Plasmodium knowlesi (W1) shows a high level of innate resistance to mefloquine as shown by recrudescence of blood-induced infection in rhesus monkeys, following oral mefloquine treatment with up to 80–160 mg/kg total dose over 3–4 days. Ketoconazole, at a dose of 75 mg/kg/day×10 days given orally to P. knowlesi-infected rhesus monkeys, exerted a suppressive antimalarial effect in 1 of the 2 monkeys as evidenced by a delayed recrudescence (on day 30) and curative action in case of the other monkey, whereas a lower dose of ketoconazole (25 mg/kg/day×10 days) failed to suppress infection effectively. When ketoconazole at a low dose (25 mg/kg/day×10 days) treatment was combined with mefloquine (20 mg/kg/day×4), 1 of the 2 infected monkeys was cured, while the combination of a higher dose of ketoconazole (75 mg/kg/day×10 days) with mefloquine (20 mg/kg/day×4), had a complete curative effect on P. knowlesi infection in all the treated monkeys (7/7). Besides exerting direct antimalarial action against a mefloquine-resistant P. knowlesi, ketoconazole also acts as an inhibitor of hepatic microsomal CYP4503A, which regulates and slows down mefloquine biotransformation to carboxymefloquine, resulting in a possible elevation of the effective plasma drug level, which seems to be responsible for elimination of the resistant P. knowlesi. The study shows a resistance reversal effect of a ketoconazole – mefloquine combination that could be exploited for the control of mefloquine-resistant malaria infection. The study shows that ketoconazole can reverse mefloquine resistance of P. knowlesi. These findings can also be applied to reverse mefloquine resistance of P. falciparum in areas where mefloquine resistance is already established. The precise role of parasite CYP450 in mefloquine resistance and its possible role, if any, in expelling the intracellular mefloquine from the resistant plasmodia still remains to be elucidated.

Murine monoclonal antibodies (mAbs) against Neospora caninum tachyzoites were produced to identify the cross-reactive antigens between N. caninum and Toxoplasma gondii. Ten mAbs recognizing cross-reactive antigens of both parasites were obtained and tentatively classified into 6 different groups based on their reactivity patterns in an indirect fluorescent antibody test and Western blot analysis. Three mAbs in group 1 recognized antigens located on the surface of parasites with molecular masses ranging from 28 to 76 kDa; one mAb in group 2 recognized antigens located on interior organelles of parasites with a molecular mass of 50 kDa; one mAb in group 3 recognized antigens located on interior organelles of parasites with molecular masses of 35 kDa and 14 kDa; three mAbs in group 4 recognized antigens located on interior organelles with a molecular mass of 64 kDa; one mAb in group 5 recognized antigens located on the surface of parasites with an unknown molecular mass; one mAb in group 6 recognized antigens located on the apical end of parasites with an unknown molecular mass. The mAbs in groups 1, 2, 3, and 5 showed inhibitory effects on the growth of the two parasites in vitro in a dose-dependent manner. A cDNA expression library prepared from N. caninum tachyzoite mRNA was immunoscreened with the mAb panel. Three kinds of proteins, protein disulfide isomerase (PDI), heat-shock protein 70 (HSP70), and ribosomal protein 1 (RP1), were identified as cross-reactive antigens recognized by mAbs in groups 2, 3, and 4, respectively. Some of the proteins could be useful in developing vaccines or drugs for controlling the diseases caused by the two parasites.

Babesia parasites generally require a defined percentage of serum in the culture medium for their in vitro growth. In this study, we attempted to culture Babesia bovis in a serum-free condition. The growth pattern and morphology of B. bovis in serum-free (plain) GIT medium were unaltered as compared to those of the standard growth condition containing 40% bovine serum in M199. When exposed to the test drugs, the parasite in plain GIT medium showed clearly lower IC50 values than those in 40% serum-containing GIT medium, indicating that several serum components may interfere with the drug bio-availability. Therefore, the serum-free culture system is useful for standardizing drug test protocols and understanding the roles of serum factors in the drug test.

Sand fly saliva plays an important role in Leishmania transmission. We characterized the host antibody response to saliva from 3 sand fly species. Specific IgG was observed in sera from experimentally bitten mice as well as in sera from individuals living in the endemic area of Leishmania tropica in Sanliurfa, Turkey. Sera of Sanliurfa inhabitants showed high IgG levels against saliva of Phlebotomus sergenti and P. papatasi, the 2 most abundant sand fly species in this area, but did not react with saliva of the New World sand fly, Lutzomyia longipalpis. Patients with active Le. tropica lesions possessed significantly higher anti-P. sergenti IgG levels than the healthy individuals from the same place while anti-P. papatasi IgG levels were equal in both groups. Major protein bands in P. papatasi and P. sergenti saliva reacted with both, human and mice sera; in P. papatasi, however, mouse IgG recognized preferentially the 42 kDa protein band while the human IgG reacted strongly with the 30 kDa band. Our data suggest that the antibody response to sand fly saliva could be used for monitoring the exposure of humans and other hosts to sand flies and might be used as a marker of risks for Leishmania transmission in endemic areas.

