Blood Collection, Processing and Quality Control Adapted procedures for Kality

Tsetse flies are fed on quality-tested fresh defibrinated blood, which has been stored in a frozen condition (Wetzel and Luger 1978). The following procedures describe the collection of animal blood in the abattoir, radiation with gamma-rays (decontamination), preservation and storage in deep freeze, quality control assurance and processing of the blood into diet for feeding tsetse flies. The procedures aim to provide nutrition at a constant quality to mass-reared fly colonies maintained over a long period for field programmes. The following is the standard procedure specifically created for the insectary at Kaliti. Before collecting the blood, all the equipment that will be in contact with blood must be thoroughly cleaned and sterilized. During the blood collection in the abattoir, the fibrin is removed by mechanically stirring the blood or by adding anticoagulants to stop the blood from clotting. If slaughtering takes place on the floor, the blood collection equipment used is selected accordingly. After collection, a sample of blood from the batch is checked for microbial contamination, and bioassayed for its nutritive value using female adult flies. All blood fed to flies must first be decontaminated, for example by treatment with radiation. Blood will then be stored in frozen conditions (-20 ⁰C) until the results from the quality control tests are ready. If results show no bacterial contamination and good nutritional value, blood will be thawed and prepared to be used.

Table of contents

Blood Collection, Processing and Quality Control ..................................................................... 1 1.

Sterilization of materials in heat sterilization oven or autoclave........................................ 5

2.

Blood Collection ................................................................................................................. 6

3.

Labelling bottles and samples............................................................................................. 9

4.

Deep freeze ....................................................................................................................... 12

5.

Blood thawing................................................................................................................... 13

6.

Sampling for 25-day feeding test (bioassay) .................................................................... 15

7.

Sterilization by radiation .................................................................................................. 17

8.

Microbial screening of the blood ...................................................................................... 19

9.

25-day feeding test: bioassay ............................................................................................ 21

10.

Proportioning ................................................................................................................ 25

11.

Sampling for microbial screening of the blood............................................................. 27

12.

Use of the database ....................................................................................................... 29

Summary of blood processing procedures

Before the collection day, the containers, stirring material and any other material that will be in contact with the blood must be sterilized in the oven over 24 hours before been used. (See Procedure 1) The collection will be done in the abattoir, using 25 litre stirring containers (See Procedure 2) that allow defibrination. Blood will be sieved and poured into 4L bottles. After driving to Kality, the 4L bottles will be labelled (See Procedure 3) and stored in the cold room (See Procedure 4). Blood now must be checked for microbial contamination, bioassayed for its nutritive value and proportioned into bottles with a manageable size to be irradiated and fed to tsetse flies. According to a programmed schedule, all the 4L bottles that have been collected on a same day will be thawed following the procedure described (See Procedure 5). Blood will be mixed into a 100L container, previously sterilized, to obtain one overall sample of the blood collected each day (See Procedure 6). This sample will be later used for the bioassay (also called 25 day feeding test) and will be composed of 25 vials labelled carefully (See Procedure 7). One single bioassay will be carried out for each collection day. After sampling for the bioassay, the blood in the 100L container is proportioned into small bottles, usually with a volume of 1L or 2L, (See Procedure 11) and labelled (See Procedure 3). The labelled bottles will be frozen until the result of the bioassay is available. During the proportioning, a sample of the blood of each bottle must be taken (See Procedure 11) and labelled (See Procedure 3). This sample must be kept together with its original bottle and will later be used for microbial screening. There are now two types of samples: the ones for the bioassay, composed by 25 vials, and the ones for the microbial screening, one Eppendorf for each bottle obtained after proportioning. The bioassay samples will be irradiated (See Procedure 8), after which, the 25 day feeding test will be conducted (See Procedure 10). Microbial contamination of the blood in the bioassay sample is assessed before starting the bioassay (See Procedure 9) to ensure that the blood in the sample is not contaminated If the quality factor of the blood is above 1, the frozen 2L bottles and its Eppendorf samples will be irradiated (See Procedure 8). Both the bottle and its sample will be irradiated in the same operation to ensure that the sample receives the same dose as the blood in the bottle. The last step is to check the bacterial contamination of the blood in the 2L bottles. For this purpose, we thaw the blood sampled in the Eppendorf tubes and conduct the microbial screening (See Procedure 9). If the number of colony forming units is below the agreed threshold, the blood in the bottle can then be marked as good quality. It will be kept in the cold store at -20°C until it is scheduled to be used for flies feeding. The whole procedures are summarized in the following flow chart

1. Sterilization of materials in heat sterilization oven or autoclave Scope: To prevent bacterial contamination, all equipment that will be in contact with blood during blood collection, blood sampling and blood proportion must be thoroughly cleaned and sterilized before being used.

