AFRICAN BEE HEALTH SURVEILLANCE SAMPLING CHECKLIST

Transcription

1 AFRICAN BEE HEALTH SURVEILLANCE SAMPLING CHECKLIST AFRICAN BEE HEALTH SAMPLING PROTOCOL The following sampling plan will be used during routine surveillance activities. To avoid cross-contamination: 1. Wear gloves at all times. 2. Change gloves or disinfect gloves between hives. 3. Change hive tools or Disinfect hive tools before use in the next hive 4. Use clean and sterile tubes, forceps, etc. Label all tubes correctly. 5. A GPS location of the sampling site should be noted. S/No. ORDER OF SAMPLING Tube selection Quantity Sampled PLACE IN 70% ETHANOL Pests and Behaviour TRANSFER SAMPLES TO LIQUID NITROGEN Chemical Ecology Pathology 1. Surveillance data collection (From Questionnaire) 2. Foraging bees 2x50ml Falcon 1 Pool of 50 & 1 pool of Small hive beetle (Aethina tumida) 50ml Falcon Collect all 15ml CryoVial 4. Large hive beetle (Oplostomus fuligineus Olivier) 50ml Falcon Collect all 5. Large hive beetle (Oplostomus haroldi Witte) 50ml Falcon Collect all 6. The greater Wax moth (Galleria mellonela) 50ml Falcon Collect all 7. The lesser Wax moth (Achroia grisella) 50ml Falcon Collect all 8. Varroa destructor 15ml CryoVial 9. Bee louse (Braula species) 15ml CryoVial 3 sugar shakes 3 sugar shakes 10. Nurse bees 50ml Falcon Pool of Worker Pupae in comb (pink-eyed stage) 2x50ml Falcon Pool of Drone Pupae in comb (pink-eyed stage) 2x50ml Falcon Pool of Dying or recently dead bee samples( to be collected only when a significant number of dead bees is present) Adult bees acting abnormally (e.g., ataxia, lethargy, jerking movements) Pollination Ecology 50ml Falcon Pool of 50 50ml Falcon Pool of Sugar ants 15ml CryoVial Collect all 16. Pseudoscorpions 15ml Falcon Collect all 17. Phorid flies 15ml Falcon Collect all 18. Wasps 15ml CryoVial Collect all 19. Beebread 15ml CryoVial 20. Honey 2x15ml CryoVial & Ziplock bag 21. Comb Wax / Beeswax 2x15ml CryoVial 5x4cm strip of comb 5x3cm strip of wax 5x1cm strip of comb 5x1cm strip of comb 5x1 cm strip of wax 5x1 cm strip of comb 5x2 strip of wax 22. Propolis 2x15ml CryoVial 10g 23. Any other? NB: Tracheal mites will be investigated from the nurse bees as need arises. Royal jelly will be collected from the hypo-pharyngeal glands of 7 day old nurse bees as need arises. 5x2 cm strip of comb in Bag 1

2 CHECKLIST FOR MATERIALS/EQUIPMENT REQUIRED TO SAMPLE AN APIARY Shared Materials & Equipment Camera 1 GPS recording device 1 Portable 100 Lt refrigerated Cool box 1 Sample ID Labels/Barcode labels 1,000 Smoker 1 Smoker Fuel (wood shavings) 1 sack Hive tools 10 Match box 1 PDA barcode scanner 1 Powder free Disposable Gloves Reusable rubber gloves L, M, S 10 pairs Garbage/waste collection bags 2 White A4 sheets 1 ream Flash lights/torch 2 Flip charts 1 Pairs of scissors 2 Masking tape 1 roll 250ml plastic cup ml Bee Mason glass jar 15 70% Ethanol (molecular grade) 5 Lt 0.5% Hypochlorite solution 5 Lt Toothpicks 1,000 6x4 ft Bed net housing 1 Aluminium foil (large) 1 roll Portable stools 2 Paper towel rolls 1 A roll of Parafilm 1 Marker pen/pencil 4 Ice packs 10 Spray bottles ethanol, distilled water 5 Distilled water 10ml containers 5 Lt 50L Liquid nitrogen canister 2 First Aid kit with antihistamines 1 MATERIALS AND EQUIPMENT REQUIREMENTS FOR SAMPLING Pathology (Systematic & Symptomatic) Pathology (Culture) Pests and Behaviour Chemical Ecology Pollination Ecology 2

