Allelic variation in 6 microsatellite markers was compared between frozen Schistosoma mansoni eggs and laboratory-passaged worms originating from the same 5 fecal samples obtained from Brazilian residents. Based on allelic richness values, the number of alleles detected per locus did not differ between egg and worm DNA templates. However, our ability to score loci differed between these DNA templates, with worms providing more scored loci per individual than eggs. Differences also existed between the worms and eggs in the identity of the specific alleles that were detected. Additionally, we observed a reduction in homozygous genotypes among laboratory-passaged worms relative to the eggs. Allelic diversity curves were calculated by genotyping all worms from a representative host sample to determine the relationship between the number of alleles detected at a locus and the number of worms genotyped. Curves for the 5 residents' worm infrapopulations for each of the loci were very similar. The equation y=19·55×ln(x)+9·992 explained the association between sampling effort (x) and number of alleles detected (y) with an R2 of 0·775. In conclusion, egg DNA templates and allelic diversity curves can benefit efforts to discern the sociological, ecological and evolutionary forces impacting the genetic diversity and disease epidemiology of human schistosomes.

Leishmania parasites are able to survive in the macrophage, one of the most hostile environments of the vertebrate host. The present study investigated how Leishmania infection influences these host cell defence mechanisms. Macrophages were infected with antimony-susceptible and -resistant Leishmania strains. Free radical production in Leishmania-infected macrophages was measured by electron paramagnetic resonance. Apoptosis was detected with fluorescence microscopy using Annexin-V FITC labelling and with Western blotting to detect caspase-3 cleavage. Independent of their drug susceptibility profile or species background, all studied Leishmania strains induced a similar increase in free radical production in macrophages. O2●− production was significantly elevated during phagocytosis of the stationary phase promastigotes. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage. Leishmania donovani infection led to phosphatidylserine externalization only in RAW 264.7 cells. After an initial burst of O2●− during phagocytosis of promastigotes, amastigotes protect themselves by decreasing the O2●− production to the basal level. An increased NO production was observed 6 h after infection. Finally, induction of cell death is probably not essential in the survival of the parasite within the macrophage.

Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

Cysts of Blastocystis ratti were produced in vitro by culturing the parasite in Iscove's modified Dulbecco's medium (IMDM) with increasing concentrations of horse serum. Yields up to 3×106 cysts/ml of culture medium were obtained after 72 h. Encystation efficiency was time, strain and inoculum size dependent. A viability of >70% was determined by flow cytometry employing fluorescein diacetate and propidium iodide staining. The presence of chitin as a cyst wall component was demonstrated by Calcofluor White M2R staining with which cystic stages showed blue fluorescence. The changes in morphology during excystation were examined by transmission electron microscopy. The cyst enlarged in size and some vacuoles appeared within the condensed cytoplasm. The vacuoles were full of inclusions and small glycogen aggregates. Coalescence of the vacuoles led to central body formation. Glycogen deposits were prominent throughout the excystation process. Some cysts divided by binary fission before the completion of the excystation.

Polistes dominulus are social wasps which are the host of the strepsipteran endoparasite Xenos vesparum. In the hibernating phase, unparasitized and parasitized wasps leave natal nests and aggregate together in sheltered quarters. In aggregations, wasps are socially active, and some individuals perform helping behaviour. Here we investigated if castrated parasitized wasps perform worker tasks in mixed aggregations. Moreover, by gas chromatography and mass spectrometry, we examined the cuticular hydrocarbons of unparasitized and parasitized wasps to evaluate if the infection alters the composition of cuticular waxes that are recognition cues in social insects. In clusters, infected females do not perform helping behaviour and they are less active than unparasitized wasps. Cuticular hydrocarbons are slightly differentiated between unparasitized and parasitized wasps but, generally, unparasitized wasps are more similar to wasps infected by Xenos females compared to wasps infected by Xenos males. Wasps infected by Xenos males do not usually survive the winter. This chemical similarity is probably a consequence of the similar physiological condition of unparasitized and female-affected Polistes wasps. At this stage, it is difficult to affirm whether these modifications are a true parasite manipulation or a consequence of infection.

