We have examined the similarities and differences in the organization of tubulin genes in New World Leishmania by restriction endonuclease digestion of genomic DNA and Southern blot analysis, using heterologous and homologous tubulin gene probes. As judged by the hybridization pattern and the restriction fragment length polymorphism (RFLP), there were large differences in both the restriction and hybridization patterns of the β-tubulin sequences between stocks of the mexicana and braziliensis complexes. There were similarities in the hybridization patterns of different speciesof the mexicana complex. In contrast, a high heterogeneity was found between species of the braziliensis complex which includes intraspecific variation. The results suggest that this polymorphism may be associated with random mutations. The same analysis gave evidence of large differences in the β-tubulin gene restriction pattern between New and Old World Leishmania. This variation in the β-tubulin gene region was sufficient to distinguish between New and Old World Leishmania groups and between stocks of the mexicana and braziliensis complexes.

Giardia that infect humans are known to be heterogeneous but they are assigned currently to a single species, Giardia intestinalis (syn. G. lamblia). The genetic differences that exist within G. intestinalis have not yet been assessed quantitatively and neither have they been compared in magnitude with those that exist between G. intestinalis and species that are morphologically similar (G. duodenalis) or morphologically distinct (e.g. G. muris). In this study, 60 Australian isolates of G. intestinalis were analysed electrophoretically at 27 enzyme loci and compared with G. muris and a feline isolate of G. duodenalis. Isolates of G. intestinalis were distinct genetically from both G. muris (approximately 80% fixed allelic differences) and the feline G. duodenalis isolate (approximately 75% fixed allelic differences). The G. intestinalis isolates were extremely heterogeneous but they fell into 2 major genetic assemblages, separated by fixed allelic differences at approximately 60% of loci examined. The magnitude of the genetic differences between the G. intestinalis assemblages approached the level that distinguished the G. duodenalis isolate from the morphologically distinct G. muris. This raises important questions about the evolutionary relationships of the assemblages with Homo sapiens, the possibility of ancient or contemporary transmission from animal hosts to humans and the biogeographical origins of the two clusters.

The vector competences of 6 geographic strains of Aedes polynesiensis for Wuchereria bancrofti were studied using two types of experimental infections. Experimental infection of laboratory-bred mosquitoes fed on the carriers' forearms with different levels of microfilaraemia showed that microfilariae (mf) uptake was directly proportional to the carrier's mf density and, as mf densities decreased, concentration capacity of Ae. polynesiensis increased. It was also shown that infection has an important effect on mosquito mortality, and that the mortality rate differed among mosquito strains. In infections using artificial feeders, the mf uptake was closely regulated, thus showing differences in the vectorial efficiency of Ae. polynesiensis related to the geographic origin of the mosquito strain. The mosquitoes from the Society archipelago were more efficient intermediate hosts than geographically distant strains when infected with W. bancrofti from an island within the archipelago (Tahiti). Mosquito strains from the Society archipelago developed the highest proportion of infective-stage larvae and exhibited the lowest mortality rate when infected with sympatric Tahitian W. bancrofti.

Heritability of susceptibility to infection with Theileria parva was estimated from full sib families of Rhipicephalus appendiculatus ticks. Male and female ticks of 2 stocks were mated singly. Nineteen full sib families of the Muguga stock and 17 full sib families of the Kiambu stock were obtained. Nymphae of these families were fed on cattle infected with T. parva so that the ticks became replete on days 16 and 17 after infection when the blood was parasitaemic with intraerythrocytic piroplasms. The T. parva infections were assessed in the resultant adult ticks of each full sib group and the abundance of infection, the number of salivary gland acini infected/tick, was found to be the most useful parameter for analysis. Estimates of heritability of the susceptibility to infection with T. parva for the Kiambu and the Muguga tick stocks were 0·24 and 0·26 respectively. Using only the data from ticks which fed on day 16, the heritability estimates were 0·39 for the Kiambu stock and 0·59 for the Muguga stock. These results indicate that tick lines of high or low susceptibility for T. parva infection could be produced through selection.

A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.

SIV rats infected with a high dose (50000 oocysts) of Eimeria nieschulzi displayed clinical symptoms of coccidiosis such as diarrhoea (days 6 and 7 post-primary infection) and weight loss (days 6–8 post-primary infection) and were completely immune to challenge with a similar dose. The ability of rats to produce tumour necrosis factor (TNF) in vivo was enhanced during the period of oocyst excretion in the primary infection but significant production of TNF did not occur after challenge infection. Thus, TNF does not appear to be an important factor in resistance to infection with E. nieschulzi but may play some role in resistance to primary infection and in the pathology associated with E. nieschulzi infection. Parasite-specific serum IgM levels (measured by enzyme-linked immunosorbent assay) were also increased during primary infection but returned to background levels at the end of the patent period and were not affected by challenge infection. In contrast to TNF and IgM, serum concentrations of E. nieschulzi-specific IgGl, IgG2a, IgG2b, IgG2c and intestinal tissue levels of IgA did not begin to increase until after day 12 post-primary infection, reached peak levels between days 20 and 30 post-primary infection and were slightly increased by challenge infection.

