Malaria parasite developmental analyses by the nested polymerase chain reaction method: an implication for the evaluation of mosquito infection rates in epidemiological studies.

A malaria mosquito vector, Anopheles saperoi, and a non-vector, Aedes albopictus, were allowed to feed on mice infected with murine malaria, Plasmodium yoelii nigeriensis, and were subsequently monitored for the development of parasites by the nested polymerase chain reaction (PCR) method, using Plasmodium genus-specific primer pairs. The mosquitos were divided into two parts, head/thorax and abdomen, for DNA analyses. The parasite DNA and murine DNA for each mosquito were examined in parallel. In both groups of mosquitos, murine DNA was detected up to 4 days post-blood meal in both the head/thorax and abdomen. After 4 days, the murine DNA fell below detectable limits. Murine DNA and parasite DNA remained undigested for the first 4 days post-blood meal. Parasite DNA was detected in the abdomen of 25% (3/12) of Ae. albopictus on day five and 10% (1/10) on day six, after murine DNA had fallen below detectable limits. Parasite DNA was not detected in the head/thorax of Ae. albopictus on those days or afterwards in either the head/thorax or abdomen, demonstrating that the parasite detected on days 5 and 6 in the abdomen degenerated and did not develop into mature oocysts or sporozoites. In the vector An. saperoi, parasite DNA was detected continuously in the head/thorax and abdomen for many days after the murine DNA had fallen below detectable limits. The detection rate of parasite DNA in the head/thorax of An. saperoi increased gradually from day 8 post blood meal until it reached a maximum level of 71.4% (15/21 12 days post-infection. Parasite DNA in abdomen reached its maximum level of 81% (17/21) 10 days post-blood meal. The implications of these results for the design and interpretation of epidemiological surveys is discussed.

Mining microorganism EST databases in the quest for new proteins

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20 of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization

Possibility of false-positive detection for sporozoites in mosquitos (Diptera: Culicidae) by nested polymerase chain reaction using Plasmodium yoelii genomic DNA.

Anopheles stephensi Liston and An. saperoi Bohart and Ingram infected with the rodent malaria parasite Plasmodium yoelii nigeriense. They were examined 12 and 19 days after blood feeding for sporozoites in head with anterior thorax (HT) and oocysts in abdomen with posterior thorax (AB) by light microscopy and by the nested polymerase chain reaction (nested PCR-based on the amplification of the sequences of the small subunit ribosomal RNA gene). The detection rate of parasite DNA by nested PCR in HT samples 12 days after blood feeding was similar to that by microscopic method. However, in HT samples 19 days after blood feeding, the rate by the PCR method was higher than that by the microscopic method. The incidence of sporozoites in salivary glands of infected mosquitos for 12 days after blood sucking was examined by the PCR method. Parasite DNA in HT of Aedes albopictus Skuse (a non vector for the rodent malaria) as well as An. stephensi and An. saperoi was detected for up to 4 days after feeding on mouse with the rodent malaria parasites. The results indicate that when the PCR method is used for detection of sporozoites of human malaria in mosquitos collected in the field, there are possibilities of including false-positive data for mosquitos that have just or recently fed on human blood infected with malaria (erythrocytic form).

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.