Inherited bacteria that parasitically distort the pattern of sex allocation of their host, biasing allocation towards female progeny, are found in many arthropods. One such manipulation is male-killing, where male progeny of infected females die during embryogenesis. We here provide evidence for a male-killing bacterium in the coccinellid beetle, Adonia variegata. We then address 3 questions. First, is this male-killing bacterium one that is found in other hosts, or does it represent a new transition to male-killing within the eubacteria? Using the sequence of the 16S rDNA of the bacterium, we found that the male-killing bacterium is a member of the Flavobacteria–Bacteroides group, most closely related to the male-killing bacterium in another ladybird beetle, Coleomegilla maculata. Secondly, is there any evidence that this bacterium affects female host physiology? In a paired test under nutritional stress, we found no evidence for a physiological benefit to infection, and weak evidence of a physiological cost, in terms of reduced fecundity. Thirdly, is there any evidence of host involvement in the transmission of the bacterium to the germ line? We found no evidence of host involvement. Rather, bacteria migrated to the ovariole independently of host cells. We conclude that the bacterium is a parasite, and discuss how 2 different species of ladybird come to be infected with 1 lineage of bacterium, and why case studies of male-killing bacteria have generally found little evidence of any symbiont contribution to host physiological functioning.

Nitric oxide (NO) production has been suggested to be required for the development of cerebral malaria. However, the importance of this molecule for the appearance of this pathology is debated. To assess whether murine cerebral malaria is NO dependent, we investigated the course of blood-stage Plasmodium berghei ANKA (PbA) infections in inducible nitric oxide synthase (iNOS)-deficient mice. Parasitaemia, haematological alterations, survival and development of cerebral malaria were not affected by the lack of iNOS. To exclude a role of NO produced by other NOS, controls included NO suppression by oral administration of aminoguanidine (AG), a NOS inhibitor. As in iNOS-deficient mice, no difference in the parasitaemia course, survival and haematological values was observed after AG treatment. Our results indicate that NO production is not a crucial factor for the development of murine cerebral malaria.

Nitric oxide (NO) production has been suggested to play a role as effector molecule in the control of the malarial infections. However, the roles of this molecule are debated. To assess whether blood-stage parasite killing is NO dependent, we investigated the course of blood-stage Plasmodium chabaudi chabaudi (Pcc) infections in inducible nitric oxide synthase (iNOS)-deficient mice. Parasitaemia, haematological alterations, and survival were not affected by the lack of iNOS. To exclude a role of NO produced by other NOS, controls included NO suppression by oral administration of aminoguanidine (AG), a NOS inhibitor. As in iNOS-deficient mice, no difference in the parasitaemia course, survival and haematological values was observed after AG treatment. Our results indicate that NO production is not required for protection against malaria in our murine experimental model. However, C57BL/6 mice treated with AG lost their resistance to Pcc infections, suggesting that the requirement for NO production for parasite killing in murine blood-stage malaria might be strain dependent.

Using an age-fractionated RBC model, we investigated the in vitro sensitivity of artemether in β-thalassaemic RBC infected with the K1 and FC27 strains of Plasmodium falciparum and, to study the role of oxidant stress in modulating the sensitivity pattern, pro-oxidant (riboflavin) and antioxidant (vitamin E) agents were added to cultures in the presence of artemether. With the FC27 strain, the artemether IC50 doses in thalassaemic samples (whole blood and fractions) were significantly higher compared to equivalent normal RBC samples (P<0·05). However, with the K1 strain, such a significant difference was not demonstrable. The addition of vitamin E reduced the antimalarial effect of artemether in both the FC27 and K1 strains (P<0·0001). In contrast, the addition of riboflavin resulted in a significant increase in antimalarial activity (P<0·0001). This effect of the drug combinations was not influenced by the red cell type (P<0·0001) and there was no interaction between red cell type and drug type (P<0·0001). These findings show that reduced sensitivity to artemether occurs in whole blood and age-fractionated β-thalassaemic trait RBC. It appears that the RBC redox status does not influence the sensitivity to artemether.

Cysts of Blastocystis ratti were produced in vitro by culturing the parasite in Iscove's modified Dulbecco's medium (IMDM) with increasing concentrations of horse serum. Yields up to 3×106 cysts/ml of culture medium were obtained after 72 h. Encystation efficiency was time, strain and inoculum size dependent. A viability of >70% was determined by flow cytometry employing fluorescein diacetate and propidium iodide staining. The presence of chitin as a cyst wall component was demonstrated by Calcofluor White M2R staining with which cystic stages showed blue fluorescence. The changes in morphology during excystation were examined by transmission electron microscopy. The cyst enlarged in size and some vacuoles appeared within the condensed cytoplasm. The vacuoles were full of inclusions and small glycogen aggregates. Coalescence of the vacuoles led to central body formation. Glycogen deposits were prominent throughout the excystation process. Some cysts divided by binary fission before the completion of the excystation.

American trypanosamiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection in natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, the mini-exon and the 24Sα rRNA gene loci have shown that T. cruzi stocks can be clustered in 2 major phylogenetic groups: lineage 1 and lineage 2. In this report, 68 recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical areas in Rio de Janeiro, Brazil, have been typed based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, 26 stocks were from golden-lion tamarins, 31 from opossums, 2 from rodents and 1 from a three-toed sloth. Thirty (44% – 30/68) isolates were typed as lineage 1, while 36 (53% – 36/68) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, 3% (2/68) of the isolates were typed as lineage 1+lineage 2. Using these geographical regions as models of sylvatic environments, it was observed that 96% of the Didelphis marsupialis were infected by lineage 2 isolates, while all 26 golden-lion tamarins were infected by lineage 1. The results show preferential association of the 2 lineages of T. cruzi with different hosts, composing the complexity of the sylvatic cycle.

