Search

Genetic polymorphism in Plasmodium falciparum populations is important as standing genetic variability may provide the potential for populations to adapt quickly when faced with environmental changes such as introduction of new antimalarial drugs. To characterize genetic diversity in this human malaria parasite, presumably neutral microsatellite (MS) and single nucleotide polymorphism (SNP) markers were identified and compared to historic methods of antigenic marker genotyping. In P. falciparum infected samples from Papua New Guinea (PNG), the three methods were shown to be equally viable for identifying diversity, (all heterozygosities > 0.930).To examine the effect of selection pressure by chloroquine (CQ, previously successful antimalarial), on presumably neutral markers in close proximity to a SNP located in the P. falciparum CQ-resistance transporter gene that is responsible for in vitro CQ-resistance, intronic MS were examined. Maximum heterozygosity for these intronic MS in sensitive parasites was 0.879, while in resistant parasites the maximum heterozygosity was 0.232 suggesting that loss of MS heterozygosity results from hitchhiking along with the pfcrt SNP. The data suggests that these markers are not representative of whole genome diversity and should be avoided for overall population studies.For a different perspective of population diversity, 7 bi-allelic SNP markers were genotyped in PNG parasite populations. While heterozygosity was high (range: 0.956-0.980), a defined population structure was found with 80 of 128 possible multi-locus genotypes (MLG) observed. Thirteen predominant MLGs accounted for 50.5% of the samples, suggesting that recrudescence rates in PNG may be overestimated as there is a high probability of pre- and post-treatment strains being similar by chance. The remaining 49.5% of the samples contained 67 MLGs (each at frequencies < 2%), which could enable relatively rapid population adaptation to environmental change.Currently, new antimalarial combination therapies are being introduced in PNG. To monitor the efficacy, antigenic genotyping is usually employed. The SNP-assay described here was found to be a viable alternative, though increasing SNP-numbers may be necessary for better strain differentiation. SNP-based analyses provide powerful approaches for monitoring antimalarial drug and vaccine efficacy, as well as the effect of additional control measures such as bed nets on parasite strain diversity.

The objective of the study was to understand the genotypic and phenotypic variations across Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) isolates form diverse hosts and geographic locations. Multiplex PCR of IS900 loci (MPIL) fingerprint analysis of M. paratuberculosis isolates recovered from diverse hosts and geographic localities clustered 78% of bovine origin isolates into a major node, while isolates from human and ovine sources showed greater genetic diversity. Amplified fragment length polymorphism (AFLP) analysis clustered 75% of the isolates from bovine sources into 2 major nodes while those recovered from sheep or human clustered on distinct branches. This suggested that the M. paratuberculosis isolates were genotypically homogenous or were converging into a common fingerprint. To evaluate the alternate possibility that MPIL and AFLP analyses were inadequate for dissecting the M. paratuberculosis genotypes, short sequence repeat (SSR) analysis was performed on mycobacterial isolates obtained from 33 different host species and from environmental sources. Sequence based characterization of the G-repeat locus enabled differentiation of the M. paratuberculosis isolates from the major MPIL cluster into seven distinct alleles. Subsequent analysis of M. paratuberculosis isolates from human Crohn’s disease cases using two polymorphic SSR loci (G- and GGT- repeats) identified a limited number of genotypes amongst the human strains indicating an association of a few M. paratuberculosis types with the pathobiology of Crohn’s disease. SSR analysis using both polymorphic loci also enabled identification of varying levels of within-farm and between farm diversity and indicated that 7g-4ggt as the most common genotype in animals from Ohio dairy farms. To explore whether the differences in fingerprint profiles translated to variation in biological function and/or host adaptation, cDNA microarray analysis of a human macrophage cell line exposed to M. paratuberculosis isolated from cattle and human hosts was undertaken. Results indicated that the expression profiles induced by representative cattle and human M. paratuberculosis strains differed in several key inflammatory and apoptotic pathways suggesting that M. paratuberculosis strains with different genotypes induce variant transcriptional regulation. Taken together, the results of our genotypic and phenotypic analyses demonstrated that SSR analysis enabled the genetic characterization of M. paratuberculosis isolates from different host species, and provided support for a genotype-phenotype association in M. paratuberculosis infection.