SEASONAL EFFECTS OF BLOOD DIET ON TSETSE (GLOSSINA AUSTENI) PERFORMANCE MASS REARED FOR STERILE INSECT TECHNIQUE (SIT).

EFFETS SAISONNIERS DU REPAS DE SANG SUR LA PERFORMANCE DES MOUCHES TSETSE (GLOSSINA AUSTENI) ELEVEES EN MASSE POUR LA TECHNIQUE DE L’INSECTE STERILE (TIS)

Mechtilda Byamungu
Tsetse & Trypanosomiasis Research Institute,
P.O. Box 1026;
TANGA, Tanzania

Résumé

            Dans la production en masse de  tsétsé pour les programmes TIS, le régime des tsétsé (sang de qualité), entre autres, est très important. Le sang de qualité est essentiel pour le maintien en bonne santé des mouches et ce,  pour une bonne survie et une bonne production. Par conséquent, avant la collecte du sang, il faudrait tenir compte de l’état de santé des animaux abattus. En outre, les procédures de collecte de sang, la manutention et la conservation doivent respecter les conditions d’hygiène pour réduire au minimum la contamination. Cette communication porte sur des études permettant d’établir les effets saisonniers du repas de sang sur la performance des mouches (survie et production des pupes).

            L’échantillonnage a été fait dans divers abattoirs pendant différentes saisons. Le sang collecté a été examiné pour déterminer la valeur de l’hématocrite et pour faire le dépistage des bactéries. L’identification des bactéries au niveau des espèces a aussi été effectuée pour établir les espèces pathogènes. Par ailleurs, le sang a été servi pour tester les mouches et des essais biologiques de 30 jours ont été faits afin de déterminer le facteur qualité (FQ). L’échantillon de sang avec FQ > 1 est considéré comme étant bon pour les mouches. L’objet principal de la présente étude est de déterminer une bonne saison pour la collecte de sang.

Summary

            In mass production of tsetse flies for SIT programs among other factors tsetse diet (quality blood) is very important. Quality blood is vital in the maintenance of health flies with good survival and good production. Therefore, before blood collection the health status of slaughtered animals should be taken into consideration. Also blood collection procedures, handling and storage should be done under hygienic conditions to minimize contamination. This work involved studies to establish seasonal effects of blood diet on fly performance (survival and pupae production)

            Sampling was done at different abattoirs in different seasons.  Collected blood was checked for PCV value and bacterial screening tests were carried out. Also, further identification of bacteria to species level was conducted to establish the pathogenic spp.  Moreover the blood was given to test flies and 30-day bioassays were performed to determine quality factor (QF). The blood sample with a QF more than 1 was assumed good for the flies. The main purpose of this work is to establish a good season for blood collection.

Introduction:

            Tsetse fly (Glossina) rearing evolved from feeding on live hosts to a membrane feeding system (Udo Fedmann 1983). Following successful colonization of tsetse flies using membrane-feeding technique, mass rearing of this vector became a reality (Mews, et al 1976 & Msangi, et al 1995). Such development on the technique has received added emphasis because of the need to mass rear the fly for experimental work and for control/eradication using the sterile insect technique (SIT). As a result of successful eradication of tsetse flies from the Island of Zanzibar (Msangi et al 1997) other eradication programmes are being planned employing large scale SIT on mainland of Africa, for example TTRI is again in the move to mass rear flies for other African countries in need. (RAF/5/051-2002/1015)

            For successful mass production what is required is to keep the insects alive and maintain a healthy stock of insects, which will produce a surplus offspring. The offspring should be of comparable size and physiological condition to insects in their natural habitat. At this juncture blood diet among other factors is of vital importance. However maintenance of large colonies requires a considerable amount of blood and this should be of good quality for fly production. To assess the blood quality we use the measurable parameters of fly performance that include survival, fecundity and offspring size (Mews, et al 1976). In view of the increasing demand for SIT there is a need to explore ways to ensure availability of inexpensive, standard quality blood.

            The amount of blood to be collected will depend on the size of the colony to be maintained. For example at the Tanga facility, we are anticipating establishing a colony of about 10 million producing females that will produce about 1 million sterile males for weekly releases. Feeding of these flies will require about 300-400l of quality-tested blood per day. For this, about 60-80 cattle will need to be slaughtered per day.

