We screened a population of the brackish water crustacean Gammarus duebeni from the Isle of Cumbrae for the presence of vertically transmitted microsporidia. We compared 2 screening techniques; light microscopy and PCR-based detection using generic 16S rDNA microsporidian primers. Fifty percent of females from this population tested positive for vertically transmitted microsporidia. The PCR screen was 100% efficient in comparison with existing LM based screening. In addition, the PCR screen produced bands of 2 sizes suggesting that more than 1 species of microsporidian was present. Sequencing revealed 2 distinct species of vertically transmitted microsporidia; 33% of females were infected with the feminizer Nosema granulosis and 17% were infected with a new species which we provisionally designate Microsporidium sp. On the basis of sequence information, we developed a discriminatory PCR–RFLP test based on MspI and HaeIII digests. This screen allows rapid detection and discrimination of vertically transmitted microsporidia in natural field populations. We applied the PCR–RFLP screen to a second G. duebeni population from the Isle of Man. This population also hosted these 2 parasite species. In total 45% of females harboured N. granulosis and 10% harboured Microsporidium sp. No dual-infected individuals were found in either population. The occurrence of 2 vertically transmitted parasites within a population has implications for our understanding of parasite–host relationships in the field and we discuss factors affecting the dynamics of parasite–parasite competition and coexistence.

The microsporidian parasite Nosema ceranae is a common pathogen of the Western honeybee (Apis mellifera) whose variable virulence could be related to its genetic polymorphism and/or its polyphenism responding to environmental cues. Since the genotyping of N. ceranae based on unique marker sequences had been unsuccessful, we tested whether a multilocus approach, assessing the diversity of ten genetic markers – encoding nine proteins and the small ribosomal RNA subunit – allowed the discrimination between N. ceranae variants isolated from single A. mellifera individuals in four distant locations. High nucleotide diversity and allele content were observed for all genes. Most importantly, the diversity was mainly present within parasite populations isolated from single honeybee individuals. In contrast the absence of isolate differentiation precluded any taxa discrimination, even through a multilocus approach, but suggested that similar populations of parasites seem to infect honeybees in distant locations. As statistical evolutionary analyses showed that the allele frequency is under selective pressure, we discuss the origin and consequences of N. ceranae heterozygosity in a single host and lack of population divergence in the context of the parasite natural and evolutionary history.

An experiment using lysimeters suggested that the eggs of Taenia saginata and Ascaris lumbricoides survive for only a short time when applied to pasture in sewage sludge. However, a subsequent experiment which followed the survival of eggs throughout the soil profile demonstrated that some T. saginata eggs could still be found at 200 days on the soil surface, and that survival increased down the profile. Rainfall is shown to be able to wash eggs into the soil where they may be afforded protection from radiation and desiccation; this may have little epidemiological significance.

Many studies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) have reported that only a small proportion (typically <40%) of infective stages (dauers), even under apparently ideal conditions, actually infect a host. The ‘phased infectivity hypothesis’ is most frequently invoked to explain this pattern of low infection with entomopathogenic nematodes. It proposes that at a given point in time not all individuals are infectious i.e. infectiousness is delayed in some individuals. We tested experimentally several predictions based on this hypothesis. Specifically, if phased infectivity occurs, we should be able to expose dauers to increasing numbers of potential hosts until dauers no longer infect and still be able to recover viable dauers. These recovered dauers which did not infect should be infectious at some later point in time. However, our results do not support the phased infectivity hypothesis for 3 species of Steinernema: most dauers could be recovered in one sampling round when provided with sufficient suitable hosts. In contrast, Heterorhabditis bacteriophora frequently did not infect all available hosts, and infectious dauers were recovered in subsequent sampling rounds. This result is more consistent with the phased infectivity hypothesis, but further research is needed before we can be more confident in the hypothesis. For all species tested, the number of available hosts influenced population levels of nematode infectivity. This suggests that the infection status of hosts can influence whether a dauer infects. Our results indicate that phased infectivity is not a common phenomenon in entomopathogenic nematode dauers, despite the widespread acceptance of this hypothesis.

To compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS–polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31·5–34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

1. A method is described for separating and enumerating the ciliate Protozoa which occur in the large intestine of the horse.

