Publications

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

Polymerase chain reaction (PCR)-based genotyping of oocysts dissected from mosquito midguts has previously been used to investigate overall levels of inbreeding within malaria parasite populations. We present a re-analysis of the population structure of Plasmodium falciparum malaria using diploid genotypes at three antigen-encoding loci in 118 oocysts dissected from 34 mosquitoes. We use these data to ask whether mating is occurring at random within the mosquito midgut, as is generally assumed. We observe a highly significant deficit of heterozygous oocysts within mosquitoes at all three loci, suggesting that fusion of gametes occurs non-randomly in the mosquito gut. A variety of biological explanations, such as interrupted feeding of mosquitoes, positive assortative mating and outcrossing depression, could account for this observation. However, an alternative artefactual explanation–the presence of non-amplifying or null alleles–can account for the observed data equally well, without the need to invoke non-random mating. To evaluate this explanation further, we estimate the frequencies of null alleles within the oocyst population using maximum likelihood, by making the assumption that non-amplifying oocysts at any of the three loci are homozygous for null alleles. Observed levels of visible heterozygotes fit closely with those expected under random mating when non-amplifying oocysts are accounted for. Other lines of evidence also support the artefactual explanation. Overall inbreeding coefficients have been recalculated in the light of this analysis, and may be considerably lower than those estimated previously. In conclusion, we suggest that the deficit of heterozygotes observed is unlikely to indicate non-random mating within the mosquito gut and is better explained by misscoring of heterozygotes as homozygotes.

Parasite genotyping by the polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in a Karen population resident on the northwestern border of Thailand where malaria transmission is low (one infection/person/year). Plasmodium falciparum infections were genotyped for allelic variation in three polymorphic antigen loci, merozoite surface proteins-1 and -2 (MSP-1 and -2) and glutamaterich protein (GLURP), before and after antimalarial drug treatment. Population genotype frequencies were measured to provide the baseline information to calculate the probability of a new infection with a different or the same genotype to the initial pretreatment isolate. Overall, 38% of the infections detected following treatment had an identical genotype before and up to 121 days after treatment. These post-treatment genotypes were considered recrudescent because of the low (< 5%) probability of repeated occurrence by chance in the same patient. This approach allows studies of antimalarial drug treatment to be conducted in areas of low transmission since recrudescences can be distinguished confidently from newly acquired infections.

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0.21 to 0.87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

It has recently been shown using genetic markers that Ascaris in humans and pigs in Central America comprise reproductively isolated populations. We present a similar analysis for a region of China in which close association between pigs and humans has been the norm for thousands of years, and agricultural practices will result in frequent exposure to eggs from both sources. DNA fragments from selected regions of mitochondrial and ribosomal DNA were amplified by PCR and allelic forms identified following digestion with a panel of restriction enzymes, using DNA from a total of 115 individual worms from both people and pigs from 2 neighbouring villages. Significant frequency differences in both mtDNA haplotypes and the rDNA spacer were found between the 2 host-associated populations, indicating that they represented reproductively isolated populations. Mitochondrial haplotype frequencies were different from those observed in Guatemala and also from other Asian Ascaris populations, suggesting low levels of gene flow between populations. However, we found no evidence for significant heterogeneity in the genetic composition of Ascaris infrapopulations in either humans or pigs, possibly indicative of agricultural practices in China which have resulted in a random distribution of alleles within the parasite populations.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.

We describe a variety of restriction site polymorphisms in the introns of Ascaris nuclear genes and in the ribosomal DNA spacers. We use these markers, in addition to previously described mitochondrial variation, to clarify our understanding of the epidemiology of Ascaris in Guatemalan villages where humans and pigs occur in sympatry and to describe the genetic structure of host-associated Ascaris populations from world-wide locations. Intron sequences were amplified from individual worms and alleles defined by endonuclease digestion. Two loci were monomorphic, while 4 length variants and 22 point mutations were detected in the other 7 loci. Within sympatric Guatemalan populations no single locus from either the nuclear or mitochondrial genome was fixed for alternative alleles, although allele frequencies were significantly different at many loci. Phenograms constructed from multilocus nuclear genotypes of individual worms failed to reveal a single case of cross-infection, and demonstrate that divergent mtDNA genotypes are segregating within host-associated populations. On a world-wide scale, the data suggest that extant worm populations result from a single host shift, although characterization of genetic variation in additional loci will be necessary to confirm this. The direction and the geographical origin of the host shift were unresolved. Overall 65% of nuclear genetic variation was found within populations, host (human or pig) explained 18%, while geographical variation within host-associated populations explained 17%. The results (a) demonstrate the utility of introns for studying the epidemiology of parasites showing limited allozyme variation (b) suggest that programmes aiming to control Ascaris infection in the human population can safely ignore zoonotic infection from pigs and (c) illustrate the problems inherent in using single genetic markers to make inferences about the epidemiology of closely related parasite taxa.

Mitochondrial DNA (mtDNA) is finding increasing usage as a tool for studying the systematics, population genetics and epidemiology of parasitic helminths, and is generally assumed to be inherited maternally. Yet two features of Ascaris biology–fertilization by large amoeboid sperm and some novel aspects of sperm mitochondria–suggest a paternal component to mitochondrial inheritance in this organism. In this study, we compare mtDNA restriction patterns of parental worms with those of their progeny but find no evidence for paternal inheritance. We suggest that sperm-derived mitochondria are actively destroyed or outcompeted by maternal organelles in the zygote.

Cases of human Ascaris infection occur sporadically in areas such as N. America and Western Europe, where this parasite is thought to be non-endemic. Clinical case histories suggest that many of these cases may be cross-infections from pigs. I describe patterns of variation in the ribosomal DNA of Ascaris from 9 such cases. For comparative purposes, I also describe patterns of variation in parasites obtained from pigs and humans from worldwide locations. A Hae III restriction site distinguishes two classes of rDNA repeats; repeats bearing this restriction site were found in > 96% of parasites from pig populations worldwide and in all 9 worms from humans in N. America. In contrast, repeats bearing this restriction site were detected in < 2% of parasites from humans in endemic areas. The molecular data clearly incriminate pigs as the source of infection in the N. American cases. I discuss evolutionary and public health implications of incomplete host fidelity in Ascaris.