            In the TTRI case the quality of blood collected for in-vitro tsetse feeding had been a problem. For un-known reasons some blood batches collected from the same sources were good for fly production and others not.

            This study therefore, was intended for investigating the possibility that seasonal variations influence the blood quality.

Material and methods

Seasonal sampling

            Blood samples were collected from Tanga, Arusha and Dar-es-salaam abattoirs; three trips were made to each abattoir to cover different seasons (dry, rain/wet, and cool dry) of the year. The dry season is in December, January and February, the rain/wet season from March to May and the cool/dry season from June to November

Preparation of equipment for blood collection:

            Equipment for blood collection were washed thoroughly and rinsed with distilled water and dried in the oven at 80ºC for plastics and at 120ºC for 24hrs for heat resistance equipment. They were then packed  in sterilized plastic sacks one to two days before blood collection is done.

            For pre-selection of animals for slaughter, we visited the holding ground with the veterinary doctors and animals that had passed the anti-mortem were the ones that we collected the blood from.

            For the Arusha abattoir, animals for slaughter  were from Arusha and Shinyanga regions.

            For the Dar-es-salaam abattoir, the animals were brought in from different regions including Shinyanga, Arusha and Dodoma.

            For the Tanga abattoir, animals were from Arusha, and Shinyanga Regions.

Normally, animals are transported from marketing centers to the slaughterhouses by trucks or trains.

Collection procedures

            Blood was collected from the jugular vein of the slaughtered animal and precautions were taken to avoid contamination. Slaughtering was done on the floor and for Arusha abattoir the animals were later hanged. The Dar-es-salaam and Tanga abattoirs do not hang the animals after slaughtering them.

            The collected blood was de-fibrinated by continuous stirring manually during the collection processes and then sieved. It took about 1hr to collect 20 litres of blood. A sample of the sieved blood was taken using capillary tubes and centrifuged for PCV measurement at the abattoir.  The blood was portioned into two-litre containers ready for storage in the freezers at -18ºC to -20ºC. It took not more than two hours from collection to storage of blood.

            Blood collections from Dar-es-salaam and Arusha were kept frozen for two days and the following day packed in a cool box containing ice packs and transported to TTRI. For the last Dar-es-salaam blood collection in March 2005, a temperature monitor was placed in the freezer during storage and in the cool box, to monitor temperature changes during transportation. The blood collected from Tanga was immediately taken to the Institute for storage.

            The transportation time of blood from collection source to Tanga (TTRI) from Dar-es-salaam was 4.5hrs to 5 hrs, Arusha 6hrs to 7hrs and from the Tanga abattoir 10min. to 15min.

            At TTRI all the collected blood was stored in freezers kept at -18 to -20ºC and the temperature was checked frequently on temperature thermometers fixed to the freezers. The Storage room and freezers were cleaned frequently to maintain hygiene conditions. Samples of blood from each container were irradiated at 100 Krads before being subjected to bacterial screening.

Bacterial screening procedures

            Blood samples were poured in sterilized petri dishes with nutrient agar placed in a lamina flow bench. The mixture was then stirred and incubated at 37ºC. The mixture was checked for bacterial colonies after 24 and 48hrs. Bacteria colonies obtained were taken for further identification to species level. Further tests are required in order to identify pathogenic bacteria to tsetse flies.

Blood quality control test

            This bioassay determines whether a blood diet is suitable for in-vitro feeding of the tsetse flies. The bioassays were conducted for 30 days by feeding young mated females and checking for feeding responses, survival, abortions, and number of pupae at day 18, and pupae class. Surviving females were dissected at day 30 to determine the uterine content and insemination rates. The above parameters are used to calculate the Quality factor (QF) of which the acceptable value is either 1 and above for blood to be used for fly feeding.

Results:

            As per Tables I and Table  II below.

  Table I   Blood collection source, date, PCV values, average pupae weights and QF   value

Table II: Bacteria species screened from blood samples obtained from different sources

Discussion

            On seasonal sampling, it was noted that there were differences in number of slaughtered animals according to season. During the dry season more animals were brought in for slaughter while in the wet season there were few animals. During the wet season (post harvest season) few animals were available because it is a suitable time for breeding and farmers depend much on crop sales for their living and they do not sell their animals.