2. The concentration of ciliates varied in the different regions of the large intestine, being low in the caecum and highest in the left dorsal colon.

3. Two distinct populations of ciliates occurred in the large intestine of the horse, one from the caecum to the pelvic flexure and the other from the pelvic flexure to the rectum.

Certain L-amino acid esters, such as L-leucine methyl ester (Leu-OMe), can kill intracellular and isolated Leishmania amazonensis amastigotes. Killing appears to involve ester trapping and hydrolysis within an acidified parasite compartment (M. Rabinovitch and S. C. Alfieri, 1987, Brazilian Journal of Medical and Biological Research 20, 665–74). We show here by acid phosphatase light microscopic cytochemistry and by ultrastructural morphometry that megasomes, lysosome-like amastigote organelles, are the putative parasite targets of Leu-OMe. This conclusion is supported by the following observations, (a) Control amastigotes displayed a string of electron-dense, acid phosphatase-positive megasomes mostly located in the cellular poles opposite the flagellar pockets. Incubation of the amastigotes with Leu-OMe resulted in concentration-dependent swelling and fusion of the organelles as well as decreased electron density of the internal contents. These changes, which preceded parasite disruption, were followed by the progressive loss of parasite viability and the release of acid phosphatase activity into the medium, (b) Incubation of the amastigotes with L-isoleucine methyl ester, a non-leishmanicidal compound, induced only moderate fusion of the megasomes. (c) Pre-incubation of the parasites with the proteinase inhibitors antipain and chymostatin, previously shown to confer protection from Leu-OMe toxicity, nearly completely prevented the morphological changes of megasomes. (d) Exposure of amastigotes to tryptophanamide (Trp-NH2), the leishmanicidal activity of which is not reduced by antipain and chymostatin, did not result in swelling and fusion of the megasomes. This last finding suggests that different mechanisms underlie the destruction of amastigotes by Trp-NH2 and Leu-OMe. Overall, the results are compatible with the hypothesis that Leu-OMe and other amino acid esters are trapped and hydrolysed within megasomes.

Infections with either 300 infective Trichinella spiralis larvae or 400 embryonated eggs of Trichuris muris were effective in eliciting accelerated expulsion of heterologous challenge infections given 20 days after the primary infection. Accelerated expulsion could also be achieved by the administration of soluble crude worm antigen given 12 days prior to heterologous challenge or by adoptive transfer of mesenteric lymph node cells taken from mice infected with the heterologous parasite. Each species is capable of eliciting an accelerated secondary expulsion response in hosts that have been actively or adoptively immunized against the other species and these results are taken to indicate that there is a specific cross-immunity between T. spiralis and T. muris due to shared antigens. It is postulated that these shared antigens are derived from stichocyte granules.

The freshwater bryozoan, Fredericella sultana, is the main primary host of the myxozoan endoparasite, Tetracapsuloides bryosalmonae which causes proliferative kidney disease (PKD) of salmonid fish. Because spores that develop in bryozoan colonies are infectious to fish, bryozoans represent the ultimate source of PKD. Bryozoans produce numerous seed-like dormant stages called statoblasts that enable persistence during unfavourable conditions and achieve long-distance dispersal. The possibility that T. bryosalmonae may undergo vertical transmission via infection of statoblasts has been the subject of much speculation since this is observed in close relatives. This study provides the first evidence that such vertical transmission of T. bryosalmonae is extensive by examining the proportions of infected statoblasts in populations of F. sultana on two different rivers systems and confirms its effectiveness by demonstrating transmission from material derived from infected statoblasts to fish hosts. Vertical transmission in statoblasts is likely to play an important role in the infection dynamics of both bryozoan and fish hosts and may substantially contribute to the widespread distribution of PKD.

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS–PAGE. The infected cells produced a number of extra activities at pH 8·0 in the presence of Ca2+. Calcium was found to enhance the activites but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

The prevalence of the caecal threadworm Trichostrongylus tenuis in red grouse in the north of Scotland was high despite low grouse densities. Prevalence, intensity and aggregation of threadworms was higher in old than in young grouse. Infections were long-lasting: populations of adult worms could survive for over 2 years in grouse, with little mortality. Parasite egg output decreased with the age of a worm population, largely as a result of a decrease in the fecundity of ageing female worms. Seasonal variations in worm fecundity were also evident.However, there was no evidence of an intensitydependent decrease of worm fecundity with increasing worm numbers in either captive or wild grouse. The long life and high reproductive capacity of T. tenuis probably contribute to its effective transmission and high prevalence.