Equipment and Materials: The following equipment will be cleaned and sterilized:  Materials to be used in blood collection: o 25 litre containers, including caps, side inlet and tap. o Paddle / stirrer o Hose o Funnel  Materials to be used in blood sampling: o Eppendorf tubes (if not stored in proper sterile conditions)  Materials to be used in blood proportion: o 2 litre bottles, including caps o Sieves The equipment will be sterilized either using a heat sterilizing oven or an autoclave. Procedure: 1. 2.

3.

Clean the blood-collection equipment. Depending on the dimensions of the equipment, select:  heat sterilizing oven for large equipment: Sterilize the equipment by exposing it to dry heat (at 80°C for polyethylene (PE) for at least 3 hours, and glass at 120°C)  autoclave for small equipment: 15 minutes at 103.45 kPa (15 psi). When not in use, to minimize microbial contamination, store all sterilized equipment in autoclave bags or in an ultraviolet (UV) cabinet.

Fig. 1. Sterilizing oven.

2. Blood Collection Before collection, make arrangements with the responsible authorities of the abattoir to ensure that the place, time and amount of blood to be collected are agreed upon. Alert the collection team (a minimum of three persons) to prepare for the work; note that some heavy lifting may be required. It is an advantage for the workers to have a good knowledge of basic biology and tsetse rearing procedures, and have the ability to handle mechanical equipment. In addition, the workers must be able to make sound judgments regarding the condition of animals (age and health) at the abattoir. The team must wear protective gear (white clothing, rubber boots and helmets). Before arrival at the abattoir, all equipment that will be in contact with blood must be thoroughly cleaned and sterilized. The equipment used to collect blood (Fig. 1 and 2) depends on the amount of blood obtained on one occasion. The frequency of collection depends on the size of the tsetse colony and the amount of space available to store frozen blood at -20°C. In general, during one collection operation, it is economical to obtain as much blood as possible because transport, human labour and quality-control measures become cheaper with increasing quantity. If the ambient temperature is high or the storage facility is a long way from the collection point, it may be necessary to cool the tank of blood. Using two or three collection sets, blood can be collected from all slaughtered animals even if the rate of slaughter is high, e.g. 100 animals in 5–6 hours. Freezing slows down, but does not stop completely, the degradation of blood. It has been demonstrated that blood kept frozen at -20°C for many years retains its nutritive characteristic. If animals are slaughtered on the floor, the blood collection equipment used is selected accordingly. Blood is collected from the cut neck of the animal in a bucket, and then immediately poured into a 25-l container for defibrination or into a container with anticoagulants.

Scope: Collection of a large volume of blood in an abattoir where animals are hung by their hind legs and pulled up with a chain, with their heads hanging down

Equipment and Materials:   

   



Vehicle (for transport to the abattoir, carrying equipment) Deep-freeze chests (or storage space maintained at –20°C) Containers (screw-capped, 20–25 l, polyethylene (PE), with a wide neck 63 to 100 mm diameter to allow a paddle/stirrer to be inserted) (The screw cap is equipped with a stand to hold the stirrer in an upright position. The container has a side inlet with a screw attachment for a plastic hose.) The container has a tap at the bottom that will be used to pour on the blood into the 2 litres bottles (Fig. 1). Hose (plastic, made of transparent silicone, with strong walls (4–5 mm thick, inside diameter 4 cm, outside diameter 5 cm), 1.5 m long to lead from the funnel to the inlet on the side of the container, should be autoclavable) Funnel (plastic or stainless steel, upper diameter 25–40 cm, with an outlet of 4 cm to fit the hose) Paddle (consists of a stainless steel rod of 1-cm diameter on which the oar-blade/stirrer is mounted) The size of the stirrer is determined by the dimensions of the mouth opening and the depth of the container. The stirrer should agitate the full volume of liquid, reaching close to the bottom of the container. The stirrer can be a mechanical handheld drill, an electric household drill or a battery-driven drill. If an electric drill is used, it should have a speed regulator and switch to control the driving speed. The drill should be strong enough to agitate a volume of 10–20 l of highly viscous liquid. When using electric stirring equipment, a ≥50-m length of waterproof extension cable is necessary to reach a power supply in the abattoir.

Procedure: 1. 2. 3. 4. 5.

6.

