Axenic cultivation and pathogenic assays of acanthamoeba strains using physical parameters

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity [0.5 M and 1 M] of non-nutrient agar plates were performed. One hundred fiftymicrol of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 micro L of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37degreeC and 42degreeC. All plates were monitored for 24 h, 48 h and 72 h. Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature [42degreeC] and osmolarity [1.5 M] and thus they were classified as weak pathogens. Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.

Variation of the chloroquine resistance transporter [crt] gene in chloroquine-resistant and chloroquine-sensitive plasmodium berghei

The emergence and spread of chloroquine resistant Plasmodium falciparum in the world stimulated some investigators to consider different aspects of chloroquine resistance in human and rodent Plasmodia. Using animal Plasmodia, particularly primate and rodent Plasmodia can be useful model for human Plasmodia studies. In this study we have tried to consider and compare the sequence of chloroquine resistance transporter [crt] gene among chloroquine-resistant and chloroquine-sensitive strains of Plasmodium berghei. This experimental study was performed at the Malaria Laboratory of School of public health. DNA was extracted from two strains of P. berghei which their resistance and sensitivity had been demonstrated in mice with treatment by chloroquine. By using specific primer for crt gene some parts of this gene were amplified by PCR, and obtained fragments were then sequenced and compared. There were considerable differences in crt gene between two strains. Sequenced 1212 bp of crt gene fragment in the two strains showed 43 differences at nucleotides level and 16 differences in presumed coding amino acids. crt can be addressed as a considerable gene which involves in induction of resistance to chloroquine in P. berghei, as P. falciparum. The results increased such a promise that considering crt gene in chloroquine-sensitive and chloroquine-resistant P. berghei can prepare suitable and helpful fields for more understanding the molecular aspects of chloroquine-resistance in Plasmodia and reversing the effectiveness of 4-aminoquinolines particularly chloroquine for treatment of drug resistant Plasmodia