The behaviour of oncomiracidia of the monogenean skin parasite Entobdella soleae, after hatching without chemical stimulation, has been studied in vertical, 4 cm high chambers illuminated from above. Most freshly hatched larvae are photo-positive and swim to the top of the vessel, where they remain actively swimming. Occasionally larvae become photo-negative and make a brief excursion to the bottom of the vessel. As the larvae grow older, these photo-negative excursions become more frequent and larvae spend progressively longer in the photo-negative phase of behaviour, but, even after many hours, photo-positive vertical excursions are still occurring. If this pattern of alternating photo-positive and photo-negative vertical movements occurs in the natural environment, these vertical movements, coupled with horizontal transport of larvae by water currents, would provide a search pattern for the host, the diurnally-inactive, bottom-living flatfish Solea solea. When eggs are stimulated to hatch in the chambers with urea, large numbers of larvae emerge within a few min, and it was observed that during the period of about 30 min after stimulation, many larvae are photo-negative. When eggs on the sea bottom are stimulated to hatch by mucus or urine from a nearby host, such behaviour would greatly enhance chances of contact with the fish. When eggs are stimulated to hatch by means of urea in the absence of light, there is evidence that the oncomiracidia are geo-negative. It has been shown that larvae are able to extricate themselves in total darkness from sediment containing buried eggs and that, in an experimental situation illuminated only by an infra-red beam, larvae readily attach themselves to the lower skin of soles within 2 min of hatching. There is evidence that, in aquaria, some invasion of the host takes place from below.

The cellular mucosal and systemic effectors of gilthead sea bream (GSB) (Sparus aurata) involved in the acute immune response to the intestinal parasite Enteromyxum leei were studied in fish experimentally infected by the anal route. In the intestinal inflammatory infiltrates and in lymphohaematopoietic organs (head kidney and spleen) of parasitized fish, the number of plasma cells, B cells (IgM immunoreactive) and mast cells (histamine immunoreactive) were significantly higher, whereas the number of acidophilic granulocytes (G7 immunoreactive) decreased, compared with non-parasitized and unexposed fish. These differences were stronger at the posterior intestine, the main target of the parasite, and no differences were found in the thymus. In non-parasitized GSB, the percentage of splenic surface occupied by melanomacrophage centres was significantly higher. These results suggest that the cellular response of GSB to E. leei includes proliferation of leukocytes in lymphohaematopoietic organs and recruitment into intestines via blood circulation involving elements of innate and adaptive immunity. Acidophilic granulocytes and mast cells presented opposite patterns of response to the parasite infection, with an overall depletion of the former and an increased amount of the latter. Some differences between both cell types were also detected in regard to their granule density and cell morphology.

1. Neodiplostomum intermedium Pearson is recorded from four new hosts; as an adult from the water rat, Hydromys chrysogaster Geoffroy, and as a metacercaria (diplostomulum), from tadpole and adult of an undescribed tree frog, Hyla sp., tadpole of (Hyla latopalmata (Günther) Mixophyes fasciolatus Günther and frog of an unidentified leptodactylid.

2. The life cycle of Neodiplostomum intermedium was followed experimentally; the hosts were: Pettancylus assimilis (Petterd), a fresh-water limpet, as first intermediate host; tadpole of Hyla pearsoni Copland as second intermediate host; Hyla caerulea (Shaw) a tree frog, and Hemisphaerodon gerrardi Peters, the pinktongued skink, as paratenic hosts; and Rattus assimilis (Gould) and laboratory rats as definitive hosts.

3. Descriptions are given of the morphology of the miracidium, mother sporocyst, daughter sporocyst, cercaria, and diplostomulum, with special reference to the structure of the miracidium and of the cercarial tail.

4. Observations are given on the embryology of the miracidium, hatching of the egg, transformation of the miracidium into the mother sporocyst with special reference to the germinal cells, the route and manner of escape of cercariae from the snail host, the development of the diplostomulum with special reference to the reserve excretory system, and the movements of diplostomula during metamorphosis of the tadpole host.