3 Shared Materials & Equipment Marker pens (Cryogenic) 4 MATERIALS AND EQUIPMENT REQUIREMENTS FOR SAMPLING Pathology (Systematic & Symptomatic) Pathology (Culture) Pests and Behaviour PPE (Bee suit + gloves + footwear) 2 sets 2 sets 2 sets 2 2 Laptop Clinical Signs Checklist (1 for each apiary) 1 1 Insect vacuum aspirator (BeeVac) Sterile Insect vacuum collection chambers Chemical Ecology Pre-labelled Sterile 50ml Falcon tubes Pre-labelled Sterile 15ml wide-mouth CryoVials Pollination Ecology Ziplock bags (A4 size) Sterile capping-scratcher Blunt-nosed stainless steel forceps (serrated tips) Small brush 5 5 Sampling spatula Sterile scalpel blade ml falcon-tube rack Chinese style grafting tools Nunc CryoFlex tubing 2,000 Heat Gun for Cryoflex Tubing 1 Sterile soil Sterile cow dung Perforated plastic rearing bowls with lids 10 Glass containers with lids (for pests handling) 20 A rectangular metal bowl (30 cm x 20 cm) Tablespoons 2 Icing sugar 5kg 5kg 2kg/apiary Sterile Cotton wool 1 3

4 ITEM 1. Individual hive Surveillance data (From Questionnaire) DETAILED DESCRIPTION OF SAMPLING S TITLE: Gathering Hive Information PURPOSE: To record notifiable changes in the hive so as to update the information in the questionnaire which is relevant to surveillance. Name of apiary Climate/weather of apiary location (meteorological data) Apiary conditions and Husbandry Practices Description of the environment of the apiary (within 10 km radius foraging range) Major forage plants in the environment of the apiary (within 10 km radius foraging range) Environmental stressors Bee Colony population Dynamics (adults, brood and honey production) Feral Colonies Pests Predators Observed disease symptoms in honeybees 2. Foraging bees TITLE: Sampling Procedure for Foraging Bees PURPOSE: To collect foraging honeybees with pollen visibly attached to the pollen pocket as they return to the hive. Properly put on bee suit. Ensure that no entry points for adult honey bees exist Wear fresh disposable gloves or disinfected reusable rubber gloves. Light the bee smoker. Sterilize hive tools. Record the hive identity (e.g. read the hive coordinates using the barcode scanner). Place a new (unused) sterile Insect Vacuum Collecting Chamber onto the Insect vacuum aspirator. Block the hive entrance using masking tape. The foraging bees will accumulate at the entrance. Prepare sterile two pre-labelled 50 ml falcon tubes (for Pathology, Chemical Ecology). Aspirate 50 and 20 bees returning with pollen loads (for each tube) by using portable insect vacuum aspirator. Remove and cap the collection tube containing bees and replace with an empty one. (Ensure the collected bees are at the bottom of the tube before uncapping). Transfer the 20 and 50 bees into their respective sterile pre-labelled 50 ml tubes. Place the falcon tubes with the bee samples back into their respective ziplock bags. Remove the used Insect Vacuum Collecting Chamber from the Insect vacuum aspirator. 4

