Factors which affect adhesion of cultured Trypanosoma congolense bloodstream forms to mammalian feeder cells have been examined. Using an in vitro binding assay, the initial events following interaction of trypanosomes with bovine aorta endothelial (BAE) cells were monitored by both light- and electron microscopy. Metabolic inhibitors and other biochemicals were incubated with either cells or parasites, to test whether any inhibited the process. Our findings suggest that adhesion of the parasites is an active process requiring metabolic energy from the trypanosomes, but not from endothelial cells. We also provide data suggesting that T. congolense bloodstream forms possess a lectin-like domain, localized at distinct sites on their flagellar surface, which interacts with specific carbohydrate receptors, most likely sialic acid residues, on the endothelial cell plasma membrane. We also suggest that the cytoskeletal protein actin is probably involved in this interaction.

Lymphatic filariasis (LF), which is highly endemic in 73 countries worldwide, is targeted for elimination by 2020. The strategy for achieving this goal is based on 4 sequential programmatic steps: mapping, Mass drug administration (MDA) implementation, post-MDA surveillance and verification of LF elimination. All 4 stages of the implementation process are dependent on the availability of user friendly and highly sensitive rapid diagnostic tools. By the end of 2012, 59 countries had completed mapping for LF and Eritrea was the only country yet to start the process. Rolling out new diagnostic tools to facilitate the mapping process will enable an accelerated shrinking of the LF map to zero endemic countries by 2020. When the Global Programme to Eliminate Lymphatic Filariasis was launched in 2000, diagnostic tools for LF were limited to clinical examination, detection of microfilaria (MF) by microscopy in night blood samples and detection of antibodies to native-antigen preparations. There has been a significant improvement in the traditional LF diagnostic methods in recent years and some new tools are now available. This paper provides an update on the human diagnostic tests available for LF and their current applications as tools in mapping and transmission monitoring. The values of entomological indicators and parasite detection and speciation methods applied to vector populations are also discussed.

Development of the inflammatory response of rainbow trout to experimental infections with Diphyllobothrium dendriticum plerocercoids is described using light and electron microscopy. The cellular response to plerocercoids occurred within 2 weeks post-infection (p.i.). This was followed by an increase in leucocyte numbers during weeks 3–6 p.i., with full encapsulation of plerocercoids by week 6 p.i. Neutrophils were the first leucocytes to engage the developing plerocercoid, followed by large influxes of macrophages which transformed into epithelioid cells. With longer times p.i. the accumulation of different leucocyte types increased, and a blood vascular network developed. Full development of the composite cyst was characterized by fibroplasia, particularly at the periphery of the cyst, and the subsequent deposition of a collagenous tissue matrix. Enzyme-linked immunosorbent assay (ELISA) examination of serum samples taken over the 20 week period showed that specific anti-D. dendriticum antibody titres were first detected at 5 weeks p.i. and increased to a maximum by 11 weeks p.i.

The hatching mechanism of the liver fluke egg was investigated and a new interpretation put forward. Light stimulates the miracidium and, when fully activated, the miracidium is responsible for altering the permeability of the membrane on the internal concave surface of the viscous cushion. Prior to expansion the cushion (a fibrillar mucoprotein complex) is in a dehydrated or semihydrated state. The change in permeability of the enclosing membrane allows the fluid egg contents to permeate into the cushion. The fibrillar matrix becomes hydrated and an increase in volume occurs. This compresses the miracidium and sacs and an internal pressure is set up which gradually increases until the operculum finally ruptures and flies back violently. The cushion and miracidium are then expelled by the hypertonicity of the egg contents.

I would like to thank Professor O. W. Richards, in whose department this work was carried out, also Dr F. Call and Professor B. G. Peters for their many helpful criticisms. I am also grateful to Miss Mary Morris for assistance with the electron microscopy.