5. The haploid chromosome number is ten, as determined from squashes of testes. One paratype and a series of experimental adults have been compared with and found different from Neodiplostomum (Fibricola) sarcophilus n.comb. The orthography and formal proposing of the names of the species of Fibricola transferred to Neodiplostomum by Pearson (1959b) are corrected.

The writer wishes to thank Dr M. J. Mackerras, Queensland Institute for Medical Research, for generously supplying water rats; Professor J. F. A. Sprent, University of Queensland Veterinary School, for his criticism of the manuscript; Mr K. Webber and his sons for their assistance in catching rats and for permission to collect snails, frogs and tadpoles from their streams; and Mr R. J. Ballantyne for technical assistance. This study was supported by a grant from the Rural Credits Fund of the Commonwealth Bank of Australia.

Variation in the immunogenicity of 3 isolates of Trichinella spiralis was assessed by the parameters of adult worm recovery, mast cell, eosinophil and antibody responses in mice of defined response phenotype. The levels of the protective, inflammatory and immune responses induced by infection differed between the isolates. Isolates showed considerable variation in the capacity to elicit mast cell and eosinophil responses. All induced increases in parasite-specific antibody, levels of total (IgGAM) antibody and of IgM and IgG isotypes rose steadily after infection, but there were significant differences in levels of response. The IgGAM response was correlated with the number of worms present, i.e. the greatest response was seen in low responder (C57BL/10) mice infected with the longest-surviving isolates. All isolates elicited specific IgG1 and IgG2a antibodies after infection, although, again, there were isolate-specific differences in the levels and kinetics of response. Levels of these isotypes were always higher, although not significantly so, in high-responder NIH mice. Low-responder mice showed higher IgE serum levels than high-responder mice after infection, one isolate giving much higher IgE values than the other two.

The diagnosis of parasitic worm (helminth) infections requires specialized laboratory settings, but most affected individuals reside in locations without access to such facilities. We tested two portable microscopic devices for the diagnosis of helminth infections in a cross-sectional survey in rural Côte d'Ivoire. We examined 164 stool samples under a light microscope and then re-examined with a commercial portable light microscope and an experimental mobile phone microscope for the diagnosis of Schistosoma mansoni and soil-transmitted helminths. Additionally, 180 filtered urine samples were examined by standard microscopy and compared with the portable light microscope for detection of Schistosoma haematobium eggs. Conventional microscopy was considered the diagnostic reference standard. For S. mansoni, S. haematobium and Trichuris trichiura, the portable light microscope showed sensitivities of 84·8%, 78·6% and 81·5%, respectively, and specificities of 85·7%, 91·0% and 93·0%, respectively. For S. mansoni and T. trichiura, we found sensitivities for the mobile phone microscope of 68·2% and 30·8%, respectively, and specificities of 64·3% and 71·0%, respectively. We conclude that the portable light microscope has sufficient diagnostic yield for Schistosoma and T. trichiura infections, while the mobile phone microscope has only modest sensitivity in its current experimental set-up. Development of portable diagnostic technologies that can be used at point-of-sample collection will enhance diagnostic coverage in clinical and epidemiological settings.

We studied the pattern of infection and the inter-annual variation and individual factors affecting the infection of 2 species of nasopharyngeal bot flies, Cephenemyia auribarbis and Pharyngomyia picta (Diptera: Oestridae), in a population of Iberian red deer (Cervus elaphus hispanicus) from south central Spain (10 annual periods between 1990 and 2003). Mean prevalence±S.E.95%CI of infection was 35·19±4·24% (n=486). The frequency distribution of the parasites was markedly aggregated (K: 0·213, mean abundance±S.D.: 5·49±12·12). Parasite load of Oestridae peaked at calf and subadult age groups and declined thereafter, which suggests that acquired immunity may be acting. In common with other host–parasite relationships, male hosts were found to have higher prevalence and abundance levels than females. The prevalence of P. picta was positively affected by the presence of C. auribarbis whereas the intensity of infection of P. picta was negatively affected by the presence of C. auribarbis. Intensity of P. picta in concomitant infections with C. auribarbis was lower than in pure P. picta infections, whilst the intensity of C. auribarbis infections did not change. This provides good evidence of interspecific competence, which could be dealt with by parasites by means of asynchronous life-cycles and different maturation periods. Weather also affects the dynamics and transmission rates of these parasites. Previous annual rainfalls positively affected the level of infection with oestrids. Yearly autumn rainfalls affected positively P. picta, possibly due to an effect on the pupal stage survival. Infection of Oestridae affected body condition in calves and subadults, suggesting that oestrids could have sublethal effects on Iberian red deer. Future research is needed to investigate the effect of parasites on the dynamics of the Iberian red deer.