Molecular detection of Babesia species in apparently healthy cattle within an endemic region was carried out in order to determine the prevalence of carriers and the geographical distribution of Babesia bigemina and Babesia bovis in Maputo Province, Mozambique. Samples from 477 animals at 5 localities were analysed using 2 techniques, the semi-nested hot-start PCR and the reverse line blot (RLB) assay. With the semi-nested hot-start PCR, detection of B. bigemina ranged between 30% and 89%, and of B. bovis between 27% and 83%. The RLB assay was comparatively less sensitive in this study and detection of B. bovis ranged from 0% to 17%, and B. bigemina was not detected at all by this technique. Analysis of new sequences of the 18S rRNA gene revealed that the current B. bigemina RLB probe is not specific for the identification of isolates in Mozambique. The RLB assay, however, resulted in the detection of 8 other haemoparasite species belonging to the genera Babesia, Theileria, Anaplasma and Ehrlichia. 18S rRNA gene sequences from the Theileria spp. were identified, and a phylogenic tree constructed with these sequences yielded a heterogeneous T. mutans-like group. In conclusion, infection with B. bigemina and B. bovis is endemic in Maputo Province, but rates of transmission vary. Furthermore, mixed infections with the haemoparasites responsible for several tick-borne diseases in cattle are common in Mozambique.

Understanding how individual parasite traits contribute to overall fitness, and how they are modulated by both external and host environment, is crucial for predicting disease outcome. Fungal (chytrid) parasites of phytoplankton are important yet poorly studied pathogens with the potential to modulate the abundance and composition of phytoplankton communities and to drive their evolution. Here, we studied life-history traits of a chytrid parasite infecting the planktonic, bloom-forming cyanobacterium Planktothrix spp. under host genotype and thermal variation. When expressing parasite fitness in terms of transmission success, disease outcome was largely modulated by temperature alone. Yet, a closer examination of individual parasite traits linked to different infection phases, such as (i) the establishment of the infection (i.e. intensity of infection) and (ii) the exploitation of host resources (i.e. size of reproductive structures and propagules), revealed differential host genotype and temperature × host genotype modulation, respectively. This illustrates how parasite fitness results from the interplay of individual parasite traits that are differentially controlled by host and external environment, and stresses the importance of combining multiple traits to gain insights into underlying infection mechanisms.

Morphological, histopathological, and immunological studies of seven strains of rickettsial disease have placed them in two widely separated groups, for which typhus and spotted fever seem the most logical names.

Diseases of the spotted fever group are carried by ticks (Arachnoidea) and characterised by distinctive histopathological lesions—thrombonecrosis of arterioles and venules, with micro-organisms of definite and characteristic morphology, invading smooth muscle cells, as well as endothelium, mesothelium, and macrophages. Tissue culture studies show massive invasion and distention of cell nuclei, while cytoplasmic invasion of host cells is relatively sparse.

Diseases of the typhus group are carried by lice and fleas (Insecta) and characterised by the facts that the intracellular parasite which causes them invades only endothelium and mesothelium, distends the cytoplasm of its host cells without ever invading nuclei, and in guinea pigs causes only proliferative endangeitis without thrombonecrosis.

In the spotted fever tick, organisms are intranuclear as well as intracytoplasmic, invade nearly all types of tissue, and are transmitted hereditarily. In typhus-infected lice and fleas, organisms are intracytoplasmic only, infect only the lining cells of the gut, and are not transmitted hereditarily.

Neither focal brain lesions nor scrotal sac exudation is of importance in the differential diagnosis, since both occur in certain strains of spotted fever, as well as in typhus. Definite necrosis of the scrotum is of importance, since it is dependent upon the above-described arteriolar thrombonecrosis and is peculiar to spotted fever.

A review of the literature shows that rickettsial diseases and their etiological agents are being classified in an inaccurate and unjustifiably complex manner, specific names often being given to organisms without the presentation of evidence sufficient to establish them as new varieties.

A plea is made for the study of unclassified or unsatisfactorily classified diseases in all parts of the world by the methods described, and for their classification on the basis of morphologic and histopathologic observations on insect and mammalian hosts and of immunological studies on the latter, rather than on variations in clinical pictures in man and in experimental animals.

The criteria for the differential diagnosis between typhus and spotted fever are presented in tabular form and illustrated wherever possible. All reported rickettsial diseases as well as their etiological agents are classed on the basis of the criteria presented, and, with the exception of the incompletely studied mite-borne group, the available evidence strongly suggests that all are varieties of typhus and spotted fever.

The morphological, histopathological, and immunological differences between typhus and spotted fever are so great that these two diseases, as well as their causative organisms, should be widely separated from one another, and given definitely distinguishing names.

It is suggested that the well-established name, Rickettsia prowazeki, be applied to intracytoplasmic micro-organisms in Insecta causing all diseases of the typhus group, and that Dermacentroxenus rickettsi (the name originally given by Wolbach) be applied to hereditarily transmitted micro-organisms invading nuclei in Arachnoidea and in tissue cultures, causing all diseases of the spotted fever group. Specific varieties, when established by the demonstration of definite and constant biological differences, should be indicated by variety names, as Rickettsia prowazeki mooseri (for endemic flea-borne typhus in all parts of the world).