            One special thing about Dar-es-salaam is that after slaughtering they cut through the heart vena cava to allow quick draining of blood from the animals. This gives a good chance for blood collection and to some extent can minimize contamination.

            There were no marked differences between the PCV (average) values from one season to another. They were above the minimum value and the Dar-es-salaam collection had higher values. QF values were just above the acceptable value with one blood collection from Arusha having a value of 0.9, which is below standard. However there was no reflection of a season effect.  Pupae weights ranged from 21mg to 23mg and there were no indication that they were affected by the seasons.

            Bacteria screening indicated that there were five groups: Bacillus spp were 9, Staphylococcus spp were 3, Acinetobacter spp 4, Pseudomonas spp 1 and Streptococcus spp 1. These species need to be re-cultured, amplified and then tested to determine if they are pathogenic to tsetse flies. There is not much information in the literature concerning bacteria isolates that are pathogenic to tsetse flies. According to Kaaya, G.P. and Darji, N. (1989) some Bacillus spp and Pseudomonas spp caused mortalities in laboratory-reared flies. Also Kaaya, G.P an Okech, M.A (1990) found the bacteria of genera Serratia, Enterobacter, Aeromonas to be pathogenic to laboratory-reared flies.  Also Geest and Bariac (1982) found Bacillus thuringiensis to be pathogenic to G. pallidipes. The mortalities on laboratory-reared flies depended on the species of bacteria, the dose ingested and time post-ingestion. Therefore, is a great need to conduct tests to check if the isolates detected here are pathogenic to tsetse flies.

 Further work

• Sub-culturing of isolates and providing them to tsetse flies to check if they are    
• Pathogenic.iding them to tsetse flies to check if they are pathogenic

• More sampling will be conducted to countercheck the findings with the obtained results.

• Collaboration with other institutions like Sokoine University of Agriculture (SUA) to check on chemical contamination and parasites for blood samples with low QF values.

Acknowledgement

• We acknowledge the assistance provided by veterinary doctors in respective districts.
• Also we appreciate the assistance provided by our Director Dr. A. Msangi during the period.
• Finally the IAEA efforts to provide funds for this work are appreciated.

References

Kaaya, G.P. and Darji, N. (1989).  Mortality of Adult tsetse, Glossina morsitans morsitans caused by entomopathogenic bacteria. Journal of invertebrate Pathology 54(1): 32-38

Kaaya, G.P..and Okech, M.A (1990).  Microorganisms associated with tsetse in nature: preliminary results on isolation, identification and pathogenicity. Insec Asci, Appl. 11, 443-448

Feldmann, U. (1993) Guidelines for rearing of tsetse flies using the membrane feeding Technique, Seibersdolf laboratory Austria

Mews, A.R., Baumgartener, Luger, H., D. amd Offori, E.D. (1976).  Colonisation of G. m. morsitans  Westwood  (Diptera Glossinidae) in the laboratory using in vitro feeding. Bull. Ent. Res, 65, 631-642

Msangi, A., Kiwia, N., Mramba, F., Kitwika, W., Malele, I., Byamungu, M., Kasilagila, G., Dyck, A.V. and Parker, A. (1995).  Eradicatoin of Glossina austeni Newstead on Unguja Island (Zanzibar) by the sterile Insect Technique.  2. Mass production of G.austeni for SIT application in Zanzibar. Second FAO/IAEA Seminar for Africa, Zanzibar Tanzania, 27 November –1st December 1995.

Msangi, A., Kiwia, N., Mramba, F., Kitwika, W., Malele, I., Byamungu, M. and Kasilagila, G. (1997). Eradication of Glossina austine  Newstead on Unguja Island Zanzibar by the sterile insect technique Mass Production & Quality assessment  of sterile flies.

RAF/5/051-2002/1015 Sterile insect technique for Area-wide Tsetse and Trypanosomiasis Management – The provision of Service for tsetse mass rearing and supplies of sterile flies in support of the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC)