Recovery of Leptospires in Short- and Medium-Term Cryopreservation Using Different Glycerol and Dimethyl Sulfoxide Concentrations

Abstract

Cryopreservation is a recognized method for the maintenance of Leptospira collections. Although cryoprotectants are commonly used in order to prevent or reduce the adverse effects of freezing, there is no consensus regarding the protocols of cryopreservation. This study aimed to compare cryopreservation protocols for Leptospira using different glycerol and dimethyl sulfoxide (DMSO) concentrations. Leptospira interrogans serovar Icterohaemorrhagiae, L. interrogans serovar Bratislava, and L. borgpetersenii serovar Hardjo were used as the experimental strains. For each strain, three protocols were tested using 5% and 10% glycerol and 2.5% DMSO. For each protocol, 12 tubes containing 1.5 mL of serovar were frozen at −70°C on the same day. An aliquot of each serovar/protocol was thawed once a month throughout 1 year. The viability of leptospires was evaluated by the recovery of those at days 7, 14, and 21 after thawing. Although no significant difference was found among the leptospiral recovery rates for the 9 serovar/protocols tested, DMSO (2.5%) was shown to be slightly better than glycerol, and its use should be encouraged as a cryoprotectant for leptospires.

Introduction

Leptospiral isolates are usually maintained at 28–30°C in semisolid media [Fletcher or semisolid Ellinghausen-McCullough-Johnson-Harris (EMJH)] with periodic subculture into fresh media at intervals of ≤2 months.¹ However, this preservation method has important drawbacks, since it is time-consuming and the regular handling of live cultures increases the risk to laboratory personnel.

In addition, it is well-known that the virulence of pathogenic leptospires is gradually lost after several passages in culture media.^2,3 This may impair experimental studies, particularly those focused on virulence or on host–pathogen interactions. Thus, considering the advantages of cryopreservation, it is assumed that freezing is particularly useful for studies on leptospirosis pathogenesis or vaccine development, when strains must remain virulent after thawing.²

Conventional methodologies used for long-term preservation of other microorganisms, such as freezing of isolates at −70°C or in liquid nitrogen (LN₂) with cryoprotectants, have shown valuable results.^1,3,4 Although cryoprotectants are commonly used in order to prevent or reduce the adverse effects of freezing, there is no consensus regarding the protocols of cryopreservation. This subject has recently been a point of discussion on the International Leptospirosis Society mail list, and discrepancies on employed protocols by different laboratories worldwide became evident.

There are different recommendations regarding the concentration of the initial inoculum, type and concentration of cryoprotectants, as well as the freezing method. Currently, the two most commonly used cryoprotectants for Leptospira cryopreservation are glycerol and dimethyl sulfoxide (DMSO).^3,5,6 While some authors have reported good results using glycerol,^1,7 other studies have suggested a higher toxicity of this cryoprotectant and have recommended the use of DMSO.^5,6

Thus, the aim of this study was to compare the recovery of leptospires in short- and medium-term experiments using different glycerol and DMSO concentrations.

Materials and Methods

Preparation of cultures

The most prevalent strains in dogs, horses, and cattle,⁸ respectively, Leptospira interrogans serovar Icterohaemorrhagiae (reference strain Verdun), L. interrogans serovar Bratislava (reference strain Jez Bratislava), and L. borgpetersenii serovar Hardjo (reference strain Sponselee), were studied. These strains underwent 15, 27, and 9 passages onto culture media (EMJH) prior to cryopreservation, respectively. All strains belonged to the collection of Veterinary Bacteriology Laboratory of Universidade Federal Fluminense (originally from Institut Pasteur, Paris, France). They were maintained in liquid EMJH medium (Difco, BD Diagnostics, Sparks, MD) and incubated at 28–30°C for 7–10 days. In order to perform the freezing procedure, it was established that each tube should be in late logarithmic growth at a density of 1 × 10⁷ leptospires/mL, based on counting in a Petroff-Hausser chamber.⁹

Freezing protocols

Three protocols were tested: (i) EMJH added to 5% glycerol, (ii) EMJH added to 10% glycerol, and (iii) EMJH added to 2.5% DMSO. For each protocol, 12 tubes containing 1.5 mL of each serovar were immediately frozen at −70°C on the same day.

Recovery of leptospires

Once a month (over 12 months), one tube of each strain/protocol was thawed at room temperature for 30 min and then transferred to a 5 mL EMJH liquid media tube. Tubes were incubated in a bacteriological incubator (BOD, Eletrolab, São Paulo, Brazil) at 28–30°C and analyzed under dark-field microscopy at 7, 14, and 21 days after thawing.

