 addressed in the following risk assessment.
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169 According to a 2010 report by the Codex Alimentarius Commission, studies on
170 the food safety aspects of edible insects remain limited. As insects are rich in nutrients,
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171 they can create the perfect conditions for the survival and/or growth of pathogenic
172 microorganisms. However, food processing such as heat treatment as well as proper
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173 storage may eliminate these risks (van der Spiegel, Noordam, & van der Fels-Klerx,
174 2013). Pathogenic bacteria can come into contact with the cricket powder (or powder
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175 from other edible insects) if the powder is prepared in an unhygienic manner (i.e., poor
176 food handling) and in poor sanitary conditions (i.e., lack of basic infrastructure, potable
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177 water, proper storage and preparing food on/with contaminated surfaces/equipment and
178 in areas near latrines, sewers or garbage dumps). If the pathogenic bacteria are capable
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179 of surviving in such conditions and multiplying to sufficient numbers, they can cause
180 severe illness in undernourished children (WHO, 2012). Therefore, a risk assessment
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181 would be extremely beneficial to estimate the human health risks associated with the
182 production, processing and consumption of these foods.
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183
184 1.4 Purpose of the Microbiological Qualitative Risk Assessment
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185 This qualitative risk assessment (QRA) aims to determine the potential
186 microbiological health risks associated with the consumption of cricket powder by
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187 infants and children aged 6-23 months to prevent or treat undernutrition in Siem Reap,
188 Cambodia. It follows the Codex principles and Guidelines for Risk Assessment
189 (FAO/WHO, 2009a) and covers any potential microbiological hazards associated with
190 the consumption of cricket powder by the infants and children, as listed above. Risk
191 management strategies to prevent or decrease any identified health risks are also
192 provided. Moreover, this QRA is based on the ‘Funky Foods’ project by World Vision
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193 Canada (as discussed above), where crickets are raised and processed into a powder in
194 the household.
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196 2 HAZARD IDENTIFICATION AND CHARACTERIZATION
197 While studies on the food safety aspects of insects are limited, the research that is
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198 currently available indicates that insects can harbour numerous hazards including
199 allergens, mycotoxins, pesticides, heavy metals and alkalis, natural toxins, pathogenic
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200 microorganisms such as Staphylococcus spp., Bacillus spp., Campylobacter, Listeria,
201 Pseudomonas, Micrococcus, Acinetobacter, Proteus, Escherichia, spore-forming
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202 bacteria, and fungi such as Aspergillus, Penicillium, Fusarium, Chaetomium, Mucor,
203 Mucorales, Alternaria, Drechslera, and Phoma (Adeduntan, 2005; Amadi, Ogbalu,
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204 Barimalaa, & Pius, 2005; Banjo, Lawal, & Adeyemi, 2006; Banjo, Lawal, Fasunwon, &
205 Alimi, 2010; Braide et al., 2011; Freye, 1996; Garofalo et al., 2017; Green, Broome,
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206 Heinze, & Johnston, 2001; Klunder, Wolkers-Rooijackers, Korpela, & Nout, 2012;
207 Nishimune, Wantanabe, Okazaki, & Akai, 2000; Saeed, Dagga, & Saraf, 1993;
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208 Simpanya, Allotey, & Mpuchane, 2000; van der Spiegel et al., 2013). For the purposes
209 of this paper and taking into account all the potential microbial and microbial related
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210 toxic hazards associated with cricket powder, and Opinions of EFSA Scientific
211 Committee (2015), the French Agency for Food, Environmental and Occupational
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212 Health & Safety (2015) and The Scientific Committee of the Federal Agency for the
213 Safety of the Food Chain (2014), the following major foodborne pathogens were
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214 considered for this microbiological qualitative risk assessment: Bacillus cereus,
215 Escherichia coli, Campylobacter spp., Clostridium perfringens, Cronobacter sakazakii,
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216 Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. The subsequent
217 subsections identify and characterize these major pathogenic hazards, which will be used
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218 in conjunction with an exposure assessment to inform a QRA.

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220 2.1 Bacillus cereus
221 Bacillus cereus is a Gram-positive, rod shaped, spore-forming, motile, facultative
222 anaerobe that is ubiquitous in nature, commonly found in soil and associated with
223 improper food handling/storage and improper cooling of cooked foodstuffs (EFSA,
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224 2005a; FSANZ, 2013; McCabe-Sellers & Beattie, 2004; Vilas-Bôas, Peruca, & Arantes,
225 2007). It has the ability to form endospores that are heat-tolerant and can survive
226 exposure to severe conditions such as radiation, chemicals and desiccation (EFSA
227 BIOHAZ Panel, 2016). Foods that are processed by drying or heating also can contain B.
228 cereus spores (Notermans & Batt, 1998).
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229 Bacillus cereus is an opportunistic pathogen and causes two types of foodborne
230 illness: (1) emetic (vomiting) foodborne intoxication and (2) diarrheal foodborne
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231 infection (EFSA, 2005a; FSANZ, 2013; Notermans & Batt, 1998). Emetic foodborne
232 intoxication is caused by the ingestion of a ring-formed peptide, heat-stable toxin named
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233 cereulide. This toxin is produced when B. cereus contaminated food is mishandled,
234 leading to the formation of toxins, that results in nausea, vomiting and abdominal
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235 cramping usually within 1 to 5 h after ingestion (EFSA, 2005a; FSANZ, 2013;
236 Notermans & Batt, 1998). Diarrheal foodborne infection is caused by the ingestion of
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237 food contaminated with B. cereus cells or spores. The spores can germinate at favourable
238 conditions in the food and produce vegetative Bacillus cells. The diarrheal infection is
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239 characterized by abdominal pain and watery diarrhea, usually occurs within 8 to 16 h
240 after ingestion of contaminated food (EFSA, 2005a; Notermans & Batt, 1998).
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241 Generally, adults under the age of 19 years and the elderly above the age of 60 years
242 develop more severe symptoms including bloody diarrhea, which can lead to fatalities in
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243 some cases (EFSA, 2005a).

244 Since B. cereus is found in soil, starchy foods such as rice, potatoes, peas, beans
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245 and spices are common sources of the organism. Foods that have been associated with
246 the diarrheal syndrome include meat, milk, vegetables, herbs and spices, fish, and cake.
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247 While foods associated with the emetic syndrome include rice, and rice products, potato,
248 pasta, semolina, cheese, spaghetti with tomato sauce, pasta salad, asparagus sauce, lamb,
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249 and ready-to-eat (RTE) foods (FSANZ, 2013; Glasset et al., 2016).
250 To control B. cereus, it is imperative that foods and leftovers be refrigerated at ≤
251 4ºC. A combination of high pressure and high temperature can also be used to inactivate
252 resistant bacterial spores that cause diarrheal infection (EFSA BIOHAZ Panel, 2016).
253 However, for the case of emetic foodborne intoxication, currently no known control
254 method to inactivate cereulide toxins is available (EFSA BIOHAZ Panel, 2016).
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255
256 2.1.1 Dose-response Assessment
257 Currently, no known human dose response curve is available for the emetic toxin
258 or diarrheal infection. Recently, EFSA BIOHAZ Panel (2016) and FSANZ (2013)
259 reported that most cases of the emetic and diarrheal illnesses have been associated with
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260 greater than 105 CFU/g of B. cereus in food with some cases ranging to 108 CFU/g for
261 the diarrheal infection. However, for both types of illness, as low as 102 CFU/g
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262 depending on whether the food was heated is sufficient to cause infection (EFSA
263 BIOHAZ Panel, 2016). In addition, cases of illness have occurred with the ingestion of
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264 between 103 and 105 CFU/g (EFSA, 2005a; EFSA BIOHAZ Panel, 2016; FSANZ, 2013;
265 Granum & Lund, 2006; Ripabelli, McLauchlin, Mithani, & Threlfall, 2000).
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267 2.1.2 Foodborne Outbreaks
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268 Overall, 413 foodborne outbreaks of B. cereus was reported in the EU and non-
269 Member States from 2007 to 2014 with 6,657 human cases, 352 hospitalizations and no
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270 deaths (EFSA BIOHAZ Panel, 2016). In contrast, during a similar time period, only two
271 B. cereus outbreaks were reported in Canada from 2008 to 2014, with 24 human cases
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272 and no hospitalizations (Belanger, Tanguay, Hamel, & Phypers, 2015).