Periods of low host density impose a constraint on parasites with direct transmission, challenging their permanence in the system. The microsporidium Octosporea bayeri faces such constraint in a metapopulation of its host, the cladoceran Daphnia magna, where ponds frequently lose their host population due to ponds drying out in summer and freezing in winter. We conducted experiments aimed to investigate the mechanisms of transmission of O. bayeri, and discuss how these mechanisms could contribute to the parasite's permanence in the system. Spores accumulate in the fat cells and the ovaries of the host, and vary in morphology, possibly corresponding to 3 different spore types. Horizontal transmission occurred through the release of spores from dead hosts, with the proportion of infected hosts depending on the spore dose. Further, spores are able to persist outside the host both in dry and wet conditions. Vertical transmission occurred to both parthenogenetic and sexual offspring. The former were invariably infected, while the sexually produced resting eggs (=ephippia) had a less efficient transmission. The parasite may be carried by the ephippia, and thus disperse to new ponds together with the host. Together, these mechanisms may allow the parasite to endure periods of harsh environmental conditions both outside and inside the host.

We studied the role of Trypanosoma cruzi reinfection in regard to inflammatory and cytokine response at the inoculation site, lymph node and heart. We reinfected Balb/c mice intradermically into the hind foot-pad with natural infective metacyclic trypomastigotes. They were followed from 24 h to 30 days after the last reinfection. At the inoculation site 24 h after the last re-infection, the infiltrating inflammatory cells increased dramatically with respect to baseline inflammation, reaching maximum infiltrates for the third day. In contrast, parasite DNA was undetectable 24 h after inoculation, despite poor cytokine induction, only IFN-gamma, IL-12 and TGF-β were noticeable on days 7 and 15, whereas in the lymph nodes draining the inoculation site positive expression of IL-2, IL-4, IL-12 and TGF-beta were found to be induced as soon as 24 h after re-entry of parasite. In the heart, the inflammatory response increased immediately 24 h after re-entry of parasites, reaching its maximum on the 7th day and returning to baseline on day 30. In conclusion, although the inflammatory response is triggered in both compartments by re-entry of parasites, the inflammatory process returns almost to baseline after 30 days, leaving a persistent low-grade inflammation.

The question whether a stress event can have a long-lasting effect on susceptibility to parasites was studied using a freshwater bivalve clam and its crustacean parasite as a model system. Anodonta piscinalis clams were collected from 2 populations during August–September 2002. Clams were transported to the laboratory and marked. The stressed clams were subjected to low oxygen for 25 days, while the unstressed control clams were caged in their lakes of origin for the same period. Then the clams were transported to a third lake where they were exposed to natural infections by the ergasilid copepod, Paraergasilus rylovi, 11 months after the stress event. The stressed clams were more intensively parasitized. They also showed lower growth, lower reproduction and lower survival than the unstressed control clams. The results indicate that susceptibility of A. piscinalis to P. rylovi infection may be condition dependent, and that stress may have a long-lasting, increasing effect on host susceptibility to parasitism in natural populations.

Oncomelania nosophora (Gastropoda: Pomatiopsidae) is the intermediate host of Schistosoma japonicum in Japan. Although most of the snails were eliminated during the 20th century, they are still found in two areas in Japan. One area is in the Kofu Basin, including Nirasaki City, in Yamanashi Prefecture. The other is the Obitsu River Basin in Kisarazu City, Chiba Prefecture. Snails collected in Nirasaki and Kisarazu were exposed to 3 geographical strains of S. japonicum originating from Japan, China, and the Philippines. Both isolates of O. nosophora showed high susceptibility to the Japanese strain of S. japonicum (74·0%–82·2% for the Nirasaki isolate and 58·0%–56·0% for the Kisarazu isolate) and low susceptibility to the Chinese strain (0·0%–1·3% and 1·4%–7·9% respectively). In contrast, the susceptibility of the snails to the Philippine strain was significantly different (P<0·01) between the isolates (3·3%–6·6% for the Nirasaki isolate and 31·9%–75·9% for the Kisarazu isolate). To examine the differences in infectivity in detail, we conducted histological observations of snails exposed to the Philippine strain at 3 h, 1, 3, and 15 days after miracidial exposure. We found differences in the development of the parasite between the isolates of snails from early after exposure.

