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Antibiotic Honey Screening The natural choice Antibiotic Honey Screening The natural choice

The Honey Industry Explained The honey industry is broken up into different segments according The Honey Industry Explained The honey industry is broken up into different segments according to the number of hives each company has: Using the USA as an example we can see this breakdown: • 98, 400 Hobby Honey Producers • Less than 9 hives • 15, 600 Side-line Honey Producers • Less than 200 hives but more than 10 • 6, 000 Commercial Honey Producers • More than 200 hives *Please note that you will need to research your own region, as each country will differ on how their industry is divided

The Honey Industry Explained • The target market for the Evidence Investigator will be The Honey Industry Explained • The target market for the Evidence Investigator will be Commercial Producers as well as Packers and Exporters • However, as a secondary target market, we may be able to offer ELISA kits to some of the smaller companies depending on sample throughput etc. We listen to each industry and are currently providing a comprehensive antibiotic portfolio that is covering the majority of our customers testing needs

2012 Statistics on Production *New Statistics available in 2016 Global Honey Production 2012 Statistics on Production *New Statistics available in 2016 Global Honey Production

Global Honey Production (2012) Rank Country Quantity (MT) Value ($1000) 1 China 436, 000 Global Honey Production (2012) Rank Country Quantity (MT) Value ($1000) 1 China 436, 000 1, 094, 110 2 Turkey 88, 162 221, 236 3 Argentina 75, 500 189, 461 4 Ukraine 70, 134 175, 996 5 USA 66, 720 167, 426 6 Russia 64, 898 162, 856 7 India 61, 000 153, 075 8 Mexico 58, 602 147, 057 9 Iran 48, 000 120, 452 10 Ethiopia 45, 905 115, 195

Global Honey Exports (2012) Rank Country Quantity (MT) Value ($1000) 1 China 110, 158 Global Honey Exports (2012) Rank Country Quantity (MT) Value ($1000) 1 China 110, 158 215, 051 2 Argentina 75, 135 215, 147 3 Mexico 32, 040 101, 497 4 India 24, 515 59, 882 5 Germany 22, 262 127, 246 6 Vietnam 21, 538 54, 826 7 Spain 19, 661 79, 843 8 Canada 18, 325 73, 820 9 Belgium 16, 726 54, 773 10 Brazil 16, 707 52, 348

Global Honey Imports (2012) Rank Country Quantity (MT) Value ($1000) 1 USA 141, 017 Global Honey Imports (2012) Rank Country Quantity (MT) Value ($1000) 1 USA 141, 017 429, 962 2 Germany 84, 129 279, 468 3 Japan 36, 823 105, 382 4 United Kingdom 33, 231 107, 575 5 France 25, 481 92, 810 6 Spain 21, 081 48, 292 7 Belgium 20, 810 55, 841 8 Italy 15, 221 56, 112 9 Poland 14, 118 34, 611 10 Netherlands 12, 942 44, 855

Status of Honey Laboratories - Worldwide Status of Honey Laboratories - Worldwide

Status of Honey Laboratories – Worldwide 2014 Status Total Private Exporter 279 Private Laboratory Status of Honey Laboratories – Worldwide 2014 Status Total Private Exporter 279 Private Laboratory 21 Government Laboratory 72 Research Institute 21 Overall Total 393

Percentage of Laboratories by Continent Percentage of Laboratories by Continent

Background • The Use of Antibiotic Drugs in Apiculture is globally restricted • There Background • The Use of Antibiotic Drugs in Apiculture is globally restricted • There are still no MRLs set for Antibiotics in Honey • As Honey is seen as a ‘Natural Product’ consumers want for it to be antibiotic free • Constant pressure on laboratories to ensure that there are little to no antibiotics present within Honey • Analytically, exporters/ importers require certificates showing that each batch of honey is free of antibiotics NB: For any customer requesting 0 ppb for Honey testing this is methodically impossible – there will always be analytical limitations with ALL methods including LC/MS MS

Testing • Typically a certificate needs to be produced from a confirmation laboratory to Testing • Typically a certificate needs to be produced from a confirmation laboratory to show that the honey is free of antibiotics, or it is below a certain threshold • However some Honey Importers/ Exporters utilise internal screening methods in order to check the raw Honey from Bee keepers OR • as a secondary check to ensure the Honey that they are receiving is as what is provided on the confirmation certificate • Randox produce a quantitative bench top system for the internal screening of Antibiotics within Honey