On arrival at the abattoir, assemble the collection equipment and check that it functions. Negotiate with the butcher for permission to skin the neck of the animal before the jugular vein is cut. When the butcher has cut the throat of the animal, direct the flow of blood into the funnel attached to a hose leading to the stirring container. Collect as much blood as possible before terminating the procedure. While one person controls the position of the funnel, the second controls the rotation of the stirrer, adjusts it to a moderate number of revolutions, and observes the filling of the container. Up to 15 l of blood can be expected from a bull, and up to 25 l can be collected in one stirring container. After 10 minutes of stirring, the blood is defibrinated, and the stirring machine is stopped. (Meanwhile, if practical, use a second set of blood-collection equipment to collect blood from additional animals.)

7. 8. 9. 10.

Open the lid of the stirring container and remove the paddle with all the clotted fibres. Close the lid and rinse all spilled blood from the outside of the storage container. Rinse the paddle of the stirrer with clean water to free it from fibrin. As more animals are slaughtered, continue collecting blood. Using two or three sets of blood-collection equipment, blood can be collected from many animals, even if the slaughtering speed is high (100 animals in 5–6 hours). 11. Mark the 25 litre containers with an identification code (animal species, lot and year) using a water-resistant marker. 12. Rinse all equipment with cold water before loading it into the vehicle. Transport all 25 litre containers to the storage facility.

Fig. 2. Stirring equipment: mechanical handheld drill, paddle, hose, funnel and container with side inlet

Fig.3. 25 liter containers.

3. Labelling bottles and samples Scope: Labelling the samples and the different blood bottles/containers used is key in order to ensure traceability of the blood in the store and to ensure the correspondence between samples and bottles. In order to minimize typos in the labels while handwriting and to avoid difficulties when reading hand written labels, labels will be produced using a label printer and specific software. The following items need to be labelled: - Abattoir bottles, usually 4L, that come from the abattoir, before sampling and proportioning - 50 ml vials containing the samples for the bioassay - Storage bottles, usually 2L or 1L, after proportioning - Eppendorf samples for microbial screening This task must be done by the Lab Technician.

Equipment and Materials:  

Label printer labels

Procedure: A. Labelling the abattoir bottles.

The blood collected in the abattoir comes to Kality in 4L bottles that are kept in the cold room at -20°C until further processing. During this period, blood must be labelled with the following information: a. Collection date b. Bottle number / total number of bottles collected that day (example: 132, 2-32, …) 22-03-2015 / 1-32

Collection date: nd 22 of March 2015

First bottle out of 32 from the same day.

B. Labelling the 50 ml vials containing the samples for bioassay

After the blood from the thawed abattoir bottles is mixed in the 100L container, 30 samples are taken into 50 mL vials for the bioassay. Each vial must be labelled with the following information: a. Collection date: if the abattoir bottles have been stored for long time, this date can be old. b. Vial number / total number of vial of the sample (example: 1-25, 2-25, …) 22-03-2015 / 1-25

Collection date: nd 22 of March 2015

First vial out of 25 from the same day.

C. Labelling the storage bottles after proportioning

After the proportioning, storage bottles (1 or 2L) will be labelled with the following information: a. Collection date: if the abattoir bottles have been stored for long time, this date can be old. b. Bottle number / total number of bottles proportioned from the same batch (example: 1-64, 2-64, …) 22-03-2015 / 1-64

Collection date: nd 22 of March 2015

First bottle out of 64 from the same batch.

D. Labelling the Eppendorf with the samples for microbial screening

During the proportioning, individual samples of the blood of each bottle are taken into Eppendorf tubes for later microbial screening. These Eppendorf tubes must be labelled with the exact same information than the original bottle from which the blood of the sample comes.

Fig.4. Thermal label printer and labels

4. Deep freeze Scope: To preserve and store the animal blood under freezing conditions to avoid the development of microbial contamination for processing into diet for feeding tsetse flies. Deep freezing the blood will be done at two different stages: a) The day of the blood collection, after coming from the abattoir, the abattoir bottles (usually 4L bottles) will be transferred to the cold store where they will be kept until the further processing b) After proportioning, the storage bottles (usually 1 or 2L) will be labelled and stored in deep freezing conditions until the results of the quality control tests

Equipment and Materials:  

Deep-freeze chests or walking room (storage space maintained at –20°C) Blood bottles, usually with a volume of 4L for the abattoir bottles and 1 or 2L for the storage bottles.

Procedure: 1. 2. 3. 4.

Clean any rest of blood on the outside walls of the bottle. Bring all the bottles to the deep-freeze chest or walking rooms and store them in an orderly manner, with the labels easily accessible. Protective clothing must be used inside the deep-freeze walking rooms. Deep-freeze the bottles immediately at -20 C. In the case of the storage bottles, the corresponding Eppendorf tube with the sample for microbial screening must be kept together.

5. Blood thawing Scope: Thawing the blood in the abattoir bottles is required in order to mix the blood in one single batch for bioassay sampling and proportioning. Thawing the blood will also be done after all the processing is completed and the quality control tests are satisfactory. Blood freezing and thawing operations should be kept to the minimum necessary since the quality of the blood is reduced after each operation while the chances of potential contamination increase.

Equipment and Materials    

Deep-freeze chests or walking room (storage space maintained at –20°C) Abattoir bottles (usually 4L) or storage bottles (usually 1 or 2L) Refrigerator (at +4ºC) Boxes (plastic, for storage of bottles with blood)

Procedure: 1. Estimate the amount of blood that will be thawed. If the goal is to proportion it into storage bottles, all the abattoir bottles from the same collection day must be thawed at the same time to obtain a single batch for the bioassay sampling. 2. If the goal is to prepare the blood for feeding the flies after all the processing is completed, the estimation of the amount needed will be done for the next 3-4 days. This procedure will be done twice per week. Verify that the results of both the microbial test and the 25-day feeding test for the batch of blood that is intended to be used are satisfactory. 3. Transfer an appropriate number of frozen bottles of blood to a refrigerator to thaw SLOWLY at +4ºC. For example, to thaw blood for feeding flies, transfer the bottles on a Friday, planning to use some of the bottles of blood on the following Tuesday, and the remaining bottles on Thursday. 4. For bottles stored at +4ºC until Tuesday, do not place the bottles tightly together, permitting them to thaw. For bottles stored at +4ºC until Thursday, place the bottles together tightly (slows down the rate of thaw) until Monday, but on Monday separate them so that they thaw more quickly. The blood must be completely thawed when the next step in processing begins. 5. This blood is not thawed rapidly (as was once done under running cold water) because apparently rapid thawing reduces the quality of the blood. Instead, the blood is

transferred from -20ºC frozen storage and kept at +4ºC in a refrigerator for several days, allowing the blood to thaw slowly. 6. When the blood has thawed, it is ready to use in feeding flies. However, keep the bottles of blood at +4ºC until the blood is actually needed to pour onto feeding trays (and under feeding membranes). If thawed blood is not used, it can be stored for a maximum of 3 days at +4ºC. If, after 3 days, this blood has still not been used, discard it. 7. If some of the thawed blood in a bottle is used to feed flies but the remainder is not used, this unused blood can be stored at +4ºC in a separate place for up to 3 days. Discard the blood if it is not used within these 3 days. 8. If there is an urgent unforeseen need of blood, frozen bottles can be thawed under cold water. This rapid thaw should not become a routine procedure.

6. Sampling for 25-day feeding test (bioassay) Scope: To take an overall sample of the blood collected in a certain day. This overall sample will be used to run the 25 day feeding test, whose result will define the average nutritional value of the blood collected in that day. This task must be done by the Lab Technician.

Equipment and Materials:        

Scintillation vials (30, each 20 ml, polyethylene) Pipette of 25 ml and pipeter Sieve (to collect floating fibres) Graduated cylinders (250 ml, 500 ml) Dispensing bottle (1 l, with 20-ml calibrated dispenser) Funnel 100L containers Abattoir bottles (usually 4L)

Procedure: 1.

2.

3.

4.

After all the abattoir bottles containing the blood collected on a selected day have been thawed, calculate the total volume of blood of the bottles. If less than 100L, only one 100L container will be used to pool all the blood. Otherwise, estimate the number of containers that will be needed and divide the number of bottles accordingly to obtain a similar volume of blood for each container (i.e. if 128L have been thawed in 4L abattoir bottles, two 100L containers will be needed. The blood from 16 abattoir bottles will be pooled in the first container and the rest will be pooled in the second container) Knowing that 600 ml of blood are needed to run the 25-day feeding test and the number of containers that will be used to pool the blood, calculate the amount of blood that will be sampled from each container (i.e., if 2 containers are to be used, then 300 mL of each will be sampled). Place the 100 litre container close to flame source or in the laminar-flow bench (hood) (which during the previous night had a UV lamp turned on to sterilize the interior); turn off the UV lamp but keep the clean air flowing. Working with your hands and all blood and equipment inside the bench, transfer the thawed blood from the abattoir bottles into it using funnels to avoid shedding of blood. Open the tap at the bottom of the container and pour blood to the graduated cylinder to fill the quantity that has been previously calculated. Pour and sieve the blood from the graduated cylinder into the dispensing bottle and leave it inside the bench. Clean thoroughly the graduated cylinder, sieve and funnel before using them again and place them inside the bench.

5. 6. 7. 8.