273 In Canada, USA, Europe and Russia, B. cereus, Salmonella spp. and C.
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274 perfringens were the three main pathogens commonly associated with foodborne
275 outbreaks in herbs and spices (Parto, 2015). Since herbs and spices have similar
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276 harvesting and processing methods to cricket powder and are often used to season edible
277 insects, they will be considered in this risk assessment. However, no B. cereus outbreaks
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278 associated with cricket powder have been reported to-date, despite this organism being
279 isolated from other species of edible insects (Banjo, Lawal, & Adeyemi, 2006;
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280 Grabowski & Klein, 2016), such as canned silkworms (6.0 log CFU/g), frozen
281 silkworms (3.0 log CFU/g), black soldier fly (>4.0 log CFU/g) (Grabowski & Klein,
282 2017).
283
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284 2.2 Clostridium perfringens

285 Clostridium perfringens is a Gram-positive, rod shaped, spore-forming,
286 anaerobic, aerotolerant bacterium that is found in the environment and in the intestine of
287 humans and animals (Albrecht, 2002; Brynestad & Granum, 2002; FDA, 2012; Petit,
288 Gibert, & Popoff, 1999). It is ubiquitous in nature and has been isolated from
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289 environmental sources such as water, soil, sewage, dust; as well as foods such as raw
290 meat, beef, poultry, gravies, dried or pre-cooked food and spices (Brynestad & Granum,
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291 2002; García & Heredia, 2011). It forms heat-resistant endospores and such spores have
292 been shown to persist in soil, sediments and in areas subjected to fecal pollution from
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293 both humans and animals (FDA, 2012).
294 Generally, C. perfringens can survive high temperatures with growth occurring
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295 between 6-60°C; however growth occurs rapidly in less than 10 min at optimal
296 temperatures ranging from 37°C to 47°C (Albrecht, 2002; Brynestad & Granum, 2002;
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297 García & Heredia, 2011; Lindström, Heikinheimo, Lahti, & Korkeala, 2011). For this
298 reason, foods that are prepared in large quantities and/or kept warm for long periods of
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299 time before being served are prone to becoming contaminated with C. perfringens. In
300 addition, cooked food must be reheated prior to consumption to ensure that any live cells
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301 of C. perfringens present in the food are killed (Albrecht, 2002; García & Heredia,
302 2011).
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303 Generally, C. perfringens is grouped into 5 types (Types A, B, C, D, E)

304 according to the exotoxins, also known as soluble antigens, that it produces (Oakley &
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305 Warrack, 1953 as cited by EFSA 2005b). Types A, C and D are considered to be human
306 pathogens, while all of the 5 types are considered to be animal pathogens (EFSA,
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307 2005b). However, it is important to know that not all C. perfringens are capable of
308 producing the enterotoxin that causes foodborne diseases. Type A, also known as Type
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309 A diarrhea, and Type C, known as Type C human necrotic enteritis, are considered the 2
310 predominant causes of foodborne illness (EFSA, 2005b). Type A is the more common
311 form of the illness, while Type C is the serious, but rare condition (Brynestad &
312 Granum, 2002).
313 Type A diarrhea is caused by the formation of spores and an enterotoxin (which
314 is heat liable), denoted as C. perfringens enterotoxin or CPE, that is produced in the
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315 large intestine after ingesting a large number of bacterial cells (~107 per g) in
316 contaminated food (Brynestad & Granum, 2002; EFSA, 2005b).
317 Type C enteritis is caused by the production of three toxins, β-toxin, δ-toxin and
318 θ-toxin, during the growth of C. perfringens (Brynestad & Granum, 2002). Primarily, C.
319 perfringens Type C affects children with severe protein malnutrition and a diet rich in
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320 trypsin inhibitors, resulting in high mortality rates (ICMSF, 2002).
321
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323 The dose response is currently unknown, however, ingestion of ≥108 viable
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324 vegetative C. perfringens cells is sufficient to cause illness (EFSA, 2005b; Pathogen
325 Regulation Directorate and Public Health Agency of Canada, 2011). This presumptive
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326 dose implies that the vegetative cells survive the acidic conditions of the stomach and
327 subsequently form spores in the large intestine and produce enterotoxin (EFSA, 2005b).
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328 Moreover, in 2013, an outbreak of CPE was reported in Belgium in which stew leftovers
329 containing up to 6 log CFU/g of CPE was the implicated source that caused 70 cases of
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330 human illness (EFSA & ECDC, 2015a). Additionally, since the organism is highly
331 prevalent in or on most foods, EFSA (2005b) stated that most microbiological tests are
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332 of limited value. As such, it is only those results where very high numbers of the
333 organism are detected in the sample that are considered to be of value. For these reasons,
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334 it is suggestive that the dose response is quite high.

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337 Overall, the number of foodborne outbreaks associated with C. perfringens in the
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338 EU, USA and Canada are varied. In 2014, the EU reported 124 foodborne outbreaks
339 caused by C. perfringens, resulting in greater than 3000 cases of illness, over 60
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340 hospitalizations and 3 deaths (EFSA & ECDC, 2015b). This is compared to 289
341 confirmed outbreaks of C. perfringens in the USA from 1998-2010, which resulted in
342 approximately 15,208 cases of illness, 83 hospitalizations and 8 deaths (Grass, Gould, &
343 Mahon, 2013). In Canada, from 2008-2014, only 3 confirmed foodborne outbreaks were
344 reported with 111 cases of illness with zero hospitalizations and zero deaths (Belanger,
345 Tanguay, Hamel, & Phypers, 2015). The implicated sources for the majority of these
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346 outbreaks has been beef or “bovine meat and products thereof”, “other mixed red meat
347 and products thereof”, poultry and pork, suggesting that protein-rich foods are the main
348 substrate for growth of this organism (EFSA, 2005b; EFSA & ECDC, 2015b; Grass,
349 Gould, & Mahon, 2013). However, as mentioned previously, C. perfringens has also
350 been linked to foodborne outbreaks in herbs and spices in the EU, as spores of the
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351 organism are capable of surviving in dry environments, which is characteristic of dried
352 herbs and spices (Parto, 2015). To-date, no outbreaks associated with C. perfringens in
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353 cricket power have been reported despite being isolated from cricket powder, albeit at
354 low levels <2.0 log CFU/g (Garofalo et al., 2017). This confirms the reported high
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355 infectious dose discussed above.
356
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357 2.3 Cronobacter sakazakii
358 Cronobacter spp., previously known as Enterobacter sakazakii, are a group of
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359 Gram-negative, rod-shaped, non-spore forming, motile, facultative anaerobe which
360 belong to the family Enterobacteriaceae (Healy, Cooney, Brien, Iversen, & Whyte, 2010;
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361 Norberg et al., 2012). The genus consists of six species and while the natural habitat of
362 the organism is not yet known, it has been isolated from several environmental sources
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363 such as wastewater, soil, water, household dust, rodents, and flies; and from numerous
364 foods including milk, cheese, pork, poultry, beef, fish, vegetables, rice, bread, fresh
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365 herbs and dried foods including spices, herbs, nuts and seeds, cereals, powdered infant
366 formula (PIF), skimmed milk powder, herbal tea and starches (Baumgartner, Grand,
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367 Liniger, & Iversen, 2009; CDC, 2015a; Friedemann, 2007; Gurtler, Kornacki, &
368 Beuchat, 2005; Healy, Cooney, Brien, Iversen, & Whyte, 2010; Norberg et al., 2012;
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369 Pava-Ripoll, Pearson, Miller, & Ziobro, 2012).

370 Cronobacter spp. are opportunistic pathogens that can cause life-threatening
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371 disease, particularly in infants less than one year of age (Farber, 2004; Healy, Cooney,
372 Brien, Iversen, & Whyte, 2010; Pagotto & Farber, 2009). Although infections by
373 Cronobacter are rare, they are associated with a high case fatality rate of 40 to 80% in
374 neonates, low-birth-weight and immunocompromised infants (Norberg et al., 2012).
375 Most commonly, PIF contaminated during processing or preparation has been implicated
376 in Cronobacter infections, especially if plant-derived ingredients were added without a
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377 prior heat treatment step (Healy, Cooney, Brien, Iversen, & Whyte, 2010). The fact that
378 it is a human pathogen that poses the greatest risk to infants and neonates, and has been
379 isolated from PIF which is of similar consistency to cricket powder, and from edible
380 mealworms (Vandeweyer, Crauwels, Lievens, & Van Campenhout, 2017) and flies
381 (Pava-Ripoll, Pearson, Miller, & Ziobro, 2012) several of which are considered edible
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382 by Jongema (2017), makes Cronobacter of relevance for this risk assessment.
383 Since 1958, approximately 120 cases of Cronobacter infections in infants have
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384 been reported, with estimates of approximately 6 cases occurring worldwide, each year
385 (FDA, 2012; Healy, Cooney, Brien, Iversen, & Whyte, 2010). However, this number is
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386 likely an underestimate as a result of underreporting from a lack of awareness or lack of
387 resources to test for Cronobacter spp. (Farber, 2004; Yan et al., 2012). Additionally, the
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388 CDC (2015) estimates that infections among older adults are more common and milder
389 than the 4-6 infections reported in infants per year.
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390
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392 The dose response for Cronobacter has not yet been determined, although doses
393 ranging from 103 to 104 CFU/g have been discussed (FAO/WHO, 2004a; Gurtler,
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394 Kornacki, & Beuchat, 2005; Iversen & Forsythe, 2004). However, studies performed on
395 mice suggest that an unusually high number of cells (108 CFU/mouse) may be required
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396 to cause infection in infants (Gurtler, Kornacki, & Beuchat, 2005).