Why use Antibiotics? American Foulbrood / European Foulbrood: • Caused by the spore- forming Why use Antibiotics? American Foulbrood / European Foulbrood: • Caused by the spore- forming Paenibacillus larvae ssp. Larvae, is the most widespread and destructive of the bee brood diseases • Larvae up to 3 days old become infected by ingesting spores that are present in their food. Young larvae less than 24 hours old are most susceptible to infection • Spores germinate in the gut of the larva and the vegetative form of the bacteria begins to grow, taking its nourishment from the larva • The vegetative form of the bacterium will die but not before it produces many millions of spores. Each dead larva may contain as many as 100 million spores. This disease only affects the bee larvae but is highly infectious and deadly to bee brood

Varroa Mites: Why use Antibiotics? • Varroa destructor can only reproduce in a honey Varroa Mites: Why use Antibiotics? • Varroa destructor can only reproduce in a honey bee colony • It attaches to the body of the bee and weakens the bee by sucking hemolymph. In this process, RNA viruses such as the deformed wing virus (DWV) spread to bees • A significant mite infestation will lead to the death of a honey bee colony, usually in the late autumn through early spring • The Varroa mite is the parasite with the most pronounced economic impact on the beekeeping industry • It may be a contributing factor to colony collapse disorder, as research shows it is the main factor for collapsed colonies around the world

Colony Collapse Disorder (CCD): Why use Antibiotics? • A phenomenon in which worker bees Colony Collapse Disorder (CCD): Why use Antibiotics? • A phenomenon in which worker bees from a beehive abruptly disappear • While such disappearances have occurred throughout the history of apiculture, and were known by various names (disappearing disease, spring dwindle, May disease, autumn collapse, and fall dwindle disease) • The syndrome was renamed colony collapse disorder in 2006 in conjunction with a drastic rise in the number of disappearances of Western honeybee colonies in North America • The mechanisms of CCD and the reasons for its increasing prevalence remain unclear, but many possible causes have been proposed: pesticides; infections with mites; malnutrition; genetic factors; immunodeficiencies; loss of habitat; changing beekeeping practices; or a combination of factors

What Antibiotics? While there are some antibiotics that are of more importance in particular What Antibiotics? While there are some antibiotics that are of more importance in particular countries, the majority will be of interest to all companies; especially those who are importing and exporting They include: • Sulphonamides • Tetracyclines • Chloramphenicol • Quinolones • Nitrofurans • Streptomycin • Macrolides

What Antibiotics? Sulphonamides: • A group of antimicrobials widely used for therapeutic and prophylactic What Antibiotics? Sulphonamides: • A group of antimicrobials widely used for therapeutic and prophylactic treatment of animal disease • Worldwide, there are concerns that the widespread use of antibiotics, results in toxic or allergic reaction, or the development of resistance in the animal and human population • Repeated low dosage is the ideal environment within which resistance factors can develop and spread. This has reduced the effectiveness of sulphonamide use in veterinary medicine and could have serious implications for human medicine • 100 ppb for the whole Sulpha group worldwide allowance. i. e. If one is detected at 60 ppb, and another two at 30 ppb, then that’s a cumulative score of 120 ppb is a violation

What Antibiotics? Tetracyclines: • A broad-spectrum antibiotic produced by the Streptomyces bacterium, indicated for What Antibiotics? Tetracyclines: • A broad-spectrum antibiotic produced by the Streptomyces bacterium, indicated for use against many bacterial infections • Their general usefulness has reduced with the onset of bacterial resistance. However they continue to remain the treatment of choice for some specific indications. • Within the animal market they are used to treat livestock populations for disease however with the onset of resistance in different strains, legislation has been introduced to strictly regulate their use

What Antibiotics? Chloramphenicol: • Effective against a wide variety of micro organisms, but due What Antibiotics? Chloramphenicol: • Effective against a wide variety of micro organisms, but due to serious side-effects (e. g. , damage to the bone marrow, including Aplastic Anaemia) in humans, it is usually reserved for the treatment of serious and lifethreatening infections (e. g. , typhoid fever). • It is widely tested for in all meat, fish, milk and animal products • It is also reported to have Carcinogenic effects

What Antibiotics? Macrolides (Tylosin; Tilmicosin): • • • Macrolides are used to treat infections What Antibiotics? Macrolides (Tylosin; Tilmicosin): • • • Macrolides are used to treat infections such as respiratory tract infections and soft tissue infections The antimicrobial spectrum of macrolides is slightly wider than that of penicillin Unlike penicillin, macrolides have shown effective against mycoplasma, mycobacteria, some rickettsia and chlamydia Nitrofurans • Antimicrobial drug which is abused commonly in fisheries; hence lots of testing for this one. • There are four main classes of Nitrofurans: AMOZ; AHD; AOZ; SEM

