Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.

The objectives of this study were (a) to evaluate the sensitivity and specificity of fœtal Doppler indices for the prediction of abnormal fœtal heart rate (aFHR) at delivery after malaria crisis and (b) to test Doppler parameters against crisis duration for predicting aFHR. Every day during the malaria crisis, the umbilical and cerebral vascular resistance indices were measured by Doppler. These indices allowed evaluation of the amplitude of the fœtal flow redistribution induced by malaria (C/U=cerebral resistance/umbilical resistance ratio), the duration of the flow redistribution period and the hypoxic index (mean %C/U change×crisis duration). It was found that the mean duration of the flow redistribution period was: 7±2 days, mean C/U change −7%±4, hypoxic index −56±37, prematures 35%, and aFHR 17%. An hypoxic index >150 predicted occurrence of aFHR with high sensitivity and specificity (100%/91%). The highest fœtal flow disturbance (max %C/U) and the duration of the period with flow disturbance (>7 days) predicted aFHR at delivery with a sensitivity of 10% and 40% and a specificity of 77% and 78%. It was concluded that the hypoxic index was more predictive of aFHR at delivery than the amplitude or the duration (i.e. crisis duration) of the fœtal flow redistribution.

A species of Besnoitia from naturally infected rabbits from Argentina was propagated experimentally in mice, gerbils, rabbits, cats, and cell cultures. Cats fed tissue cysts from rabbits shed oocysts with a prepatent period of nine to 13 days. Sporulated oocysts were infective to gerbils, rabbits, outbred Swiss Webster and interferon gamma gene knockout mice. Bradyzoites were infective orally to gerbils and cats. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells. Schizonts were seen in the lamina propria of the small intestine of cats fed tissue cysts; the largest ones measured 52×45 μm. Schizonts were also present in mesenteric lymph nodes, livers, and other extra-intestinal organs of cats fed tissue cysts. Oocysts were 10–14×10–13 μm in size. This rabbit-derived species of Besnoitia resembled B. darlingi of the North American opossum, Didelphis virginiana with an opossum-cat cycle, but it was not transmissible to D. virginiana, and B. darlingi of opossums was not transmissible to rabbits. Based on biological, serological, antigenic, and molecular differences between the rabbit and the opossum Besnoitia, a new name, B. oryctofelisi is proposed for the parasite from domestic rabbits from Argentina.

The mechanism by which nitric oxide (NO)-dependent cytotoxicity acts against Toxoplasma gondii tachyzoites is poorly understood. An NO donor, sodium nitroprusside (SNP), was used to induce death in T. gondii tachyzoites in vitro as a model for investigating (i) whether NO is capable of inducing apoptosis-like death in tachyzoites and (ii) whether a calcium signal transduction pathway is involved. Exposure to 2 mM SNP resulted in a pattern of tachyzoite death that shares many features with metazoan apoptosis and it may involve a calcium signal transduction pathway. Motility and cell survival in these parasites showed a gradual decline with increasing levels of SNP. Features common to metazoan apoptosis are observed after exposure to 2 mM SNP. Ethylene glycol bis-(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA), Verapamil and bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid/acetoxymethyl ester (BAPTA/AM) partially increased the cell survival concomitant with decreased [Ca2+]i in cells exposed to SNP. An NO scavenger (N-acetylcysteine), the analogue of SNP (devoid of NO), inhibited the rate of apoptosis after SNP treatment compared with SNP treatment without scavenger, but alone did not induce apoptosis. Taken together, the results indicate that SNP is capable of inducing apoptosis in T. gondii tachyzoites via a calcium signal transduction pathway.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after in vitro treatment with diethylcarbamazine. One of the first morphological alterations produced by treatment with 5, 10 or 50 μg/ml of DEC was the loss of microfilarial sheaths. Drastic effects of DEC were only detected when microfilariae were exsheathed. Microfilariae treated with 5, 10 or 50 μg/ml DEC showed severely affected organelles, formation of several vacuoles mainly in the hypodermis, and cytolysis. Some microfilariae showed extreme cellular disorganization with abundance of electron-dense degenerating organelles, numerous large vacuoles, and nuclear condensation. Lamellar bodies probably related to an assembly of endoplasmic reticulum membranes were observed in some damaged microfilariae. Thus, in vitro treatment with concentrations of DEC similar to therapeutic conditions had direct effect by causing a loss of the microfilarial sheath of W. bancrofti with subsequent damage of organelles and apoptosis.

The phylogeny of representative haemozoan species of the phylum Apicomplexa was reconstructed by cladistic analyses of ultrastructural and life-cycle characteristics. The analysis incorporated 4 apicomplexans previously not included in phylogenetic reconstructions: Haemogregarina clelandi from the Brisbane River tortoise (Emydura signata), Hepatozoon sp. from the slaty grey snake (Stegonotus cucullatus), Hepatozoon (Haemogregarina) boigae from the brown tree snake (Boiga irregularis), and Haemoproteus chelodina from the saw-shelled tortoise (Elseya latisternum). There was no apparent correlation between parasite phylogeny and that of their vertebrate hosts, but there appeared to be some relationship between parasites and their intermediate hosts, suggestive of parasite/vector co-evolution.