397
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399 While outbreaks of C. sakazakii are rare, the majority of outbreaks reported in
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400 the literature to-date, have been associated with PIF from several countries including
401 Canada (3 cases), USA (24 cases), Europe (36 cases), Israel (5 cases), New Zealand (7
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402 cases) and India (2 cases) all in infants and neonates (Baumbach, Grand, Liniger, &
403 Iversen, 2009; Bowen & Braden, 2006; Caubilla-Barron et al., 2007; CDC, 2002;
404 Norberg et al., 2012; Pagotto & Farber, 2009; Ray et al., 2007). Moreover, despite being
405 isolated from edible insects as previously mentioned, no outbreaks have been reported in
406 cricket powder.
407
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408 2.4 Escherichia coli

409 Escherichia coli is a Gram-negative, rod-shaped, non-spore forming, motile
410 facultative anaerobe that belongs to the family Enterobacteriaceae (Croxen et al., 2013;
411 Farrokh et al., 2013; FDA, 2012). Normally, most E. coli are commensal (i.e., non-
412 pathogenic) and are part of the normal intestinal flora of animals and humans that act to
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413 prevent the colonization of harmful pathogens in the gut. The bacterium is also often
414 used as an indicator of fecal contamination and gaps in hygiene (Farrokh et al., 2013;
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415 FDA, 2012). However, there are six groups of E. coli that are pathogenic: (1)
416 enterohemorrhagic E.coli (EHEC) more commonly referred to as either Shiga toxin-
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417 producing E. coli (STEC) or Verocytotoxin-producing E.coli (VTEC); (2)
418 enteropathogenic E. coli (EPEC); (3) enterotoxigenic E. coli (ETEC); (4)
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419 enteroaggregative E. coli (EAEC); (5) enteroinvasive E. coli (EIEC); and (6) diffusely
420 adherent E. coli (DAEC) (Croxen et al., 2013; Farrokh et al., 2013; FDA, 2012).
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421 The risk with E. coli contamination in food or water can occur if the strains are
422 pathogenic. These strains can cause a wide range of illnesses such as diarrhea (often
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423 bloody), urinary tract infections, respiratory illness and pneumonia and hemolytic
424 uremic syndrome (HUS) (Croxen et al., 2013; Behravesh, Williams, & Tauxe, 2012).
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425 Symptoms can include stomach cramps, diarrhea (usually bloody), vomiting, low-grade
426 fever, nausea, dehydration (Behravesh, Williams, & Tauxe, 2012; PHAC, 2011) and the
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427 infection normally lasts for about 5-7 days after an incubation period of 2-48 h (Hodges
428 & Gill, 2010; PHAC, 2017a). Although the illness is self-limiting, complications such as
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429 HUS can develop in around 5% of cases, with children most commonly affected. The
430 mortality rate in children is ~5% depending on the type of strain (Baker, Rubinelli, Park,
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431 Carbonero, & Ricke, 2016).

432
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434 In general, the dose response varies depending on the pathogenic group of E.
435 coli. For ETEC, the dose response ranges from 107-1010 (PHAC, 2011), whereas for
436 DAEC, EPEC and EAEC it ranges from 105-1010, 106-109 and 108-1010, respectively
437 (Kothary & Babu, 2001). In contrast, for foodborne E. coli O157:H7 outbreaks, the dose
438 response ranges from 10-100 cells (FSANZ, 2013; Schmid-Hempel & Frank, 2007).
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439
441 Overall, from 2008-2014, the USA reported 226 foodborne outbreaks attributed
442 to E. coli with approximately 3,757 cases of illness, 826 hospitalizations and 16 deaths
443 (CDC, 2016a). Moreover, the CDC (2012) and Scallan et al. (2011) estimate that STEC
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444 and non-STEC cause an average of 265,000 illnesses each year, taking into
445 consideration estimates of under-reporting, in the USA with more than 3,600
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446 hospitalizations and 30 deaths. This is compared to approximately 17 confirmed
447 foodborne outbreaks of VTEC/STEC reported in Canada from 2008 to 2014, which
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448 caused 201 cases, 84 hospitalizations and 3 deaths (Belanger, Tanguay, Hamel, &
449 Phypers, 2015). The Public Health Agency of Canada (PHAC, 2017a) estimates about
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450 474 cases of VTEC/STEC infections are reported in Canada each year. Between 2009
451 and 2015, the EU reported 51 pathogenic E. coli related foodborne outbreaks, which
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452 resulted in 5,062 cases of illness, 2,625 hospitalizations and 54 deaths (EFSA, 2017). In
453 addition, foodborne illness caused by ETEC is one of the main causes of diarrheal-
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454 related human disease and it is estimated that there are more than 650 million cases of
455 ETEC infection worldwide each year (Johnson & Nolan, 2009).
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456 Varying strains of EHEC have been implicated in numerous outbreaks

457 worldwide. For example E. coli O157:H7 has been linked to outbreaks in hazelnuts,
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458 walnuts, ground beef, dry-cured salami, Mettwurst, unpasteurized apple juice and cider,
459 radish sprouts and alfalfa sprouts, raw and pasteurized milk, cheese, beef jerky,
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460 fermented sausage, flour and a Mexican wheat snack (Farrokh et al., 2013; Parto, 2015).
461 Other outbreaks involving E. coli O121 and O26 have been associated with flour, and in
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462 a mixed food at a popular Mexican grill restaurant (CDC, 2016b; PHAC, 2017b).
463 Although generic E. coli has been isolated from other edible insects such as
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464 grasshoppers (1.72 log10 CFU/g E.coli in fried grasshoppers and 5.32 log10 CFU/g
465 E.coli in fresh grasshoppers) (Ali, Mohamadou, Siadou, Aoudou, & Tchiegang, 2010)
466 and caterpillars (Braide et al., 2011), so far it has not been isolated from crickets to date
467 and no E. coli foodborne outbreaks have been reported from consuming crickets or
468 cricket powder.
469
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470 2.5 Listeria spp.

471 Listeria spp. are Gram-positive, rod-shaped, non-spore forming, motile,
472 facultative anaerobes that are part of the Listeriaceae family (FDA, 2012; Holt, Krieg,
473 Sneath, Staley, & Williams, 1994). Currently, the genus consists of 17 recognized
474 species (Orsi and Wiedmann, 2016). Of these 17 species, L. monocytogenes is
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475 considered to be the major human pathogen while L. ivanovii is considered to be an
476 animal pathogen, mainly affecting ruminants (FDA, 2012; FSANZ, 2013; Orsi &
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477 Wiedmann, 2016; Todd & Notermans, 2011). Since L. monocytogenes is the major
478 human pathogen, the risk assessment that follows will only focus on this species of
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479 Listeria, in addition to the other foodborne pathogens listed in Section 2.
480 L. monocytogenes is widespread in nature having been found in moist
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481 environments, soil, decaying vegetation, domestic and wild birds, fodder, water, grass,
482 meadows, forests, silage, and on farms and in food-manufacturing environments on
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483 floors, drains and wet areas (FDA, 2012; FSANZ, 2013; Todd & Notermans, 2011).
484 L. monocytogenes is only composed of 13 different serotypes, with serotypes 4b,
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485 1/2a and 1/2b causing the majority of worldwide infections. Moreover, the FDA (2012)
486 considers L. monocytogenes to be the leading cause of death from foodborne illness,
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487 despite the fact that the number of people who are infected by the pathogen is small (as
488 discussed below). Infection of humans by L. monocytogenes can cause one of two forms
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489 of human listeriosis: (1) a non-invasive gastrointestinal illness and (2) an invasive
490 gastrointestinal illness, which causes septicaemia and meningitis. The former is often
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491 self-limiting in healthy populations while the latter is associated with higher risk
492 populations such as the very young, the elderly, and immunocompromised individuals
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493 (FDA, 2012; FSANZ, 2013). Pregnant women are also at risk as the pathogen might
494 only cause flu-like symptoms to the mother, but can cause miscarriages or stillbirth of
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495 the fetus, or may cause the baby if born alive to contract bacteremia and meningitis
496 (FDA, 2012; FSANZ, 2013).
497
498
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500 Currently, the dose response of L. monocytogenes is not yet known; however,
501 studies have suggested that it varies depending on the serotype, susceptibility of the host,
502 and food matrix (FDA, 2012). The FDA (2012) reported that in pasteurized milk, <1000
503 cells were needed to cause disease in susceptible populations, whereas high levels of
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504 approximately 105 or 109 are required to cause illness in healthy individuals (Sim et al.,
505 2002). Moreover, several studies provided estimates of developing listeriosis from a
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506 single cell. The FAO/WHO (2004b) and the FDA, FSIS & USDA (2003) stated that the
507 probability of a healthy individual developing listeriosis from a single L. monocytogenes
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508 cell was 2.37 x 10-14, whereas for a susceptible population it was 1.06 x 10-12. On the
509 other hand, Chen et al. (2006) accounted for the different L. monocytogenes serotypes
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510 and determined that the probability of a susceptible individual developing invasive
511 listeriosis was 1.31 x 10-8 to 5.01 x 10-11.
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512
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514 Between 2008 and 2014, the USA reported 40 listeriosis outbreaks resulting in
515 446 cases of illness, 389 hospitalizations and 82 deaths (CDC, 2016a). In contrast,
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516 during the same time period, Canada reported only 3 L. monocytogenes foodborne
517 outbreaks, which caused 67 cases of illness, 9 hospitalizations and 22 deaths (Belanger,
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518 Tanguay, Hamel, & Phypers, 2015). The EU, during 2009-2015 reported 34 L.
519 monocytogenes outbreaks, which resulted in 471 cases of illness, 173 hospitalizations
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520 and 32 deaths (EFSA, 2017).