5 3. Small hive beetle (Aethina tumida) TITLE: Sampling Procedure for Small Hive Beetle (Aethina tumida) PURPOSE: To enumerate, collect and store small hive beetles from honeybee colonies. Place a new unused sterile Insect Vacuum Collecting Chamber onto the Insect vacuum aspirator. Light the smoker using fuel (dry& fresh grass/paper) Apply 5-10 smoke puffs at the hive entrance and pause briefly (30sec) to allow the smoke take effect Use a fresh and clean hive tool to open the hive. Gently lift up topboard/hive lid. Inspect the hive in the following sequence: topboard/hive lid, the bottom board, the frames, the brood chamber and the inner wall. Aspirate and count number of beetles crawling in each section of the hive. Transfer some small hive beetles from the Insect Vacuum Collecting Chamber into a sterile pre-labelled 50ml Falcon tube/15ml wide-mouth CryoVial. Place the falcon tubes with the bee samples back into their respective ziplock bags. Remove the used Insect Vacuum Collecting Chamber from the Insect vacuum aspirator. Record the number of beetles recovered from the top board/lid, bottom board, frames, and brood chamber walls separately on a hive card. For laboratory stock cultures, pour an aliquot of beetles into a mason jar with pollen dough. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are <30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Transfer sample aliquot to liquid nitrogen. Keep live beetles into glass containers in a shock-cushioned well-aerated space. 4. Large hive beetle (Oplostomus Olivier) fuligineus Ship in well-cushioned sample boxes prior to transportation from field site to laboratory. TITLE: Sampling Procedure for Large Hive Beetles (Oplostomus species) PURPOSE: To enumerate, collect and store large hive beetle samples collected from honey bee colonies Inspect the hive in the following sequence: topboard/hive lid, the bottom board, the frames, the brood chamber and the inner wall. Handpick and count number of large hive beetles crawling on the inside part and edges of the lid (use 2-3 persons to prevent beetles from escaping) Place the beetles into a sterile pre-labelled 50ml Falcon tube. Place the falcon tubes with the large hive beetles back into their respective ziplock bags. 5

6 Record the number of beetles recovered from the top board/lid, bottom board, frames and brood chamber walls separately on a hive card. For laboratory stock cultures, maintain a portion of beetles in plastic rearing bowls/glass jars containing either cowdung & soil or moist cotton wool only. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Transfer aliquot to liquid nitrogen. Place glass containers holding live beetles in a shock-cushioned well aerated space. Repackage field samples into well-cushioned sample boxes prior to transportation from field site to laboratory. 5. Large hive beetle (Oplostomus haroldi Witte) Same as for Large hive beetle (Oplostomus fuligineus Olivier) 6. The greater Wax moth (Galleria mellonela) TITLE: Sampling Procedure for Wax Moth (Galleria & Achroia species) PURPOSE: To enumerate, collect and store wax moth samples collected from honey bee colonies Inspect the hive in the following sequence: topboard/hive lid, the bottom board, the frames, the brood chamber and the inner wall. Handpick and count number of wax moth larvae, pupae and adults on the inside part and edges of the lid Place the samples (larvae, pupae and adults) into a sterile pre-labelled 50ml Falcon tube. Place the falcon tubes with the samples back into their respective ziplock bags. 6

7 Record the number of wax moth stages recovered from the top board/lid, bottom board, frames, and brood chamber walls separately on a hive card. For laboratory stock cultures, maintain a portion of wax moth larvae and pupae in plastic bowls/glass jars containing moist cotton wool and honey wax. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Place glass/bowls containers holding live insects in a shock-cushioned well aerated space. Repackage field samples into well-cushioned sample boxes prior to transportation from field site to laboratory. 7. The lesser Wax moth (Achroia grisella) Apply the same protocol as that described for the greater Wax moth above. 8. Varroa destructor TITLE: Sampling Procedure for Varroa Mites (Varroa destructor) PURPOSE: to enumerate, collect and store mite samples collected from honey bee colonies Gently remove a frame containing brood (replicates 1 & 2), pollen and honey (replicate 3) and shake the bees in to a metal bowl Slightly tilt the bowl to consolidate the bees in to a corner Use a 250ml plastic cup to scoop a group of bees (~300) in to the 500ml Bee Mason glass jar. Screw tightly the lid of the jar and pour 2-3 tablespoons full of icing sugar via the perforations in the lid (bee screen mesh). Vigorously shake the bees in the jar for about 30s Allow the jar to sit for about one minute. Shake the icing sugar and the dislodge mites through the perforated lid into a wide-mouth plastic bowl Add more sugar and shake until there is no more mite drop after shaking. Pick the mites from the icing sugar using a toothpick /fingers and place them into sterile pre-labelled 15ml CryoVial. Place the tubes with the samples back into their respective ziplock bags. 7

