DOI: 10.5061/dryad.gb5mkkws0
title: Leukocyte proportions in Pteropus alecto blood smears

description: The black flying fox (Pteropus alecto) is a natural reservoir for Hendra
 virus, a paramyxovirus that causes fatal infections in humans and horses
 in Australia. Increased excretion of Hendra virus by flying foxes
 has been hypothesized to be associated with physiological or energetic
 stress in the reservoir hosts. The objective of this study was to explore
 the leukocyte profiles of wild-caught P. alecto, with a focus on
 describing the morphology of each cell type to facilitate identification
 for clinical purposes and future virus spillover research. To this end, we
 have created an atlas of images displaying the commonly observed
 morphological variations across each cell type. We provide quantitative
 and morphological information regarding the leukocyte profiles in bats
 captured at two roost sites located in Redcliffe and Toowoomba,
 Queensland, Australia, over the course of two years. We examined the
 morphology of leukocytes, platelets, and erythrocytes
 of P. alecto using cytochemical staining and
 characterization of blood films through light microscopy. Leukocyte
 profiles were broadly consistent with previous studies of P.
 alecto and other Pteropus species. A small
 proportion of individual samples presented evidence of hemoparasitic
 infection or leukocyte morphological traits that are relevant for future
 research on bat health, including unique large granular lymphocytes.
 Considering hematology is done by visual inspection of blood smears,
 examples of the varied cell morphologies are included as a visual guide.
 To the best of our knowledge, this study provides the first
 qualitative assessment of P. alecto leukocytes,
 as well as the first set of published hematology reference images for this
 species. 
description: 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).  <em>P. alecto</em> 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.