Human African trypanosomiasis (HAT) or sleeping sickness is caused by protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. Despite the enormous technological progress in molecular parasitology in recent years, the diagnosis of HAT is still problematic due to the lack of specific tools. To date, there are two realities when it comes to HAT; the first one being the world of modern experimental laboratories, equipped with the latest state-of-the-art technology, and the second being the world of HAT diagnosis, where the latest semi-commercial test was introduced 30 years ago (Magnus et al.1978). Hence, it appears that the lack of progress in HAT diagnosis is not primarily due to a lack of scientific interest or a lack of research funds, but mainly results from the many obstacles encountered in the translation of basic research into field-applicable diagnostics. This review will provide an overview of current diagnostic methods and highlight specific difficulties in solving the shortcomings of these methods. Future perspectives for accurate, robust, affordable diagnostics will be discussed as well.

Thanks to modern molecular biology methods, our understanding of the impact of (endo)symbiotic bacteria on parasitic protists and helminths is growing fast. In this issue, 9 papers have been brought together that describe various facets of the relationships between these microorganisms, reveal their range and high frequency, as well as their capacity to create novel biological complexity. Comparative analyses of these host–endosymbiont interactions indicate that there may be no discrete types of relationships but rather a continuum ranging from a dispensable endosymbiont minimally integrated within the host cell to organelles, such as mitochondria and plastids that evolved into an indispensable, deeply integrated components of the cell. We hope that this series of studies on parasites and (endo)symbiotic bacteria will increase awareness about these relationships and their representation in microbial ecology models.

A virus disease of Atropa belladonna caused by a new virus is described. The virus is sap-transmissible to tobacco and other solanaceous plants and gives good local lesions on Nicotiana glutinosa and Phaseolus vulgaris. It occurs in fairly high concentration in the plant, it withstands ageing in extracted sap for 6–11 days, and is inactivated at a temperature between 75 and 80° C.

We studied the effect of density of larvae on pre-imaginal development in 2 flea species (Xenopsylla conformis and Xenopsylla ramesis) parasitic on 2 desert rodent species (Dipodillus dasyurus, adult body mass 20 g and Meriones crassus, 80 g). We predicted a decrease in duration of development with an increase in density of larvae. In general, in both flea species, duration of larva-to-pupa development decreased with an increasing larval density. In addition, this stage of development was longer in male fleas and in fleas from parents fed on D. dasyurus. The effect of larval density on larval development was manifested mainly when parent fleas fed on D. dasyurus. Duration of pupation decreased with increasing larval density only in offspring of fleas fed on G. dasyurus. In both fleas, pupation was longer in males. The effect of parent host on duration of pupation was found in X. ramesis only (longer if the host was M. crassus). Resistance of newly emerged fleas to starvation depended mainly on parent host species. Young X. conformis survived longer if their parents fed on D. dasyurus, whereas young X. ramesis survived longer if their parents fed on M. crassus. It was also found that (a) an individual flea that spent more time as a larva also spent more time as a pupa and (b) longer larval development resulted in a shorter time that a newly emerged flea was able to survive when starved.

Infestation of a new host is a crucial stage in the life-cycle of parasites, and the possibility that hosts avoid infesting contact depends, in part, on the predictability of infestation risk. Immature free stages of ticks (Acari, Ixodidae) have limited mobility and survival in the vegetation and strongly depend on host behaviours for their infestation. We studied spatial and temporal distributions of the larvae of 2 major groups of African tick species in a ranch in Zimbabwe. No difference in the abundance of Rhipicephalus evertsi evertsi larvae was found among vegetation types and during most of the seasonal cycle, and no reliable indicator of their presence on a given site was identified. Rhipicephalus appendiculatus/Rhipicephalus zambeziensis larvae are mainly found during the cool dry season, in vegetation types situated close to permanent water holes or dominated by Acacia trees, which provide key forage resources for ungulates; and several indicators of their presence were identified. For both tick groups, spatial and temporal distributions of the larvae result in an optimized contact with ungulate hosts: R. e. evertsi larvae are unpredictable and thus unavoidable by hosts, whereas R. appendiculatus/R. zambeziensis larvae are predictable but also unavoidable because they are associated with key-resources for ungulates.

In the context of a broader program dealing with the symbiotic associations of apple snails, we sampled three species that coexist in Lake Regatas (Palermo, Buenos Aires, Argentina). The population of Asolene platae, (but not those of Pomacea canaliculata and Pomacea scalaris) showed trematode larvae (echinocercariae and xiphidiocercariae) in haemocoelic spaces and connective tissue of the digestive gland. The echinocercariae resembled those of the genus Echinochasmus, but lacked sensory hairs on their body and tail; whereas xiphidiocercariae were similar to the xiphidiocercariae armatae belonging to the Opisthoglyphe type. The phylogenetical positions of these trematodes were inferred by the 28S rRNA, ITS1 and mtCOXI gene sequences. The 28S rRNA gene linked the echinocercarial sequences with the polyphyletic genus Echinochasmus (Echinochasmidae), while the xiphidiocercarial sequences were linked with the genus Phaneropsolus (Phaneropsolidae). The molecular markers used were able to distinguish two cryptic molecular entities of the single echinocercarial morphotype. Although ITS1 and mtCOXI did not allow resolving phylogeny beyond the family level because of the scarce number of sequences in the molecular databases, both cercariae (echinocercariae and xiphidiocercariae) could be distinguished by the Internal Transcribed Spacer 1 amplicon size. This is the first correlative morphological and molecular study of zoonotic trematodes in Neotropical ampullariids.