521 New Zealand, Canada, and the USA, have reported outbreaks of L.
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522 monocytogenes in salami, PIF, cantaloupe, raw milk, inadequately pasteurized milk,
523 chocolate, soft cheeses, cheese, ice cream, raw vegetables, raw poultry and meats,
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524 fermented raw-meat sausages, hot dogs, deli meats, sliced turkey, raw and smoked fish
525 and seafood, frozen vegetables, packaged salads, bean sprouts, and prepackaged caramel
526 apples (CDC, 2016a; FDA, 2012; Parto, 2015). Moreover, no foodborne outbreaks of L.
527 monocytogenes or Listeria spp. have been reported from the consumption of cricket
528 powder despite being detected (in low amounts) in the microbiota of powdered crickets
529 and dried mealworm larvae (Garofalo et al., 2017).
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530
531 2.6 Salmonella spp.
532 Salmonella spp. are Gram-negative, rod-shaped, non-spore forming, motile,
533 facultative anaerobes belonging to the family Enterobacteriaceae (Holt, Krieg, Sneath,
534 Staley, & Williams, 1994). The genus is grouped into two species: Salmonella enterica
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535 and Salmonella bongori and in total comprises of over 2500 different serovars (Grimont
536 & Weill, 2008). S. enterica subspecies I, which includes Salmonella Enteritidis,
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537 Salmonella Typhimurium, Salmonella Newport, Salmonella Javiana, and Salmonella
538 Heidelberg, cause most of the observed human illnesses (FSIS, 2010; Pui et al., 2011).
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539 Salmonella spp. are ubiquitous and found in the intestine of many animal species
540 (cattle, swine, poultry, and wild birds) and humans. They can contaminate and survive in
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541 a variety of foods with poultry, eggs, dairy products, and fresh produce being the most
542 common.
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543 A Salmonella infection can cause varying degrees of illness: (a) gastroenteritis,
544 which is in most cases self-limiting, (b) septicaemia, a systemic infection requiring
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545 hospitalization and antimicrobial treatment, (c) enteric fevers (caused by typhoidal
546 Salmonella), and (d) in rare cases, reactive arthritis. Globally, there are about 1.3 billion
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547 cases of salmonellosis every year, with a mortality rate of less than 1% in developed
548 countries and about 24% in developing countries (PHAC, 2010). Children under the age
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549 of 5 are at the highest risk of acquiring Salmonella infection, and younger children, older
550 adults and those with weakened immune systems are at the highest risk of developing
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551 severe symptoms (PHAC, 2010). The serovars involved in salmonellosis have the ability
552 to invade cells, replicate intracellularly and cause disease after they penetrate the
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553 intestinal mucosa (Giannella, 1996).

554
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556 The dose response for Salmonella varies with the serotype, immune status of the
557 patient and the level of acidity in their stomach. For gastroenteritis, the dose response is
558 approximately 103 cells (PHAC, 2010), however, this can vary greatly depending on the
559 susceptibility of the patients, serotype, and food source. For example, levels as low as
560 one cell have caused foodborne outbreaks in cheddar cheese (D’Aoust, 1985). Bollaerts
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561 et al. (2008) used modified fractional polynomial models to estimate the dose response
562 for illness from outbreak data. These models indicated that the dose response depended
563 on the combination of food-pathogen matrix for susceptible populations. In addition, in
564 the FAO/WHO risk assessment of Salmonella in eggs and broiler chickens, several
565 approaches and models to characterize the dose-response relationship for Salmonella
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566 were presented. These included: i) the beta-Poisson model fitted to the human feeding
567 trial data for Salmonella (Fazil, 1996 as cited by FAO/WHO, 2002); ii) a US model
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568 based on the use of a surrogate pathogen to describe the dose-response relationship; iii) a
569 Weibull dose-response relationship updated to reflect outbreak information using
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570 Bayesian techniques; and iv) a beta-Poisson model fitted to real outbreak data. In
571 addition to these models, the authors explored the effect of fitting the beta-Poisson
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572 model to the human feeding trial data for naive subjects only. The dose-response model
573 derived from outbreak data was considered to be the best available estimate for the
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574 probability of illness upon ingestion of a dose of Salmonella (FAO/WHO, 2002).
575
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577 Globally, Salmonella is considered to be one of the most common and major
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578 causes of foodborne illness in humans (EFSA & ECDC, 2015b). In the EU,
579 salmonellosis is considered the second most commonly reported gastrointestinal
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580 infections after Campylobacter related infections. From 2009 to 2015, the EU reported
581 1,796 foodborne outbreaks related to Salmonella, causing 27,741 cases of illness, 6,109
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582 hospitalizations and 37 deaths (EFSA, 2017). In the USA from 2008-2014, there were
583 920 Salmonella related foodborne outbreaks, which caused 25,537 cases of illness, 3,461
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584 hospitalizations and 33 deaths (CDC, 2016a). This can be compared to Canada, which
585 had 47 Salmonella outbreaks, with 2,041 cases of illness, 106 hospitalizations and 4
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586 deaths from 2008-2014 (Belanger, Tanguay, Hamel, & Phypers, 2015).
587 Salmonella spp. have been implicated in several outbreaks in Canada, USA,
588 Europe, Serbia, Russia, New Zealand and Australia related to low-moisture foods (Finn,
589 Condell, McClure, Amézquita, & Fanning, 2013; Maurer & Lee, 2005; Parto, 2015).
590 Therefore, low moisture foods such as cricket powder can likely support the survival of
591 Salmonella spp. To-date, there have not been any reported salmonellosis outbreaks
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592 linked to the consumption of edible insects, despite Salmonella spp., being isolated from
593 some insect species such as fresh raw grasshoppers (5.48 log10 CFU/g) and fried
594 grasshoppers (1.00 log10 CFU/g) (Ali, Mohamadou, Siadou, Aoudou, & Tchiegang,
595 2010).
596
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597 2.7 Staphylococcus aureus
598 Staphylococcus aureus is a Gram-positive, spherical or cocci-shaped, non-spore
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599 forming, non-motile, facultative anaerobe that is part of the family Staphylococcaceae
600 (Holt, Krieg, Sneath, Staley, & Williams, 1994; Le Loir, Baron, & Gautier, 2003). It is
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601 an opportunistic pathogen that is ubiquitous in nature and is found in air, dust, water,
602 surfaces, and in the natural flora of skin and mucous membranes of humans and animals
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603 (Argudín, Mendoza, & Rodicio, 2010; Hennekinne, De Buyser, & Dragacci, 2012; Le
604 Loir, Baron, & Gautier, 2003). It can grow between 7 and 48°C, with the optimum being
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605 37°C and at pH values between 4 and 10, with the optimum being pH 6 to 7
606 (Hennekinne, De Buyser, & Dragacci, 2012). Additionally, it can grow at an aw of
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607 between 0.83- 0.86 (Hennekinne, De Buyser, & Dragacci, 2012).

608 Although S. aureus can be eliminated from foods by heat treatment, cross-
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609 contamination of the cooked and processed foods from food handlers, or from the
610 environment after the last microbial reduction step, can create conditions for the growth
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611 of S. aureus. This can lead to the formation of staphylococcus enterotoxins (SEs) in the
612 food product, and these toxins are resistant to heat treatment, freezing, drying and low
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613 pH (Argudín, Mendoza, & Rodicio, 2010; Hennekinne, De Buyser, & Dragacci, 2012).
614 Ingestion of food contaminated with SEs can result in staphylococcal food poisoning,
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615 which is self-limiting and usually resolve within 24-48 h, however, they can be severe in
616 infants, elderly and immunocompromised individuals (Argudín, Mendoza, & Rodicio,
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617 2010; Kadariya, Smith, & Thapaliya, 2014).

