semiannual newsletter - n°13 april 2015 labinfo€¦ · 12 maldi-tof ms as an identification tool...
TRANSCRIPT
4 Bacterial typing : an indispensable step in the epidemiological monitoring of food-borne infectious diseases
7 The Asian fruit fly Drosophila suzukii: after the introduction, establishment and the first symptoms of damage, research is now also being conducted on control measures
12 MALDI-TOF MS as an identification tool for food-borne pathogens
15 The consumption of insects
18 Next Generation Sequencing to identify GMO in food and feed products
21 Atmospheric Pressure Gas Chromatography-Tandem mass spectrometry (APGC-MS/MS) for dioxins and PCBs analysis in food and feed
24 Evolution of the screening methods in the search for residues
28 Workshops & Symposia
LabinfoNewsletter for the approved food safety laboratories
SEMIANNUAL NEWSLETTER - N°13 APRIL 2015
FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels
Resp
onsi
ble
edito
r : H
erm
an D
irick
s
2
LabInfoNewsletter for the approved food safety laboratories
Editors’ groupDirk Courtheyn, Marnix De Gruyter, Conny De Schepper, Alain Dubois, Marc Evrard, Geert Janssens, Alain Laure, Kathleen Martens, Eva Wevers and Marie-Christine Wilem
Authors of this issueBert Matthijs, Pierre Wattiau, Hein Imberechts, Hans Casteels, Johan Witters, Nick Berkvens, Marie Polet, Nadine Botteldoorn, Katelijne Dierick, Marnix De Gruyter, Sander Willems, Marie-Alice Fraiture, Sigrid De Keersmaecker, Nancy Roosens, Gauthier Eppe, Georges Scholl, Jean-François Focant, Edwin De Pauw and Philippe Delahaut TranslationTranslation Service of the AgencyEditors’ group
Photographs and illustrationsSupplied by the laboratories
LayoutGert Van Kerckhove
Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: [email protected]
32
Dear reader,
Quite some things have happened since the last edition of Labinfo. Geert De Poorter has left us after a fruitful career in the FASFC and took up new challenges in the FPS Economy.
Pending the final assignment of Geert’s function to a new Director General, I accepted the function of ad interim Director General Laboratories at the request of our C.E.O. Herman Diricks. And I hardly had the time to settle in the job when the new government presen-ted its coalition agreement and the related budget cuts. Since that moment, we have worked very hard on a savings strategy providing us, the laboratories Administration, with the necessary resources to fulfil our mission and core tasks in a professional and qualitati-vely outstanding manner.
As a result of this strategy, we not only have to make serious in-house efforts, but we de-mand these efforts of our most important partners too: the national reference laboratories (NRLs) and the associations for animal disease control (DGZ and ARSIA). The discussions being held with these partners are not easy, but are carried out in a serene, open and con-structive climate. By way of this editorial, I wish to thank them and I hope together we will succeed in converting these budget cuts into an opportunity to improve all our activities. To secure the future we undoubtedly must be creative and cooperate more effectively with the NRLs as well as with the approved laboratories.
You will notice in the articles of this edition of Labinfo that there are quite some new developments concerning analytical techniques and parameters to be determined in the field of food safety.
I wish you a lot of reading pleasure with this 13th edition of Labinfo.
Bert MatthijsAd interim Director General Laboratories
EditorialPublication destinée aux laboratoires de sécurité alimentaire agréés
4
Bacterial typing : an indispensable step in the epidemiological monitoring of food-borne infectious diseasesPierre Wattiau and H. ImberechtsUnit “Foodborne and Highly Pathogenic Zoonoses”, Operational Direction Bacterial Diseases, CODA-CERVA, Groe-selenberg 99, 1180 Brussels
Detection of pathogenic bacteria at all levels of the food chain involves implementing a host of different laborato-ry methods. For each bacterium, procedures do exist that are adapted to the bacterium characteristics, the matrix to be analysed, the level of detection sensitivity that has to be reached and the desired level of characterization of the micro-organism. Enrichment, cultivation and identification are the first steps of a bacteriological analysis and provide essential information on the microbiological quality of the analysed matrices. However, these results are not very useful per se for tracing the origin of a contamination, for comparing clinical cases or for determining the common source of an infection. In order to reach this, more specialized techniques are needed to deliver the equivalent of a digital fingerprint of the bacteria. The present article provides a short overview of the most popu-lar techniques used for this purpose and their applicability in different contexts.
Traditional identification of routinely isolated bacteria generally stops at the species or subspecies level. In certain cases, bacterial subtypes can be identified by searching for particular biochemical characteristics (biotypes). In other cases, the presence of virulence or colonization factors will be screened (pathotypes), antigens on the sur-face of the bacteria will be identified with the help of antibodies (serotypes), particular genetic characteristics will be highlighted (genotypes), the toxins produced are characterized (toxinotypes), the resistance profile to bacterial reference viruses (phage types) or the electrophoretic profile of the ribosomal RNA fragmented by restriction enzymes (ribotypes) will be established. None of these “first line” methods are, however, sensitive enough to satis-factorily establish the identity or the close relationship between multiple isolates. To do this, the fine composition of the bacteria has to be probed by means of techniques that are capable of sensing the smallest differences in the macromolecules they are composed of. The following paragraphs briefly discuss the most popular DNA-based subtyping methods with their respective advantages and disadvantages.
54
PFGE (Pulsed Field Gel Electrophoresis)
Pulsed field electrophoresis allows for the establishment of a detailed fingerprint of the bacterial genome by separating very large DNA fragments. This technique makes it possible to visualize genomic differences resulting from deletions, insertions or rearrangements that differ between bacteria of the same subtype. The technique is sensitive and universal, but requires a well-trained and expensive handwork.
MLVA (Multiple Loci Variable Number of Tandem Repeats Analysis)
This technique is aimed at determining the length of short repeated sequences that are spread throughout the genomes of the majority of bacteria: since the number of repetitions show extreme variations from one bacterial strain to another, the identity or the proximity of two strains can be reliably established. Although relatively easy to implement, MLVA is not universal and a typing scheme that is specific to each subspecies has to be established and validated beforehand. The hypersensitivity of certain markers, which could wrongly lead to the conclusion that differences exist between identical bacteria, has also been reported.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
The CRISPR motifs present in about half of the bacteria consist of groups of repeated sequences that are separat-ed by unique sequences, the organization and composition of which differ among bacteria of the same species. These sequences can at the same time serve to identify the subspecies and to identify groups of individuals with-in that subspecies. Easy to implement, the usefulness of this technique is limited to bacteria that contain CRISPR motifs in their genome.
