Plasmodium falciparum parasites infecting Aotus trivirgatus erythrocytes were cultured in media (Harvard and TC199) augmented with human, foetal calf, or other sera. Conditions were established which supported growth of parasites and allowed cyclical multiplication when fresh erythrocytes (from Aotus or Homo) were added in sub-culture (mean multiplication rate: x 3). Immunoglobulin G pools, prepared from plasma collected in endemic malarious areas in Africa and from unexposed Britons, were tested for effects on the in vitro growth (measured by incorporation of tritiated leucine) and multiplication of parasites. Whilst non-immune IgG was without effect, IgG from both East and West Africa inhibited the multiplication of East African (Uganda-Palo Alto strain) parasites.

Three heat-induced genes of the infective-stage larvae of Trichinella spiralis were successfully identified by the suppression subtractive hybridization (SSH) technique. As indicated by reverse Northern blotting, 19 of 25 clones were scored as differentially transcribed in the heat-shocked infective-stage larvae. The sequencing data showed the presence of 12 different genes. Three were homologous to histone H3, histone H2B and translationally controlled tumour protein (TCTP). A 0.6 kb cDNA of histone H3 was generated by the RACE method and sequenced. It contained an open reading frame of 136 amino acids that demonstrated 94% identity with genes from Drosophila hydei. Semi-quantitative RT-PCR indicated that after heat-shock treatment, the expression levels of histone H3, histone H2B and TCTP increased 4.8, 27 and 5.7-fold, respectively. Northern analysis confirmed the upregulation of histone H3, histone H2B and TCTP transcripts. The upregulation of these genes during stress conditions has not been reported in parasitic organisms. The stress proteins may play an active role to sustain the parasite after exposure to hostile host factors.

Host tissue penetration, feeding and immune evasion by helminth parasites may be mediated by both mechanical processes and histolytic products released by the parasite. The aim of this study was to investigate potential histolytic products released during in vitro maintenance of exsheathed third (L3) and 4th larval stage (L4) and adult Ostertagia ostertagi. Therefore, the pH optima, substrate specificity, molecular size and inhibitor sensitivity of in vitro released (IVR) proteinases were analysed by spectrophotometry and substrate gel electrophoresis. It was shown that L3, L4 and adult IVR proteinases degrade a variety of protein substrates in a pH-dependent and stage-specific manner. At alkaline pH, gelatin, casein and fibrinogen were degraded by metallo- and serine proteinases. In contrast, mucin, fibrinogen, albumin and haemoglobin were degraded at acidic pH by aspartyl protease- and cathepsin L-like activity. At pH 3, the heavy chain of bovine IgG was completely degraded by an aspartyl proteinase secreted by all 3 parasitic stages. The specificity of the L3, L4 and adult Ostertagia ostertagi proteinases against the different substrates indicates variable functional requirements.

1. Attempts, made over a period of more than 6 months, to produce a strain of Plasmodium gallinaceum resistant to mepacrine failed.

2. A strain of P. gallinaceum resistant to daily doses of 0·08 mg./20 g. of pamaquin was obtained after approximately 8 months' treatment with the drug.

3. A strain of P. gallinaceum resistant to the maximum dose of paludrine which the chicks would tolerate (1·0 mg./20 g. twice daily) was obtained after 4½ months' treatment with gradually increasing doses of the drug.

4. The exo-erythrocytic parasites of P. gallinaceum formed in chicks infected with a paludrine-resistant strain are themselves resistant to the drug. It is not, however, considered probable that the development of resistance is due to the action of the drugs upon the exo-erythrocytic parasites, as the technique used was one which reduced their number to a minimum.

5. A strain of P. gallinaceum resistant to paludrine showed a normal sensitivity to mepacrine and pamaquin, but was resistant to 4430, the methyl homologue of paludrine.

6. Chicks carrying a latent infection of the normal strain of P. gallinaceum were immune to infection with the paludrine-resistant strain; and conversely chicks carrying a latent infection of the paludrine-resistant strain were immune to infection with the normal parasite.

7. A paludrine-resistant strain of P. gallinaceum retained its resistance to this drug after five cyclical passages through mosquitoes without intervening drug treatment.