618 Staphylococcus aureus can produce various types of enterotoxins in improperly
619 handled food. For the most part, it is only those strains that produce staph enterotoxin A,
620 which are associated with foodborne disease. The organism is very susceptible to
621 inactivation by heat treatment and most sanitizing agents. Therefore, the presence of this
622 bacterium and/or its enterotoxins in processed foods or on food processing equipment is
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623 generally an indication of poor sanitation. The ingestion of contaminated food can cause
624 foodborne disease after a short 1-6 h incubation time and can lead to hyper-salivation,
625 nausea, vomiting and abdominal cramping with or without diarrhea (Argudín, Mendoza,
626 & Rodicio, 2010; Kadariya, Smith, & Thapaliya, 2014; Le Loir, Baron, & Gautier,
627 2003). However, the incubation time and severity of symptoms depends on the amount
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628 of enterotoxins and the susceptibility of each individual (Hennekinne, De Buyser, &
629 Dragacci, 2012).
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630
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632 The dose response of staphylococcal enterotoxin (SE) is reported differently in
633 various studies. Some authors suggest that 0.5 µg to < 1.0 µg of SE is enough to cause
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634 staphylococcal food poisoning, while others suggest that 10-20 µg of SEs is required to
635 cause disease (FDA, 2012; Hennekinne, De Buyser, & Dragacci, 2012; Kadariya, Smith,
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636 & Thapaliya, 2014). However, approximately 105 to 108 CFU/g of S. aureus needed to
637 produce doses of SE that cause illness in humans (Seo and Bohach, 2007 and Montville
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638 and Matthews, 2008 as cited by FSANZ, 2013).

639
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641 In the EU from 2009 to 2015, there were 270 foodborne outbreaks related to
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642 Staphylococcus enterotoxins, which resulted in 4,649 cases of illness, 662

643 hospitalizations and 0 deaths (EFSA, 2017). In terms of just Staphylococcus outbreaks,
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644 the EU during the same time period reported 73 outbreaks, with 698 cases of illness, 161
645 hospitalizations and 2 deaths (EFSA, 2017). On the other hand, from 2008 to 2014, the
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646 USA reported 102 Staphylococcus foodborne outbreaks, which caused 2,376 cases of
647 illness, 115 hospitalizations and 1 death (CDC, 2016a). This was compared to Canada
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648 which, from 2008 to 2014, reported 2 Staphylococcus outbreaks that caused cases of 23
649 illness, 1 hospitalization and 0 deaths (Belanger, Tanguay, Hamel, & Phypers, 2015).
650 Like Salmonella, S. aureus is capable of surviving in low moisture foods, and dry
651 environments as numerous countries including Canada, USA, Europe, and Australia
652 have reported outbreaks of S. aureus in chocolate, rice, grains, diet supplements, milk
653 and milk products such milk powder, cheddar cheese, pasta, cereal, rice noodles, fried
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654 rice, jam, salads, meat and meat products, bakery products and cakes (Argudín,
655 Mendoza, & Rodicio, 2010; Bennett, Walsh, & Gould, 2013; D’Aoust, 1985; Kadariya,
656 Smith, & Thapaliya, 2014; Parto, 2015). Moreover, some researchers have isolated S.
657 aureus from edible insects such as larvae of rhinoceros beetles (Banjo, Lawal, &
658 Adeyemi, 2006) and from monarchs of the Niger Delta (Amadi et al., 2005), and
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659 Staphylococcus spp. from caterpillars of emperor moths (Braide et al., 2011), from dried
660 Mediterranean field crickets (Grabowski & Klein, 2016), and the microbiota (in low
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661 amounts) of dried and powdered crickets, dried locusts and dried mealworm larvae
662 (Garofalo et al., 2017). However, currently, no foodborne outbreaks have been reported.
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663
664 3 EXPOSURE ASSESSMENT
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665 3.1 Potential Modes of Contamination
666 Contamination of edible crickets can occur during any step in the food chain.
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667 These steps include rearing, harvesting, washing, drying/roasting, grinding, storage, and
668 during preparation of the porridge itself. Moreover, soil is an important source of
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669 contamination for bacterial endospores, especially when insects are farmed in containers
670 containing soil (Banjo, Lawal, & Adeyemi, 2006; Klunder, Wolkers-Rooijackers,
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671 Korpela, & Nout, 2012). Although the crickets that are being considered for this risk
672 assessment are not reared in soil, they are reared outdoors, underneath the house. In fact,
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673 most Cambodian houses are raised with the kitchen being positioned outdoors below the
674 house to control for heat (Personal Communication, World Vision Canada, 2017).
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675 Therefore, there is a potential that crickets can become contaminated with soil during
676 their harvesting or from soil particles in the air (Baumgardner, 2012). Aside from soil,
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677 crickets and other edible insects can also be exposed to microbial hazards from dust
678 (EFSA, 2015). Likewise, the feed used for rearing, the water used for washing them, and
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679 the environmental conditions during drying and grinding can also expose crickets to
680 pathogens. Additionally, contact surfaces used for harvesting, drying/roasting, grinding
681 of the cricket powder, and preparation of porridge or storage can act as potential sources
682 of contamination. Similarly, adding the powder into products of high aw can increase the
683 risk of growth of pathogenic microorganisms, such as in the case of adding cricket
684 powder to porridge (Schaarschmidt et al., 2016).
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685 Studies examining the incidence and/or levels of B. cereus, C. perfringens,

686 Cronobacter spp., E. coli, L. monocytogenes, Salmonella spp. and S. aureus in cricket
687 powder have not been reported. Moreover, only a handful of studies have examined the
688 microbial quality of other edible insects (Ali, Mohamadou, Siadou, Aoudou, &
689 Tchiegang, 2010; Amadi, Ogbalu, Barimalla, & Pius, 2005; Banjo, Lawal, & Adeyemi,
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690 2006; Braide et al., 2011; EFSA, 2015; Garofalo et al., 2017; Grabowski & Klein, 2017,
691 2016); and even fewer have assessed the overall microbial safety of edible insects
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692 (Vromman, Thiry, Herman, & Huffel, 2015). The hazards associated with edible insects
693 depend on the type of insect, rearing environment and feed, and final processing (raw,
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694 roasted, dried or deep-fried). Although insects are taxonomically distant from traditional
695 sources of animal protein (i.e., pork, beef, poultry, venison, lamb, etc.) and the risk
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696 associated with them is low, contamination can occur from waste, unhygienic treatment
697 and food handling (EFSA, 2015).
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698
699 3.2 Likelihood of Growth
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700 The microflora of insects, as discussed in section 2, can pose a health risk to
701 consumers. Several studies have isolated Enterobacteriaceae and pathogenic organisms
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702 from different types of edible insects such as locusts, mealworms, raw silk worms and
703 crickets (Banjo, Lawal, & Adeyemi, 2006; EFSA, 2015; Grabowski and Klein, 2016;
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704 Klunder, Wolkers-Rooijackers, Korpela, & Nout, 2012).

705 For example, Klunder, Wolkers-Rooijackers, Korpela, & Nout (2012) examined
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706 the microbial composition of mealworms and crickets that were either fresh or processed
707 (boiled, roasted) and were able to isolate Enterobacteriaceae and spore-forming bacteria
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708 from fresh insects and spore-formers from boiled insects. They found that roasting was
709 not enough to inactivate the Enterobacteriaceae (Klunder, Wolkers-Rooijackers,
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710 Korpela, & Nout, 2012). Banjo, Lawal, & Adeyemi (2006) analyzed the gut and surface
711 of the beetle, Oryctes Monoceros, and found S. aureus and B. cereus. They concluded
712 that insects can come into direct contact with soil during drying, and hence can become
713 contaminated with B. cereus present in the soil, and by S. aureus from the handling of
714 beetles during processing (Banjo, Lawal, & Adeyemi, 2006). Belluco et al. (2013)
715 reviewed studies examining the microbial quality of different species of insects.
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716 Coliforms and S. aureus were isolated from several insects, however, Salmonella was
717 not. Similarly, Grabowski & Klein (2016) examined dried, powdered, deep-fried and
718 cooked Mediterranean field crickets and superworms. They found that dried and
719 powdered crickets and superworms had a higher microbial load than when fried.
720 Additionally, the powered and dried crickets and superworms were positive for B. cereus
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721 and the Enterobacteriaceae, but negative for Salmonella, E. coli and S. aureus,
722 potentially due to proper hygienic conditions being followed during the processing step
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723 (Grabowski & Klein, 2016).
724 The crickets that are being considered for this risk assessment are sundried
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725 before being ground, which aids in controlling the growth of pathogens. However, many
726 foodborne pathogens (including B. cereus, C. perfringens, Cronobacter and Salmonella)
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727 have the ability to survive in dry foods (aw < 0.83) and have been isolated from dry
728 foods (Beuchat et al., 2013).
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729 Furthermore, insects can act as vectors for pathogens such as Salmonella and E.
730 coli O157:H7 and can transfer these pathogens from contaminated environments to the
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731 consumer. Several challenge studies have shown the transfer of these pathogens from
732 infected livestock to insects. In one such study by Holt, Geden, Moore, & Gast (2007),
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733 Salmonella-free houseflies were released into rooms containing hens challenged with S.
734 Enteritidis and the houseflies rapidly became infected with S. Enteritidis after 48 h.
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735 Following this, the authors introduced these now contaminated flies orally to fasted S.
736 Enteritidis-free hens and monitored for shedding of the organism for 3-weeks. Overall,
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737 the authors noted that one third of the hens showed intestinal colonization of the
738 bacterium and, as such, suggested that contaminated flies might be considered a source
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739 for transmission of Salmonella to a flock (Holt, Geden, Moore, & Gast, 2007). In
740 another study, houseflies challenged with E. coli O157:H7 showed colonization of the
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741 bacteria in their gut, with excretion of the organism occurring for a period of three days
742 post-inoculation (Kobayashi et al., 1999). Therefore, not only can these pathogens
743 colonize the gut of insects, they can also be excreted following colonization. However,
744 such instances are rare, especially if insects are reared and processed in hygienic
745 conditions. Therefore, the main food safety concern with edible insects is during the
746 post-harvest period (Belluco et al., 2013).
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747 The quantitative or qualitative analysis of the microbial reduction in crickets