8 Count the number of Varroa mites in the bowl obtained during all three shakes, and record on the hive card. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Place tubes holding insects in a ziplock bag and store in a Refrigerated Cool box at 4 C. 9. Bee louse (Braula species) Repackage field samples into well-cushioned sample cool boxes prior to transportation from field site to laboratory. TITLE: Standard Operating Procedure (SOP) For Bee Louse (Braula species) Enumeration, Collection and Storage PURPOSE: To enumerate and store bee louse collected from honey bee colonies Gently lift up topboard/hive lid. Gently remove a frame containing brood (replicates 1 & 2), pollen and honey (replicate 3) and shake the bees in to a metal bowl Slightly tilt the bowl to consolidate the bees in to a corner Use the plastic cup to scoop a group of bees (~300) in to the 500ml Bee Mason glass jar. Screw tightly the lid of the jar and pour 2-3 tables spoon full of icing sugar via the perforations in the lid (bee screen mesh). Vigorously shake the bees in the jar for about 30s Allow the jar to sit for about 1 min Shake the icing sugar and the dislodge lice through the perforated lid unto a white sheet of paper (A4) Add more sugar and shake until there is no more louse drop after shaking. Place the samples (bee louse) into a sterile pre-labelled 15ml CryoVial. Place the tubes with the samples back into their respective ziplock bags. Count the number of Varroa mites on the A4 sheet obtained during all three shakes, and record on the hive card. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Place tubes holding insects in a ziplock bag and store in a Refrigerated Cool box at 4 C. 8

9 Repackage field samples into well-cushioned sample cool boxes prior to transportation from field site to laboratory. 10. Nurse bees TITLE: Procedure for Sampling of old Nurse Bees PURPOSE: To sample nurse bees for disease surveillance. Place a new (unused) sterile Insect Vacuum Collecting Chamber onto the Insect vacuum aspirator. Prepare a sterile pre-labelled 50ml Falcon tube/15ml wide-mouth CryoVial. Scoop 50 nurse bees from the combs directly in two sterile pre-labelled 50ml Falcon tubes. Place the falcon tubes with the bee samples back into their respective ziplock bags. Place the ziplock bags in a Refrigerated Cool box at 4 C for 10 mins to knock out bees. For only - Add alcohol (70% ethanol) into the sample vial to preserve the bees. 70% Ethanol 11. Worker Pupae (Pink-eye stage) TITLE: Sampling of Worker Pupae (pink-eye stage) PURPOSE: To sample honeybee pink-eyed pupae for disease surveillance. Select frames with days old worker pupae (Pink-eyed to gray-eyed with ivory body). With the aid of a sterile capping-scratcher, uncap the brood cells. Select frames with days old worker pupae (Pink-eyed to gray-eyed with ivory body). Using a sterile scalpel blade, excise two 5x2cm sections with worker pupae and gently place them into the first sterile pre-labelled 50 ml Falcon tube one on top of the other (do not crush them in). Repeat the above process for the second tube. Place the tubes with the samples back into their respective ziplock bags. Do not re-use the forceps and capping-scratcher before they are sterilized Record any abnormal appearance 9

10 12. Drone Pupae (Pink-eye stage) Sample days old drone pupae (Pink-eyed to gray-eyed with ivory body) as described for the worker pupae above. 13. Dying or recently dead bee samples TITLE: Sampling of Dying or Recently Dead Bee Samples PURPOSE: To sample dying or recently dead honeybees for disease diagnosis. Prepare a sterile pre-labelled 15ml wide-mouth CryoVial. Collect 50 dying or recently dead bees into a sterile pre-labelled 15ml wide-mouth CryoVial. Place the falcon tubes with the bee samples back into their respective ziplock bags. Remove the used Insect Vacuum Collecting Chamber from the Insect vacuum aspirator. 14. Adult bees acting abnormally (e.g., ataxia, lethargy, jerking movements) TITLE: Sampling of Adult Bees Acting Abnormally PURPOSE: To sample adult honeybees acting abnormally for disease diagnosis. Prepare a sterile pre-labelled 15ml wide-mouth CryoVial. Collect two sets of 50 adult bees acting abnormally into a sterile pre-labelled 50 ml Falcon tube. Place the falcon tubes with the bee samples back into their respective ziplock bags. Remove the used Insect Vacuum Collecting Chamber from the Insect vacuum aspirator. 10