MLST (Multiple Loci Sequence Typing)
This method consists in determining the nucleotide sequence of a number of genes (5 to 10) spread throughout the genome and coding for ancillary proteins. Its degree of sensitivity varies strongly depending on the bacterial species considered and its implementation is relatively long and costly. The method is very useful for taxonomic and phylogenetic studies but is of little interest when it comes to tracing epidemics.
WGS (Whole Genome Sequencing)
The purpose of WGS is to determine the full nucleotide sequence of a bacterial genome. Most often the sequence is first obtained in the form of a multitude of fragments with lengths ranging from 100 to 250 nucleotides de-pending on the technique and which are determined several times for the same genome. The overlaps between fragments and/or their relative positions compared to a reference sequence can be used to assemble the whole genome sequenced or a part of it. WGS techniques, the popularity of which continues to increase and the costs of which continue to decrease, are the most sensitive fingerprinting techniques. By means of appropriate data processing, these techniques allow to assess the degree of relationship between different bacteria. However, their routine use is still for the future, even if there is little doubt that these techniques will sooner or later replace the other methods.
6
SNP typing (Single Nucleotide Polymorphism typing)
Nucleotide sequence data can be used to infer variable positions in the genome of bacteria belonging to the same subspecies. When interrogated at numerous positions within the same bacterial genome, these polymor-phic nucleotides provide a complex genetic signature which is unique to the bacterial strain considered. This method requires no sophisticated instrumentation compared to WGS and its cost is moderate. Platforms of the micro-array type are the preferred instruments to interrogate multiple polymorphic nucleotides or remarkable genetic characters simultaneously. Numerous micro-array formats exist, depending on whether they are com-posed of small DNA ‘spots’ printed on the surface of microscopic glass slides or of microbeads covered with DNA and suspended in a liquid. Figure 1. Typical profile of a bacterial typing experiment obtained by means of PFGE (A) by interrogating SNPs using a DNA micro-array of the glass-slide type (B) and by interrogating SNPs using polystyrene microbeads ana-lysed in a flow cytometer (C).
A
B
C
76
The Asian fruit fly Drosophila suzukii: after the introduction, establishment and the first symptoms of damage, research is now also being conducted on control measures Hans Casteels, Johan Witters and Nick BerkvensInstitute for Agricultural and Fisheries Research, Eenheid Plant – GewasbeschermingBurg. Van Gansberghelaan 96 bus 2, 9820 Merelbeke
Introduction
One of the most well-known fruit flies in our regions is Drosophila melanogaster (family of Drosophilidae). This “common fruit fly” can be found during the summer months on (over)ripe fruit and on fruit and vegetable waste in our GFT (Vegetable Fruit and Garden waste) containers. Despite the fact that its presence is often a nuisance to humans, this little fly is harmless. In addition, the larvae of this species are useful. Together with the larvae of many other Drosophilidae, they play an important role in our ecosystem, since they contribute to the degradation of all kinds of organic materials. Worldwide, more than 3,800 fruit fly species are documented; what’s the harm in one more?
And yet, in the case of the Asian fruit fly, Drosophila suzukii, also known as the Suzukii fly, this could have a severe economic impact on our fruit farming sector in the long term. This fruit fly is one of the most feared insect pests in the European small fruit and stone fruit cultivation sector; in the United States and Southern Europe, cultivators have incurred revenue losses of more than 50%. Contrary to our native common fruit fly, which affects overripe or damaged fruit, this fruit fly also affects unripe fruit. Because infestation is not visible during the first days after the eggs have been laid, since the larvae feed on the fleshy part on the inside of the fruit, there is a considerable risk that infected fruit enters the market, with all the possible consequences for our fruit cultivators, auction organiza-tions and supermarkets.
The problem of the Asian fruit fly will be discussed in chronological order, from the first time the fly was spotted in a private garden in Ostend (2011) to the occurrences of the species in fruit plantations in the period 2012-2014, whether or not with (economic) damage and providing solutions to the field by submitting an IWT LA-project demand.
8
Origin and introduction
D. suzukii is of Asian origin; the fly was first discovered in Japan in 1916. Around 1930, D. suzukii could be found in countries such as China and Korea, and later also in Myanmar, Pakistan, Taiwan and Thailand. After its introduction in California in 2008, the little fruit fly started a victory march throughout North America. Nowadays, the exotic insect can be found in large parts of the United States and in Canada. Around the same time, the fly was also introduced in Europe. To aggravate matters, the little fly easily adapts to different European climate types, ena-bling a quick geographical spread. The fly was successively found in Italy (2009); France, Slovenia and Spain (2010); Switzerland, Germany and Belgium (2011) and in Portugal, the United Kingdom and the Netherlands (2012). The increasing international trade in (infected) fruit is responsible for the spread over large distances and probably lies at the basis of introduction in Belgium and other European countries. Further spreading in Belgium is a conse-quence of the domestic trade on the one hand, and the migration of the adult flies on the other hand. Since this harmful fruit fly has already become wide-spread throughout Europe, quarantine measures are no longer an option.
Life cycle
The reproduction of this species of fruit flies happens really quickly. As for most insects, its development depends on the temperature; under favourable climatic conditions, the complete life cycle only encompasses 1 to 2 weeks (25-30 °C). This short life cycle allows the flies to produce multiple generations each year (in Japan up to 13 generations per year have been recorded). The males are characterized by a distinctive black spot on both wings (picture 1).
Picture1: male D. suzukii (picture ILVO)
After mating, the females lay an average of 350 eggs by means of a typically strongly chitinized and serrated ovi-positor (picture 2), which enables them to penetrate the peel of hard and unripe fruit.
