In vitro viability of purified microfilariae
The viability of cryopreserved mf was compared between traditional PVP-DMSO and several commercially available cryopreservation media. Microscopic observation revealed that mf cryopreserved with CultureSure exhibited the highest motility (χ2 = 721.2, df = 7, P < 0.0001, AR = 8.5 on 0 dpt; χ2 = 648.5, df = 7, P < 0.0001, AR = 10.51 on 1 dpt and χ2 = 333.3, df = 7, P < 0.0001, AR = 12.19 on 3 dpt) (Fig. 1a). Mf cryopreserved with LaboBanker exhibited the second highest motility (AR = 7.67 on 0 dpt, 9.88 on 1 dpt, and 6.55 on 3 dpt), and Mf in CryoScarless exhibited the lowest motility (AR = -24.03 on 0 dpt, -20.06 on 1 dpt, and -10.12 on 3 dpt). When mf motility was compared based on moderately active and highly active levels, mf in CultureSure (t = 6.634, df = 2.214, P = 0.017 on 0 dpt; t = 6.682, df = 3.118, P = 0.0061 on 1 dpt and t = 6.76, df = 3.835, P = 0.0029 on 3 dpt), LaboBanker (t = 4.495, df = 3.934, P = 0.011 on 0 dpt; t = 5.944, df = 3.373, P = 0.0068 on 1 dpt and t = 4.493, df = 3.997, P = 0.011 on 3 dpt) and Cellbanker (t = 5.024, df = 3.571, P = 0.0099 on 0 dpt; t = 6.184, df = 2.383, P = 0.016 on 1 dpt and t = 3.02, df = 3.698, P = 0.043 on 3 dpt) media all exhibited significantly higher motility on 0, 1 and 3 dpt than mf preserved in traditional PVP-DMSO media. Mf in CryoScarless medium showed significantly lower motility compared to PVP-DMSO on 0 (t = 18.87, df = 2.116, P = 0.0022) and 1 dpt (t = 10.61, df = 2.071, P = 0.0078).
The viability of mf on 3 dpt was quantified based on the ability of PI to penetrate dead or dying cells but not living cells. CultureSure-cryopreserved mf showed significantly lower PI values compared to all of the other cryopreservation media (χ2 = 161.3, df =7, P < 0.0001, AR = 5.7) (Fig. 1b). The mf cryopreserved in PVP-DMSO (AR = 3.59), LaboBanker (AR = 2.87) and Bambanker (AR = 2.53) showed significantly lower PI positive rates (Fig. 1b). These results are supported by fluorescent imaging of mf PI staining in each medium (Fig. 1c). Translating our results, to do a parallel with other published studies, the total motile parasite ratio of CultureSure-cryopreserved mf after thawing (97.2%, Fig. 1a) was considered equivalent or slightly better than the reported for cryopreserved mf using 5% DMSO medium (78.8–91.6%) [5] or PVP-DMSO (> 95%) [6]. From these experiments, it is clear that CultureSure is the optimal cryopreservation medium tested for the preservation of D. immitis mf.
Parasite concentration levels were a factor influencing motility in the various media. In general, motility of mf cryopreserved at a high concentration (c.15,000 mf/ml) was significantly greater than mf cryopreserved at a low concentration (c.3000 mf/ml). This was true for mf cryopreserved in PVP-DMSO on 0 to 3 dpt (t = 4.451, df = 3.499, P = 0.015 on 0 dpt; t = 36.17, df = 2.929, P < 0.0001 on 1 dpt and t = 11, df = 2.414, P = 0.004 on 3 dpt), in CultureSure on 1 (t = 4.708, df = 3.817, P = 0.010) and 3 dpt (t = 3.805, df = 2.865, P = 0.035) and in LaboBanker on 0 (t = 4.748, df = 3.291, P = 0.014) and 3 dpt (t = 6.522, df = 2.901, P = 0.008) (Fig. 1d). These results suggest that cryopreservation of mf should be carried out at the higher mf concentration to retain better viability.
In vitro viability of microfilariae in mf-infected total blood
Cryopreservation of D. immitis mf-infected total blood is especially important in clinical settings. The motility of mf in mf-infected total blood was compared over a range of blood volumes in CultureSure (1 ml total volume) and with purified mf (c.25,000 mf/ml) (Fig. 2a). The results were comparable to purified mf cryopreservation, in most cases. Significant differences were observed between purified mf and mf-infected total blood volumes of 400 µl on 0 dpt (q = 5.431, df = 12, P = 0.022) and 300 µl on 3 dpt (q = 5.38, df = 12, P = 0.024). In addition, a significant difference was seen for mf-infected volumes of 200 µl and 400 µl on 0 dpt (q = 5.048, df = 12, P = 0.035). Analysis of PI staining of mf on 3 dpt, demonstrated that mf-infected total blood at a volume of 200 µl showed significantly lower PI positive values (χ2 = 12.19, df =5, P = 0.032, AR = 2.36, Fig. 2b), whereas the 300 µl sample showed the highest PI staining values (AR = 1.98, Fig. 2b). These findings were supported by fluorescent imaging (Fig. 2c). Altogether, these results suggest that cryopreservation is optimal with 200 µl (c.25 mf/µl) of infected blood brought to a total volume of 1 ml with CultureSure.
