Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39°C in the vertebrate host to 20°C) and a concomitant pH increase (from 7·3 in mouse blood to 8·0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise non-permissive pH of 7·3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogenesis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.

To study congenital transmission of Toxoplasma gondii during acute and chronic infections, 4 pregnant Sprague-Dawley rats were each fed 10000 oocysts of the VEG strain. Toxoplasma gondii was recovered from 33, 55, 83 and 57% of rats (F1) when dams were inoculated at 6, 9, 12 or 15 days of gestation, respectively. Progeny of 15 congenitally infected female rats were examined for T. gondii. Toxoplasma gondii was recovered from tissues of 1 of 155 rats (F2) born to congenitally infected dams. A total of 4 (F2) females were mated; 0 of 40 (F3) rats born to them were infected. None of the acutely infected 4 dams that had given birth to congenitally infected litters produced congenitally infected offspring during the second pregnancy. Thus, unlike mice, evidence for repeated congenital transmission of T. gondii in the rat was found in <1% of cases. Of the 16 congenitally T. gondii infected pups with demonstrable tissue cysts, 5 were seronegative (<1[ratio ]4) in the Sabin-Feldman dye test and 5 were seronegative (<1[ratio ]20) in the modified agglutination test by the use of whole formalinized tachyzoites and mercaptoethanol.

The persistence of Toxoplasma gondii tissue cysts in organs of cats (definitive host) and rodents (intermediate hosts) was studied. Nine cats, 12 rats, and 12 mice were fed T. gondii oocysts and their organs were digested in pepsin and then bioassayed for bradyzoites in mice. Of 9 cats killed 37 or 51 days after feeding 102 (2 cats), 103 (3 cats) or 104 (4 cats) oocysts of the VEG strain, tissue cysts were found in each cat; in the tongue of 9, in the heart of 5, in the brain of 4, and in the eyes of 1 cat. The dose had no effect on the distribution of tissue cysts in cats. Twelve rats were each fed 105 oocysts of the VEG strain of T. gondii and killed 21, 29, 64 or 237 days later. At each time-period, 11 tissues of 3 rats were pooled and bioassayed in mice. Tissue cysts were found in the brain, skeletal muscle, heart and kidneys of rats at each killing time; in the lungs, intestines, and mesenteric lymph nodes in 3 of 4 instances; in the tongue, liver, and eyes in 2 instances and in the spleen in 1 instance. Also, using the same procedures and sampling the same 11 tissues as used for rats, tissue cysts were seen in all organs except in the tongue and liver of 3 mice killed on day 82 after feeding the VEG strain. In 9 mice (3 with each strain) fed oocysts of the ME-49, GT-1, or P89 T. gondii strain and killed 62–130 days later, tissue cysts were found consistently only in the brain. Thus, in rats and mice, most tissue cysts were found in the brain and rarely in the tongue. This was in marked contrast to the distribution of tissue cysts in cats.

This study was performed in order to define Schistosoma mansoni antigens that are able to function as modulator agents in the granulomatous hypersensitivity to parasite eggs in BALB/c and C57BL/6 mice. A fraction of S. mansoni, designated PIII, derived from adult worm antigen preparation (SWAP) was obtained using anion-exchange chromatography on an FPLC system. Immunization of mice with PIII in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant induced an immune response in these animals as determined by ELISA and spleen cell proliferation assays against S. mansoni antigens SEA, SWAP and PIII. In addition, PIII caused a significant degree of protection against a challenge infection in immunized mice as observed by the decrease on worm burden recovered from the portal system. We also showed that PIII profoundly inhibited the vigorous anamnestic granulomatous response to eggs in the liver and lungs. This suppression correlated with a significant decrease in granuloma size. From these results we conclude that the PIII preparation contains antigens that can mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs.

A novel method for the isolation of schistosome eggs and miracidia from livers of mice infected with Schistosoma japonicum or S. mansoni is described. The method employed collagenase B to degrade the interstitial matrix of mouse liver tissue, after which the schistosome eggs were separated from the liver cells by 2 single-step density centrifugations through Percoll. Using this procedure sufficient quantities of miracidia were obtained to generate a cDNA library. Southern blot analysis demonstrated that miracidia isolated by this method were free from contaminating host DNA.

This paper describes the first localization study of the 14·7 kDa fatty acid-binding protein in Schistosoma japonicum (Sj-FABPc) using transmission electron microscopy. A polyclonal antibody raised against recombinant Sj-FABPc was used in combination with a colloidal gold marker to determine the distribution of the protein within adult parasites. Sj-FABPc was localized within lipid droplets below the subtegumental region of the male parasite. Additionally, Sj-FABPc was present in the vitelline droplets of the vitelline glands of female parasites. There were no detectable levels of Sj-FABPc on the surface or within the tegument of male or female parasites. Possible functions of Sj-FABPc within S. japonicum and the relevance of these immunolocalization findings in light of the recent reports that the homologue Sm-FABPc is an important anti-S. mansoni vaccine target molecule are also discussed.

Forty-one Trypanosoma cruzi stocks isolated from the Chilean vectors Triatoma infestans and Triatoma spinolai were characterized by pulse-field gel electrophoresis and Southern blotting with the cruzipain gene, and by schizodeme analysis of kinetoplast DNA with EcoRI and Msp I. Seven parasite groups were found by molecular karyotype which correlate with schizodeme and multilocus enzyme electrophoresis, supporting the concept of clonal propagation for Trypanosoma cruzi. A predominant T. cruzi stock was isolated from domiciliary T. infestans in several geographical areas of Chile. In contrast, other frequently found genotypes were circulating in the sylvatic and domestic transmission cycles of specific geographical areas. The greatest heterogeneity of T. cruzi stocks was found among sylvatic T. spinolai where at least 4 genotypes were obtained from a sample of 16 T. cruzi stocks.