Tsetse flies (Glossina morsitans) were fed on the blood of mice containing any one of 5 variable antigen types (VATs) of Trypanosoma brucei AnTAR 1 serodeme. The VATs of the metacyclic trypanosomes subsequently detected in the flies' saliva probes were investigated using monospecific antisera to AnTAR 1 VATs in indirect immunofluorescence and trypanolysis reactions; these sera included 3 raised against AnTats 1.6, 1.30 and 1.45, previously identified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6·0–8·3%; AnTat 1.30, 13·7–18·2%; AnTat 1.45, 2·0–8·0%), regardless of the age of the fly or the ingested VAT. As these 3 VATs accounted for no more than 30% of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes.

The reactions of the house-fly larva to four environmental factors, namely temperature, air humidity, smell and light have been studied in the Laboratory. It was hoped that the results would shed some light on the general behaviour of the larva in its normal habitat. A comprehension of this behaviour will assist in understanding the ecology of the species, and hence facilitate its control. In order to analyse the behaviour of the larva, its reactions (other than to light) were first studied in relation to one factor at a time. An attempt was then made to test experimentally its reactions to various combinations of factors.

Perforated plastic cylinders (hair curlers) were implanted subcutaneously in rabbits for the collection of tissue fluid. Using this technique, a study was made of the chemical constituents of tissue fluid and serum in rabbits infected with Trypanosoma (Trypanozoon) brucei, as compared with uninfected, control animals. Changes were caused by the infection in the concentrations of proteins, electrolytes, urea, creatinine, cholesterol and pyruvate, and in the activities of the enzymes aspartate transaminase, creatine phosphokinase and alkaline phosphatase.Antitrypanosomal antibody, detectable by the indirect fluorescent antibody technique, was shown to penetrate the tissue fluid and to reach a titre of about one-fifth of that in the serum. Renal failure, probably resulting from allergic reactions, was shown to be a contributory cause of death in rabbits infected with T. (T.) brucei.

While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0·32±0·07 μM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.

The strongest evidence for host specificity of mammalian trypanosomes comes from parasites of the subgenus Trypanosoma (Herpetosoma). Laboratory studies have shown that T. (Herpetosoma) species will not infect an alternative host. However, this has not been demonstrated in wild populations. We screened 560 bank voles (Clethrionomys glareolus) and 148 wood mice (Apodemus sylvaticus) for trypanosomes by PCR amplification of the 18S rRNA gene. In total, 109 (19%) bank voles and 12 (8%) wood mice were infected. A HaeIII restriction site was discovered that could be used to discriminate between T. (H.) evotomys of the bank vole and T. (H.) grosi of the wood mouse. All the parasites in the bank voles were identified as T. (Herpetosoma) evotomys by RFLP-PCR. Out of the 12 wood mouse infections 10 were due to T. grosi. Two of the wood mice were infected with parasites with a novel genotype that was most similar to those of T. evotomys and T. microti of voles. Fifty-six fleas collected from the rodents were also screened for trypanosomes; 9 were infected with T. evotomys and 1 with T. grosi. One of the fleas infected with T. evotomys was collected from a wood mouse.

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12–24h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24h. The data indicate that at 48h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1×10−5m) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1×10−6m), a sodium ionophore and ammonium chloride (NH4Cl) (5×10−2m), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1×10−3m) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

1. The enzymic activity was studied in the following Dermatophyte fungi: Sabouraudites radiolatus; S. lanosus; S. audouini; Trichophyton tonsurans; Grubyella schoenleinii. Both the normal and pleomorphic forms of S. radiolatus were studied comparatively.

2. An active proteolytic enzyme is present in all the species. This enzyme is active in an alkaline medium and can hydrolyse intact proteins (casein) with the production of free amino-acids (tryptophane), and is very similar to trypsin in its behaviour.

3. Pepsin is not present in any of these fungi.

4. The amount of the proteolytic enzyme present varies in the different species, and it is particularly abundant in S. radiolatus.

5. A keratolytic enzyme was not found in any of these fungi.

6. A lipolytic enzyme, lipase, which readily splits tributyrin into fatty acids, is present in all the species. It is about equally strong in all of them.

7. Urease is present in all, with the exception of T. tonsurans.

8. None of them contains invertase, inulase, lactase or zymase.

9. Maltase and diastase are present in all. They are strongest in S. lanosus and T. tonsurans and are weakest in S. radiolatus.

10. The species with most proteolytic activity have least carbohydrases, and conversely.

11. Amygdalase is present in all the species.

12. The normal form of S. radiolatus has greater proteolytic activity than the pleomorphic form. The pleomorphic form has strong urease and amygdalase, both of which are very weak in the normal form. Otherwise the enzymic activity of the two forms is similar.