What Antibiotics? Quinolones: • A group of broad-spectrum antibiotics which are highly potent and What Antibiotics? Quinolones: • A group of broad-spectrum antibiotics which are highly potent and at one time they were considered relatively safe. However, they can have potentially troublesome and irreversible side effects. For example, numerous case reports (since 1965) have implicated their use in cases of spontaneous tendon ruptures and tendon damage, especially with the concurrent use of a systemic corticosteroid Aminoglycosides (Streptomycin; Dihydrostreptomycin): • • • Aminoglycosides are a group of antibiotics that are effective against certain types of bacteria. Streptogramins are a class of antibiotics. Members include: • Pristinamycin • Quinupristin/ dalfopristin

Maximum Residue Limits http: //www. cseindia. org/userfiles/Antiboitics_Honey. pdf Maximum Residue Limits http: //www. cseindia. org/userfiles/Antiboitics_Honey. pdf

Key Honey Accounts Key Honey Accounts

Optional Extras for AM III Sample Preparation mi. Vac Sample Evaporator • As part Optional Extras for AM III Sample Preparation mi. Vac Sample Evaporator • As part of the AMIII procedure there is the requirement to evaporate off the sample in a late stage of the preparation • The mi. Vac sample concentrator Duo allows samples to be evaporated off without the need of a fume hood within a contained environment

Optional Extras for AM III Sample Preparation Rotina Centrifuge • Required to separate the Optional Extras for AM III Sample Preparation Rotina Centrifuge • Required to separate the sample for the extraction of the Ethyl Acetate layer • Two sizes available –Rotina 320 R • 4 x 43 ml tubes • 24 x 15 ml tubes –Rotina 380 R • 20 x 43 ml tubes • 28 x 15 ml tubes • Variable temp -20 C to +40 C • Fast cool function • Easy rotor changing

Optional Extras for AM III Sample Preparation Stuart Vortexer • Required to help dissolve Optional Extras for AM III Sample Preparation Stuart Vortexer • Required to help dissolve the honey (a roller is also acceptable) • Variable speed control (200 -2500 rpm) • Intermittent or continuous mode

Optional Extras for AM III Sample Preparation • 20 – 200 µl and 200 Optional Extras for AM III Sample Preparation • 20 – 200 µl and 200 – 1000 µl Pipettes are available, as well as pipette tips • Multichannel and stepper pipettes are also available • Vials and other glassware an imperative part of sample preparation • Test tubes and test tube racks can also be provided if required Controls can also be purchased for QC in 3 x 1 ml kits for each array

AM I Advantages (EV 3843) • Low Limits of Detection compared to competitors • AM I Advantages (EV 3843) • Low Limits of Detection compared to competitors • Individual Sulphonamide identification along with Trimethoprim and Dapsone • Trimethoprim is commonly found with Sulphonamides as it helps with the absorbance of this drug family • No PABA interference in honey resulting in excellent specificity • Para-Amino. Benzoic Acid is structurally similar to Sulphonamides, therefore some systems can mistakenly detect PABA as Sulphas – resulting in false positives

AM II Advantages (EV 3524 A/B) • Low Limits of Detection compared to competitors AM II Advantages (EV 3524 A/B) • Low Limits of Detection compared to competitors • Four major antibiotic groups detectable using one honey sample • Generic detection of all major Tetracycline metabolites and epimers • No other platform can detect these drug residues simultaneously

II International Symposium on Bee Products Annual meeting of Evaluation and Validation of a II International Symposium on Bee Products Annual meeting of Evaluation and Validation of a Biochip Multi-Array technology for the screening of antibiotic residues in honey according to the European guideline for the validation of screening methods Valerie Gaudin*, Celine Hedou, Eric Verdon the International Honey Commission Bragança, Portugal Anses, Laboratory of Fougeres, European Union Reference Laboratory (EU-RL) for Antimicrobial and Dye Residue Control in Food-Producing Animals 9 -12 September 2012 This study was ran in accordance with the 657/2002/EC Directive – a copy of this Directive and the Poster presented by ANSES can be provided upon request

AM III Advantages (EV 3695) • Only major screening technology that simultaneously tests for AM III Advantages (EV 3695) • Only major screening technology that simultaneously tests for all 4 Nitrofuran metabolites • Excellent limits of detection compared to competitors and confirmatory methods • Sample preparation and screening can be performed in one day • Derivatisation step is only 2 hours (instead of 24 hours)

AM III CAP only Advantages (EV 3738) • Excellent Limits of Detection • Sample AM III CAP only Advantages (EV 3738) • Excellent Limits of Detection • Sample preparation and screening can be performed in one day • Offered for those customers not wishing to run Nitroimadazoles screening