11 15. Sugar ants TITLE: Sampling of Sugar ants PURPOSE: To sample sugar ants for disease diagnosis. Prepare a sterile pre-labelled 15ml wide-mouth CryoVial. Sweep a stream of sugar ants using a small brush into the tube Place the tube with the sugar ants back into their respective ziplock bags. Pest & Behaviour samples Collect samples for species identification in the laboratory. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Pest & Behaviour samples Pest & Behaviour samples Store samples in 70% molecular grade ethanol in falcon tubes. Transport in ziplock bags at room temperature 16. Pseudoscorpions TITLE: Procedure for Sampling Pseudoscorpions PURPOSE: To enumerate, collect and store Pseudoscorpions collected in honey bee colonies Gently lift up the brood chamber along with the hive lid. Visually inspect the bottom board for pseudoscorpions Collect any pseudoscorpions seen using an aspirator Place the samples (pseudoscorpions) into a sterile pre-labelled 15ml Falcon tubes. Place the tubes with the samples back into their respective ziplock bags. Pests and Behaviour samples should be preserved in 70% molecular grade ethanol. Count the number of pseudoscorpions and record on the hive card Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist 11

12 Repackage field samples into well-cushioned sample boxes prior to transportation from field site to laboratory. Repackage field samples into well-cushioned sample cool boxes prior to transportation from field site to laboratory. 17. Phorid flies TITLE: Procedure for Sampling Phorid flies PURPOSE: To enumerate, collect and store phorid flies collected in honey bee colonies Light smoker using fuel (dry& fresh grass/paper). Properly put on bee suit. Ensure that no entry points for adult honeybees exist. Apply 5-10 smoke puffs at hive entrance and pause briefly (30sec) to allow the smoke take effect. Gently mount mosquito net over 1-2 honey bee colonies overnight Collect bees trapped within the net the following morning and maintain alive in laboratory Use a vacuum pump to collect foragers returning and leave the hive Rear puparia to adults in Petri dishes at C Count the number of phorid larvae from dead bees and record. Hand over individuals to pathology group and preserve the rest in 70% ethanol Hand over all samples when individual arthropods are < 30 Collect enumeration and identification data recorded by the Pests & Behaviour scientist Maintain collected bee samples on water/honey in the lab for a period of two weeks for emergence of phorid larvae from dead bees 18. Wasps Follow the same procedure as for the pseudoscorpions 19. Beebread TITLE: Sampling of Beebread PURPOSE: To sample Beebread for surveillance. Select a frame with fresh beebread not entombed beebread (beebread covered by propolis). Fresh beebread appears dry, grainy and sometimes colourful in the cells usually surrounded by brood frames. Use the blunt end of a sterile scapel and insert it all the way to the bottom of the cell and rotate the stick all the way around the cell scraping the beebread as you go. Scoop from each side of the comb (10 cells/comb spatially spread across the face of the comb (for Chemical Ecology and Pollination) and place the sample into sterile pre-labelled 15 ml wide mouth CryoVials. 12

13 Using a sterile scalpel blade, excise 5x1cm strip of comb with beebread (for Pathology) Transfer the strip of comb with beebread into sterile pre-labelled 15ml wide-mouth CryoVials. Place the tubes with the samples back into their respective ziplock bags. Pollination Ecology Samples Pollination Ecology Samples Pollination Ecology Samples 20. Honey: TITLE: Sampling of Honey PURPOSE: To sample Honey for pesticides and disease surveillance. Select a frame with mature (capped) honey. Excise two5cm x 1cm and one 5x2 cm strip of combed honey using a sterile scalpel blade. Transfer each of the two 5x1 cm of honey comb into a sterile pre-labelled 15ml wide-mouth CryoVial and the 5x2 cm honey comb strip (preferably mature honey) into a ziplock bag. Place the tubes with the samples back into their respective ziplock bags. Pollination Ecology Samples Pollination Ecology Samples Pollination Ecology Samples Ship at 4 C 13