748 during the processing steps has not been reported. Klunder, Wolkers-Rooijackers,
749 Korpela, & Nout (2012) examined the microbial load (Enterobacteriaceae and spore-
750 forming bacteria) of roasting and boiling, either individually or in combination. They
751 found that boiling eliminated the Enterobacteriaceae, however, it did not eliminate the
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752 spore-forming bacteria. Roasting and boiling of crickets was unable to inactivate spore-
753 forming bacteria, therefore, posing a potential food safety challenge. Grabowski & Klein
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754 (2017) examined the microbiology (Enterobacteriaceae, staphylococci, Bacillus spp.
755 Salmonella, Listeria monocytogenes, E. coli, yeasts and molds) of processed insects such
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756 as deep-fried and spiced, cooked in soy sauce, dried and powdered insects. Overall,
757 Salmonella, E. coli, S. aureus, and L. monocytogenes were not isolated from any of the
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758 samples. They noted that the dried and powdered insects exceeded the recommended
759 bacterial counts as compared to those insects that were cooked or deep-fried. As such,
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760 the authors recommended a final heating step for dried and powdered insects
761 (Grabowski & Klein, 2017).
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762
763 4 RISK CHARACTERIZATION
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764 There is not enough information to produce a quantitative estimate of the true
765 probability of microbial pathogen contamination in cricket powder at the time of
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766 consumption. The information on the identified hazards in cricket powder is obtained
767 from studies of insects of other species in addition to the limited studies of crickets.
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768 Furthermore, there are no quantitative studies that have determined the level of microbial
769 hazards at each processing step in the production of cricket powder. Therefore, the
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770 characterization of risks into categories (see Tables 4 and 5) involves various
771 assumptions and uncertainties that are listed in numbers 1-13 below. These
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772 assumptions/uncertainties also include where contamination is likely to occur.

773 1. Crickets are raised outdoors in a soil-free environment (with paper egg
774 cartons), but where soil may be introduced through the air or unclean hands.
775 This can lead to subsequent contamination if conditions are dry and soil
776 particles are distributed in the air. Moreover, mosquito netting is used to
777 prevent intrusion by ants and other pests, but it does not prevent access to
24
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778 birds and potentially rodents. As such, this is a risk factor for contamination.
779 In addition, rearing of crickets may be done in paper egg cartons, which
780 could pose a risk for contamination with Salmonella and Campylobacter. It
781 is likely that the eggshells come in contact with poultry feces, which can
782 then transfer onto the paper cartons if the eggs are not washed before
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783 packaging.
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784 2. Crickets are fed leaves, vegetable scraps and chicken feed. Feeding crickets
785 with chicken feed or with feed intended for other ruminant/non-ruminant
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786 animals poses another risk, as it is likely that this type of feed can infect
787 crickets with pathogenic organisms such as Salmonella spp. (Crump, Griffin,
788 & Angulo, 2002; Jones, 2011). If the leaves or vegetable scraps are not
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789 handled in a hygienic manner they can also become contaminated, especially
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790 if they are left outside in contact with soil or in unclean containers and then
791 subsequently fed to the crickets without prior washing and heat treatment.
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792 3. Crickets are killed by suffocation instead of boiling for 5 min (ANSES,
793 2014). Suffocation only kills the crickets, and does not inactivate the
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794 potential pathogenic organisms that they may be carrying.

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795 4. Sun drying/roasting helps in reducing the aw and in limiting the survival of
796 pathogens. However, pathogens such as E. coli, Cronobacter spp., and
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797 Salmonella spp., can survive the drying process. Additionally, drying insects
798 in the open can expose them to the spores of B. cereus and C. perfringens
799 that are present in dust and soil.
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AC
800 5. The cricket powder is made at an in-home setting and ground outdoors in a
801 mortar and pestle that was not cleaned between uses. Likewise, grinding the
802 crickets outdoors can potentially expose powder to soil particles in the air
803 (Baumgardner, 2012), especially if the ground is dry and the soil easily
804 disturbed.
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805 6. The porridge is rice-based and processed in a home setting in conditions

806 where contamination is likely to occur.
807 7. Bacillus cereus – The likelihood of occurrence of B. cereus cells and its
808 spores in dried and powdered insects is moderate, occurring regularly, as
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809 seen in the study by Grabowski & Klein (2016, 2017). However, the typical
810 dose required for intoxication by B. cereus is high (i.e., greater than 105
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811 cfu/g). The possibility of B. cereus reaching that level is very low to low, if
812 all the processing steps are correctly followed and the porridge undergoes a
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813 rolling boil for 5 min prior to consumption.
814 8. Clostridium perfringens Type A and Type C – The dose response is likely
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815 higher than 106 cells/g and despite being isolated from cricket powder, the
816 likelihood of occurrence of C. perfringens cells and its spores in cricket
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817 powder is very low to low, as long as the product is cooked thoroughly
818 (rolling boil for 5 min) prior to consumption.
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819 9. Cronobacter sakazakii – Since Cronobacter has been isolated from

mealworms and flies, albeit not from crickets, the likelihood of occurrence
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820
821 of Cronobacter in cricket powder is negligible to very low if boiled for 5
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822 min prior to consumption.
823 10. Enterohemorrhagic E. coli – The likelihood of occurrence of E. coli after

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824 boiling is extremely low. However, if any cells do survive, adverse effects
825 from E. coli infection may occur, especially if E. coli strains of the
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826 STEC/VTEC variety are present, as the dose response is very low at ~10
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827 cells. Moreover, like Salmonella spp., since E. coli has been isolated from
828 edible insects such as grasshoppers and caterpillars, and from the intestinal
829 tracts of animals, one can assume that there might be a low level of
830 contamination in crickets, despite no studies having isolated E.coli from
831 crickets.
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832 11. Listeria monocytogenes – The likelihood of occurrence of L. monocytogenes

833 after boiling is extremely low. However, as is the case with E. coli, if any
834 cells do survive, adverse effects from L. monocytogenes infection may
835 occur.
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836 12. Salmonella spp. – It has been found in other insects such as locusts,
837 grasshoppers and mealworms. Therefore, one can assume that there might be
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838 a low level frequency of contamination in crickets, despite no studies having
839 isolated Salmonella from crickets.
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840 13. Staphylococcus aureus - Based on the amount of contact (i.e., handling time)
841 required for the production of cricket powder and making porridge, one
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842 could assume that the likelihood of occurrence of S. aureus in cricket
843 powder occurs regularly, if the powder is not boiled before consumption and
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844 hands are not washed prior to preparing the porridge. Since high levels of S.
845 aureus (105-108 CFU/g) are needed to cause toxin formation, the risk of this
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846 occurring is low especially if the powder is boiled before consumption.

847 Moreover, given the fact that Staphylococcus spp., has been isolated from
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848 dried and powered crickets, as well as dried locusts and mealworm larvae,
849 one can assume that there is a low level of contamination of the bacterium in
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850 crickets.
851
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852 The risk rankings in the Tables that follow, are qualitative, in that the numbers
853 assigned are based on the probability (P) and impact (I) of illness should microbial
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854 contamination occur. The probability (or likelihood) of occurrence of microbial

855 contamination (Table 1) is magnified by the impact (or severity of consequences) of that
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856 occurrence (Table 2) and results in the risk rating (R) (Table 3).
857 Based on the assumptions and uncertainties listed above and in Tables 1-3, the
858 following is the overall risk of illness from consumption of porridge containing cricket
859 powder that has not been subjected to boiling prior to consumption and is contaminated
860 with the foodborne pathogens listed in Table 4: (i) for B. cereus, C. perfringens Type A,
861 Salmonella spp., and S. aureus, the risk of illness is moderate; (ii) for C. perfringens
27
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862 Type C, C. sakazakii, Enterohemorrhagic E. coli and L. monocytogenes, the risk of