The fine structure of the flame cells, tubules and excretory pore of the miracidium of Fasciola hepatica are described. The tubules are formed from the flattened surfaces of cells rolled round and joined at their tips by a desmosome. Vesicles are present in the wall of the tubule, possibly indicating secretory activity. Clubshaped microvilli are present only in the terminal region near the excretory pore. Possible functions of the system are discussed.

Until recently, almost all studies of leishmaniasis epidemiology were qualitative and descriptive. But now that the natural history of many Leishmania parasites is quite well known, there is growing interest in quantitative analysis. In this paper I use mathematical models in conjunction with field data to try to answer a wider range of questions than has previously been possible with descriptive techniques, and to sharpen some of the outstanding questions for laboratory workers. This is done with reference to the persistence and resilience of canine leishmaniasis, the maintenance of virulence poly morphisms in Leishmania populations, and the possible existence of cycles of human kala-azar. I conclude by posing a set of problems under three headings: diagnosis of infection (as distinct from disease), natural immunity to Leishmania infection in the vertebrate host, and genetic variation in the parasite population. Some solutions from the laboratory can be found in the companion paper by Black (1992).

The migration of schistosomula through the lungs of mice has been investigated at the ultrastructural level. On Days 4 and 7 post-infection schistosomula were located only within the vasculature. Newly arrived Day 4 schistosomula were highly convoluted and completely occluded vessels measuring approximately 15–25μm in diameter. By Day 7 parasites were located in pulmonary capillaries, often causing considerable distension. The subtegumentary fibrous interstitial layer was absent in elongated lung schistosomula. The minimum diameter of such parasites was 8μm, and their tegument was usually unridged and flattened against the vascular endothelium, thus resulting in close apposition of endothelial and tegumental membranes. The tight fit of schistosomula in the pulmonary capillaries implies that migration through them is both a strenuous and slow process. The migratory delay in the lungs after arrival from the skin occurs because dimensional changes are necessary for successful capillary migration.

Novel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

Aquaculture, including both freshwater and marine production, has on a world scale exhibited one of the highest growth rates within animal protein production during recent decades and is expected to expand further at the same rate within the next 10 years. Control of diseases is one of the most prominent challenges if this production goal is to be reached. Apart from viral, bacterial, fungal and metazoan infections it has been documented that protozoan parasites affect health and welfare and thereby production of fish in marine aquaculture. Representatives within the main protozoan groups such as amoebae, dinoflagellates, kinetoplastid flagellates, diplomonadid flagellates, apicomplexans, microsporidians and ciliates have been shown to cause severe morbidity and mortality among farmed fish. Well studied examples are Neoparamoeba perurans, Amyloodinium ocellatum, Spironucleus salmonicida, Ichthyobodo necator, Cryptobia salmositica, Loma salmonae, Cryptocaryon irritans, Miamiensis avidus and Trichodina jadranica. The present report provides details on the parasites’ biology and impact on productivity and evaluates tools for diagnosis, control and management. Special emphasis is placed on antiprotozoan immune responses in fish and a strategy for development of vaccines is presented.

Killing of metacestodes by normal or post-infection macrophages and the regulation of this activity by cytokines were studied in vitro. The protoscolecidal activity of normal macrophages against Echinococcus granulosus was inhibited by a product of naive T-enriched lymphocytes co-cultured with protoscoleces (PSC). By contrast, supernates from co-cultures of Mesocestoides corti tetrathyridia (MCT) and T-enriched or B-enriched normal lymphocytes increased killing of MCT by normal macrophages. Larvicidal activity (against both PSC and MCT) was enhanced by high concentrations of macrophage-activating factors produced by Con A-stimulated rat lymphocytes (Con A-LK), but was reduced by low concentrations of these factors.