The reduction in worm burden obtained by treatment of Schistosoma mansoni with praziquantel and oxamniquine was greater in mice with heavy infections than in relatively lightly infected animals. The reduction in worm burden achieved by each drug correlated with the size of the pre-treatment worm burden (r2 = 0·82 and 0·81 for praziquantel and oxamniquine, respectively). Intensity of infection did not affect the degree of tegumental damage and drug-induced antigen exposure on worms recovered soon after treatment with praziquantel. However, praziquantel-treated worms from mice with heavy infections had significantly more murine antibody attached to the treated-worm surface than worms from praziquantel-treated lightly infected mice. Heavily infected mice had greater levels of circulating anti-worm antibodies than lighter infected mice. The correlation between infection intensity and cure rates achieved by praziquantel and oxamniquine may thus be a reflection of the higher litres of relevant antibody in heavily infected mice mediating death of drug-treated worms.

This study examines the ability of an assay which measures the amount of a schistosome specific antigen (CAA) in the host circulation to reliably reflect relative worm burden. Mice were infected with 5 species of schistosome with a range of infection dose. The levels of serum CAA increased during schistosome maturation. In all species tested CAA levels correlated well with adult worm burden once the parasites achieved sexual maturity and remained relatively stable during the establishment of egg production. The amount of CAA produced varied between species but within each species CAA levels were proportional to worm numbers: no density-dependent effects on CAA levels were observed even when mice carried worm burdens that were very large relative to host size. T-cell deprivation of the host had no effect on the CAA/worm burden relationship in either Schistosoma mansoni or S. haematobium infections and the CAA equilibrium was unaltered in intact mice when reduction of worm fecundity occurred. These data support the use of the CAA as an accurate and robust estimate of relative schistosome burden in man.

The monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region. The nucleotide sequences reported in this paper have been submitted to the EMBL Data Library under accession numbers Z26942 (G. salaris), Z35128 (G. derjavini) and Z35129 (G. truttae).

The behaviour of cercariae was recorded on video at 10, 20 and 30 °C and a combination of population and individual behavioural patterns studied. Initially cercarial populations were extremely active, individuals alternating between swimming and sinking behaviour. As they aged the percentage sinking and resting increased resulting in the establishment of a vertical distribution in the water column. An increased environmental temperature reduced the active life-span of the parasites. Over time cercarial swimming rates declined. Swimming rates increased with environmental temperature and as a result of a shadow stimulus. As cercariae aged, the duration of swimming events decreased. Post-stimulation swimming periods were significantly longer than pre-stimulation periods. Pre- and post-stimulation swimming distances also declined with age. Post-stimulation swimming distances were significantly longer than pre-stimulation distances. The sinking rates of cercariae decreased with age while sinking periods and distances increased. Data for proportional time spent swimming and the ratio of sinking to swimming events indicated that as cercariae aged, less time was spent swimming. The frequency of swimming/sinking events declined with age. The implications of this behaviour for dispersal and transmission are discussed.

To test the hypothesis that the relatively high developmental temperature threshold of the parasitic nematode Elaphostrongylus rangiferi in the intermediate snail host is an adaptation to minimize larval mortality during winter, an experiment was set up in which snails of the species Arianta arbustorum were experimentally infected with the parasite. The snails were divided into 3 groups known to contain 1st, 2nd or 3rd-stage larvae, and incubated at 3 °C for an experimental period of 18 weeks. First-stage larvae showed a significantly higher survival rate within snails than 2nd or 3rd-stage larvae. We also found that snails carrying 1st-stage larvae survived better than snails with other larval stages. It is concluded that if the nematode has started development before the hibernation, this has a real and significant effect on the risk of dying. The high developmental threshold is therefore likely to be an adaptation to reduce the chance of hibernating as developing larvae during long periods of low temperatures.

This paper demonstrates that the establishment and growth of the filarial nematode parasite, Litomosoides carinii, is reduced in pyridoxine-deficient cotton rats. Young cotton rats were assigned to one of three dietary groups: vitamin B6-deficient cotton rats (B6 –AL) were fed a pyridoxine-free diet ad libitum; pair-fed controls (B6 + PF) were fed the same amount of pyridoxine-free diet as animals in the deficient group and given daily oral supplements of 100 μg pyridoxine; and pyridoxine-sufficient controls (B6 + AL) were fed the pyridoxine-free diet ad libitum and supplemented daily with 100 μg pyridoxine. Half of each group was infected with 50 L3 of L. carinii by subcutaneous injection 8 weeks after the start of the experimental feeding period. B6-deficient cotton rats ate less (P < 0·001) and gained less weight (P < 0·001) than B6-supplemented controls. The levels of microfilaraemia in deficient animals, measured weekly throughout the experiment by taking blood smears, was significantly lower than in supplemented animals (P < 0·001). The deficient rats became latent for L. carinii at 20 weeks post-infection, whereas there was patent microfilaraemia in rats in the other dietary groups until the end of the experiment. Smaller (P < 0·001) and fewer (P < 0·05) adult worms were recovered from the pleural and abdominal cavities of deficient animals than from either pair-fed or sufficient controls at autopsy 28 weeks post-infection.

We combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.