High tolerance to repeated cycles of freezing and thawing in different Trichinella nativa isolates

Trichinella nativa is the most frequent Trichinella species in arctic wildlife and also the predominating species seen in Norwegian fauna. The adaptation of T. nativa to a cold climate is reflected by the well-documented freeze tolerance of its muscle larvae. The ability of the larvae to survive repeated freezing and thawing events has not however been elucidated and was investigated in the present study, using an Alaskan isolate and two isolates from coastal and inland Norway, respectively. Each T. nativa isolate was inoculated in foxes (Vulpes vulpes) and the muscle tissue obtained after 20 weeks was minced and divided into freezer bags. In the initial part of the study, the bags were exposed to either continuous freezing (-5 degrees C) for up to 7 weeks or freezing (-5 degrees C) for up to 7 weeks with seven overnight (+21 degrees C) thawing events. Once a week a bag was removed from each group, the meat was digested and muscle larvae isolated. In vitro assessment of larval viability was carried out based on larval motility (active vs non-active) and morphology, coiled (alive) or C-shaped (dead). Larval infectivity was subsequently bioassayed in mice, administering 500 larvae per mouse. The mice were euthanised 4 weeks post inoculation, the muscle digested and larvae per gram (lpg) and reproductive capacity index (RCI) were calculated. During the second part of the study, some of the minced fox muscle, exposed to the initial freeze protocol, was stored for a further 23 weeks at -18 degrees C prior to in vitro and in vivo assessment of larval viability and infectivity. The study demonstrated that Trichinella isolates originating from carnivores from higher northern latitudes expressed highest tolerance to freezing and that temperature fluctuations around freezing point, for up to 7 weeks, had little effect on larval infectivity. A negative effect of the initial repeated freeze-thaw events could be demonstrated once the larvae were exposed to longer periods of subsequent deep freezing. Furthermore, it was demonstrated that larval morphology and motility are not suitable for the assessment of infectivity of Trichinella larvae. It was concluded that bioassay in mice was the only suitable method currently available for assessing larval viability.