African sleeping sickness is a parasitic disease transmitted through the bites of tsetse flies of the genus Glossina. We constructed mechanistic models for the basic reproduction number, R0, of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, respectively the causative agents of West and East African human sleeping sickness. We present global sensitivity analyses of these models that rank the importance of the biological parameters that may explain variation in R0, using parameter ranges based on literature, field data and expertize out of Uganda. For West African sleeping sickness, our results indicate that the proportion of bloodmeals taken from humans by Glossina fuscipes fuscipes is the most important factor, suggesting that differences in the exposure of humans to tsetse are fundamental to the distribution of T. b. gambiense. The second ranked parameter for T. b. gambiense and the highest ranked for T. b. rhodesiense was the proportion of Glossina refractory to infection. This finding underlines the possible implications of recent work showing that nutritionally stressed tsetse are more susceptible to trypanosome infection, and provides broad support for control strategies in development that are aimed at increasing refractoriness in tsetse flies. We note though that for T. b. rhodesiense the population parameters for tsetse – species composition, survival and abundance – were ranked almost as highly as the proportion refractory, and that the model assumed regular treatment of livestock with trypanocides as an established practice in the areas of Uganda experiencing East African sleeping sickness.

The acquired immunity of twenty-eight splenectomized and twenty-three intact cattle following primary inoculation with either of two strains (the Y and the R strain) of Babesia bigemina was investigated. Some of these animals were carriers, while others had ceased to be infected. Observations were made on the response of splenectomized animals challenged up to four times with the original infecting (homologous) strain and finally with the heterologous strain.

In the splenectomized animals, a total of sixty-three homologous challenges produced parasitaemias in only twenty-seven instances, and these were generally delayed and of lower intensity than in control animals. Fifteen negative transmission tests performed shortly after thirty-two challenges showed that it was difficult to re-establish the homologous strain in some animals that had achieved self-cure. Substantial homologous immunity had developed within 16 weeks of the primary infection in animals which were no longer carrying B. bigemina.

All twenty-one splenectomized animals subjected to heterologous challenge developed well-marked parasitaemias. Nine of these had been highly immune to homologous challenge 2 weeks earlier. Eight of the twenty-one were carriers at the time of challenge and these showed much lower parasitaemias than the self-cured animals which almost invariably were acutely affected.

Challenges of twenty-three intact steers confirmed that the two strains differed immunologically. Six of twelve challenged homologously failed to show detectable parasitaemia, whereas all eleven challenged heterologously developed parasitaemias.

The pathogenic Theileria species Theileria parva and T. annulata infect bovine leukocytes and erythrocytes causing acute, often fatal lymphoproliferative diseases in cattle. The parasites are of interest not only because of their economic importance as pathogens, but also because of their unique ability to transform the leukocytes they infect. The latter property allows parasitized leukocytes to be cultured as continuously growing cell lines in vitro, thus providing an amenable in vitro system to study the parasite/host cell relationship and parasite-specific cellular immune responses. This paper summarizes important advances in knowledge of the immunobiology of these parasites over the last 40 years, focusing particularly on areas of relevance to vaccination.

The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests that seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. One-dimensional PAGE gels showed 40–50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5–7 μg of seminal protein and with only 60 μg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between 2 laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionization tandem mass spectrometry revealed that 26·5% of the proteins had matches among arthropods in databases and 14·5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.

The whole life cycle of Paramphistomum microbothrium Fischoeder, 1901, found in cattle of Kenya, East Africa, has been established experimentally.

In laboratory conditions, the eggs hatched miracidia on the 14th to 16th day, if they were kept in water at a temperature of 26–28° C.

In a snail, Bulinus alluaudi (Dautzenberg), kept at the temperature of 18–20° C., the miracidium developed into a sporocyst, the elongated body of which, containing young rediae, reached a length of 3·6 mm. in about 2 weeks.

The first rediae which emerged from a sporocyst were observed on the 14th day, and on the 20th day the first generation rediae began to produce second-generation rediae. From the 28th day onward the first-generation parent rediae ceased to produce daughter rediae and began to develop cercariae only. This period of production of cercariae by the redia lasted about 30 days, and when the life of the first-generation rediae drew to its close, the old rediae developed a few daughter rediae again.

