Improved protocol for aseptic collection and handling procedures of bovine blood diet in areas with special contamination challenges for use in tsetse rearing

摘要

Importation of blood to support tsetse feeding in Africa is costly and marred by delays, quarantine restrictions and other logistic problems,1 while chemical contamination is likely to be encountered in tsetse diet from drug residues arising from treatment of animal diseases in Africa. Further, blood diet collection procedures are not standardized risking the health of the donor animals. We established a blood collection procedure of bovine blood for in vitro tsetse feeding in areas with special contamination challenges that is safe for both donor animals and tsetse flies. Six Orma Boran steers (registered at the Kenya Stud Book, Boran Cattle Breeders Society 2009, Kenya) aged between 12 and 16 months supplied from Kenya Agricultural Research Institute–Trypanosomiasis Research Centre (KARI-TRC), delivered on foot, thoroughly washed with water using a soft brush (PVC, LG Harris and Co (EA) Ltd, Nairobi, Kenya) to remove possible pesticide contamination, de-wormed with 10% albendazole and acclimatized for one week before collection of blood could start. The criteria for the selection of steers were healthy looking animals above six months of age and animals recently exposed to treatment, especially antibiotics, were excluded.2 The animals were confined in a well-ventilated, fly-proof barn house partitioned into eight metallic cubicles each measuring 3 x 4.3 x 2.5 (height) m with concrete floor and wood chippings provided as bedding material. The steers were kept in two adjacent cubicles in groups of three animals each separated by round metal cross bars. The animals were kept in a locked room under natural light; room temperature and humidity were not regulated. The steers were maintained on 9–10 kg/animal/day dry fodder (Hay, KARI-TRC, Kikuyu, Kenya), 1 kg/animal/day concentrates (Ranch cubes?, Unga Feeds Ltd, Nairobi, Kenya), 2 kg block/animal/month mineral salts (Maclick?, Cooper Kenya Ltd, Nairobi, Kenya), 5 kg/animal/day green fodder (Nappier grass, KARI-TRC). Mains tap untreated water was provided ad libitum. Blood collection from the six steers was carried out once a month for a period of four months. Animal handling protocols and procedures used were reviewed and approved by the KARI-TRC Institutional Animal Care and Use Committee. Body weight, pulse rate, rectal temperature, respiratory rate, rumen movement and packed cell volume were monitored before and after the experiment and found to have no clinical abnormalities. Prior to each bleeding session, the steers were transferred to the restraining crush (Tubar? Cattle Crush, manufactured by HJ Urry & Son Ltd, Alveley, Bridgnorth, Shropshire, UK) situated within the barn house. After the study, the steers were recruited back to the resident herd at KARI-TRC. Plastic, glass and metallic ware used were sterilized and stored as described earlier,2 while personnel wore waterproof overalls, sterile gloves, face masks and head caps. Blood collection conformed to guidelines described earlier3 to avoid animal exsanguination. We collected blood from the same steer once a month provided 10% of the total blood volume was not exceeded. Bleeding on a weekly basis should not exceed 7.5% of the total blood volume. Total blood volume was calculated by multiplying circulating blood volume (60 mL/kg) by the animal's body weight.3 Fresh blood was aseptically collected by jugular venepuncture4 over a period of four months. The distal end of the jugular vein was cleaned with methylated spirit, blocked with the thumb to ensure engorgement then punctured using a sterile 14-gauge needle and blood allowed to freely flow via a foot long delivery tube into a 2 L conical flask containing 800 g of 5–8 mm diameter glass beads with continuous swirling for at least 10 min to ensure complete de-fibrination. The processing and bacteriological screening of the blood diet was carried out as described elsewhere,2,4 but with modification. The standard nutrient agar plated Petri dishes were incubated at 36 ±