Take the empty 100L container to the washing area and place the next container inside the bench. Repeat the same steps until the blood sample from the last 100L container has been added to the dispensing bottle. 600 ml of blood must have been collected in this bottle. Shake the blood gently in the bottle and use the dispenser (or the 25 ml pipette) to portion into 20-ml aliquots in 30 sterile vials. Label the vials as described in Procedure 3 with the following information: a. Collection date: if the abattoir bottles have been stored for long time, this date can be old. b. Vial number / total number of vial of the sample (example: 1-25, 2-25, …) 22-03-2015 / 1-25

Collection date: nd 22 of March 2015

9.

First vial out of 25 from the same day.

Record the previous data electronically (in a database or spread sheet) and in a hardcover record book. 10. Store the vials in deep freeze before irradiating them the next day. The vials are ready to start the 25-day feeding test. 11. This operation must be done by the Lab Technician in a laminar flow cabinet. All the equipment used must have been sterilized previously in the oven/autoclave, following the appropriate procedure described in #1

Fig.5. Scintillation vials

7. Sterilization by radiation This procedure describes the sterilization (decontamination) of animal blood, after collecting blood at an abattoir, for processing into diet for feeding tsetse flies. Access to a gamma radiation source is a prerequisite for sterilization at 1–1.5 kGy. Since the radiation chamber of the Gamma Cell 220 currently in use in Kaliti is reduced, bottles with a maximum capacity of 2 litres will be used to store and irradiate the blood in a frozen state. Samples for bioassay and microbial screening will also be irradiated before conducting the tests. Scope: Sterilization of the frozen blood stored in the 2-litre bottles and of the samples for bioassay and microbial screening using gamma radiation

Equipment and Materials:     

Gamma radiation source Blood (frozen) in 2L bottles Blood samples in 20 mL vials for bioassay Blood samples in Eppendorf tubes for microbial screening Irradiation basket (cylindrical metal box) for bioassay samples

Procedure for blood irradiation 1.

2. 3. 4. 5. 6.

7. 8.

Transport the frozen blood (in 2L bottles) from storage walk-in freezers to the irradiation facility. The Eppendorf tube with the sample for microbial screening must be properly fixed to the bottle. Both the sample and the bottle must be irradiated in the same operation, to ensure that both receive the same dose. Open the gamma cell, put the 2-litre bottle into the irradiation chamber and close the chamber. Close the doors of the irradiation chamber with special care with the sliding latch. Set the exposure time (according activity of the source) for 1 kGy. Activate the irradiation from the outside remote panel. After the exposure is finished, get back into the irradiation room, open the Gamma Cell, and remove the 2-l bottle, Label the irradiated bottle with a thermal label printed with the following information:  Irradiation date  Operator  Signature of the operator Put the bottle together with the other irradiated bottles and place a new nonirradiated bottle in the chamber. Repeat until all bottles have been irradiated.

9. Transport the blood in a frozen condition back to the store. 10. Store the blood frozen until use or further processing and testing. Special attention is required to avoid mixing the non-irradiated bottles with the irradiated ones. The elapsed time with the bottles out of the freezer must be kept as low as possible. If thawing of the external layer of blood is observed, reduce the thawing time by irradiating fewer bottles at a time. Irradiation must be done under the appropriate supervision of the Radiation Protection Officer (RPO) and following the strict regulations of the Ethiopian Irradiation Protection Authority. Irradiation must be done by authorized personnel.

Procedure for samples irradiation 1. 2. 3. 4. 5. 6. 7.

Take the sample vials for the 25-day feeding test from the deep-freezer chest, put them in the irradiation basket and bring them to the irradiation facility. Check that vials are properly labelled. Open the gamma cell, put the basket into the irradiation chamber and close the chamber. Set the exposure time (according to the date) for 1 kGy. Press the start button. Retrieve the vials when irradiation is complete. Store the vials in a deep-freeze chest in the lab. They are ready to start the bioassay.

8. Microbial screening of the blood Scope: Microbial screening of blood to decide if the blood is suitable for tsetse feeding. Microbial screening has to be done for each feeding bottle (usually 2L bottles) that has been stored in the walk in freezer to ensure that the bottle is not contaminated. The blood sampled for the bioassay will also be tested for bacterial contamination before the bioassay is started. In this case, only one test will be conducted for each set of bioassay samples. In both cases, the samples have been irradiated to reduce the bacterial load before the microbial screening is conducted. Equipment and Materials:            

Laminar-flow bench (hood) Bunsen burner Balance Magnetic stirrer/hot plate Incubator Bacterial-colony counter/viewer Petri dishes (glass, autoclaved) Distilled water (autoclaved) Nutrient agar Syringe and needle (1 ml, disposable) Erlenmeyer flasks (various sizes) Eppendorf with the blood corresponding to the bottle to be tested

Procedure: 1.

