Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.

Isospora belli, Cyclospora cayetanensis as well as several species of microsporidia are recognized as emerging protozoan pathogens of humans. All are obligate intracellular parasites, with Isospora and the microsporidia being primarily associated with immunocompromised hosts. Cyclospora is a cause of traveller's diarrhoea, and is responsible for water-borne and food-borne outbreaks of disease. Drug treatment is available for these infections. Improved diagnostic methods including the autofluorescence of I. belli and C. cayetanensis oocysts have assisted in the routine detection of these pathogens. Since the recognition of immunosuppression due to HIV infection, microsporidia have become recognized as important human pathogens with a continuing expansion of the parasite-associated clinico-pathological spectrum. The small size, intracellular nature and poor staining properties with many histological stains result in under-reporting of microsporidial infections. Trichrome stain and optical brighteners are used to detect spores in faeces, urines, respiratory secretions and other aspirates. Electron microscopy remains an important diagnostic method but its sensitivity is relatively poor. Molecular techniques should overcome current diagnostic limitations. The ability to extract DNA and amplify by PCR directly from clinical samples has increased the usefulness of molecular methods. Restriction fragment length polymorphism analysis of amplicons can be used to determine genus, species and strain types of various microsporidia. Increased specificity is required in primer design because current primers used for amplifying non-microsporidian DNA also amplify microsporidian DNA. Diagnosis and pathogen characterisation rely increasingly on PCR-based approaches, and the sequence analysis approach becomes increasingly feasible and affordable. However, robust, reliable and sensitive methods are still required for dissecting pathogenesis, epidemiology, transmission routes and sources of infections.

Cryptosporidium research has focused on the development of infection control, and effective therapy that has thus far been hampered by the inability to culture Cryptosporidium in vitro. Other limitations include inadequate animal models, cumbersome screening procedures for chemotherapeutic approaches and a lack of tools for genetic manipulation. These limitations can, however, be eased by the improvement and focused development of in vitro cultivation. The ability to culture relevant Cryptosporidium isolates in vitro and to propagate the life cycle stages that are responsible for causing disease in an infected host is still a critical link. This ability will facilitate other relevant approaches, e.g., the ability to knockout genes and the application of broader screening for drug discoveries and vaccine developments, in combination with new discoveries on the parasite's basic biology, genetic manipulation and new life cycle stages. Success in this effort represents an essential step towards significant progress in the control of cryptosporidiosis.

Aggregation of parasites amongst hosts is important for the epidemiology of vector-borne diseases because hosts that support the majority of the vector population are responsible for the majority of pathogen transmission. Ixodes ricinus ticks transmit numerous pathogens of medical importance including Borrelia burgdorferi s.l. and tick-borne encephalitis virus. One transmission route involved is ‘co-feeding transmission’, where larvae become infected via feeding alongside infected nymphs. The aggregation of ticks on hosts leads to an increase in the number of larvae feeding alongside nymphs, increasing the transmission potential via this route. The basic reproduction number, R0, can be used to identify whether a pathogen will become established if introduced. In the current study we use previously published tick, and pathogen, specific data to parameterize an R0 model to investigate how the degree of aggregation of ticks on hosts affects pathogen persistence. The coincident aggregated distribution permitted the establishment of tick-borne encephalitis virus but did not influence whether B. burgdorferi s.l. became established. The relationship between the k-exponent of the negative binomial distribution and R0 was also defined. Therefore, the degree of aggregation of ticks on small mammal hosts has important implications for the risk to human health in a given area.

The small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.

Several recent studies have discussed the interaction of host life-history traits and parasite life cycles. It has been observed that the life-history of a host often changes after infection by a parasite. In some cases, changes of host life-history traits reduce the costs of parasitism and can be interpreted as a form of resistance against the parasite. In other cases, changes of host life-history traits increase the parasite's transmission and can be interpreted as manipulation by the parasite. Alternatively, changes of host's life-history traits can also induce responses in the parasite's life cycle traits. After a brief review of recent studies, we treat in more detail the interaction between the microsporidian parasite Edhazardia aedis and its host, the mosquito Aedes aegypti. We consider the interactions between the host's life-history and parasite's life cycle that help shape the evolutionary ecology of their relationship. In particular, these interactions determine whether the parasite is benign and transmits vertically or is virulent and transmits horizontally.