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel in 5–15% of cured cases of kala-azar (KA) after months or years of treatment in India. Molecular differences are reported to exist between the KA and PKDL isolates which may underlie the diversity in clinical manifestations of the disease. Here, arbitrary primed-PCR (AP-PCR) has been used for genetic fingerprinting of parasite isolates from dermal lesions of PKDL patients (n=14) and compared with bone-marrow derived parasites from KA patients (n=3). All isolates showed an identical AP-PCR pattern with 4 arbitrary primers. Further, AP-PCR was exploited to identify the stage regulated genes of the parasite. Six polymorphic fragments were identified in PKDL in comparison with KA isolates, and were subjected to Northern blot analysis. Five polymorphic fragments represented transcribed sequences; 4 out of 5 drew differential expression in pro- and amastigote stages, although the expression was comparable between PKDL and KA isolates. The study led to the identification of genes, which exhibit stage-regulated expression in Leishmania donovani derived from PKDL or KA patients, including a putative phosphodiesterase, DEAD box RNA helicase, iron superoxide dismutase b (fesodb) and a hypothetical protein. Demonstration of transcripts of DEAD box RNA helicase in PKDL and KA diseased tissues implicates its role in disease pathogenesis.

Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -β were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin–receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -β were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.

An attempt was made to evaluate the immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) as a DNA vaccine, for human lymphatic filariasis. BmTGA was cloned and characterized in the DNA vaccine vector pVAX1. Further, the tissue distribution study of the DNA construct, pVAX-TGA was carried out in mice and the DNA vaccine was shown to be efficiently distributed to all the organs, was accessible to the immune system, and at the same time was metabolized quickly and did not pose problems of toxicity. Intramuscular immunization in mice showed significant antibody production and splenocyte proliferation upon antigenic stimulation. The immune responses were biased towards the Th1 arm, as evaluated in terms of isotype antibody distribution and cytokine profile. Thus, analysis of the humoral and cellular immune responses indicated that BmTGA is a potent immunogen. However, protection studies as determined by the micropore chamber method using live microfilarial larvae, showed that the DNA vaccine could confer only partial protection in the mouse model. We conclude that despite the induction of sufficient humoral and cellular immune responses, BmTGA as a DNA vaccine could not confer much protection against subsequent challenge and other aspects of the immune responses need to be further investigated.

1. Observations have been made on the life cycles of Cercaria paracauda, Iles, G. tetraglandis, Iles, and C. Apatemon gracilis minor, Yamaguti. The development of the tetracotyle metacercaria of C. tetraglandis has been followed and also that of the metacercaria and adult of A. g. minor. The pathology and distribution of the metacercariae of the last two species within the leech, Erpobdella testacea (Sav.) has been studied.

2. It was noted that development of the metacercariae within the same host and resulting from the same infection was not simultaneous and that it was improbable that a 100 % encystment of the metacercariae would occur. Temperature was found to have a marked effect on the length of time required for encystment.

3. Data are given on the infectivity and excystation of the metacercariae of A. g. minor and on the biology and morphology of the adult of this subspecies.

4. Stress is placed on the importance, in the systematics of the digenetic trematodes, of considering both the morphological and biological characters of the adult and those of the related larval stages.

The egg of Ixodes ricinus L. is described, and the increase in size during development is noted. It is suggested that the increase is due to the absorption of water. The egg wall consists of exo- and endochorion with a thin vitelline membrane, which becomes thicker as development proceeds and assumes a laminated appearance. The secretion layer of Gené's organ is described, and appears to function as a cement for holding the eggs together in the cluster. Chemical tests show it to be of a waxy nature.

It is deduced from experimental evidence that the egg shell is not absolutely rigid, and the weight of evidence favours the view that the shape of the egg shell is maintained by the internal pressure of the egg contents, and the elasticity of the shell.

The development processes as seen in the living egg are described. Hatching from the egg shell is brought about by a muscular force acting on a limited area at the posterior pole of the egg.