The dry weight and nitrogen content of Hymenolepis diminuta were studied after a period of 15 days‘ growth in rats receiving different diets. The special diets were fed during the last 7 days of this period, that is, while the major part of growth is occurring but before shedding proglottids.

Replacement of all the protein (casein) of the diet by carbohydrate resulted in greater development of the worm. However, replacement of casein by fat had no effect on worm development, indicating that removal of protein is not a significant factor, whereas addition of carbohydrate is effective.

Since exchange of casein by the nutritionally incomplete protein zein did not affect worm development significantly, protein quality appears not to be a factor in growth of H. diminuta.

The addition of tryptophan and lysine to the meal containing zein, in order to raise its nutritional value to that of casein, did not influence the growth of the parasite. However, the feeding of the same amounts of these two amino acids at times other than those of giving zein depressed the growth of the parasite. This was not due to type of protein (zein) fed in the rest of the diet, since a similar phenomenon occurs when casein is the dietary protein, or when a protein-free diet rich in carbohydrate is given. Addition of these amino acids, singly, either to diets containing protein or deficient in protein, had significant actions on worm growth, which was sometimes stimulated and in other circumstances reduced.

The nitrogen balance of the host was unaffected by the worm burden.

We are deeply indebted to Dr C. A. Hopkins for providing facilities, for infection of rats with the cestode, and for his helpful advice. Miss M. Cowan provided skilled technical assistance during the feeding of the animals and analysis of the host and parasite tissues. One of us (D.F.M.) gratefully acknowledges the Award of a Commonwealth Bursary from the Royal Society and the Nuffield Foundation.

Iron overload in BALB/c mice by treatment with ferric ammonium citrate promotes the hepatic development of Plasmodium yoelii in vivo and in vitro. This was the result of increased penetration of the parasite into hepatocytes since no effect was observed on parasite transformation or maturation. These results could explain why in endemic regions iron supplementation led, in certain studies, to an increase in clinical episodes of malaria and in the prevalence of malaria infection.

The development of cerebral lesions in Plasmodium berghei-infected mice was dependent on the Strain of mice and the size of the infectious inoculum. In particular, C57B1/6J mice develop cerebral lesions when infected with low numbers of parasitized erythrocytes. By increasing the number of parasites in the infectious inoculum, the percentage of animals that develop cerebral malaria is decreased. Varying degrees of protection against the development of cerebral malaria can be obtained by several methods of immunization. (1) Injection of mice with large numbers of disrupted parasitized erythrocytes 1 or 2 weeks before the challenge infection (protection up to 70%). (2) A 2-day immunizing infection given 9 or 14 days before the challenge infection (protection up to 85%). (3) Injection of mice with plasmodial exoantigen preparations 1 week before the challenge infection (variable protection-rate, up to 100%). In all mice protected against cerebral malaria, parasitaemia is not affected by the immunizing treatment, indicating that protective mechanisms against cerebral malaria and parasitaemia are independent.

Trichobilharzia ocellata cercariae attach readily to the foot skin of their duck host, but poorly to preen-gland contents. The attachment to duck foot disappears when the skin surface lipids are extracted, and can be restored by reapplication of the lipids to the skin. Hydrophilic skin extracts are without any effect. Thin-layer chromatographic fractionation of duck-foot skin surface lipids reveals cholesterol and ceramides as attachment stimuli. A stimulation of cercarial attachment by these hydrophobic host signals is supported by the host identification pattern of the cercariae, which secures a close contact with encountered substrates.

Mice infected with Nematospiroides dubius generate weaker immune responses to concurrently administered sheep red blood cells (SRBC), than non-infected controls. The experiments described in this paper demonstrate that both adult and larval stages of N. dubius cause non-specific immunodepression of the response to SRBC. Mice which had been infected with larvae exposed to 25 krad. of irradiation, which prevents development to the adult luminal stages, produced as weak haemagglutination responses to SRBC as mice infected with normal worms even when SRBC were administered 6 weeks after infection. The removal of adult N. dubius by treatment with pyrantel 9, 11 and 15 days after infection with normal larvae did not restore the host's ability to respond to SRBC given on day 14. It was only when the mice had been without worms for 17 days that their capacity to respond normally to SRBC was restored. Mice infected with 60 or 400 transplanted adult worms produced depressed haemagglutination and plaque-forming responses to concurrently injected SRBC when compared with normal or sham-operated controls. The significance of these results is discussed in relation to the possible role of non-specific immunodepression in facilitating the survival of N. dubius in the host.