863 illness is serious. However, when considering the health status of the infants and
864 children population in Cambodia, it is not unrealistic to assume that low numbers of B.
865 cereus, C. perfringens Type A, C. perfringens Type C, and S. aureus can cause illness.
866 As such, for these organisms, this risk rating changes from moderate to low to moderate
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867 and from serious to low to moderate.
868 Following the same procedure as above, the following is the overall risk of
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869 illness from consumption of boiled (rolling boil for 5 min) porridge containing cricket
870 powder contaminated with the foodborne pathogens stated in Table 5: (i) for S. aureus,
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871 the risk of illness is low; (ii) for B. cereus, C. perfringens Type A, C. sakazakii,
872 enterohemorrhagic E. coli, L. monocytogenes and Salmonella spp., the risk of illness is
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873 low to moderate; (iii) for C. perfringens Type C, the risk of illness is moderate to
874 serious.
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875
876 5 CONCLUSIONS & RECOMMENDATIONS
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877 The QRA suggests that if porridge fortified with cricket powder is boiled prior to
878 consumption, the food safety risk to undernourished infants and children 6-23 months of
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879 age is low for S. aureus; low to moderate for B. cereus, C. perfringens Type A, C.
880 sakazakii, enterohemorrhagic E. coli, L. monocytogenes and Salmonella spp.; and
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881 moderate to serious risk for C. perfringens Type C. However, if the fortified porridge is
882 not boiled, the food safety risk is increased.
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883 Therefore, to diminish the overall risk, the following recommendations are
884 provided:
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885 (1) It is imperative that crickets are reared in a controlled, strict containment
886 system that limits microbial hazards. Since crickets will be reared outdoors, it
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887 is necessary that access to the crickets by birds and rodents and their
888 subsequent feces be prevented. Simply using mosquito netting will not prevent
889 this.
890 (2) Paper egg cartons used for rearing should be new and not previously used.
891 These paper egg cartons should be replaced when worn or any soiled matter is
892 visible on the cartons.
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893 (3) Crickets should be fed vegetable scraps that are handled and stored in a
894 hygienic manner before feeding. The vegetable scraps should also not come
895 into contact with soil. Moreover, if chicken feed is to be used, it should
896 undergo a final heat treatment step before being fed to the crickets.
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897 (4) Crickets should either be boiled for 5 min following suffocation or slaughtered
898 by boiling for 5 min. Afterwards, these dead crickets should be placed on
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899 trays/racks and dried in a manner such that access to the outside environment
900 is prevented. This will ensure that most pathogenic organisms are killed and
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901 recontamination does not occur (Klunder, Wolkers-Rooijackers, Korpela, &
902 Nout, 2012).
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903 (5) After drying, crickets should be sundried (for approximately 3 days) in a
904 container where exposure to B. cereus, C. perfringens, E.coli, Cronobacter
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905 spp. and Salmonella spp. from the outside environment is prevented from
906 occurring.
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907 (6) Following sun drying, the cricket powder should be processed in a hygienic
manner, ensuring that every effort is made to control contamination and/or
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908
909 cross-contamination. When grinding the crickets, the mortar and pestle should
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910 be thoroughly cleaned and dried before use. Since grinding is done outdoors,
911 one should ensure that the process is conducted in a manner where the powder
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912 does not come into contact with soil or soil particles from the air (this is
913 especially important in dry conditions).
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914 (7) Cricket powder should be stored in separate clean and dried containers that
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915 ensure no added moisture or access to by pests. If rice is used as a drying

916 agent, it must be thoroughly cleaned and heat-treated before being added to
917 the powder. For example, dry rice grains that undergo a simple heat treatment
918 such as dry roasting on a pan is suitable as a drying agent. Glass and plastic
919 containers are preferred over plastic bags, and if cracks are seen in the glass or
920 plastic, then new containers should be used.
29
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921 (8) The fortified porridge with cricket powder should be boiled (a rolling boil for
922 5 min) prior to consumption, and every attempt should be made to prevent
923 recontamination after boiling.
924 (9) Simply consuming cricket powder that is sprinkled on rice porridge should be
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925 avoided. Instead, the cricket powder should be mixed with the rice or
926 vegetable porridge, and then boiled (rolling boil for 5 min) prior to
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927 consumption.
928 (10) When processing cricket powder and porridge all equipment, containers,
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929 utensils and surfaces should be thoroughly cleaned and dried before use. If
930 dishes are dried in the sun and not indoors, one needs to ensure that the dishes
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931 are dried in such a way that they do not become contaminated with soil or
932 animal droppings, etc.
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933 (11) Since hands are the last line of defense in the chain of transmission of
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934 gastrointestinal pathogens from either hand-to-mouth or from food/water

935 handling, it is critical that hands are washed with soap and water throughout to
limit potential contamination with pathogenic bacteria. After hand washing
D
936
937 with soap or ash, hands should be dried not by using clothing (as this can
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938 result in recontamination of hands), but by using clean cloths or clean paper
939 towels (Hoque, Mahalanabis, Pelto, & Alam, 1995; Montville, Chen, &
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940 Schaffner, 2002) or air-dried (CDC, 2015b; Jensen, Schafner, Danyluk, &
941 Harris, 2012; Todd, Michaels, Smith, Greig, & Bartleson, 2010).
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942 (12) Data on the use of ash as an alternative for soap is limited. However,
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943 particularly in poor areas where soap is not available or too expensive to
944 acquire, hand washing with ash and water has been suggested to be better than
945 hand washing with water alone (Anuradha, Yasoda Devi, & Prakash, 1999;
946 Hoque & Briend, 1991; Hoque, Mahalanabis, Pelto, & Alam, 1995;
947 Bloomfield & Nath, 2009). However, it should be noted that ash can contain
948 materials that pose a toxicity risk depending on the source material, i.e., ash
949 can be the resultant product of the burning of coal, wood fibre, dry leaves,
30
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950 fodder, cow dung cakes, waste crops and other kinds of solid waste matters
951 (Bloomfield & Nath, 2009). As such, using non-toxic materials for producing
952 ash and ensuring that it does not become contaminated is essential. For
953 example, in Cambodia, wood and leaves are used to produce ash (Personal
954 Communication, World Vision Canada, 2017).
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955
956 6 ACKNOWLEDGMENTS
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957 We would like to thank Barbara Main and Jungsun Sharon Jo at World Vision
958 Canada very much for providing their excellent support and guidance throughout this
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959 whole project.
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1385 Table 1. Description of the likelihood of illness given the occurrence of microbial contamination
Likelihood or Probability Category Description
PT
Negligible Extremely rare that consideration is not warranted
Very Low Very rare but cannot be excluded
Low Rare but does occur
RI
Medium Occurs regularly
High Occurs very often
Very High Occurs almost certainly
SC
1386 (Source: Monaghan et al., 2017).
1387
U
1388 Table 2. Description of the severity of illness should microbial contamination occur
AN
Severity or Description
Consequence
Category
M
Insignificant No effect on health
Minor No direct effect on health or a minor temporary impact (i.e., feeling ill for a few days without any overt symptoms), self-limiting
D
Moderate Minor to moderate impact on health (e.g., nausea, diarrhea, vomiting, mild stomach cramps), usually no hospitalization
TE
Major Moderate to major health effects (e.g., septicemia, bloody diarrhea) with possible hospitalization
Critical Severe health effects, chronic sequelae or permanent reduction of health, hospitalization and possibly death
1389 (Source: FAO/WHO, 2009b; FAO, 2009).
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1390 Table 3. Risk Rating (R) = Likelihood (P) x Severity (I)*

Severity/Consequence (I)
PT
Critical Major Moderate Minor Insignificant
Likelihood (P) 5 4 3 2 1
Very High 6 30 (Severe Risk) 24 (Severe Risk) 18 (Serious Risk) 12 (Moderate Risk) 6 (Low Risk)
RI
High 5 25 (Severe Risk) 20 (Serious Risk) 15 (Serious Risk) 10 (Moderate Risk) 5 (Low Risk)
Medium 4 20 (Serious Risk) 16 (Serious Risk) 12 (Moderate Risk) 8 (Moderate Risk) 4 (Low Risk)
SC
Low 3 15 (Serious Risk) 12 (Moderate Risk) 9 (Moderate Risk) 6 (Low Risk) 3 (Low Risk)
Very Low 2 10 (Moderate Risk) 8 (Moderate Risk) 6 (Low Risk) 4 (Low Risk) 2 (Low Risk)
Negligible 1 5 (Low Risk) 4 (Low Risk) 3 (Low Risk) 2 (Low Risk) 1 (Low Risk)
U
1391 (Source: modified from ICMSF, 2002; FAO/WHO, 2009b; FAO, 2009; Monaghan et al., 2017).
AN
1392 *The risk ratings are classified into 4 categories: Low Risk (1-7), Moderate Risk (8-14), Serious Risk (15-21) and Severe Risk (22-30).
1393
M
1394
D
TE
C EP
AC
50
ACCEPTED MANUSCRIPT
1395 Table 4: Overall qualitative health hazard risk assessment for cricket powder that has been added to porridge and not boiled prior to
1396 consumption by infants and children 6-23 months of age*
PT
Organism D-Kill Step Likelihood of Likelihood of Severity or Risk Rating
(i.e., a rolling Occurrence Multiplication Needed Consequence
boil for 5 min) for Infection to Occur**
RI
Bacillus cereus No Medium Yes Moderate Moderate Risk***
Clostridium perfringens Type A No Medium Yes Moderate Moderate Risk***
SC
Clostridium perfringens Type C No Medium ND‡ Critical Serious Risk
Cronobacter sakazakii No Low No Critical Serious Risk
Enterohemorrhagic Escherichia coli (EHEC) No Low No Critical Serious Risk
U
Listeria monocytogenes No Low No Critical Serious Risk
Salmonella spp. No Low No Major Moderate Risk
AN
Staphylococcus aureus No Medium Yes Moderate Moderate Risk***
1397 * This would also apply to cases where cricket powder is added to the porridge after the porridge is boiled.
M
1398 ** This takes into consideration whether multiplication of the organism can occur and if enough time exists (before consumption) for the organism to
1399 reach potentially hazardous levels.
D
1400 *** Even considering the health status of the infants and children population in Cambodia, it is unlikely that low numbers of B. cereus, C. perfringens
TE
1401 Type A, and S. aureus would cause illness. As such, for these organisms, this risk rating would change from moderate, to low- to-moderate, when
1402 considering the likelihood of multiplication needed for infection to occur.
EP
‡
1403 ND – Not Determined. There is not enough information to make a determination of multiplication for C. perfringens Type C. As such, given that C.
1404 perfringens Type C primarily affects individuals with severe protein intake, which is typical for the target population of this risk assessment, the risk
C
1405 rating for this organism remains as a serious risk.

