Effect of refrigeration and freezing on survival of Toxoplasma gandii tissue cysts

Abstract

Ingestion of Toxoplasma gondii tissue cysts is a major route of infection with this parasite in nature. The viability of brain cysts of the Me49 strain of T. gondii after storage at the usual refrigerator and freezer temperatures of 4°C and -20°C, respectively, was studied by bioassay in mice. The source of brain cysts were mice infected with 10 cysts 8 weeks previously. Brains were homogenized in saline and cysts counted; aliquots assessed to contain 2000 cysts each were stored either at 4°C for 5, 6, 7, 8, 10 and 12 weeks or at -20°C for 24, 48 and 72 hours. At these time points, each aliquot was inoculated into 4 fresh mice (500 cysts per mouse), 2 each by the subcutaneous (s.c.) and peroral (p.o.) routes. All inoculated mice were examined for the presence of either tachyzoites in peritoneal exudates of animals dying within the first 3 weeks after inoculation, or brain cysts in animals that died later than 3 weeks after inoculation or in 6-week survivors. These findings were confirmed by serology. The results showed that infection was induced with cysts stored at 4°C for 5, 6, and 7 weeks, while storage for 8 weeks and longer resulted in complete loss of viability. Cysts stored at -20°C were able to induce infection after 24 and 48h of storage, but not after 72h. Since not all cysts are destroyed simultaneously, as shown by the decreasing percentage of animals infected with inocula stored at either temperature for increasing periods of time, and viability is not completely lost after 48h of storage at -20°C even in a small volume of homogenized tissue, these results suggest that the time of freezing meat for human consumption should be prolonged over 48h as a measure of prevention of T. gondii infection.

U radu su prikazani rezultati ispitivanja vremena preživljavanja tkivnih cista Toxoplasma gondii čuvanih na temperaturama običnog frižidera (4°C) ili zamrzivača (-20°C). Kao izvor cista korišćeni su miševi inficirani sa 10 cista soja Me49; posle 8 nedelja, životinje su žrtvovane, mozgovi homogenizovani i ciste prebrojane. Alikvoti od po 2000 cista su odlagani u frižider na 5, 6, 7, 8, 10 ili 12 nedelja, ili u zamrzivač na 24, 48 i 72 sata. Posle isteka odgovarajućeg vremena, svaki alikvot je inokulisan u po 4 miša, i to u 2 subkutano a u 2 per os. Inokulisani miševi su ispitani na infekciju T. gondii, tako što je kod životinja koje su ugibale u toku prve 3 nedelje po inokulaciji pregledan peritoneaini eksudat na tahizoite, a kod životinja koje su ugibale kasnije kao i kod preživelih koje su žrtvovane posle 6 nedelja pregledan mozak na tkivne ciste. Direktni nalaz je potvrđen i serološki, dokazivanjem specifičnih IgG antitela pomoću direktnog aglutinacionog testa. Rezultati su pokazali da je moguće inficirati miševe cistama koje su čuvane 5, 6 i 7 nedelja na 4°C, pri čemu se procenat inficiranih miševa smanjivao sa svakom nedeljom stajanja, a da su ciste koje su stajale 8 i vise nedelja u potpunosti izgubile vijabilnost. S druge strane, bilo je moguće izazvati infekciju miševa cistama koje su bile zamrznute na -20°C tokom 24 pa i 48, ali ne i 72 h. Međutim, podatak da i posle 48 h zamrzavanja u malom volumenu homogenizovanog tkiva jedan broj cista zadržava vijabilnost, sugeriše potrebu da se u cilju prevencije toksoplazmatske infekcije meso za ljudsku ishranu zamrzava duže od uobičajeno u literaturi navođenih 24-48 h. .