Mix 2 g of agar with 100 ml distilled water in a 200-ml glass Erlenmeyer flask closed by cotton wool, stir gently and boil. 2. Autoclave the fluid agar at 125°C for 15 minutes. 3. Put the flask of agar in a water bath at 40°C. 4. Clean working bench and hands with antiseptic or sterilizing fluid. 5. Place sterile Petri dishes on the bench. 6. Wipe the outer surface of all containers before placing them on the bench. 7. Ignite the Bunsen burner. 8. Label the Petri dishes on the bottom of the smaller half with the blood code, and have one Petri dish for air and one Petri dish for nutrient agar. 9. Open the Petri dishes briefly, flame the open parts, and close them immediately. 10. Make sure the agar in the Erlenmeyer flask is between 40–45°C.

11. Lift the lids of the Petri dishes, and add 13 ml agar to the empty Petri dishes. Aim at an agar-layer thickness of ¼-⅓ of the internal depth of the dish. 12. Again lift the lids slightly from the Petri dishes and pour in 1-ml samples of blood from the Eppendorf tubes. 13. Stir gently for about 15 seconds. 14. Wait until the mixture of agar and blood has solidified, and then turn the Petri dish upside down. 15. Incubate the inoculated Petri dishes in the upside-down position at 37°C for 72 hours. 16. Check the agar layers for bacterial colonies after 48 and 72 hours. 17. Record the number of bacterial colonies found per sample. 18. If, after 72 hours, the irradiated blood contains NOT more than 10 colonies, the corresponding bottle of blood is ready to be thawed, provided the result from the 25day feeding test of its corresponding batch is satisfactory. 19. If the blood has more than 10 colonies, discard this bottle of blood, and take a new bottle and repeat the test. 20. Record the previous data electronically in the spread sheet prepared for this purpose and in a hardcover record book.

Fig.6. Laminar flow bench with the equipment used for the microbial screening

Fig.7. Petri dishes with nutrient agar ready for incubation

9. 25-day feeding test: bioassay These procedures describe the quality assurance and data recording for blood used to feed large colonies of tsetse. The aim is to maintain high-quality mass-reared colonies of tsetse over long periods. During collection and processing, blood can become contaminated with bacteria and chemicals. Under the influence of physiology, nutrition and disease, the composition of blood in animals varies among individuals of the same species. Due to these factors, a quality-control procedure was developed, and biological criteria for evaluating the suitability of blood used to feed flies were identified. Microbial screening of blood is done using blood from one 2-l can from each batch brought back to the laboratory after collection, and also on blood after decontamination. The second bacterial screening is carried out to confirm the efficacy of the sterilization procedure. On average, 96% of microbial contamination is eliminated by irradiation at 1 kGy. A 25-day feeding test was developed with the aim of having a simple numerical system that adequately summarizes and combines the various data obtained from pupal production and dissections. It is expressed as the quality factor (QF). For irradiated blood, a QF of >1 is acceptable. The assumption is that the larva/pupa will develop into a viable fly. Since both the female adult and the larva within the uterus are dependent on the same source of food, it follows that the availability of blood is one of the most important factors affecting reproductive physiology in tsetse. For the blood quality feeding test, a minimum quantity of 600 ml of blood is necessary, and is handled in the following way. A sample for bacterial testing is taken from the storage container, and blood foreseen to be used later for colony feeding is treated precisely in the same way as the test amount. The diet or a mixture of blood is portioned into 20 ml aliquots in 30 sterile vials. The vials are closed, and exposed to 1 kGy gamma radiation. After irradiation, another sample is taken for bacteriological screening to confirm decontamination. Once the result of the bacterial screening after irradiation is found to be acceptable, the blood is ready to be fed to the flies.

Entomology Unit, Joint FAO/IAEA Programme IAEA Agriculture Laboratory Seibersdorf Record Sheet for Tsetse Blood Diet Quality Control

BLOOD CODE:

BC06076

date collected 20.04.06

storage

Test-No. Year

date collected

storage

date processed

pH

additives

osm. pr.

mOsm

MICROBIAL SCREENING cont. A

0 Sp/ml

SURVIVAL & FECUNDITY

Sp/ml

Sp/ml

G. pallidipes Test

dead st bl

diet before g

cont. B

(CFU/ml) diet after g

Sp/ml

DISSECTIONS

species

fem. surv.

N 1

day

DATE

1

5.5.06

30

2

6.5.06

30

3

7.5.06

30

4

8.5.06

30

5

9.5.06

30

6

10.5.06

30

7

7 8

11.5.06 12.5.06

30 30

8 9

females:

males: date EM

date MAT

date SEP

2 date EM

3 5

other remarks:

6

9

13.5.06

30

10

14.5.06

30

No. of.

No. of.

11 12

15.5.06 16.5.06

30 30

pupae prod.

abortions in dish

13

17.5.06

30

14

14 15

18.5.06 19.5.06

30 30

15 16

16

20.5.06

30

17

17

21.5.06

30

18

22.5.06

2

28

15

19

19 20

23.5.06 24.5.06

1

27 27

3 1

20 21

21

25.5.06

27

1

22

22

26.5.06

27

23

23

27.5.06

27

24

24 25

28.5.06 29.5.06

27 27

2

3

27

22

1

26 26 26

1 2 7

26 26

6

S 26 27 28

30.5.06 31.5.06 1.6.06

29 30

2.6.06 3.6.06

S

4

Sp

4

10

weight class distribution A

B

C

D

11 E

12 13

18

S

0

0

0

0

0

25 26 27 28 29 30

S

38

0

6 2006

0

0

0

0

Day 25 FS 18

28

PT

22

PB

0

PD

0

E+I

0

AB

0

QF

FS 25

27

PA

0

PC

22

PE

0

II + III

0

BL

0

QFC

1.24

Day 30 FS 18

28

PT

38

PB

0

PD

0

E+I

0

AB

0

QF

FS 30

26

PA

0

PC

38

PE

0

II + III

0

BL

0

QFC

Fig.8. Template of the excel file to record and calculate the blood quality factor

1.70

UT

Scope: 25-day feeding test Equipment and Materials:             

Scintillation vials (30, each 20 ml, polyethylene) Dispensing bottle (1 l, with 20-ml calibrated dispenser) Deep-freezer Laminar-flow bench (hood) Foil (aluminium, to cover autoclaved or sterilized glassware) Magnetic stirrer (to agitate blood — volumes between 100 and 1000 ml) Petri dishes (for nutrient agar plates) Syringes (1 ml, sterile, disposable) Incubator Bacterial colony-viewing/counting device Teneral female flies (30, mated in the appropriate way, and caged at 15 per standard 11cm-diameter cage) Radiation source Stereomicroscope

Procedure: 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

Thoroughly clean and sterilize all parts of equipment that have come into contact with blood. Sterilize in an oven by exposure to 80°C (polyethylene) or 120°C (glass and metal) for 3 hours or in an autoclave at 120°C for 30 minutes, and then store properly to minimize the risk of microbial contamination. At emergence, put 15 teneral females and 15 teneral males each in two standard 11cm-diameter cages. Mate flies in the appropriate way. Feed flies daily. For 25 days, feed from the same batch of blood. Check for mortality, and dissect any dead female — examining for uterine content, insemination, and mating scars where applicable, and record. After 8 days, place the cages on a dish, and thereafter examine the dishes for abortions under the stereomicroscope on days 10, 15, 20 and 25 from the time of emergence. Collect larvae and pupae. Allow larvae to pupate, and record individual weight. If a pupal size-sorting machine is available, at the termination of the test, sort by class all pupae, count, and note the various weight classes. Dissect all females surviving 25 days, and examine for mating scars, insemination, uterine content, follicle next in ovulation sequence (FNOS), and reproductive abnormalities such as blockage of the oviduct. Calculate the quality factor.

14. Record the previous data electronically in the spread sheet prepared for this purpose (see figure 10) and in a hardcover record book.

Parameters used to calculate the blood quality factor (QF) (e.g. Fig. 10): Parameter

Explanation

First reproductive cycle: FS 18 number of females surviving on day 18 FS 25 number of females surviving on day 25 PT total number of produced pupae PA number of A-class pupae PB number of B-class pupae PC number of C-class pupae PD number of D-class pupae PE number of E-class pupae Second reproductive cycle: E+I number of inseminated females on day 25 with early pregnancy stage in utero II + III number of inseminated females on day 25 with late pregnancy stages in utero BL number of inseminated females on day 25 with oviduct blockage AB number of inseminated females on day 25 that aborted, empty uterus, follicle next in ovulation sequence (FNOS) is not mature Calculation of the blood quality factor (QF): QF =

positive parameters from first reproductive cycle + positive parameters from second reproductive cycle – negative parameters from first reproductive cycle – parameters from second cycle / number of females

QF = [FS 25 + PT + (PB x 0.3) + (PC x 0.4) + (PD x 0.5) + (PE x 0.6) + (E + I x 0.3) + (II + III x 0.6) – (PA x 0.2) – (AB x 0.5) – (BL x 1.0)] [FS 18 + FS 25] A QF value of 1.0 is the minimum acceptable value. For laboratories where tests are run regularly, experience shows that dissections can be omitted at day 25 without affecting the overall value, but the formula for calculating the QF value is modified (see below). An EXCEL spreadsheet to calculate the QF is available at the Entomology Unit, FAO/IAEA Agriculture and Biotechnology Laboratories, Seibersdorf, Austria. To calculate the QFC (calculated quality factor), use the following formula: QFC = [PA*11 + PB*17 + PC*19 + PD*20 + PE*22] [FS 18*23.86 + 0.616] An EXCEL spreadsheet to calculate the QFC is available at the Entomology Unit, FAO/IAEA Agriculture and Biotechnology Laboratories, Seibersdorf, Austria.