New technological methods, such as rapidly developing molecular approaches, often provide new tools for scientific advances. However, these new tools are often not utilized equally across different research areas, possibly leading to disparities in progress between these areas. Here, we use empirical evidence from the scientific literature to test for potential discrepancies in the use of genetic tools to study parasitic vs non-parasitic organisms across three distinguishable molecular periods, the allozyme, nucleotide and genomics periods. Publications on parasites constitute only a fraction (<5%) of the total research output across all molecular periods and are dominated by medically relevant parasites (especially protists), particularly during the early phase of each period. Our analysis suggests an increasing complexity of topics and research questions being addressed with the development of more sophisticated molecular tools, with the research focus between the periods shifting from predominantly species discovery to broader theory-focused questions. We conclude that both new and older molecular methods offer powerful tools for research on parasites, including their diverse roles in ecosystems and their relevance as human pathogens. While older methods, such as barcoding approaches, will continue to feature in the molecular toolbox of parasitologists for years to come, we encourage parasitologists to be more responsive to new approaches that provide the tools to address broader questions.

A prior systematic review on the efficacy of halofuginone (HFG) treatment to prevent or treat cryptosporidiosis in bovine calves was inconclusive. We undertook an updated synthesis and meta-analyses on key outcomes for the treatment of calves with HFG. Evaluated outcomes were oocyst shedding, diarrhoea, mortality and weight gain. Experiments had to describe results for same age animals in contemporary arms. Most doses were 100–150 mcg kg−1 day−1. Results were subgrouped by study design, experiments with the lowest risk of bias and lack of industry funding. Eighteen articles were found that described 25 experiments. Most evidence came from randomized controlled trials in Europe. Significantly lower incidence of oocyst shedding, diarrhoea burden and mortality was reported when treatment started before calves were 5 days old. Most studies reported on outcomes for animals up to at least 28 days old. Publication bias was possible in all outcomes and seemed especially likely for diarrhoea outcomes. Beneficial results when HFG treatment was initiated in calves older than 5 days were also found. Prophylactic treatment to prevent cryptosporidiosis is effective in preventing multiple negative outcomes and is beneficial to calf health and will result in a reduction of environmental contamination by Cryptosporidium oocysts.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µm, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca++. We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

Infections with Ascaris lumbricoides, Triclmris trichiura and the hookworm species are often found in the same communities and individuals. Hosts infected by more than one species are potentially at risk of morbidity associated with each infection. This paper describes the use of a probabilistic model to predict the prevalence of multiple-species infections in communities for which only overall prevalence data exist. The model is tested against field data, using log-linear analysis, and is found to be more effective at estimating the numbers of multiple infections involving hookworms than those involving only A. lumbricoides and T. trichiura. This latter combination of infections is found, in half the communities examined, to be more common than expected by chance. An age-stratified analysis reveals that the degree of interaction between these two infections does not alter significantly with age in the child age classes of a Malaysian population.

In the system investigated the spleen was essential in most rats for the acquisition of immunity and its maintenance in the presence of the piroplasm.

It is stressed that model immunological systems involving the role of the spleen do not simulate the complexity of a parasitic infection. The roles of the spleen as an antibody producer, and as a site of phagocytosis and haemopoiesis are discussed in relation to the control of Babesia rodhaini by the rat. The possibility that the piroplasm may be able to undergo antigenic variation is considered.

The factors responsible for the reactivation of a Neospora caninum latent infection are unknown, but it is postulated that the maternal immune response could be altered during pregnancy. The immune response was investigated in N. caninum chronically infected mice during successive pregnancies as well as in non-pregnant infected mice and mice infected when pregnant. Vertical transmission was demonstrated in chronically infected mice after the first pregnancy but the rate of fœtal infection fell after further pregnancies. Non-pregnant chronically infected mice showed a marked specific proliferative response and an IgG2a isotype preferential secretion. During the course of the first pregnancy, no significant modification of the immune response was recorded. After 2 successive pregnancies, the specific cellular response showed a significant fall whereas Th2 cytokine mRNA expression was noted. At the same time, IgG1 secretion increased to reach the IgG2a level. At the third delivery, a partial restoration of the proliferative response was observed. The reactivation of N. caninum chronic infection during pregnancy does not seem to be consecutive to an immunodepression. Nevertheless, pregnancy could favour parasite multiplication in utero after an occasional spontaneous release of bradyzoites.

A new species of ascaridoid nematode, Baylisascaris tasmaniensis, is described from three marsupial carnivores of Tasmania, namely Sarcophilus harrisi, Dasyurus viverrinus, and Dasyurops maculatus. The new species is differentiated from other species in the genus Baylisascaris and the phylogenetic affinities within the genus are discussed in relation to the origin of the placental and marsupial carnivores.