The writer is indebted to Mr Robert David, of Longlands Farm, Pyle, Dr A. D. Lees, of the Agricultural Research Council, and Mr Simeon, of the Glamorgan War Agricultural Executive Committee, for supplies of material; to Mr T. W. Tyssul Jones, M.Sc., for his assistance in checking the measurements of the egg dimensions, and to Mr L. Cowley, M.Sc., Assistant Curator (Department of Zoology), National Museum of Wales, and Dr P. Tate, Molteno Institute of Parasitology, Cambridge, for reading the original script and for making many useful suggestions and modifications.

To test the homogeneity of 18S sequences within Trypanosoma cruzi, riboprint profiles were separated by temperature gradient gel electrophoresis (TGGE). Upon interpretation of melting curves of fragments within a riboprint profile, there appeared to be two 18S sequence types within each stock examined. Two similar types were also observed within outgroup taxa Trypanosoma conorhini, Trypanosoma rangeli and Leishmania braziliensis. From DNA hybridization studies, these fragments were shown to have homology to the 18S V1 region. There are therefore two 18S V1 regions, differing in sequence, present in all taxa examined. When only a single 18S sequence is used to represent each taxa for phylogenetic inference, comparisons may be between paralogous and not orthologous copies of this region, such that, inferred relationships may merely reflect a gene history. This seriously questions the current molecular phylogeny of these protozoa using 18S data.

Myxozoans are widespread and common endoparasites of fish with complex life cycles, infecting vertebrate and invertebrate hosts. There are two classes: Myxosporea and Malacosporea. To date about 2500 myxosporean species have been described. By comparison, there are only five described malacosporean species. Malacosporean development in the invertebrate hosts (freshwater bryozoans) has been relatively well studied but is poorly known in fish hosts. Our aim was to investigate the presence and development of malacosporeans infecting a diversity of fish from Brazil, Europe and the USA. We examined kidney from 256 fish belonging variously to the Salmonidae, Cyprinidae, Nemacheilidae, Esocidae, Percidae, Polyodontidae, Serrasalmidae, Cichlidae and Pimelodidae. Malacosporean infections were detected and identified by polymerase chain reaction and small subunit ribosomal DNA sequencing, and the presence of sporogonic stages was evaluated by ultrastructural examination. We found five malacosporean infections in populations of seven European fish species (brown trout, rainbow trout, white fish, dace, roach, gudgeon and stone loach). Ultrastructural analyses revealed sporogonic stages in kidney tubules of three fish species (brown trout, roach and stone loach), providing evidence that fish belonging to at least three families are true hosts. These results expand the range of fish hosts exploited by malacosporeans to complete their life cycle.

The study was designed to determine comparatively the prognostic value of immunoblotting and ELISA in the serological follow-up of young cystic echinococcosis (CE) patients exhibiting either a cured or a progredient (non-cured) course of disease after treatment. A total of 54 patients (mean age 9 years, range from 3 to 15 years) with surgically, radiologically and/or histologically proven CE were studied for a period up to 60 months after surgery. Additionally, some of the patients underwent chemotherapy. Based on the clinical course and outcome, as well as on imaging findings, patients were clustered into 2 groups of either cured (CCE), or non-cured (NCCE) CE patients. ELISA showed a high rate of seropositivity 4 to 5 years post-surgery for both CCE (57·1%) and NCCE (100%) patients, the difference found between the two groups was statistically not significant. Immunoblotting based upon recognition of AgB subcomponents (8 and 16 kDa bands) showed a decrease of respective antibody reactivities after 4 years post-surgery. Only sera from 14·3% of CCE patients recognized the subcomponents of AgB after 4 years, while none (0%) of these sera was still reactive at 5 years post-surgery. At variance, immunoblotting remained positive for AgB subcomponents in 100% of the NCCE cases as tested between 4 and 5 years after surgical treatment. Immunoblotting therefore proved to be a useful approach for monitoring post-surgical follow-ups of human CCE and NCCE in young patients when based upon the recognition of AgB subcomponents.

Regional differences in the tegumentary tissue of Moniezia expansa, as revealed at the light-microscope level by histological and histochemical techniques, are described and evidence for secretory activity by the interproglottidal glands is presented.

In very immature proglottides the interproglottidal glands are at the ‘precryptic’ stage. Gland cells may be differentiated from other tegumentary cells by their high RNA content and in certain gland cells the presence of an alcian blue staining material.

In mature proglottides the glands consist of rosette-like clusters of cells around crypt-like intuckings of the tegument. Two types of cells are found in the gland, small alcian blue-staining cells which are most numerous in the neck region of the crypt, and larger cells, the predominant gland cells, which do not stain with alcian blue but possess non-specific esterase activity. No other tegumentary cells in Moniezia exhibit this activity. Esterase and phosphatase activity is found in the tegument and crypt of the glands and in the interproglottidal folds.

The non-enzyme histochemistry confirms and extends the observations of previous workers.

Cytochrome oxidase and succinic dehydrogenase were detected in the tegumentary cells and tegument. Very strong reactions were given in the neck and scolex, with a progressive diminution of activity posteriorly along the strobila. Very low activities were recorded in the tegument of the glands.