Scanning and transmission electron microscopy of Trypanosoma congolense epimastigotes attached to a plastic substratum shows them to elaborate a complex flagellum filament system and plaque with a highly organized structure. Non-ionic detergent extraction of these cells shows that the resulting cytoskeletons remain attached to the plaque. The subpellicular corset of microtubules can be removed by salt or Ca+2 treatment leaving the axoneme, paraflagellar rod, associated filaments and the plaque. Neither of these treatments therefore removed the plaque-associated material from the substratum. Analysis of these fractions by SDS–polyacrylamide gel electrophoresis reveals an abundant 70 kDa protein that is highly enriched in the salt extracted ‘minimal plaque’ structures and appears likely to be a major constituent of this structure. These studies reveal that the complex filament and microtubule systems of the cytoskeleton involved the attachment of trypanosomes to substrata and have established a method of biochemical fractionation of the structures and components involved.

The melanization of Dirofilaria immitis microfilariae in Armigeres subalbatus haemolymph in vitro is a two-step process. Firstly, the microfilariae are encased in a transparent capsule, then the capsule material is melanized later. Benzamidine HCl and p-amidinophenylmethanesulfonyl fluoride, both serine protease inhibitors, inhibited the deposition of the transparent capsule material and melanization. Diethyldithiocarbamate, a phenoloxidase inhibitor, did not prevent the deposition of the transparent capsule material but it did inhibit melanization. m-Hydroxybenzylhydrazine, a dopa decarboxylase inhibitor, had no inhibitory action on the deposition of the transparent capsule material. However, the time for the transparent capsule to become melanized was delayed due to the presence of m-hydroxybenzylhydrazine in the test system. Immunocytochemical localization showed that prophenoloxidase and/or phenoloxidase was present in the transparent capsule material. These results suggest (1) that prophenoloxidase cascade is essential in the melanization of microfilariae in mosquitoes and its putative activation behaviour is similar to that of other insects and (2) that the deposition of transparent capsule material is a result of the activation of prophenoloxidase by serine proteases and the melanization of the transparent capsule material is due to the presence of active phenoloxidase in capsule.

Different populations of the whitespotted rabbitfish, Siganus sutor, were examined for ectoparasites: adults from the Mombasa area (sampled in December 1990) and different age classes (adult, subadult and juveniles) from Gazi Bay (sampled in December 1992 and August 1993). The most common gill parasites were: the monogeneans Pseudohaliotrema sp., Tetrancistrum sigani and Microcotyle mouwoi, the copepods Hatschekia sp., Pseudolepeophtheirus sp. and juvenile Caligidae, and prazina larvae of the isopod Gnathia sp. Adult siganids had a higher parasite load than subadults. Juvenile rabbitfish did not harbour any gill parasites. Temporal differences in the parasite load of subadult rabbitfish were observed for M. mouwoi (highest in the December samples) and for juvenile Caligidae (highest in August). The microhabitat of the 5 most common gill parasites was species specific. Most parasite species showed distinct site preferences with respect to both gill arches and gill sectors, within the gill arches. Niche breadth of the different gill parasite species was independent of the abundance of any of the other species present. However, niche breadths of M. mouwoi, Tetrancistrum sp. and Hatschekia sp. increased with their own abundance. This suggests that interspecific competition for space is low and that intraspecific factors could play an important role in the microhabitat choice of these gill parasites. The hypothesis that niche restriction leads to higher intraspecific contact and an enhancement of chances to mate was tested on 2 monogenean species, Pseudohaliotrema sp. and Tetrancistrum sigani. Their highly aggregated distribution over the gill filaments, leading to increased intraspecific contact, is consistent with the hypothesis.

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) ([ges ]40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3% routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin–Cohn's fraction V) promoted the growth of B. divergens with high PPE (>30%) close to those obtained in RPMI–10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.

A new method has been developed to investigate behavioural diapause in adult Rhipicephalus appendiculatus ticks. It is based on a system of gauze columns in which the activity of the ticks can be monitored in the laboratory. An experiment was conducted, involving different photoperiodic conditions during the nymph-to-adult moulting and pre-questing periods, with the aim of comparing the results obtained by the in vitro method with those of the standard in vivo method. Comparable results were obtained with both methods.

A cDNA was isolated from a cDNA expression library using serum prepared against a high molecular mass fraction of Fasciola hepatica excretory–secretory products. The full-length cDNA encodes a member of the recently described peroxiredoxin antioxidant family. Peroxiredoxin could be the major hydrogen peroxide removing antioxidant in F. hepatica since this parasite does not express a catalase and expresses little glutathione peroxidase activity. This novel antioxidant may be involved in functions such as protection against reactive oxygen species (ROS) generated by metabolic processes and/or protection of the parasite against ROS released by immune effector cells.

The defensive glands of beetles, Tenebrio molitor, infected with metacestodes (cysticercoids) of Hymenolepis diminuta are everted less frequently upon stimulation, and contain less toluquinone (methylbenzoquinone) and m-cresol, than glands of uninfected controls. These differences, as shown in predation trials with wild rats, increase the likelihood that both cysticercoids and beetles will be ingested by the tapeworm's definitive host. This is the first documented case of a parasite inhibiting the chemical defence of an intermediate host, and one of only a few reports of parasite-induced manipulation of host biology supported by empirical evidence implicating facilitated parasite transmission between host species.