98
Picture 2: ovipositor female (picture ILVO)
The deposited eggs are usually difficult to detect on the harvested fruit, making it virtually impossible for any farmer, auction house or supermarket to check the apparently intact fruit. After 12 to 72 hours, the eggs of the fruit eating larvae hatch. There are three larval stages followed by a pupal stage that takes place inside the fruit or on the surface of the fruit. After hatching, the adult flies leave the fruit they grew in. Because of its high level of fertility and short generation time, this fly is able to reproduce very quickly. How the flies hibernate in our regions is not completely known yet. However, these flies are however known to still be active at lower temperatures than their European counterparts, which explains why they can be seen until late autumn.
Host plants and damage
Almost any plant with a thin peel, both cultivated and wild plants, can serve as a host plant; but these fruit flies have a strong preference for blackberries, raspberries, strawberries, blueberries and cherries. In addition to these species, larvae can also develop in several other kinds of fruit. Even though the damage has remained limited in the country of origin due to the presence of natural enemies, the establishment of this fly in Belgium may cause important revenue losses in the short term. This is the case anyway in regions with a comparable moderate climate where a small number of generations can cause considerable damage. The extensive list of host plants, among which also a number of wild species such as the may-bush, elderberry, rose hips and wild berries, make it difficult to control this fly. Just like any other species of fruit flies, the suzuki fly is also able to develop on overripe or rotting fruit, also in our GFT containers.
The first step toward knowledge development: monitoring
After the Suzuki fly was first observed in a private garden in Ostend (September 2011), the FASFC set up a moni-toring network by means of attraction traps (Droso trap, Biobest) at different locations, to confirm the presence of D. suzukii in our country (pictures 3 & 4). In addition to fruit plantations, a number of fruit auctions, storage rooms and packing rooms were also monitored. In 2012 and 2013, no suzukii flies were found at the latter locations. Pc-fruit, CRA-W and GFW (Groupement des Fraisiéristes Wallons) also monitored the presence of this fly in a number of fruit plantations.
10
Picture 3: monitoring on blackberries grown under covers (picture FASFC)
Picture 4: monitoring in cherry orchards (picture FASFC)
The entomological lab of the Diagnostic Center for Plants (ILVO) is responsible for detecting and identifying harm-ful insects and mites in all samples that are taken by the FASFC in the framework of plant protection. During the monitoring campaign, all captured insect materials were examined on the presence of suzuki flies using stereo microscopes; the confirmation of their presence also took place in the entomological lab which, as a National Reference Laboratory-Plant Diseases, is also accredited for insects.
In 2012, samples were taken at a total of 53 locations (cherry, strawberry, raspberry, blueberry and plum). The presence of D. suzukii was confirmed in 14 of these locations (26%). The highest numbers were found in cherry trees in Flanders, but adult D. suzukii were also found in plums, strawberries, raspberries and blueberries. In Wal-lonia, most of the flies were caught in the raspberry cultivation under cover. It is remarkable that the suzuki flies were observed late in the season; with one exception (in January, in Gembloux), the first flies were only observed from mid July until the end of December. In 2013, the monitoring network was extended to 108 locations (among which blackberry, grape, wild cherry and pear). Flies were found at 76 locations (70%) and again quite late during the growing season (early August). At some locations, minimal negligible damage could also be observed. The observations in 2013 showed that the spread of this fruit fly increased in size, both in Flanders and in Wallonia. In most cases, small numbers of flies were found, but in one (neglected) cherry orchard, hundreds of specimens were caught during the growth season. The spectrum of host plants in which the fly was found also extended even further. The majority of flies were found in cherry plantations, but the fly was also detected in strawberries, raspberries, blackberries, grapes and in several other berries and also in wild cherries. In the late fall of 2013, the larvae of D. suzukii also appeared to be present in unpicked pears. In 2012 and 2013, the first adult flies were not caught until the second half of July-August; the majority of the catches did not take place until late autumn (Octo-ber- November). Even at low temperatures, flies continued to be caught. The question remains to what extent the harsh winter conditions in February 2012/March 2013 had negative effects on a fast(er) population development/
1110
growth. Adults hibernate in diapause in sheltered hiding places (greenhouses, compost, warehouses, nearby vegetation) and after the winter a new population build-up is necessary. The life cycle of the flies is relatively long (90 to 160 days) and they are already active at temperatures above 8°C. The suzuki fruit fly has a long reproduction season, which is beneficial for a strong population growth. The observations by pcfruit and CRA-W continued in 2014 at more than 90 locations. The observation of flies during winter and early spring of 2014 was remarkable. The very mild winter of 2013-2014 has probably played a part in this. There were only 3 frost days; the average temperature was 6.3 °C, contrary to 3.6°C under normal circumstances. This probably explains why suzuki flies were detected in monitoring traps during the winter and spring of 2014. In June 2014, cherries were found to be damaged and in the course of July, there was an increasing amount of damaged berries, blackberries, strawberries and raspberries. Also in other European countries, the level of infestation is greater than in previous years.
Additional research
At the moment, it is difficult to assess the implications of these catches in the near future for all small/ soft fruit cultivations (strawberries, berries, raspberries, grapes, etc.) and also stone fruit (cherries). Damage to fruit has remained limited up to this point, but is on the increase. Of course, ILVO Crop Protection continues to monitor the “flights”’ of the flies. In the framework of the above-mentioned problem, the Test Centre Fruit Cultivation has sub-mitted an IWT project demand which was also approved. In the first place, this project will provide an in-depth study on a number of key characteristics of the suzuki fly, such as its phenology, population dynamics, hibernation capacity/strategies, preference for a certain variety of fruit,... In doing so, survival capability under more extreme conditions (cold/heat tolerance) deserves particular attention. For the effective control of the suzuki flies that are already present at the premises of fruit businesses, the focus will go to the optimal elaboration of “mass trapping” and “attracting and killing” techniques. Since cultivation hygiene is also a crucial concept in controlling this harm-ful fruit fly, an innovative solution will be developed for the practical disposal of fruit waste and for killing the life stages of the fly that are potentially present. In this respect, the development of a compost container in which the different life stages of the fly can be killed efficiently, and in which the first stages of composting can already take place, is being considered. This way, after a short period of time in the container, the infected fruit can be com-posted further without any risks.
Conclusion :
The results of the monitoring show that the suzuki fly can survive in cultivated crops/wild species and that it is apparently highly resistant to lower temperatures. Despite the fact that a number of products have an approval to be used against this fruit fly (spinosad, dimethoate and lambda-cyhalothrin), applying a chemical treatment is not easy due to possible residues in the harvest and the presence of the fruit fly in wild fruit varieties. Therefore, a quick extermination is not an option; this exotic species is here to stay, likely to expand its territory even further. Consequently, additional research is needed to develop useful control strategies to prevent further spreading and economic damage to the fruit cultivation sector.
