In vitro culture and isoenzyme analysis of giardia lamblia.

Abstract

Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.