Leukocyte proportions in Pteropus alecto blood smears

This study was carried out in accordance with guidelines for animal care and handling under Griffith University Animal Ethics Committee (Approval ENV/10/16/AEC) and Montana State University IACUC Committee (#201750). P. alecto were captured between June 2018 and July 2020 at two roost sites in Queensland, Australia: one in Redcliffe and one in Toowoomba (Table S1). Redcliffe is a coastal suburb approximately 30 kilometers northeast of Brisbane. Toowoomba is a small inland city 700 m above sea level in the Great Dividing Range, approximately 120 kilometers west of Brisbane. These roost locations are spaced approximately 160 kilometers apart and are continuously occupied by P. alecto. Bats were captured, examined, sampled, and released within their roost site. Bats were captured pre-dawn in mist nets and anesthetized by a veterinarian or veterinarian-trained technician using 5% isoflurane in oxygen at 800 mL/min, followed by administration of 1.5% isoflurane in oxygen at the same rate once animals were fully anesthetized. Physical examination was conducted to identify age, sex, and body condition, as well as any macroscopic injuries or abnormalities. Age was estimated as juvenile (less than 1 year old), subadult (pre-reproductive, ~1-2 years old), and adult (greater than ~2 years old) based on morphometric measurements of forearm length, body mass, tooth appearance, and reproductive maturity. Distinction between adults and subadults was made based on penis and testes size and development in males, and pregnancy (via abdominal palpation) or evidence of past suckling (based on nipple protrusion and balding around nipples) in females. All bats were marked by painting the claws on one hind limb with colored nail lacquer to identify recently captured bats and avoid resampling during the consecutive days of capture sessions. Bats captured from May 2019 onwards were also marked with a subcutaneous RFID Passive Integrated Transponder (PIT tag, or ‘microchip’; ZD Tech Group, China) inserted between the scapulae. After sampling, each bat was monitored for recovery from anesthesia for at least 30 minutes; good grip ability and airway stability were confirmed before release. Prior to PIT tag insertion, a maximum of 2.5 mL of blood was drawn from the cephalic or uropatagial vein, with samples not exceeding 0.6% of body mass. Blood smears were prepared on-site upon sample collection using blood drawn directly from the syringe, without the use of anticoagulant agents. Multiple smears were made for each bat when blood volume allowed. Smears were dried at ambient temperature and fixed in 100% methanol for three minutes. The samples were then stored at room temperature and out of UV light for up to two years until analysis at Montana State University (Import Permit No. 20200728-2504A). Blood smears were stained with the commercial Romanowsky stain variant DipQuik (Jorgensen Laboratories,Loveland, CO, 80538, USA). Shape, length, texture, and cell monolayer of each smear were examined for quality, and only medium and high-quality smears were analyzed. Smears with uneven distribution of leukocytes, damage or poor stain quality, and high numbers of reactive, unidentifiable leukocytes were not analyzed. The morphological characteristics of leukocytes, erythrocytes, and thrombocytes in the monolayer were assessed using an AmScope E5 Biological Series microscope. Standard leukocyte differentials were performed manually by counting 100 different leukocytes in the monolayer of each smear. Up to three differential counts (up to 300 leukocytes total) were performed on each smear to ensure at least 2/3 of the monolayer was examined. To avoid inconsistencies in identification of cells that could bias results, a single laboratorian conducted all smear examinations.