The gene coding for α tubulin has been isolated by the polymerase chain reaction and sequenced from 2 isolates of Neospora caninum (Nc-Liverpool and Nc-SweB1)The nucleotide sequence data reported in this paper are available from the GenBank database under the accession numbers AF508031 (N. caninum) and AY169962 (H. heydorni).. The data show that the gene, as in Toxoplasma gondii, is single copy and contains 3 exons and 2 introns and is identical in sequence in the 2 isolates studied. Comparison of the predicted protein sequence shows it to be identical to the α tubulin protein encoded by the T. gondii gene. The majority of the nucleotide substitutions that have occurred during the evolution of the T. gondii and N. caninum genes from their common ancestor have occurred in the third codon position. A partial coding sequence for α tubulin was also obtained from Hammondia heydorni and compared to other α tubulin sequences from Apicomplexa. The results show the sequences of the T. gondii, N. caninum and H. heydorni α tubulin genes to be similar but not identical in sequence, thereby providing new evidence that N. caninum and H. heydorni are genetically distinct species.

Groups of nymphal Rhipicephalus appendiculatus Muguga, having a mean of 1 or 9 Theileria parva Muguga-infected salivary gland acini per tick, were kept under quasi-natural conditions at an altitude of 1950 m or 20°C at a relative humidity of 85% in the laboratory and their survival and infection prevalence and abundance determined over time. Theileria parva infections for both categories of ticks survived in the nymphal ticks for 50 or 26 weeks post salivary gland infection under quasi-natural or laboratory conditions respectively. There was a distinct decline in infections in the more heavily infected nymphae under both conditions of exposure, reflecting an apparent density dependence in parasite survival. Nymphal ticks having an average infection level of 1 infected salivary gland acinus per tick, survived for up to 69 or 65 weeks post-repletion under quasi-natural or the laboratory conditions respectively. Nymphae having an average infection level of 9 infected salivary gland acini per tick survived for a similar duration under each of the 2 conditions. The infection level of 9 infected salivary gland acini per tick did not seem to significantly affect the survival of the tick vector compared to those having an average of 1 infected salivary gland acinus per tick.

Small nuclear ribonucleoproteins (snRNPs) are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(ω)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica.

To assess the potential role of IgA antibody in expulsion of the nematode of the genus Trichinella from the intestine, a panel of IgA monoclonal antibodies (mAbs) were produced from the mesenteric lymph node cells from BALB/c mice orally vaccinated with irradiated muscle larvae of Trichinella britovi. One IgA mAb, HUSM-Tb1, formed immunoprecipitates on the surface of live muscle larvae, and by immunohistochemistry reacted with their stichocytes and cuticular surface, but not with those tissues of the adult stage or newborn larvae. Intraperitoneal injection of BALB/c mice with this mAb 5 h before challenge conferred a high level of protection (more than 95%) against T. britovi infection, when 2·0 mg of specific IgA/20 g body weight was given to a mouse. The same treatment produced a similar effect in SCID mice lacking functional T- and B-cells, indicating no requirement of synergistc T-cell factors for the effect. Passive transfer of the mAb at the time of challenge or later showed less or no effect upon worm expulsion. It is concluded that the mucosal IgA response, when adequately induced, can impede the establishment of infective Trichinella parasites in the mouse intestine.

The Echinococcus granulosus genome was searched for microsatellites using 8 different repeated oligonucleotides as probes (GT15, CT15, AT15, CG15, CAT10, CAA10, CGG10 and CATA10). Southern blot experiments revealed that DNA regions containing GT, CAA, CATA and CT repeats are the most frequent in the E. granulosus genome. AT and CG probes showed no hybridization signal. Two loci containing CA/GT (Egmsca1 and Egmsca2) and 1 locus containing GA/CT (Egmsga1) repeats were cloned and sequenced. The locus Egmsca1 was analysed in 73 isolates from Brazil and Argentina whose strains were previously characterized. Brazilian isolates from cattle strain and Argentinean isolates from camel strain were monomorphic and shared the allele (CA)7. Argentinean isolates of sheep and Tasmanian sheep strains shared 2 alleles [(CA)8 and (CA)10] with Brazilian isolates of sheep strain. The allele (CA)11 was found only in Brazilian isolates of sheep strain at a low frequency. The Brazilian and the Argentinean sheep strain populations were tested for the Hardy-Weinberg equilibrium, and only the former was in agreement with the expectations. No polymorphism was found among individual protoscoleces from a single hydatid cyst, validating the utilization of pooled protoscoleces from 1 cyst, grouped as an isolate, in population studies. This work describes for the first time the isolation and characterization of microsatellites from E. granulosus.