In a longitudinal study in a UK upland site, 38% of adult sheep were detected as infected with the tick-borne bacterium Ehrlichia phagocytophila by PCR of blood samples. Infection prevalence declined significantly with sheep age but varied significantly and non-linearly with the number of adult Ixodes ricinus ticks feeding per sheep. These findings suggested that under conditions of natural repeated tick-borne challenge sheep remain partially susceptible to re-infections, but the likelihood of re-infection depended on the numbers of feeding ticks. Transmission efficiency from sheep to immature ticks also varied significantly and non-linearly with the number of adult ticks feeding per sheep: transmission efficiency was almost zero in sheep with low adult tick infestations rising to 30% at certain levels of adult tick infestation. Infection intensity in infected engorged immature ticks also varied with the number of adult ticks feeding per sheep, but neither prevalence nor intensity of infection in engorged ticks were related to sheep blood PCR result. These findings suggest that variation in the numbers of ticks feeding per sheep may influence E. phagocytophila transmission by direct effects on transmission at the tick–host interface.

Serology is an important tool for the diagnosis of alveolar echinococcosis (AE) in humans. In order to improve serodiagnostic performance, we have developed an in vitro-produced Echinococcus mulilocularis metacestode vesicle fluid (EmVF) antigen for application in an immunoblot assay. Immunoblot analysis of EmVF revealed an abundant immunoreactive band triplet of 20–22 kDa, achieving a sensitivity of 100% based on the testing of sera from 62 pre-operative and pre-treatment cases of active and inactive AE. Thus, the EmVF-immunoblotting allowed the specific detection of cases seronegative by the Em2- and/or EmII/3–10-ELISA, usually attributable to abortive, inactive cases of AE. The specificity of the EmVF-immunoblotting did not allow discrimination between AE and cystic echinococcosis (CE) but was 100% with respect to non-Echinococcus parasitic infections or cancer malignancies. Based on the findings of this study, it is recommended that the current ELISA test combination (Em2- and II/3–10-ELISA) be complemented with EmVF-immunoblotting, allowing an improved diagnosis of both clinical and subclinical forms of AE, including those associated with E. multilocularis-specific antibody reactivities not detectable by ELISA.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ixodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 °C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.

Varroa destructor is considered the most damaging parasite affecting honeybees (Apis mellifera L.). However, some honeybee populations such as the savannah honeybee (Apis mellifera scutellata) can survive mite infestation without treatment. It is unclear if survival is due to resistance mechanisms decreasing parasite reproduction or to tolerance mechanisms decreasing the detrimental effects of mites on the host. This study investigates both aspects by quantifying the reproductive output of V. destructor and its physiological costs at the individual host level. Costs measured were not consistently lower when compared with susceptible honeybee populations, indicating a lack of tolerance. In contrast, reproduction of V. destructor mites was distinctly lower than in susceptible populations. There was higher proportion of infertile individuals and the reproductive success of fertile mites was lower than measured to date, even in surviving populations. Our results suggest that survival of savannah honeybees is based on resistance rather than tolerance to this parasite. We identified traits that may be useful for breeding programmes aimed at increasing the survival of susceptible populations. African honeybees may have benefited from a lack of human interference, allowing natural selection to shape a population of honeybees that is more resistant to Varroa mite infestation.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts – including four managed species – from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Deer are important blood hosts for feeding Ixodes ricinus ticks but they do not support transmission of many tick-borne pathogens, so acting as dead-end transmission hosts. Mathematical models show their role as tick amplifiers, but also suggest that they dilute pathogen transmission, thus reducing infection prevalence. Empirical evidence for this is conflicting: experimental plots with deer removal (i.e. deer exclosures) show that the effect depends on the size of the exclosure. Here we present simulations of dynamic models that take into account different tick stages, and several host species (e.g. rodents) that may move to and from deer exclosures; models were calibrated with respect to Ixodes ricinus ticks and tick-borne encephalitis (TBE) in Trentino (northern Italy). Results show that in small exclosures, the density of rodent-feeding ticks may be higher inside than outside, whereas in large exclosures, a reduction of such tick density may be reached. Similarly, TBE prevalence in rodents decreases in large exclosures and may be slightly higher in small exclosures than outside them. The density of infected questing nymphs inside small exclosures can be much higher, in our numerical example almost twice as large as that outside, leading to potential TBE infection risk hotspots.