10.Proportioning Scope: To proportion blood pooled in the 100L containers during the bioassay sampling into storage bottles (usually 2L) so that they can be irradiated with the Gamma Cell.

Equipment and Materials  



Funnels (metal, small) Storage bottles (usually 1 or 2L volume), preferably made of heat- and cold-resistant plastic, that are heat-sterilized before use and able to withstand prolonged storage at 20°C. The lid must have a screw cap with a rubber gasket that can be tightly closed in order to allow hosting the Eppendorf tube with the sample for microbial screening. Laminar-flow bench (hood)

Procedure: 1. 2. 3. 4. 5. 6. 7. 8. 9.

After the sample for the 25-day feeding test has been taken, leave the 100L container inside the laminar flow bench. Place a 2-litre bottle under the tap of the 100L container and a funnel if necessary in between booth. Open the tap at the bottom of the 100L container and fill the bottle with approximately 200 ml of blood. Shake the bottle gently for 20 seconds and let the blood be in contact with the walls of the bottle Take a sample in a disposable eppendorf tube of 1.5 ml as described in procedure #11 Fill up the bottle of blood and close the pressure cap Fix the eppendorf to the center upper part of the pressure cap with tape Close the screw tap tightly Label each bottle and the eppendorf with the following information: a. Collection date: if the abattoir bottles have been stored for long time, this date can be old. b. Bottle number / total number of bottles proportioned from the same batch (example: 1-64, 2-64, …) 22-03-2015 / 1-64

Collection date: nd 22 of March 2015

First bottle out of 64 from the same batch.

10. Deep-freeze the bottles and its bacteriology samples together immediately at -20 C. Sampling and proportioning must be done at the same time. 11. Thoroughly clean and sterilize all equipment that was in contact with blood, and keep until required again. 12. Record the previous data electronically (in a database or spread sheet) and in a hardcover record book. 13. This operation must be done by the Lab Technician in a laminar flow cabinet. All the bottles and equipment used must have been sterilized previously in the oven/autoclave, following the appropriate procedure described in #1.

Fig.9. Blood storage bottle with different capacity.

11.Sampling for microbial screening of the blood Scope: To take a blood sample of each of the storage bottles used in the proportion operation to run the microbial contamination test. This task must be done by the Lab Technician.

Equipment and Materials:   

Pipette Sterile tips Eppendorfs tube 1,5 ml

Procedure: 1.

This procedure is done in parallel with the proportion procedure. During the proportion procedure of the blood into storage bottles, one blood sample of each bottle has to be taken in order to run the microbial contamination test. The aim of this test is to check the sterility of the bottle, so, in order to ensure that the sample is representative, the procedure to take the sample will be as follows: 2. Fill the bottle with approximately 200 ml of blood. 3. Shake it gently for 20 seconds and let the blood be in contact with the walls of the bottle 4. Take the sample (1 ml) in a disposable eppendorf tube of 1.5 ml using a pipette with sterile tip. 5. Fill up the bottle of blood and close the pressure cap 6. These Eppendorf tubes must be labelled with the exact same information than the original bottle from which the blood of the sample comes. 7. Fix the eppendorf to the center upper part of the tap with tape 8. Close the screw tap tightly 9. Deep-freeze the bottles and its bacteriology samples together immediately at -20 C. Sampling and proportioning must be done the same day. 10. Thoroughly clean and sterilize all equipment that was in contact with blood, and keep until required again. 11. Record the previous data electronically (in a database or spread sheet) and in a hardcover record book. 12. This operation must be done by the Lab Technician in a laminar flow cabinet. All the bottles and equipment used must have been sterilized previously in the oven/autoclave, following the appropriate procedure described in #1.

Fig.10. Eppendorf tube 1.5 ml

Fig.11. Pipette with sterile tip

12. Use of the database

A database has been specifically developed to allow a better storage and management of the information related to the collected blood following the current procedures adapted for Kality. The database intends to be user friendly. The users’ manual of this database can be freely downloaded from the website of the Insect Pest Control Section.