140 EFFECTIVENESS OF WASHING PROCEDURES FOR REMOVING BRUCELLA ABORTUS FROM IN VIVO-DERIVED WOOD BISON EMBRYOS

Abstract

Endemic brucellosis threatens wild herds of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park, the largest genetic reserve of wood bison in the world. The overall goal of our project was to produce and preserve disease-free embryos for the purpose of conserving the genetic diversity of this species. The aim of the present experiment was to determine the effectiveness of washing procedures for removing Brucella bacteria from in vivo-derived wood bison embryos exposed in vitro to the pathogen. Wood bison cows were given 300 mg im of Folltropin diluted in 0.5% hyaluronan on the day of follicle wave emergence (Day 0) and 100 mg im of hyaluronan on Day 2, and then given 2500 IU im of hCG on Day 5 and inseminated 12 and 24 h later. Embryos were collected on Day 13. The experiment was done in 6 replicates (n = 4 bison/replicate) and an average of 9 embryos/replicate were collected. Zona pellucida-intact embryos were kept in holding medium (PBS + 2% fetal calf serum) and transported to a Biosafety Level 3 laboratory at the International Vaccine Centre, University of Saskatchewan. Embryos were transferred through 5 aliquots of holding medium to remove any contaminant before exposure to Brucella. Embryos were divided equally into 2 Petri dishes (representing later wash groups with v. without antibiotics) containing 2.7 mL of holding medium (n = 2 to 7 embryos per dish/replicate). In a Class II biosafety cabinet, Brucella abortus biovar 1 (1 × 10⁷ to 1 × 10⁹ CFU mL^–1 in 0.3 mL) was added to each Petri dish and incubated for 2 h at 37°C in 8% CO₂. A sample of holding medium was taken before exposure and after incubation for culture as negative and positive controls, respectively. After incubation, embryos in each Petri dish were subjected to a 10-step washing procedure (according to the IETS Manual, 2010) using wash medium (PBS + 0.4% BSA) without antibiotics or with antibiotics (100 IU mL^–1 of penicillin + 100 μg mL^–1 of streptomycin). The embryo wash medium was cultured at wash steps 1, 3, 6, and 9. After the tenth wash, the zona pellucida of each embryo was ruptured mechanically using a glass pipette and embryos were cultured individually. Culturing of samples was done on sheep blood agar and specific identification of Brucella organisms was done by PCR. Brucella abortus was detected in 3 embryos from the group washed in medium without antibiotics (3/27), whereas all embryos washed in medium with antibiotics were culture negative (0/27). Brucella abortus was not detected in wash media after the third wash in either group (with or without antibiotics). In summary, Brucella abortus was removed from 89% of in vitro-exposed wood bison embryos using the washing procedure without antibiotics, and from 100% using the washing procedure with antibiotics. Results validate the embryo washing technique for producing Brucella-free wood bison embryos.

Thanks to the Canadian Food Inspection Agency for the field strain of Brucella abortus, Bioniche AH for Folltropin and embryo collection supplies, Merck AH for hCG (Chorulon), and Intervac/VIDO for technical and logistical support.