12
MALDI-TOF MS as an identification tool for food-borne pathogens
Marie Polet, Nadine Botteldoorn and Katelijne DierickNRL Food microbiology, Rue Juliette Wytsmanstraat 14, 1050 Brussels
At the beginning, confirmation and identification of bacteria in microbiology were mainly based on morphology. The biochemical tests in test tubes emerged later, which were afterwards miniaturized. PCR was later developed, followed by the MALDI-TOF MS (i.e. matrix-assisted laser desorption-ionization – time of flight mass spectrometry). MALDI-TOF MS has already been used for about twenty years in different fields of applications especially in chem-ical laboratories for the detection of different molecules like sugars, nucleic acid and proteins. More recently the technique is more and more used for veterinary diagnosis and in the environmental field, en is now booming in the microbiological laboratories as a good identification system. At global level, there are nowadays around 1000 installations in the clinical and pharmaceutical sectors and about a hundred ones in the food sector.
As far as food safety is concerned, this technique meets the necessary requirements for confirmation and identifi-cation of food-borne pathogens. MALDI-TOF MS is faster than the usual microbiology methods but has the same discrimination level, it is less expensive and requires less technical expertise than the genotyping methods. More-over, MALDI-TOF MS can also be used in routine laboratories of food microbiology, where the time factor plays a key role since these laboratories mostly work with the food industry. This technique can also be used in the case of food-borne outbreaks.
The MALDI-TOF MS technology in microbiology, can identify microorganisms to the species level. The principle is based on ionisation of the bacterial proteins by a laser beam and on creation of typical peaks (spectrum). By means of a spectra database, the related software searches for the correspondence with the bacterium species according to a reliability index between both spectra. The MALDI-TOF MS does however not give information on the serotype and pathogenicity of the species (ex: Vibrio parahaemolyticus).
A mass spectrometer is typically composed of 3 elements: an ion source (MALDI), a separation of the molecules (the TOF) and the detection.
MALDI-MS:
The analyte is first co-crystallized with small organic compounds (matrix) in order to be protected against direct contact with the ionizing beam and to avoid its degradation. These compounds will absorb the UV laser radiation and transfer the energy to the proteins that become positively ionized. The generated ions will free themselves from the proteins. The charged molecules (+1) are then accelerated in an electric field.
1312
TOF:
After the ions went through the electric field, they enter a tube not exposed to an electric field and are separated according to their mass-charge. The ions reach different speeds because the big molecules move more slowly than the small ones. A time-of-flight analyzer will then detect each passing ion and create the peaks on the spec-trum, which will afterwards be compared with a database. Some components of the spectrum are specific to a genus, others to a species or even to a subspecies. According to the sample quality and purity and to the number of reference spectra in the database, the identification of the bacterium takes a few seconds or a few minutes.
Figure 1: Working principle of a MALDI-TOF mass spectrometer
All sorts of food microorganisms can be screened by following the same protocol.
The preparation of the test sample is a critical step in the MALDI-TOF MS analyses. Indeed, in order to get reliable and reproducible identification results, it is necessary that the growth conditions of the bacterium (time and incubation temperature, culture medium) stay the same through the different tests. The test starts from a pure culture of bacteria. This culture can be used as such or be subjected to a pre-treatment. The tests must be carried out with fresh bacterial cultures because the stress caused by the cold or by a nutrient deficiency could alter the protein composition and so have an impact on the test result.
14
The quality of the database is also a critical point for a precise identification of the bacteria. The many surveys on identification of bacteria with MALDI-TOF MS have been carried out on clinical samples and the requirements specific to the food microbiology laboratories are usually not taken into account. Only a few reports do study the MALDI-TOF MS applied to food microbiology. Some surveys reveal that the major obstacle in the identification of food isolates is related to the database, which has to be regularly updated with reference spectra coming from food strains.
Table 1: The pros and cons of MALDI-TOF MS applied to food microbiology
PROS CONS
-Sample preparation is universal for bacteria, yeasts and moulds
- Purchasing the device is expensive
- Protocol is simple - Fresh cultures for the analyses
- Low operating costs (consumables, staff ) - Influence of growth medium on test result?
- No waste -The database must be large enough to discriminate properly very related bacteria, such as E. coli and Shigella spp
- Very rapid result -Does not give any information on the pathogenicity of the species (ex: Vibrio parahaemolyticus)
- The database can easily be expanded -The species which are problematic for identification with 16S are sometimes also problematic with this technique
The MALDI-TOF MS will be integrated in the identification tests within the current revision of ISO 7218, besides the biochemical galleries, nucleic probes and agglutination tests.
To conclude, the MALDI-TOF MS is a promising tool for the identification of bacteria in the routine laboratories of food microbiology. The simple protocol, the rapid results and the low test costs are the main advantages of this technique, which is certainly fitted for a use in the food safety field.
Littérature:
http://www.biomerieux.com/fr/spectrometrie-de-masse-maldi-tof Pavlovic, M., et al. “Application of MALDI-TOF MS for the Identification of Food Borne Bacteria.” Open.Microbiol.J. 7 (2013): 135-41
1514
The consumption of insects
Marnix De GruyterFLVVG, Braemkasteelstraat 59, 9050 Gentbrugge
Introduction
The consumption of insects: to the majority of us Westerners this might appear strange or even repulsive. For centuries, we have been relying on our domesticated animal species to provide us with meat and other useful products, such as wool, leather and milk. However, for a large part of the world population, eating insects isn’t that unusual. At least 2 billion people have included insects as a part of their daily diets, mainly in tropical regions. It is estimated that about 1,900 different insect species are eaten worldwide. These insects belong to a wide variety of classes. More than 31% of the insects consumed belong to the Coleoptera (beetles), 18% are caterpillars belong-ing to the Lepidoptera (butterflies) and 14% are Hymenoptera (such as bees and wasps). But grasshoppers and termites are also often on the menu.
Why did this dietary custom continue to exist in the tropics and hasn’t it gained a foothold in Western society? There are some explanations for this. First and foremost, Western society has grown estranged from nature. In our society, insects are rather seen as pests that threaten crops and transmit diseases. In tropical regions, however, they have become a part of the natural environment and culture (e.g. for medicinal purposes). Insects play a mi-nor role in our culture, with the exception of a number of insects that produce products that are useful to us, like silk worms and honey bees. Moreover, there is also an aversion to eating insects from an aesthetic point of view, or because it’s associated with primitive behaviour. This explains the lack of interest from the agricultural world.
Why should we eat insects anyway?
Nevertheless, eating insects offers a lot of advantages compared to our traditional livestock. The world currently hosts 7 billion people, a population that is expected to have grown to about 9 billion within the foreseeable fu-ture. More than one billion of these people will suffer from famines. This might also undermine countries’ political stability. The growing world population will exert a lot of pressure on the environment. Traditional livestock farm-ing is very polluting. Only second to energy production, livestock farming is the largest producer of greenhouse gases. Livestock farming requires considerable quantities of water. The production of 1 kg of meat requires 3,682 litres of water.
16
This is completely different for insects. Insect farming requires less space and water. Compared to cattle, insects have a better feed-to-meat conversion rate: crickets only need 2 kg of feed to produce 1 kg of body weight. This barely produces any greenhouse gases. Farming insects on human waste can help to curb environmental pollution (however, this offers the disadvantage that heavy metals are taken up by the insects). Insects are easy to breed and they reproduce quickly. Actually, no large or expensive installations are needed. In Thailand, crickets are reared in ordinary metal containers with a layer of rice waste on the bottom. Plastic bottles are used to supply drinking water. The containers are covered with a fine mesh net (mosquito net) to prevent insects from escaping. The females lay their eggs in bowls filled with sand and burned rice husks. The bowls are removed and placed in another metal bin. Each bowl is covered with rice to ensure a constant hatching temperature. The bowls had to be surrounded by water to prevent predator ants from entering. This non-industrial production can be sufficient to supply food for an entire village. The low installation costs entail that the underprivileged can also participate in the production process.
In addition, insects are very nutritious. Most insects contain more than 60% of proteins, which are easily digestible for humans. The fat content varies strongly, from 7 to 77 g/100 g of dry weight. The cholesterol level is usually a bit lower than in meat, but insects contain more essential fatty acids (linoleic acid and linolenic acid). The calo-rific value of insects varies between 293 and 762 kcal/g dry weight. In addition, many insects prove to be rich in essential minerals such as zinc, copper, iron and vitamins such as thiamine and riboflavin. Consequently, regarding their nutritional value, insects can be compared to other food of animal origin such as meat, fish and crustaceans. Moreover, they have much higher fibre content.
Potential dangers
Despite all of the advantages, there are also a number of issues. Insects are rich in nutrients, which makes them an attractive nutrient medium for micro-organisms. Just like humans, they have an intestinal flora. This flora can contain different pathogens, such as Salmonella and Camphylobacter. There is evidence that these bacteria sur-vive less than 72 hours and consequently do not constitute a serious threat. Insect pathogens sometimes belong to different taxonomic groups than human pathogens. They have completely different life cycles than human pathogens, so they do not pose any threat as such.
The most important aspect regarding micro-organisms is not as much the intestine flora of the insect itself, but rather the safe storage and conservation of the products. Research on meal-worms has shown that Enterobac-teriaceae and spore producing bacteria could be isolated from fresh insects. Boiling the insects for 5 minutes eliminated Enterobacteriaceae, but spore producing organisms were found to survive this process. It is recom-mended to store the insects in a cool place at a temperature of 5 to 7 °C. At these temperatures, spoilage of fresh as well as cooked insects can be prevented for a time span of 2 weeks. Roasting insects did not appear to kill all of the Enterobacteriaceae. Fungi are also a probable source of danger. Some species of fungi produce mycotoxins in large quantities. This way, frequently eating insects can be dangerous. The spread of fungi can be countered by storing them in a cool place or by means of a drying process.
It is well-known that some insects may contain parasites. Some parasites use insects as a vector. An important example of this is Trypanosoma, a parasite that causes Chagas’ disease. Research has shown that the parasite can also infect human beings via oral transmission and the link has been made with the consumption of raw insects. Insects can also be carriers of other dangerous parasites such as Entamoeba histolytica or Toxoplasmosis. Conse-quently, eating insects is not without danger.
1716
Another danger of eating insects is chemical contamination. Pesticides are the main cause of chemical contam-ination. All pesticides are potentially dangerous to human beings, especially if insects are harvested in the wild instead of being farmed, the latter method allowing for better monitoring. In general, chlorinated pesticides are no longer present in large quantities, but organophosphorous components may constitute a potential danger.
Like mushrooms, some insects can contain very toxic components, while others are free of toxins. Insects use these toxins as a repellent. They can get these substances from plants they live on and they can subsequently store these in special organs. Such insects can pose a potential threat. Some of the substances, however, lose their active ingredient when the insects are boiled. Insects like bees and ants produce their own poisons. These substances are neutralized by our digestive tract. Researchers have come to the conclusion that insects living on plants that are edible for human beings, are usually safe to consume, but that others are potentially dangerous. In some cases, the organs that contain the toxic components can be removed. In East Italy, it is a custom to eat larvae of butterflies of the genus Zygaena. These larvae, however, contain cyanogenic glycosides, from which hydrogen cyanide could be liberated. That’s why consumers only eat the sweet-tasting crops which are free of toxic ingredients.
It is important to note that some insects may also contain components that have an effect on the intake of vita-mins (so-called anti-nutritional components). Eating caterpillars of the species Anaphe venata seemed to hamper the intake of thiamine, which might cause a deficiency after some time. Especially in regions where there is an inadequate food supply, this might cause problems.
Finally, insects can also take up and store heavy metals. Large amounts of arsenic and lead were found in some insects. This might be linked to the insect’s living conditions. Those who preferably live on the soil, possibly also take in more toxic metals. These toxic metals can find their way into the food chain when the insects are used as feed for poultry for example.
These potential dangers should be somewhat nuanced. Many issues can be avoided by handling insects in a hygienic manner, as is already the case for cattle. Rearing insects on ‘farms’ allows for better monitoring. Parasites are for example bound to their natural environment for their cycles. The farming of insects also lowers the intake of metals. Insects can be bred selectively and for breeding purposes, one could also consider only those species known to be free of harmful components. A certain degree of monitoring will always be necessary, as is already the case at present for other foodstuffs.