Cercariae began to emerge from the first-generation rediae 30 days after exposure of the snail to miracidia. The cercariae left the parent redia in a very immature state and further development occurred in the liver of the snail. The emergence of cercariae from the infected snail began on the 43rd day after exposure to miracidia. Shortly after emerging from the snail, the cercariae attached themselves to vegetation and encysted.

Development of rediae of the second, third, fourth and apparently more successive generations followed an identical course to that outlined for the rediae of the first generation. As a result the successive generations of rediae maintain the infection going in an intermediate host and the infected snails were continually shedding cercariae as long as they lived. In the laboratory experiments the life span of some of the infected snails exceeded a year.

In cattle infected experimentally P. microbothrium reached maturity and began passing out the eggs about a 100 days after the encysted cercariae were fed to the animals.

In Europe, the zoonotic cycle of Babesia microti has not been determined so far. Recently, B. microti was detected in Ixodes ricinus ticks in Slovenia by using molecular methods. In order to investigate the mammalian hosts of B. microti in Slovenia we collected 261 small mammals representing 11 species. They were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-rDNA). The bank vole (Clethrionomys glareolus) and yellow-necked mouse (Apodemus flavicollis) were infected with B. microti. The prevalence rate was 15·9% for C. glareolus and 11·8% for A. flavicollis. Nucleotide sequences of amplified portions of B. microti nss-rDNA from C. glareolus and A. flavicollis were indistinguishable from each other and identical with those previously described in I. ricinus ticks collected in Slovenia. The results of this study represent molecular evidence of B. microti in small mammals in Europe.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S–5S–18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.The nucleotide sequence data reported in this paper are available in the EMBL database with the accession number AJ238400.The commercial use of the diagnostic test presented here is protected by a patent. This patent is pending upon the application number P9801511.

Understanding the frequency distribution of parasites and parasite stages among hosts is essential for efficient experimental design and statistical analysis, and is also required for the development of sustainable methods of controlling infection. Nematodirus battus is one of the most important organisms that infect sheep but the distribution of parasites among hosts is unknown. An initial analysis indicated a high frequency of animals without N. battus and with zero egg counts, suggesting the possibility of a zero-inflated distribution. We developed a Bayesian analysis using Markov chain Monte Carlo methods to estimate the parameters of the zero-inflated negative binomial distribution. The analysis of 3000 simulated data sets indicated that this method out-performed the maximum likelihood procedure. Application of this technique to faecal egg counts from lambs in a commercial upland flock indicated that N. battus counts were indeed zero-inflated. Estimating the extent of zero-inflation is important for effective statistical analysis and for the accurate identification of genetically resistant animals.

Total DNAs, isolated from a range of taeniid cestodes (Taenia solium, T. saginata, T. pisiformis, T. crassiceps, T. hydatigena, T. ovis, T. multiceps and T. taeniaeformis), have been subjected to restriction enzyme digestion, Southern transfer and hybridization analysis using cloned fragments of the ribosomal RNA gene of Schistosoma mansoni. Substantial inter-specific genetic differences have been revealed on the basis of characteristic hybridization patterns for each of the taeniid cestode species. Furthermore, a random genomic DNA library has been constructed in the vector plasmid pAT153 using DNA extracted from a pig isolate (Indian origin) of T. solium. A panel of taeniid cestode DNAs including DNA from Echinococcus granulosus, has been used in conjunction with hybridization and restriction enzyme analysis to identify in the library a single recombinant plasmid with a T. solium-specific insert (coded pTS10) and two recombinant plasmids with T. solium inserts having selective specificities for T. solium and T. ovis (coded pTS17) and T. solium, T. saginata, T. ovis and T. multiceps (coded pTS28). These recombinant plasmids and the cloned fragments of the ribosomal RNA gene of S. mansoni have been used in restriction endonuclease, Southern transfer and hybridization analysis to detect intra-specific genetic variation in cysticerci of T. solium from India, Mexico and Zimbabwe. In addition, pTS10 and pTS17 have been used in a simple dot-blot assay to distinguish T. solium from T. saginata.