The pond snail Lymnaea stagnalis is an intermediate vector for the liver fluke Fasciola hepatica, a common parasite of ruminants and humans. Yet, despite being a disease of medical and economic importance, as well as a potentially useful comparative tool, the genetics of the relationship between Lymnaea and Fasciola has barely been investigated. As a complement to forthcoming F. hepatica expressed sequence tags (ESTs), we generated 1320 ESTs from L. stagnalis central nervous system (CNS) libraries. We estimate that these sequences derive from 771 different genes, of which 374 showed significant similarity to proteins in public databases, and 169 were similar to ESTs from the snail vector Biomphalaria glabrata. These L. stagnalis ESTs will provide insight into the function of the snail CNS, as well as the molecular components of behaviour and response to parasitism. In the future, the comparative analysis of Lymnaea/Fasciola with Biomphalaria/Schistosoma will help to understand both conserved and divergent aspects of the host-parasite relationship. The L. stagnalis ESTs will also assist gene prediction in the forthcoming B. glabrata genome sequence. The dataset is available for searching on the world-wide web at http://zeldia.cap.ed.ac.uk/mollusca.html.

This study evaluated a relatively new molecular technique, serial analysis of gene expression (SAGE), as a tool for quantifying gene expression in the ovine abomasal nematode Haemonchus contortus for which there is relatively limited (~20% gene coverage) sequence information. SAGE technology generates data that are both qualitative and quantitative and, as such, compliments other functional genomics approaches such as EST analysis and micro-array. Prior to embarking on large-scale comparisons, the present study was initiated to establish (i) how well SAGE and EST data taken from the same life-cycle stage would compare, (ii) how easily SAGE tags could be assigned to genes given that the genome sequence is not available and (iii) whether it would be possible to extend the sequences of the SAGE tags to facilitate their identification. Of 2825 tag sequences analysed from adults harvested 28 days post-infection, the identity of the encoding gene could be ascribed to 63% of the tags. The relative abundance of these genes, arbitrarily categorized on the basis of function, was comparable with that of an EST dataset also from adults (n=2317). In addition, tag sequences could be readily extended and thereby identified using a tag-based primer and Reverse Transcription-PCR.

Trichinella spiralis infection causes the transformation of infected muscle cells, which leads to nurse cell formation. To search for the candidate genes responsible for nurse cell formation, cDNA microarray analysis of muscle tissues was performed before and after Trichinella infection. The Atlas mouse 1.2 cDNA expression microarray revealed the expression profiles of 1176 known genes. Out of these, 311 gene expressions were detected in normal and/or infected muscles. After the infection, 184 out of the 311 genes increased in expression by more than 3-fold. These included genes responsible for cell differentiation, proliferation, cell cycle and apoptosis. Thus this study suggested candidate genes for further investigation to dissect the molecular mechanisms of nurse cell formation.

The host-parasite relationship is exquisitely specific. In exploiting the host niche, a variety of helminth parasites have been shown to directly manipulate their hosts' immune responses. We assessed the ability of a whole-worm extract of Hymenolepis diminuta to modulate immune cell activation. Immune cells isolated from human blood or rodent spleens were activated with the T cell mitogen, concanavalin A (Con A)±H. diminuta extract and cytokine production (i.e. IL-2, -4, -10, -12) and proliferation assessed by ELISA and [3H]thymidine incorporation 24 and 72 h post-treatment, respectively. Co-treatment with the H. diminuta extract (100 μg protein/ml) virtually abolished Con A-induced immune cell proliferation, which was not due to increased apoptosis. Boiling of the worm extract reduced its anti-proliferative effect and fractionation indicated that a >50 kDa component was predominantly responsible for the inhibition of Con A-induced immune cell proliferation. Cytokine determinations revealed that the H. diminuta extract significantly reduced Con A-stimulated IL-2 and IL-4, but enhanced the production of IFNγ, IL-12 and IL-10. The increased IL-12 was due to an LPS contaminant in the extract and a helminth-derived ‘IL-12’-like peptide that bound in the ELISA and Western blots. In contrast, a H. diminuta–derived factor directly stimulated IL-10 production by murine splenocytes, and contaminating LPS synergistically enhanced the production of IL-10. Thus, H. diminuta has the potential to block stimulated T cell proliferation and, by inhibiting IL-4 and promoting IL-10 production, may bias the immune environment towards one of immunoregulation and away from IL-4 dominated T helper 2 type events.