Statistics

Data for recovery of the isolates after the use of each protocol were analyzed using SPSS Statistics for Windows, version 20 (IBM Corp., Armonk, NY). In order to evaluate the developed protocols, McNemar (two samples) and Cochran's Q (K samples) tests were used. Additionally, the chi-square test was used to analyze 2 × 2 contingency tables in each single moment of the study for recovery data. A confidence interval of 95% was used (P < 0.05) to determine significance.

Results

Regardless of the strain, time of freezing, or protocol, all strains were successfully recovered at the end of the experiment (D21; Table 1).

Table 1.

Recovery Rate of Frozen (−70°C) Leptospires on Days 7, 14, and 21 After Thawing to Each Serovar/Protocol All Over 12 Months

		5% Glycerol: Day			10% Glycerol: Day			2.5% DMSO: Day
Species/serovar	Month after freezing	7	14	21	7	14	21	7	14	21
L. interrogans serovar Icterohaemorrhagiae	1	+	+	+	+	+	+	+	+	+
	2	+	+	+	+	+	+	+	+	+
	3	+	+	+	+	+	+	+	+	+
	4	+	+	+	+	+	+	+	+	+
	5	+	+	+	+	+	+	+	+	+
	6	–	+	+	–	+	+	+	+	+
	7	–	+	+	–	+	+	–	+	+
	8	–	–	+	–	–	+	–	+	+
	9	–	–	+	–	–	+	–	+	+
	10	–	–	+	–	–	+	–	+	+
	11	–	–	+	–	–	+	–	+	+
	12	–	–	+	–	–	+	–	+	+
L. borgpetersenii serovar Hardjo	1	+	+	+	+	+	+	+	+	+
	2	+	+	+	+	+	+	+	+	+
	3	+	+	+	+	+	+	+	+	+
	4	+	+	+	+	+	+	+	+	+
	5	+	+	+	+	+	+	+	+	+
	6	+	+	+	+	+	+	+	+	+
	7	+	+	+	+	+	+	+	+	+
	8	+	+	+	+	+	+	+	+	+
	9	+	+	+	+	+	+	+	+	+
	10	+	+	+	+	+	+	+	+	+
	11	+	+	+	+	+	+	–	+	+
	12	+	+	+	–	+	+	+	+	+
L. interrogans serovar Bratislava	1	+	+	+	+	+	+	+	+	+
	2	+	+	+	+	+	+	+	+	+
	3	+	+	+	+	+	+	+	+	+
	4	+	+	+	+	+	+	+	+	+
	5	+	+	+	+	+	+	+	+	+
	6	+	+	+	+	+	+	+	+	+
	7	–	+	+	+	+	+	+	+	+
	8	+	+	+	+	+	+	+	+	+
	9	–	+	+	+	+	+	+	+	+
	10	+	+	+	+	+	+	+	+	+
	11	–	–	+	–	–	+	–	+	+
	12	–	+	+	–	–	+	–	+	+

+, presence of pure Leptospira culture after thawing; –, absence of Leptospira culture after thawing.

Although not significantly different, a slight positive effect in leptospires growth was observed when using DMSO.

It is noteworthy that L. interrogans serovars Icterohaemorrhagiae and Bratislava needed at least 3 weeks of incubation to present pure growth after 8 and 11 months of freezing, respectively.

Discussion

Glycerol has been reported to be 10 times more toxic than DMSO to leptospires,⁶ which may explain the observed difference on Leptospira recovery rate. This tendency was clearly observed in tubes that were kept for >9 months, and was particularly evident for Icterohaemorrhagiae strains.

Concentrations of glycerol >5% may result in cellular toxicity to leptospires.⁷ Furthermore, it has been reported that L. interrogans serovar Copenhageni (same group of Icterohaemorrhagiae) grew poorly after freezing with glycerol at concentrations >10% compared with DMSO.⁵

It is noteworthy that since this study was conducted with strains that presented high passages onto culture media, phenotypic or genotypic evaluation of virulence after thawing was not possible. Despite that, good resilience of the tested strains was demonstrated in the short and medium terms. Moreover, although the outcomes are in agreement with other studies,^1,4 the possible differences among the leptospiral species regarding their cryopreservation remains to be better elucidated.

In conclusion, although no significant difference among the leptospiral recovery rates for the 9 serovar/protocols tested was found, DMSO (2.5%) was shown to be slightly better than glycerol, and its use should be encouraged as a cryoprotectant for leptospires.

Footnotes

Acknowledgments

This study was supported by CNPq and FAPERJ, Brazil. W.L. is a CNPq fellow.

Author Disclosure Statement

No conflicting financial interests exist.

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