AM IV Advantages (EV 3878 A/B) • Excellent Limits of Detection • Sample preparation AM IV Advantages (EV 3878 A/B) • Excellent Limits of Detection • Sample preparation and screening can be performed in one day • Individual identification of Aminoglycosides and Macrolides • More elaborate and extensive test for Aminoglycosides and Macrolides than AMII

AM V (EV 4027) Nitroimidazoles Array • Dimetridazole • Ronidazole • 2 -Hydroxymethyl-1 -methyl-5 AM V (EV 4027) Nitroimidazoles Array • Dimetridazole • Ronidazole • 2 -Hydroxymethyl-1 -methyl-5 -nitro-1 H-imidazole (HMMNI) • Metronidazole *Main Residue of Interest* • Metronidazole-OH (MNZOH) • Ipronidazole-OH (IPZOH) • Tinidazole • Propenidazole • Ternidazole • Carnidazole

Cross Selling – Enzymatic Tests We are in the final stages of establishing several Cross Selling – Enzymatic Tests We are in the final stages of establishing several enzymatic tests for the Honey Industry using the RX Altona • Glucose/ Fructose • Diastase (Amylase) • Colouration Instrumentation for the Kits e. g. going through the final stages of validation for this equipment We are also adding to the portfolio of ELISA and Biochip tests currently on offer • Pesticides Array will be released 4 th Quarter 2016

- Honey customers in new territories do not want to be the first to - Honey customers in new territories do not want to be the first to implement new technology - Some can be unaware of our level of support - They have a right to feel insecure about this Case Studies: New Territories The Negatives BUT - Essentially they are exporters/ Importers - They are used to dealing with all Honey throughout the globe. - Randox can supply references for new customers - companies E. g. Famille Mary in November requested two references - we supplied Bulgarian and German references to facilitate their decision - There is the possibility of 3 month demo periods with demo analysers based on minimum sale of trial kits (in European countries) - Worked very well for us in Romania recently with Tremot Dobre

- Case Studies: New Territories Honey Customers are Rivals and Competitors - They want - Case Studies: New Territories Honey Customers are Rivals and Competitors - They want to be the FIRST - They pride themselves on being at the forefront of supplying the most reliable and best Honey their country has to offer - This is reflected in their Quality Approach and essentially the techniques that are employed in the lab The Positives SO - You don’t necessarily have to be the cheapest ……just the best - We have 64 Honey exporters globally - Covering 26 Countries - Randox is at the forefront on Honey testing and we will continue to break into new territories and current ones using this strategy - But we need to offer more

Sales Probes 1. Do they have an internal laboratory that they use for screening Sales Probes 1. Do they have an internal laboratory that they use for screening drug residues? o This is the most important point to find out, if they do – there is an opportunity for RFD to present our technology o If they do they may be using ELISA, which is similar to what we offer 2. What type of analysis do they perform? o Confirmatory methods – HPLC, LC/MS-MS, GC/MS-MS o Screening methods - CHARM, ELISA (r. Biopharm, Biooscientific, Europroxima etc. ) 3. What analytes are they testing? o Sulphonamides, Tetracyclines, Quinolones, Chloramphenicol etc.

Sales Probes 4. How many samples do they test each year? o This will Sales Probes 4. How many samples do they test each year? o This will help decide whether Biochip or ELISA is the best option for them 5. Does the company export, if so to what countries, or even what companies? o Knowing countries can help us discuss the different MRL’s or regulations between different countries, enforcing the need for testing o If they are exporting to one of our current customers, this will be a good reference point for them

Sales Probes 6. Does the company send for external analysis? o Most companies probably Sales Probes 6. Does the company send for external analysis? o Most companies probably will send to either Intertek or QSI, if so they will be spending a lot of time and money on this – we can highlight the cost and time saving aspect of using the Investigator to first screen samples; this will reduce the number of samples that need to be sent for confirmation 7. How much do they spend on drug residue testing? o Some companies will be more willing than others to provide this information to us, but it acts as a good comparison tool

Frequently Asked Questions Cost This is one of the most common sales blocks. We Frequently Asked Questions Cost This is one of the most common sales blocks. We try to be as competitive as possible however some competitors cannot be beaten on kit price. If this happens we stress our benefits – lower limits of detection, easier sample preparation, quicker analysis time, ability to test for multiple residues simultaneously, a wider testing portfolio and very little peripheral materials required. All these benefits will help to reduce time, costs and labour requirements; where competitors fail to do so. In comparison to Intertek and other confirmatory methods, our technology is just a screening method and therefore positive samples will need to be sent for confirmation. However we need to remember to emphasis is the reduction in those samples that need to be sent for confirmation if the customer is first screening with our technology. The Evidence Investigator has no false negatives and less than 5% false positives, which is low in comparison to other screening methods.