Vector infectivity and survival of cryopreserved microfilariae
Infectivity of mosquito vectors with D. immitis mf cryopreserved with CultureSure or LaboBanker was compared with traditional PVP-DMSO cryopreservation. Non-cryopreserved, fresh mf were used as a positive control. The survival rate of mosquitoes fed LaboBanker cryopreserved mf was significantly higher compared to mosquitoes fed CultureSure (χ2 = 4.761, df = 1, P = 0.029) or PVP-DMSO (χ2 = 4.086, df = 1, P = 0.043) cryopreserved mf on 13 dpi (Fig. 3a). The number of L3 in mosquitoes fed CultureSure cryopreserved mf tended to be higher than that of the L3 for mf cryopreserved in PVP-DMSO (t = 1.728, df = 53.64, P = 0.090) and fresh (t = 1.897, df = 56.14, P = 0.063) mf (Fig. 3b). Although different mosquito colonies and mf infectious inocula were used, the mf infectivity reported here was similar to the one previously reported for PVP-DMSO cryopreserved mf [6]. The ratios of mean L3 numbers in individual mosquitoes/feeding inoculum concentration were 0.85 and 0.77 for PVP-DMSO cryopreserved mf in our study and a previous study, respectively [6]. Remarkably, this ratio, determined for CultureSure was 1.03. The number of L3 of mosquitoes fed LaboBanker cryopreserved mf was significantly lower compared to the other treatments (Fresh: t = 5.868, df = 71.41, P < 0.0001; PVP-DMSO: t = 5.67, df = 45, P < 0.0001 and CultureSure: t = 6.496, df = 42.96, P < 0.0001). These results indicate that CultureSure is an optimal medium for D. immitis mf cryopreservation. Lok et al. [5] demonstrated that 6.0– 20.0% of mf cryopreserved in 5% DMSO medium reached the L3 stage when inoculated to mosquitoes using the enema method. The estimated average blood volume ingested by Ae. aegypti per blood meal was 2.3–2.6 µl [12, 13]. In our study, assuming a blood intake of 2.5 µl (18 mf/µl) per mosquito, the L3 development ratio was calculated as 41.8% (18.8 L3/45 mf), suggesting CultureSure is better than 5% DMSO medium for the cryopreservation of D. immits mf.
Importance of DMSO in cryopreservation media
DMSO appears to be an important component in cryopreservation media for D. immitis mf. In the present study, most of the commercial cryopreservation media examined yielded a positive performance, except for CryoScarless, which was the only medium that did not contain DMSO. DMSO is known to inhibit ice crystal formation during the freezing process and it is commonly used for mammalian cell cryopreservation [14]. Selection of a DMSO-containing medium seems critical for successful cryopreservation of D. immitis mf.
Justification and practical applications for using cryopreserved microfilariae
Preparation of D. immitis-infected animals and fresh mf is a bottleneck for in vivo mosquito vector experiments. Here, we demonstrated that cryopreserved mf could be used for in vivo mosquito experiments instead of fresh mf, thereby eliminating the need to maintain infected animals. The concentration of cryopreserved mf fed to mosquitoes in mf-infected blood was adjusted to 3-fold higher levels than non-cryopreserved, fresh mf. Under these conditions, the number of L3 for the mosquitoes was almost equivalent to that of fresh mf.
Standardization of D. immitis mf cryopreservation method is essential for the study of drug-resistant D. immitis. Macrocyclic lactones have been widely used to prevent heartworm infections for the past three decades [15]. Since the emergence of macrocyclic lactone resistance in D. immitis in 2005 [16], numerous field and laboratory-induced isolates have been studied [17–19]. These isolates are essential for further studies to completely understand drug resistance in D. immitis. However, it is quite expensive and time-consuming to maintain each of these isolates by continuous alternate passages in mosquitoes and dogs. Although cryopreservation methods are not without limitations, such as the need for continuous and relatively frequent replenishment of liquid nitrogen levels, they offer unique advantages over continuous biological maintenance. In addition and importantly, cryopreservation allows the reduction of animal use (fewer dogs are needed), respecting the ethics in animal experimentation, and makes the continuous maintenance of an insectary for mosquito production expendable.
Cryopreservation of D. immitis mf has practical applications for veterinary science education [20]. Currently, diagnosis of D. immitis infection is usually performed with a commercial antigen detection kit; however, false negatives are common [21]. Adequate skills need to be developed to ensure mf detection in clinical settings. The supply of D. immitis mf available for veterinary education curricula is limited due to the difficulty of maintaining live animals infected with D. immitis mf. We believe our results validate the use of a commercial cryopreservation medium for D. immitis mf, overcoming the current issues.