Next Generation Sequencing to identify GMO in food and feed products
Sander Willems1*, Marie-Alice Fraiture1 ,2*, Sigrid C. J. De Keersmaecker1 and Nancy H Roosens1
1 Platform Biotechnology and molecular Biology, Scientific Institute of Public Health, rue J. Wytsmanstraat 14, 1050 Brussels, Belgium2 Instituut voor Landbouw- en Visserijonderzoek (ILVO), Eenheid Technologie & Voeding (T&V), Burg. Van Gansber-ghelaan 115, 9820 Merelbeke, Belgium *equal contribution
The growing number and diversity of GMOs present on the market makes the use of the current for GMO analysis gold standard real-time PCR technology more and more complex and time-consuming in order to detect a GMO in food and feed samples. Indeed, an increased number of screening and event specific methods (target-ing the GM element(s) and the junction between the GM cassette and the host genome, respectively) has to be developed and used by the enforcement laboratories in order to cover all the authorised GMOs in a country. In addition, the real–time PCR strategy implies the prior knowledge of the sequence, at least partial, of the GM cassette. Collecting these sequences for unauthorized GMOs is challenging and designing each corresponding method is extremely time-demanding even impossible regarding the numerous possible GM elements found in unauthorized GMOs. This poses a major problem as GMOs remain undetectable when no method targeting the GM element has been used in the tested sample. Recently, to take up the challenge of GMO detection in food and feed matrices, Next Generation Sequencing (NGS) allowing massive parallel DNA fragment sequencing resulting in millions of sequencing reads, has been proposed as a promising technology.
Figure 1: Massive DNA fragments (reads) from a GMO, produced with NGS technology.
1919
In this context, the Scientific Institute of Public Health (WIV-ISP) has started a pioneer study financed by the project UGMMONITOR (SPF convention RF 11/6242) and EPIGMO (Ylieff ). This study has first used the NGS tech-nology to assess its potential applicability to detect and identify a GMO (figure 1) in different types of alimentary matrices, i.e. grains of 100% GM rice, grains of 10% GM rice and 100% GM rice noodles (processed food) (figure 2). Secondly, to evaluate the potential use of NGS by “bioinformatics laymen” the data were analysed by using two different platforms: an easy-to-use commercial platform (CLC Genomics Workbench), without the need for a thorough bioinformatics background, and an “in-house” platform allowing greater control of the workflow and parameters and as a consequence demanding a higher level of expertise in bioinformatics. Thirdly, a conceptual statistical framework was developed and applied to estimate the amount of reads necessary to be able to detect and identify several common GMOs at concentrations representative of “typical” food and feed matrices.
Figure 2: Types of rice matrices used in this study: rice grains (a) and “home-made” rice noodles produced from rice grains (b).
Our results showed that it is possible to use NGS to identify and characterize all the types of samples envisaged in this study. The analysis requires only prior knowledge of the sequence, at least partial, of the GM cassette and of the host genome used as reference during mapping of the sequencing reads. Therefore, the NGS strategy allows to use a standardized approach for any type of GMO, in contrast with the specific method development and use that need to be designed for each GMO individually when using the real-time PCR approach. Moreover, a pro-cessed matrix such as rice noodles, yielding degraded DNA after DNA extraction from the sample, is not an issue in using the NGS technology (Illumina).
The study highlights also that the development of new user-friendly specific visualization software is necessary to efficiently analyse and deliver the knowledge to the user, especially in the present context of lack of bioinformat-ics expertise in the enforcement laboratories.
2020
The conceptual statistical model has indicated that a large genome like that of wheat requires a higher number of sequence reads (i.e. a higher coverage or larger sequencing depth), resulting in larger costs per sample but at a price affordable for an “enforcement lab” when the GMO is present at 100%. However, detecting small amounts of GM DNA (1%) in a plant DNA mixture is at the present time impossible when considering the cost and the complexity of the analysis.
The present study offers preliminary information about some major strengths and weaknesses of the NGS tech-nology that need to be addressed before consideration of any routine use of NGS in GMO analysis. It is concluded that NGS has the potential of solving current problems in the GMO detection. However, before any implementa-tion in routine, extended research projects and validation guidelines are necessary.
Detailed results of this research were submitted for peer-reviewed publication.
Acknowledgments
This research is funded by the Federal Public Service Health, Food Chain Safety and Environment (convention RF 11/6242) through the Project UGMMONITOR. The authors would like also to thank Emmanuel Guiderdoni (CIRAD, UMR AGAP, Biological Systems department, Montpellier, France) for his kindness to provide rice grains.
212020
Atmospheric Pressure Gas Chromatography-Tandem mass spectrometry (APGC-MS/MS) for dioxins and PCBs analysis in food and feedGauthier Eppe*, Georges Scholl, Jean-François Focant and Edwin De PauwCART University of Liège, Allée de la Chimie 3, B-6c Sart-Tilman, B-4000 Liège, BELGIUM
Introduction
Two years ago we discussed in this journal the use of GC-MS/MS as a new potential option for confirmation of PCDD/Fs and DL-PCBs analysis in the framework of official food and feed control. Later, this new confirmatory approach has been extended to the auto-control of food and feed business operators. In this paper, we are taking stock of the different tools to perform dioxin analysis with mass spectrometers other than the so-called high-res-olution MS sector instruments (HRMS), with a specific focus on Atmospheric Pressure Gas Chromatography-Tan-dem mass spectrometry (APGC-MS/MS). PCDD/Fs and PCBs analysis by GC-MS/MS is not a new concept, the first scientific papers relating the development of the method date from the nineties, mainly by using ion trap mass spectrometers. The method was able to easily detect trace levels of PCDD/Fs and PCBs in environmental samples. The lack of sensitivity to achieve sub-part-per-trillions (ppt) levels necessary for food and feed analysis made the technique inadequate and out of business for this particular analytical application. The new generation of bench-top triple-quadrupole MS recently launched on the market by several manufacturers has truly made a difference. The first studies and validations performed in food and feed highlighted the potentialities of the technique leading in 2012 to the amendment of the EU legislation by integrating the GC-MS/MS as confirmatory method (252/2012/EC)1. At this stage, it is important to emphasize that only the last generation of triple quadruple is able to meet the performance criteria of the EU legislation. Thus, it means that the risk succumbing to the tempta-tion for ‘non-dioxin’ laboratories to start this very particular analysis, not equipped with the latest generation of triple quadrupole is high, with little chance to succeed. Indeed, the new EU Regulation amending 252/2012/EC (589/2014/EC)2 is rather performance-based oriented, indicating that a confirmatory method can only be used if the method has demonstrated by a complete validation that all the quality and performance criteria listed in the above mentioned EU document have been met. Numerous analytical studies were performed within the Europe-an-National Reference laboratories (EU-RL/NRL) network to assess the capability of the GC-MS/MS method to be used as confirmatory method over the last two years. Depending on the GC-MS/MS used, most of these studies demonstrated the feasibility to achieve comparable analytical performances to GC-HRMS at low ppt levels.