Frequently Asked Questions Validation Anses and Lune de Miel have validated various arrays to Frequently Asked Questions Validation Anses and Lune de Miel have validated various arrays to screen for honey in accordance with the EU Directive 657/2002/EC (The European Guideline for the Validation of Screening Methods). In addition to this we are an ISO 13485 Accredited Manufacturer (an International Organization for Standardization (ISO) standard that represents the requirements for a comprehensive quality management system for the design and manufacture of medical devices) In addition to the various validations for the machine and kits, each individual laboratory can apply for an ISO 17025 Accreditation from DG SANCO, an independent European company We are also working on getting AOAC Accreditation, however this is a very lengthy process

Frequently Asked Questions Expertise required Our Technical Support Team are fantastic at the training Frequently Asked Questions Expertise required Our Technical Support Team are fantastic at the training they provide and all aspects of the analysis is explained in great detail. In addition to this the team provide 24/7 support and will happily run remote diagnostics sessions with the customer, provided the customer has an internet connection. Here the Technical Support Scientist will log on to the Investigator software remotely and diagnose any problems that they are having. The team also have the ability to manually process results if 6010 errors occur (this is when the camera has not taken the correct image of the biochip, meaning the software cannot calculate the residue concentration in accordance with the Relative Light Units obtained. Some laboratories are also concerned about how skilled their workers need to be in order to run the Evidence Investigator successfully, we assure each individual that the sample preparation and analysis is extremely straight forward and easy to follow – it requires less expertise than the likes of the HPLC or LC/MS-MS protocol.

Frequently Asked Questions Do we have any rapid tests? Some companies may be more Frequently Asked Questions Do we have any rapid tests? Some companies may be more interested in a rapid test to quickly detect whethere any residues present as they may not need to worry about actual specifics. However we do not currently provide any sort of rapid test. We try to sell Biochip Array Technology as the “middle point” between rapid tests and confirmatory methods. Rapid tests will not provide the customer with the detail and precision that the Evidence Investigator will; we believe that people should and do favour the Investigator as a better and more comprehensive screening method – saving them time and money in the long run.

Frequently Asked Questions Can our kits be used on any other machines, or can Frequently Asked Questions Can our kits be used on any other machines, or can our machine use any other kits? Our ELISA kits can be used on other Plate Readers and Plate Washers, and the opposite of this the Plate Washers and Plate Readers we provide can be used in conjunction with other kits. However, this is only really an advantage for us in the following way – if a site is already running competitors ELISA tests, we have the opportunity to introduce our ELISA kits for use on the existing Plate Reader. This can also be a “foot in the door” to introduce the Evidence Investigator at a later stage if their sample throughput increases. Our Biochip kits are unique to the Evidence Investigator, so they cannot be used on any other equipment and no other kits will work on our machine.

Frequently Asked Questions Do we test for pesticide residues? No, we do not have Frequently Asked Questions Do we test for pesticide residues? No, we do not have a pesticides kit at the minute, but this is something we are developing Does the colour of the honey affect results? For the most part – no, the colour of the honey does not affect the results. However, if the honey is particularly dark in appearance, the results may be less reliable than a lighter honey. This is because the darker the honey, the more chance of a higher background.

Frequently Asked Questions Does the floral type of the honey influence results? R&D are Frequently Asked Questions Does the floral type of the honey influence results? R&D are constantly working on validating different floral types for use with our Antimicrobial Arrays. We currently have between 30 and 40 floral types validated for us on the Evidence Investigator and this number is continually growing. In comparison to this, some of our competitors’ machines (CHARM for example) provide false positives when screening certain floral types due to interferents (Bonveti et al).

Frequently Asked Questions Is the Limit of Detection the same as the Limit of Frequently Asked Questions Is the Limit of Detection the same as the Limit of Quantification? This is not something that we need to know off by heart, or even how to work out, however the following is a useful paragraph to be able to provide customers with if they ask the question. The Limits of Detection (LODs) provided with our kit inserts are determined as the mean of 20 negative samples + 3 standard deviations. This would mean that at this specified LOD there is less than 5% false positives. At this moment we are working with AOAC for certification; they determine the LOD as mean of 20 negative samples + 3 standard deviations and the LOQ as the mean of 20 negative samples + 10 standard deviations. Therefore in this case the LOQ would be higher than LOD.