14 21. Comb Wax TITLE: Sampling of Comb Wax PURPOSE: To sample comb wax for pesticides and disease surveillance. Select an empty frame (with no honey, beebread or brood). The sampled area is to be selected with regard to its cleanliness whatever its colour (new or old wax). Excise three sets of 5x1cm strip of wax (approximately 15 g) using a sterile scalpel blade. Transfer the wax into a sterile pre-labelled 15ml wide-mouth CryoVial. Place the tubes with the samples back into their respective ziplock bags. Record whether the wax sample is new (clear) or old (dark). 22. Propolis TITLE: Sampling of Propolis PURPOSE: To sample Propolis for pesticides and disease surveillance. Using a plain hive tool, scrape propolis from hive walls, frame bars, isolating material, around hive entrances and different cracks. Transfer approximately 15 g of propolis into a sterile pre-labelled 15ml wide-mouth CryoVial. Place the tubes (no of tubes) with the samples back into their respective ziplock bags. Record whether the wax sample is new (clear) or old (dark). 14

15 23. Any other Note the protocol used as appropriate. 24. Closing of the hive and work in the apiary Using the Hive sampling checklist, ensure the correct number of samples have been collected in each section. Return inspected frames into an empty brood chamber. Assemble the hive to its original state. Put back the hive lid. Discard disposable gloves Disinfect hive tools Disinfect re-useable rubber gloves Disinfect any other re-useable tools NB: After sampling from all hives in the apiary, clear out all the debris into the garbage bag. 15

16 SYMPTOMATIC BEES SAMPLING CHECKLIST AFRICAN BEE HEALTH SAMPLING PROTOCOL The following sampling plan will be used during routine surveillance activities. To avoid cross-contamination: 1. Wear gloves at all times. 2. Change gloves or disinfect gloves between hives. 3. Change hive tools or Disinfect hive tools before use in the next hive 4. Use clean and sterile tubes, forceps, etc. Label all tubes correctly. 5. A GPS location of the sampling site should be noted. S/No. ORDER OF SAMPLING Tube selection Quantity Sampled TRANSFER SAMPLES TO LIQUID NITROGEN 1. Clinical Signs Checklist (1 for each apiary) 2. Foraging bees 50 ml Falcon Pool of Nurse bees 50 ml Falcon Pool of Drone Pupae in combs (pink-eyed stage) 2x50 ml Falcon Pool of Worker Pupae in combs (pink-eyed stage) 2x50 ml Falcon Pool of Dying or recently dead bee samples 50 ml Falcon Pool of Adult bees acting abnormally (e.g., ataxia, lethargy, jerking movements) NB: Use the protocol previously described for each sample. 50 ml Falcon Pool of 50 SAMPLING CHECKLIST FOR BEE PATHOGEN CULTURE The following sampling plan will be used during routine surveillance activities. To avoid cross-contamination: 1. Wear gloves at all times. 2. Change gloves or disinfect gloves between hives. 3. Change hive tools or Disinfect hive tools before use in the next hive 4. Use clean and sterile tubes, forceps, etc. Label all tubes correctly. 5. A GPS location of the sampling site should be noted. S/No. NATURE OF SAMPLE Tube selection Quantity Sampled TRANSFER SAMPLES TO LIQUID NITROGEN 1. Clinical Signs Checklist (1 for each apiary) 2. Bacterial culture material 15ml CryoVial Pool of Viral culture material 15ml CryoVial Pool of Fungal culture material 15ml CryoVial Pool of Nosema culture material 15ml CryoVial Pool of 50 NB: - Pre-select the appropriate sample material (adult bee, brood or pest) for each pathogen based on the laboratory results. - Use the protocol previously described for each sample. 16