Key words: host-parasite interaction, life-history, life cycle, coevolution.

A simple and robust method for the isolation of gastro-intestinal nematode eggs from faeces is described that uses both salt- and sugar solutions for flotation. Application of this ‘salt–sugar’; isolation method to large numbers of faecal samples of adult dairy cows indicates a 3- or 4-fold reduction in the proportion of e.p.g.-negative cows relative to studies that used other techniques for egg isolation. The procedure detects more eggs than the Wisconsin flotation method in replicate samples and in spiked egg-free faeces. The number of recovered eggs in spiked faecal samples is linear over a range of egg concentrations, and the transparent faecal preparations that result from the protocol can be stored as digital images which can be used as input for an efficient automated egg-counting procedure. The increased rate of processing of faeces combined with the large reduction of the percentage of e.p.g.-negative cows allows more accurate analysis of large numbers of adult or resistant animals for studies of nematode parasitism.

Urinary schistosomiasis is an important source of human morbidity in Msambweni, Kenya, where the intermediate host snail, Bulinus nasutus is found in ponds and water pools. In the past, aquatic habitats in the area have been studied separately; however, recent collections of B. nasutus snails and shells indicated that many of these ponds are in fact connected during and following sufficient rains. Satellite imagery and a geographical information system (GIS) were used to survey the main water courses and potential drainage routes, to locate potential source populations of snails and to determine probable snail dispersal routes. The 2 water bodies implicated as being the most important Schistosoma haematobium transmission foci in the area were found to differ in their degree of connectivity to other B. nasutus source habitats. One pond becomes connected even after normal rains, while the other pond requires prolonged rains or flooding to become connected with source habitats. Consequently, the transmission foci differ in their susceptibility to snail population control measures. Spatially explicit dispersal models that consider the spatial and temporal patterns of connectivity between aquatic habitats will contribute to improved snail surveillance and more focused control for urinary schistosomiasis at a local level.

Controlled human malaria infections (CHMIs) are a powerful tool to assess the efficacy of drugs and/or vaccine candidates, but also to study anti-malarial immune responses at well-defined time points after infection. In this review, we discuss the insights that CHMI trials have provided into early immune activation and regulation during acute infection, and the capacity to induce and maintain immunological memory. Importantly, these studies show that a single infection is sufficient to induce long-lasting parasite-specific T- and B-cell memory responses, and suggest that blood-stage induced regulatory responses can limit inflammation both in ongoing and potentially future infections. As future perspective of investigation in CHMIs, we discuss the role of innate cell subsets, the interplay between innate and adaptive immune activation and the potential modulation of these responses after natural pre-exposure.

Since its early introduction as a marker for the immunodiagnosis of hydatid disease, antigen 5 (Ag5) has been regarded as one of the more relevant antigens of Echinococcus granulosus, and it is still widely used in different confirmation techniques. In this work we prepared 2 recombinant forms of the antigen, namely, rAg5 (corresponding to the unprocessed polypeptide chain of the antigen) and rAg5-38s (corresponding to its 38 kDa subunit). Their antigenicities were compared to that of the native antigen using a human serum collection. There was a major drop in the reactivity of the sera, particularly against rAg5-38s, which was confirmed by analysis of the cross-reactivity of 2 panels of monoclonal antibodies specific for rAg5-38s and the native antigen. Using the chemically deglycosylated native antigen, we demonstrated that the reduced antigenicity of the recombinants is due to the loss of the sugar determinants, and not to their misfolding. Inhibition experiments using phosphorylcholine confirmed that this moiety also contributes to the reactivity of the antigen, but to a much lesser extent. The presence of immunodominant highly cross-reactive glycan moieties in the Ag5 molecule may involve a parasite evasion mechanism.