APGC-MS/MS
Among the different possibilities to analyze trace levels of dioxin and PCBs in food and feed, Atmospheric Pressure Chemical Ionization (APCI) is a soft chemical ionization technique that produces abundant molecular or pseu-domolecular ions [M+.] by charge transfer (Figure 1) or by protonation [M+H]+. Although the first developments of APCI sources have been used to interface MS with liquid chromatography (LC), the ionization interface can also be connected to GC. The reduced fragmentation when using APCI makes the technique suitable for generating se-lective MS/MS fragments in the second quadrupole of the MS and sensitive Multiple Reaction Monitoring (MRM) transitions for numerous analytical targeted applications in MS/MS mode. This is in contrast to traditional Elec-
22
tron Ionization (EI) at 70eV used in GC/MS, which generally suffers from extensive fragmentation and where the selection of the precursor ion is always a compromise between selectivity and sensitivity. In some cases, with less stable molecules, it might complicate the application of a quantitative MS/MS approach due to the lack of specif-ic/abundant precursor ions. Recently, developments in APGC-MS/MS has resulted in very sensitive analytical appli-cations for polycyclic aromatics hydrocarbons (PAHs)3, pesticides4, polybrominated diphenylethers (PBDEs)5 and PCDD/Fs and PCBs6. For instance, Kotz et al. showed that the absolute amount of 2,3,7,8-TCDD detected by APGC-MS/MS, by selecting two precursor ions, e.g. 320 and 322 m/z [M+.], giving the two product ions 257 and 259 m/z [M-COCl.]+ (Figure 1), injected on GC column can go down below 10 femtograms (1fg = 10-15 g). It provides comparable sensitivity obtained with sector instruments (HRMS) based on standard calibration solution injected. For comparison with EI source GC-MS/MS, Kotz and co-workers have also reported of 50 fg absolute amount of 2,3,7,8-TCCD on column7. In an other study with a EI-GC-MS/MS, Fürst and co-workers achieved the same level of performances. The lowest calibration point for PCDD and PCDF congeners was 100 fg injected on-column with an excellent linearity from 0.1 to 10 pg injected on column8. L’Homme et al. reported an instrumental limit of quan-tification (iLOQ) of 0.016 pg/µL for 2,3,7,8-TCDD (80 fg on GC column) with also a EI-GC-MS/MS instrument9. All these works showed, on a limited number of samples, sufficient sensitivity for monitoring maximum and action levels for PCDD/Fs and PCBs in food and feed.
Figure 1: Analysis of 2,3,7,8-TCDD by APGC-MS/MS
A substantial gain using APCI compared to EI has been observed. The analytical performances, limited to instrumental injec-tions, are most likely comparable because both PCDD/Fs and PCBs give rise to intense molecular ions by electron ionization. It is not the case for all the pollutants, it can be much better in favor of APCI as reported by Portolés and co-workers for pyre-throid insecticides4, where extensive fragmentation of those pesticides occurs in EI mode.
2322
The sensitivity of the detection system is obviously pivotal for this type of analytical applications but femtograms of PCDD/Fs and PCBs injected from calibration solution are not real samples; measuring routinely at sub-ppt levels PCDD/Fs and PCBs is the true challenge and it requires the upmost sensitive detection system but the extraction and clean-up processes both play an important role. It definitely has influences on the quality and reliability of the analytical results. APGC-MS/MS or EI-GC-MS/MS techniques have demonstrated their potentialities on real samples at the levels of interest but long-term stability and performances in routine condition use still need to be assessed to completely assert that those instruments can be considered equal to magnetic sector instruments in terms of food and feed control under the EU legislation.
References: 1. Commission Regulation (EU) No 252/2012 of 21 March 2012 repealing Regulation (EC) No 1883/2006 (OJ L 84, 23.3.2012, p. 1–22)2. Commission Regulation (EU) No 589/2014 of 2 June 2014 repealing Regulation (EC) No 252/2012 (OJ L 164, 3.6.2014, p. 18–40)3. Domeño, C., et al., (2012). Journal of Chromatography A, 1252(0): 146-154.4. Porteles T, Mol J.G.J; Sancho J. V., Hernandez F., (2012) Analytical Chemistry 84,9802-9810. 5. Geng D, Jogsten IE, Kukucka P, Hagberg J, Roos A, van Bavel B, (2014) Organohalogen Compd 76 in press6. Kotz A, Traag W, Winterhalter H, Malisch R, Dunstan J (2013) Organohalogen Compd 75, 678-6817. Kotz A, Malisch R, Wahl K, Bitomsky N, Adamovic K, Gerteisen I, Leswal S, Schachtele J, Tritschler R, Winterhalter H (2011) Organohalogen Compd 73 : 688-6918. Sandy C, Fürst C, Bernsmann T, Baumesiter D (2011) Organohalogen Compd 73: 1370-13719. L’Homme B, Scholl G, Eppe G, Focant JF (2014) Organohalogen compd, 76 in press
24
Evolution of the screening methods in the search for residuesPhilippe DelahautCER Groupe - Département Santé, Rue du Point du Jour 8, 6900 Marloie, Belgique
The aim of a screening method consists in detecting the possible presence of residues in the production chain. Positive samples should be quickly identified, with a minimum number of purification and extraction stages. The percentage of false positive results must be low, whereas false negative results are to be avoided.