The discrete typing units (DTUs) of Trypanosoma cruzi that infect domestic dogs and cats have rarely been studied. With this purpose we conducted a cross-sectional xenodiagnostic survey of dog and cat populations residing in 2 infested rural villages in Pampa del Indio, in the humid Argentine Chaco. Parasites were isolated by culture from 44 dogs and 12 cats with a positive xenodiagnosis. DTUs were identified from parasite culture samples using a strategy based on multiple polymerase-chain reactions. TcVI was identified in 37 of 44 dogs and in 10 of 12 cats, whereas TcV was identified in 5 dogs and in 2 cats –a new finding for cats. No mixed infections were detected. The occurrence of 2 dogs infected with TcIII –classically found in armadillos– suggests a probable link with the local sylvatic transmission cycle involving Dasypus novemcinctus armadillos and a potential risk of human infection with TcIII. Our study reinforces the importance of dogs and cats as domestic reservoir hosts and sources of various DTUs infecting humans, and suggests a link between dogs and the sylvatic transmission cycle of TcIII.

Plasmodium malariae, a protozoan parasite that causes malaria in humans, has a global distribution in tropical and subtropical regions and is commonly found in sympatry with other Plasmodium species of humans. Little is known about the genetics or population structure of P. malariae. In the present study, we describe polymorphic genetic markers for P. malariae and present the first molecular epidemiological data for this parasite. Six microsatellite or minisatellite markers were validated using 76 P. malariae samples from a diverse geographical range. The repeat unit length varied from 2 to17 bp, and up to 10 different alleles per locus were detected. Multiple genotypes of P. malariae were detected in 33 of 70 samples from humans with naturally acquired infection. Heterozygosity was calculated to be between 0·236 and 0·811. Allelic diversity was reduced for samples from South America and, at some loci, in samples from Thailand compared with those from Malawi. The number of unique multilocus genotypes defined using the 6 markers was significantly greater in Malawi than in Thailand, even when data from single genotype infections were used. There was a significant reduction in the multiplicity of infection in symptomatic infections compared with asymptomatic ones, suggesting that clinical episodes are usually caused by the expansion of a single genotype.

Of 414 Bermuda fishes, 148 belonging to 13 species harboured Digenea and 11 belonging to 6 species Aspidogastrea, Acanthocephala or nematodes. In a previous paper 69 of these fishes belonging to 20 species were found to contain cestodes. The number of infected species was greatest in the case of cestodes and the number of infected individuals in the case of Digenea. The latter is probably due to the closer proximity, for reasonably long periods, of the various hosts in the life-cycles. Of the 17 species of Digenea 3 are new host records, 3 new locality records and 4 new host and new locality records. New records were found, also, in the other groups of helminths. The digenean fauna of Bermuda fishes most closely resembles that of Dry Tortugas, Florida. Digenea and cestodes were not usually found in the same families of fishes. Cestode larvae were abundant in the deep-water Serranidae and Lutianidae which had no Digenea, the latter being common in Clupeidae, Hae-mulidae and Belonidae, which do not harbour cestodes and which occur, mostly, in the inner reefs and shallows.

The functional morphology of the apical disk, tentacles and rhynchus of Alcicornis carangis is described. The tentacles are invaginable and concerned with attachment.

I am most grateful to Dr W. H. Sutcliffe Jun., Director of the Bermuda Biological Station, for the excellent facilities provided and to The Shell Grants Committee and The National Science Foundation for grants which made the work possible. I would also like to express my gratitude to all those who kindly helped in the capture of fishes and to Mrs M. Fisher for technical assistance.