The sheep ked has been largely eradicated in the UK but persists in the feral Soay sheep of St Kilda in the Outer Hebrides. Sheep keds transmit Trypanosoma melophagium, but parasitaemias are typically cryptic and this trypanosome has not been recorded in the St Kilda sheep. Trypanosomes were detected by PCR in preserved keds and were also found in gut smears from live keds; one infected gut was used to establish the trypanosome in vitro. Examination of the morphology of bloodstream forms from culture confirmed its identity as T. melophagium. Most keds were found to harbour the trypanosome, particularly those collected from lambs. DNA was extracted from preserved keds and from trypanosomes grown in vitro. Sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene and the spliced leader transcript showed the T. melophagium sequences to be very similar to those from T. theileri. A partial sequence of the ked SSU rRNA gene was also obtained. The close genetic relationship of T. melophagium and T. theileri suggests that T. melophagium represents a lineage of T. theileri that adapted to transmission by sheep keds and hence became a specific parasite of sheep.

Notocotylus malhamensis n. sp. is described from the caecum of the bank vole (Myodes glareolus) and the field vole (Microtus agrestis) from Malham Tarn Nature Reserve in North Yorkshire, UK. In total, 581 specimens were collected from rodents trapped at a wetland site (Tarn Fen) between July 2010 and October 2011 with a prevalence of 66·7% and mean intensity of 94·6 in the bank vole and 50% prevalence and a mean intensity of 4·3 in the field vole. This species appears to be most closely related to other previously described Notocotylus species infecting rodents in Europe but differs principally by the metraterm to cirrus sac ratio (1:1·5–1:1·2) in combination with a densely spinulated cirrus, simple caeca and a greater number of ventral glands in the lateral rows (14–17). The use of molecular differentiation was of limited use in this study due to a paucity of relevant information in the DNA sequence databases. However, the complete ITS1-5.8S rDNA-ITS2 and partial 28S gene sequences have been generated to provide a definitive tool for identification of this species in future studies. As far as we know this is the first report of a notocotylid infection in M. glareolus in the UK.

The data needed to develop analytical models of trypanosomiasis transmission have become available only recently. By making some simplifying assumptions, models of the dynamics of the disease in vector, cattle and wild mammal populations can be constructed in order to determine criteria for successful disease control by mass and targetted chemotherapy, and by vector control. The heterogeneity in transmission due to tsetse fly feeding preferences and the variability of immunological characteristics among the vertebrate hosts account for differences in prevalence of Trypanosoma vivax and T. congolense, and also lead to an increase in the basic reproductive rates of the parasites and a corresponding decrease in the vector population density threshold for disease eradication or persistence. The long life-span of the vectors relative to the duration of the parasites' developmental period lead to high infection rates in the vector and high values of R0. The efficacy of chemotherapeutic regimes depends on the relationship between treatment rate and the duration of prophylaxis conferred by the drugs used. The model's predictions of the effects of vector control are shown to be in broad agreement with published field data for Mkwaja Ranch, Tanzania. Vector control programmes are frequently blighted by reinvasion, and the implications of this are discussed in terms of a model for fly immigration. With immigration of vectors, the disease is always endemic, though the infection rate in the fly population is modified by the effect of differential mortalities inside and outside the controlled area on cohorts of incubating flies. Sensitivity analysis of the model, using Monte-Carlo methods, enables an assessment of the relative importance of the parameters to be made. The results emphasize the need for studies of the wild animal reservoir to be carried out alongside entomological surveys. The relative accuracy with which field measurements need to be made in order to minimize the uncertainty in predictions of trypanosomiasis prevalence is discussed.

In this review we have given an account of the various methods which are available to determine the size of virus particles. In § IV we have endeavoured to bring the ultrafiltration method into agreement with other methods by suggesting a different factor for converting pore size to virus size from the factors commonly used. Throughout we have recognized the probability that most viruses are hydrated in solution and have distinguished between the size and molecular weight in solution and the size and molecular weight when dried.