The current screening methods for the detection of residues are mainly enzyme-linked immuno-assays of the ELISA type (Enzyme Linked ImmunoSorbent Assay) and dipstick systems for controls at the production site.
The key elements of those kinds of assays are the antibody or receptor, the labelled or not labelled antigen and the detection system. It is by improving those various elements that new methods can be developed.
a) Antibody
Most of the antibodies used for in vitro diagnosis are polyclonal or monoclonal antibodies. There are other alter-natives such as recombinant antibodies, nanobodies, aptamers, MIP’s (Molecular Imprinted Polymer) or receptors intended to produce the ligands essential to the antigen binding. The main developments in this field consist in trying to obtain ligands that are binding to several molecules of the same group in order to get a multiresidue sys-tem, which is particularly interesting during the screening. At present some antibodies make it possible to identify all β-agonists in one determination. Assay kits able to quantify all quinolones or the whole set of sulfamides in one test are available as well.
Receptors have the advantage of binding according to a biological or pharmacological activity. Receptors able to bind with the molecule groups of β-lactames or tetracyclines are already available. The future developments in the field of binders will mainly be intended to producing reagents with a broad spectrum and a good affinity.
2524
b) Detection methods
The detection of residues such as antibiotics in foodstuffs of animal origin is often carried out by using screening tests such as tests of microbiological inhibition, ELISA’s or immuno-chromatographic tests (Figure 1). During the last decennium some tests have been improved and new quick tests have been developed, following various trends:• Response times – In order to save time, new tests have been developed with a testing duration of maximum
3 minutes or less. • Multiplicity – Most of quick tests detected only one substance, or only substances belonging to one class
of antibiotics (essentially β-lactames). Today, generic quick tests are available for the detection of 4 classes of antibiotics or more.
• MRL – The detection capacity of quick tests has been improved during the last years, and is better appropriate to the needs of the agri-food market.
• Room temperature – Some producers of kits have been focussing on a test that could be carried out at room temperature, so that no heating system had to be used.
Figure 1: illustration of an immunochromatographic test
26
Besides that race towards an immediate and multiplexed test, which is adapted to official detection thresholds and does not require any equipment, some other approaches based upon new technologies are gradually appearing. Technologies such as micro-fluidics, surface plasmon resonance (SPR), optical biosensors and biochips are already or will soon be available for residue analysis.
Among those technologies, flow cytometry makes it possible to detect several residues of contaminants in food-stuffs at the same time. For this purpose we bind specific or generic “binders” (such as antibodies) as well as the hapten at the surface of fluorescent microparticles. Those particles can be of different sizes and fluorescences.So we can have, in the same determination, beads of 4 or 5 microns in size and about 25 different fluorescence in-tensities, generated by fluorochromes in various proportions in each bead. At the surface of each bead, a different hapten is bound. Multiplexing is obtained by multiplying beads in the same test.
The main challenge is the production of binders and the development of a generic method to extract com-pounds with sometimes very different physico-chemical characteristics. At the other side, the main advantage in comparison with classical ELISA’s is the multiplexing. The technological developments make the prospect of using portable sensors with a high-throughput analysis a possibility.
Within the CER Groupe, in the framework of a EUREKA project, we are at present finalizing the development phase of a system for detecting 10 classes of antibiotics in meat at the same time.
Numerous developments in the field of biosensors appeared in recent years. The biosensor is an analytic system that makes it possible to measure a change in the concentration of a molecule at the surface of a detector. This measurement is performed in real time, without having to use any markers. The main advantages of this technique are the quick response time, the reproducibility and the absence of interfer-ence with the matrix, so that the technique makes it possible to follow up interactions between a specific antigen and the corresponding antibody.
As a conclusion, we may expect to have still quicker and more reliable methods available for the detection of residues in the next coming years. A not inconsiderable element to be taken into consideration is the cost price for the sector.
2726
28
The trainings for the approved laboratories organized by the FASFC in co-operationwith the National Reference Laboratories are available on the website of the FASFC
(www.favv.be > Business Sectors > Laboratories > Trainings).
The schedule is updated regularly, it is therefore recommended to check the websitefrom time to time.
Other interesting workshops and symposia are mentioned below.
Workshops & Symposia
Date Subject Place More information (website)
13-17.04.2015IDF/ISO Analytical Week 2015 & Final Optimir scientific and expert meeting
Namur, Belgium www.idf-iso-analytical-week.org
20-22.04.201410th Conference RME2015Food Feed Water AnalysisInnovations and breakthroughs!
The Netherlands http://www.bastiaanse-communication.com/rme2015/
20-22.04.2015 2015 IAFP European Symposium on Food Safety Calls
Cardiff, Wales https://www.foodprotection.org/europeansymposium/
25-28.04.2015 25th European Congress of Clinical Microbiology and Infectious Diseases
Copenhagen, Denmark
http://www.eccmid.org/
21-22.05.2015
AOAC Europe – NMKL – NordVal International Symposium 2015: “Food Labs in Crystal Ball; Future Challenges in Food Analysis”
Stockholm, Sweden
Jointly organised by AOAC Europe, NMKL and NordVal International.http://www.aoaceurope.com/
June 2015Global Forum on Genetically Modified Wheat
http://www.bastiaanse-communication.com/html/ upcoming.html
7-11.06.20156th Congress of European Microbiologists (FEMS 2015 Congress)
Maastricht, Nederland
http://fems-microbiology.kenes.com/
16-19.06.2015SASKATOON INTERNATIONAL WORKSHOP on VALIDATION and REGULATORY ANALYSIS
Calgary, Alberta, Canada
http://www.saskval.ca/
22-24.06.2015World Congress and Expo on Applied Microbiology
Frankfurt, Germany
http://www.jmbfs.org/conference/world- congress-and-expo-on-applied-microbiology/
8-12.09.20159th International Conference on Predictive Modelling in Foods (ICPMF)
Rio de Janeiro, Brazil
http://icpmf9.com/
15.09.2015 Mass Spectrometry in Food and Feed II Gent, Belgium www.voeding.kvcv.be
8-9.10.2015 20th Conference on Food Microbiology Brussels, Belgium http://www.bsfm.be/
3-6.11.20157th International Symposium on Recent Advances in Food Analysis (RAFA 2015)
Prague, Czech Republic
www.rafa2015.eu
2928