The intracellular cycle of Trypanosoma cruzi in mammalian host cells involves the differentiation of dividing amastigote forms into flagellated trypomastigote forms. The mechanism(s) regulating the growth and differentiation of the intracellular parasites is (are) not known. The number of parasites in infected cells can be several hundred and may be enough to induce apoptosis, a suicide-like death programme, generating products (e.g. nuclear proteins) that could function as signals to initiate the differentiation of amastigotes into trypomastigotes. Murine fibroblasts infected with T. cruzi were examined during a 5-day course of infection for evidence of apoptosis. However, characteristics of apoptosis, including degeneration of nuclear structure, condensation of chromatin, loss of plasma membrane integrity, or the cleavage of DNA into nucleosomal fragments, were not observed. Therefore, it is unlikely that products resulting from host cell apoptosis function to induce parasite differentiation. The possibility that T. cruzi might inhibit host cell apoptosis by increasing intracellular levels of Bcl-2, an endogenous inhibitor of apoptosis, was then investigated. Analysis of infected cells by flow cytometry did not demonstrate a significant amount of intracellular Bcl-2. This suggests that if the parasite is inhibiting host cell apoptosis, it is by a method that does not involve increasing levels of Bcl-2.

Tick-borne encephalitis (TBE) virus has a highly focal distribution through Eurasia. Endemic cycles appear to depend on the transmission of non-systemic infections between ticks co-feeding on the same rodent hosts. The particular features of seasonal dynamics and infestation patterns of larval and nymphal Ixodes ricinus, but not Dermacentor reticulatus, from 4 regions within TBE foci in Slovakia, are such as to promote TBE virus transmission. The distributions of larvae and nymphs on their principal rodent hosts are highly aggregated and, rather than being independent, the distributions of each stage are coincident so that the same ca. 20% of hosts feed about three-quarters of both larvae and nymphs. This results in twice the number of infectible larvae feeding alongside potentially infected nymphs compared with the null hypothesis of independent distributions. Overall, co-feeding transmission under these circumstances brings the reproductive number (R0) for TBE virus to a level that accounts quantitatively for maintained endemic cycles. Essential for coincident aggregated distributions of larvae and nymphs is their synchronous seasonal activity. Preliminary comparisons support the prediction of a greater degree of coincident seasonality within recorded TBE foci than outside. This identifies the particular climatic factors that permit such patterns of tick seasonal dynamics as the primary predictors for the focal distribution of TBE.

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S. japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna. Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18–24 h. Redial stages were visible inside in vitro-transformed mother sporocysts after 12–16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14–20 days post-cultivation. Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150–170 μm. Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells. Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period. Unlike Schistosoma spp., in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F. magna was dependent on Bge cell products. Less than 5% of F. magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge). However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively. This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments. Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions. This is the first report of in vitro development of rediae from miracidia for a digenetic trematode.

Onchocerciasis (‘River Blindness’), caused by the filarial nematode Onchocerca volvulus is of major public health importance in West Africa. Ivermectin, a drug originally developed for veterinary use, is now being incorporated in control strategies but whilst it has potent efficacy against L1 larvae (microfilariae), ivermectin is not lethal to adult (L5) O. volvulus, nor to adults of the related cattle parasite O. ochengi. We have exploited this model to determine if ivermectin has prophylactic activity against naturally transmitted, O. ochengi infections in a controlled, prospective study in northern Cameroon. Calves were treated monthly with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months. None of 15 treated calves developed adult worm infection, whereas 5/6 untreated controls became infected (P<0·001) with a total of 54 O. ochengi nodules, and all 5 developed patent microfilaridermia. These results have significant implications for the use of ivermectin in humans, and suggest that strategic chemotherapy at times of maximal transmission will confer prophylactic as well as therapeutic benefits.

To examine levels of heat shock proteins (HSPs) in host responses to helminth infection, rats were infected with Trichinella spiralis. The kinetics of HSP25, HSP60, HSP70 and HSP90 production in the liver and muscle of infected rats was compared with that of non-infected controls. HSPs were detected using electrophoretic analysis of fixed amounts of proteins (0·02 mg) and then blotting and incubation of membranes with polyclonal anti-HSP25 antibody, or monoclonal antibody against HSP60, or HSP70 or HSP90. Quantitation of blotted separated polypeptides reactive with the specific anti-HSP antibodies was achieved using an image analyser. Enhancement of HSP25 production was observed in the liver of infected rats, whereas muscle from the same rats exhibited enhanced production of HSP25 and HSP60 one day after infection only. These data indicate that HSPs levels can be used successfully to measure stress injury brought about by helminth infection in organs and tissues of the host.

In the present study, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was developed to identify 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory–secretory (E–S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the HinfI or DdeI endonuclease restriction of the 2800 bp fragment; T. nativa by the RsaI restriction of the 2800 bp fragment, or by the AluI restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the AluI restriction of the 1250 bp fragments, and can be discriminated between them by the SspI restriction of the 2800 bp fragment; T. pseudospiralis by the MspI restriction of the 372 bp fragment; T. nelsoni by the HhaI or AluI restriction of the 2800 bp fragment; Trichinella T5 by the HhaI restriction of the 2800 bp fragment; Trichinella T6 by the AluI restriction of the 1250 bp fragment; and Trichinella T8 by the SspI or RsaI restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi, Trichinella T5 and T6.