Female beagles (2-4 years old) were experimentally infected with D. immitis SF1 strain originally isolated in Japan  and reared under a veterinarian’s administration. Aedes aegypti Liverpool (LVP)-OB strain, kindly provided by Dr. R. Maeda, the Obihiro University of Agriculture and Veterinary Medicine (OUAVM), susceptible to D. immitis infection was maintained at 27 ˚C and >80% humidity on a diet of 5% fructose and a 12:12 h light-dark cycle.
Mf purification and cryopreservation
Cryopreservation was initially tested using purified D. immitis mf isolated from heparinized blood of an experimentally infected dog. The mf were purified using a PD-10 gel purification column (GE Healthcare, Amersham Place, England) equilibrated with RPMI 1640 Medium (Gibco 2120429, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1x penicillin-streptomycin (Gibco 15140122) [8, 9]. Purified mf were centrifuged at 1,000g for 10 min at room temperature. The supernatant was discarded and the pelleted mf were resuspended in 1 mL of cryopreservation medium (ca. 3,000 or 15,000 mf/tube) and transferred to cryogenic tubes (Nunc 119546, Thermo Fisher Scientific). Samples were stored in freezing containers (Corning® CoolCell® LX, Corning, NY, USA) overnight at -80 ˚C before being transferred into liquid nitrogen, and stored until analysis. Cryopreservation media used in this study were as follows: PVP-DMSO (0.004 M PVP, average molecular weight of 40,000 and 6% DMSO) in phosphate buffered saline (PBS) [6, 10], CultureSure® Freezing Medium (CultureSure, FUJIFILM Wako, Osaka, Japan), Cellbanker® 1 (Cellbanker, Zenoaq Resource, Shiga, Japan), LaboBanker 1 (LaboBanker, Kurabo, Osaka, Japan), Cos banker (Cosmo Bio, Tokyo, Japan), Bambanker® (GC Lymphotec, Tokyo, Japan), CryoScarless® DMSO-Free (CryoScarless, Bio Verde, Kyoto, Japan), and RPMI 1640 (Table 1).
In vitro motility assay
Cryopreserved mf were thawed rapidly by swirling in a water bath at 37℃. Immediately after thawing, the mf were suspended in 10 mL of RPMI 1640 medium and centrifuged at 1,500g for 10 min at room temperature. The pelleted mf were resuspended in 1 mL of RPMI 1640 medium containing 5% fetal calf serum, transferred to 12-well cell culture plates (Nunc), and incubated in 5% CO2 in air at 37 °C for 3 d. The motility of mf was assessed microscopically on 0, 1, and 3 d post thawing (dpt) (n ≥ 3), and subjectively scored as inactive or dead (-), less active (+), moderately active (++), and highly active (+++) . The results were statistically analyzed to rank the various media using a chi-square test and adjusted residuals (AR). Absolute AR values were considered significant at AR ≥ 1.96. The difference between PVP-DMSO and each cryopreservation medium was analyzed using Welch’s t-test.
Cryopreservation of D. immitis mf-infected total blood
Fresh, heparinized blood (50-400 µL) was collected from D. immitis-infected dogs (ca. 25,000 mf/mL) and mixed with CultureSure up to 1 mL, and then cryopreserved as described above. Differences in motility between purified mf and mf-infected total blood were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test.
Propidium iodide (PI) staining assay
To evaluate any physical damage caused by cryopreservation, cultured mf were stained with PI (2 µg/mL) (Molecular probes, Eugene, Orfegon, USA) for 10 min on 3 dpt. The stained mf were washed with PBS by centrifugation at 1500g for 10 min at 4 °C, and then subjected to fluorescence microscopy observation using the same exposure time in each experiment. Methanol-fixed mf were used as a positive control. The results were statistically compared using a chi-square test and calculated AR.
In vivo third stage larva (L3) development assay
Mf cryopreserved with commercial CultureSure, LaboBanker media, and traditional PVP-DMSO were used to determine the infectivity of mosquitoes in vivo. Thawed mf was fed to female Ae. aegypti on 7 d post-emergence for 2 h by artificial membrane feeding using Parafilm (Bemis Flexible Packaging, USA). Blood feeding experiments were completed within 4 h post-thawing. The concentration of thawed mf was adjusted to 18 mf/µL using heparinized blood collected from a healthy dog. Non-cryopreserved fresh blood (6 mf/µL) was collected from an infected dog and used as the control. Fully fed mosquitoes were selected under CO2 anesthesia and maintained for 13 d. Mosquito survival was monitored every day. At 13 d post-infection (dpi), all surviving female mosquitoes were dissected under a stereomicroscope to investigate the number of L3. Mosquito survival was analyzed by chi-square test, and the L3 count was analyzed by Welch’s t-test.
All statistical analyses were performed using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). A significant difference was defined by p < 0.05 and p < 0.10 was considered a marginal difference.