AC
1406
1407
51
ACCEPTED MANUSCRIPT
1408 Table 5: Overall qualitative health hazard risk assessment for cricket powder that has been added to porridge and boiled prior to
1409 consumption by infants and children 6-23 months of age
PT
Organism D-Kill Step Likelihood of Severity or Risk Rating
(i.e., a rolling Occurrencea Consequence
boil for 5 min)
RI
Bacillus cereus Yes Very Low to Low Moderate Low to Moderate Risk
Clostridium perfringens Type A Yes Very Low to Low Moderate Low to Moderate Risk
SC
Clostridium perfringens Type C Yes Very Low to Low Critical Moderate to Serious Risk
Cronobacter sakazakii Yes Negligible to Very Low Critical Low to Moderate Risk
Enterohemorrhagic Escherichia coli (EHEC) Yes Negligible to Very Low Critical Low to Moderate Risk
U
Listeria monocytogenes Yes Negligible to Very Low Critical Low to Moderate Risk
Salmonella spp. Yes Negligible to Very Low Major Low to Moderate Risk
AN
Staphylococcus aureus Yes Negligible to Very Low Moderate Low Risk
a
1410 The likelihood of occurrence takes into consideration studies that have isolated these organisms in either crickets or other edible insects and if they
M
1411 were capable of surviving and multiplying (to potentially hazardous levels) in the porridge fortified with cricket powder, after being subjected to a
1412 rolling boil for 5 min.
D
TE
C EP
AC
52
ACCEPTED MANUSCRIPT
1413 Supplementary Material

1414
REARING CRICKETS – should be done under strict controlled conditions, preventing access to by
1415 birds/rodents, with the use of new, not previously used egg cartons, and feeding vegetable scraps that
1416 have not been contaminated with any soil. Any chicken feed should be heat treated before feeding.
1417
1418 AT FACTORY AT HOME
PT
(World Vision Canada Pilot Project)
1419
1420
Kill crickets by suffocation Kill crickets Kill crickets by
RI
1421
by suffocation boiling x 5 min
1422
1423 Clean crickets with
SC
running water x 3 Clean crickets with
1424
running water x 3 times
1425
U
1426 Boil cleaned dead crickets x 5 min
Boil cleaned dead
1427
AN
crickets x 5 min
1428 Dry crickets in solar oven at 80ºC for ~24
h or until constant weight is reached
1429
Roast crickets on a frying pan using a low
M
1430 flame for 15 min

Grind crickets into powder using a clean
1431
electric grinder indoors to reach
1432 appropriate mesh size
D
Sun-dry crickets for 1 to 3 days (until dry)

1433
TE
1434 Store powder in plastic bags, then

Pound dried crickets using a clean mortar
1435 place into boxes for transport
and pestle to make powder
1436
EP
1437
Store powder in a clean and dry air-tight
1438 glass or plastic container
C
1439
1440 Home use of cricket powder
AC
1441
1442
Mix powder in a cooked porridge before feeding
1443 Figure 1: Current process of preparing cricket powder, following harvesting at the factory. The
1444 at- home process will be advocated in future projects. Both the at-factory and at-home flow
1445 charts includes the recommendations discussed in the conclusions section of the risk assessment.
1446 The box in dotted lines indicates that the roasting step may not be performed.
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Accepted Manuscript

Qualitative risk assessment of cricket powder to be used to treat undernutrition in

K. Walia, A. Kapoor, J.M. Farber

To appear in: Food Control

Received Date: 12 February 2018

1 Qualitative risk assessment of cricket powder to be used to treat undernutrition in

29 enterohemorrhagic E. coli, L. monocytogenes and Salmonella spp.; and a moderate to

35 procedures and using a heat treatment to further reduce the hazard.

37 Keywords: Edible insects; Microbial hazards; Food safety; Foodborne pathogens;

54 Malnutrition in children directly contributes to diseases, disorders, and in severe

58 Key factors that contribute to micronutrient deficiencies (i.e., undernutrition due

86 (FAO & ILSI, 1997; WHO, 2012).

88 one example of a potential solution to address the issue of undernutrition amongst

92 Entomophagy, the eating of insects, is a traditional practice in many nations. To-

218 in conjunction with an exposure assessment to inform a QRA.

243 some cases (EFSA, 2005a).

272 and no hospitalizations (Belanger, Tanguay, Hamel, & Phypers, 2015).

284 2.2 Clostridium perfringens

303 Generally, C. perfringens is grouped into 5 types (Types A, B, C, D, E)

334 it is suggestive that the dose response is quite high.

336 2.2.2 Foodborne Outbreaks

369 Pava-Ripoll, Pearson, Miller, & Ziobro, 2012).

396 to cause infection in infants (Gurtler, Kornacki, & Beuchat, 2005).

398 2.3.2 Foodborne Outbreaks

408 2.4 Escherichia coli

431 Carbonero, & Ricke, 2016).

433 2.4.1 Dose-response Assessment

456 Varying strains of EHEC have been implicated in numerous outbreaks

470 2.5 Listeria spp.

499 2.5.1 Dose-response Assessment

520 and 32 deaths (EFSA, 2017).

553 intestinal mucosa (Giannella, 1996).

555 2.6.1 Dose-response Assessment

576 2.6.2 Foodborne Outbreaks

607 between 0.83- 0.86 (Hennekinne, De Buyser, & Dragacci, 2012).

617 2010; Kadariya, Smith, & Thapaliya, 2014).

638 and Matthews, 2008 as cited by FSANZ, 2013).

640 2.7.2 Foodborne Outbreaks

642 Staphylococcus enterotoxins, which resulted in 4,649 cases of illness, 662

685 Studies examining the incidence and/or levels of B. cereus, C. perfringens,

704 Klunder, Wolkers-Rooijackers, Korpela, & Nout, 2012).

747 The quantitative or qualitative analysis of the microbial reduction in crickets

772 assumptions/uncertainties also include where contamination is likely to occur.

794 potential pathogenic organisms that they may be carrying.

805 6. The porridge is rice-based and processed in a home setting in conditions

819 9. Cronobacter sakazakii – Since Cronobacter has been isolated from

822 min prior to consumption.

823 10. Enterohemorrhagic E. coli – The likelihood of occurrence of E. coli after

832 11. Listeria monocytogenes – The likelihood of occurrence of L. monocytogenes

846 occurring is low especially if the powder is boiled before consumption.

854 contamination occur. The probability (or likelihood) of occurrence of microbial

862 Type C, C. sakazakii, Enterohemorrhagic E. coli and L. monocytogenes, the risk of

915 ensure no added moisture or access to by pests. If rice is used as a drying

934 gastrointestinal pathogens from either hand-to-mouth or from food/water

973 Journal of Food Safety, 25, 193–197.

979 occupational-health-safety-use-insects-food-and Accessed 1 October 2017.

981 on bacterial contamination. The Indian Journal of Pediatrics, 66(1), 7–10.

1000 the American Board of Family Medicine, 25(5),734-744.

1007 Institute of Medicine (US). Improving Food Safety Through a One-Health

1008 Approach: Workshop Summary. Washington (DC): National Academies Press

1029 income countries. The Lancet, 382(9890), 427–451.

1032 Forum on Home Hygiene. http://www.ifh-homehygiene.org Accessed 18 December

1041 alcinoe. Journal Ecolology Natural Environment, 3(5), 176–180.

1054 11-15/index.html Accessed 1 October 2017.