In § VII we have given formulae suitable for interpreting centrifugation and diffusion data when the possibility of hydration is contemplated.

It is evident that this complication, added to that of shape, makes it necessary for several measurements by different methods to be made before one can claim to know the size of a virus. For this reason, only in the cases of three viruses have we thought the data sufficiently adequate to enable the size and shape and molecular weight of the Virus, both dry and hydrated, to be stated. These three viruses, tobacco mosaic, tomato bushy stunt and vaccinia respectively, are separately discussed in § X.

It will be clear from the preceding sections that, while the position regarding our knowledge of the absolute sizes of viruses is far from satisfactory, there has been amassed a large amount of data bearing on this subject. We should, however, point out that we have found it necessary to select what we consider to be the best experimental data in some cases and that there may be conflicting ideas expressed by various authorities. Frampton (1942) has studied the electron microscope photographs published by Stanley & Anderson (1941) and Anderson & Stanley (1941) and arrives at an entirely different estimate of the length of tobacco mosaic virus. Kausche, Pfankuch & Ruska (1939) reported one value for the length of this virus which is approximately half that given by Stanley & Anderson. Electron photomicrographs published by von Ardenne (1940) and Holmes (1941) for what are probably strains of the same virus, also suggest that the values given should not be taken as absolute. Frampton (1939 a, b), on the basis of diffusion and viscosity experiments and the stream birefringence of this virus, has suggested previously that it forms a gel at any concentration and therefore cannot be said to have a size. Lauffer (1940) has given reasons for supposing this argument to be incorrect. Bernal & Fankuchen (1941a) have discussed the possibility of tobacco mosaic virus particles being shorter than the value taken from Kausche et al. (1939) and conclude that in the plant itself the particle may be as short as 100 mμ.

In obtaining values of size arid shape from electron microscope data we have made the assumptions, which may not be correct, that long, thin viruses shrink in width rather than in length on drying and that almost spherical viruses contract approximately evenly in all directions. At the moment there would seem to be no method of proving or disproving the truth of these assumptions, but we believe it unlikely that drying will result in such a gross change in shape that it would invalidate our calculations. For instance, in the case of haemocyanin from Helix pomatia, it seems improbable that, on drying, an already anhydrous ellipsoidal molecule of 66 × 15·32 mμ would contract in length and expand in-width to form a sphere of some 24 mμ diameter.

In our treatment of hydration we have found it necessary to regard the density and volume of ‘bound’ water as being the same as that of water in bulk, which may not be entirely true. However, we regard the total volume occupied by water in cases of great hydration, as shown by tomato bushy stunt virus, as being not markedly smaller than that of the same mass of free water. It is, nevertheless, a well-established fact that in certain cases, gelatin for example (Svedberg, 1924), a small contraction in volume does take place when dry protein is added to water. This phenomenon does not, however, necessitate the assumption that the water of hydration, is denser than ordinary water, and can be explained in other ways.

The viscosity of solutions of viruses, especially the rod-shaped plant viruses, has attracted much attention as a method of finding frictional and axial ratios of viruses (Frampton, 1939 a, b; Lauffer, 1938; Loring, 1938; Neurath, Cooper, Sharp, Taylor, Beard & Beard, 1941; Kobinson, 1939 a, b; Stanley, 1939), but, in addition to the lack of experimental verification of the formulae used, in many cases (Robinson, 1939 a, b; Frampton, 1939 a, b) the formulae have been applied to experimental results obtained in circumstances which exclude the fundamental postulates on which the formulae are based. For this reason we have omitted a detailed discussion of such methods.

It would appear that in order to obtain evidence as to the size of a virus it is desirable to study the virus in as purified a form as possible and also to show that when ‘homo-geneous’ preparations are obtained, they do not consist merely of macromolecular substances contaminated with a small quantity of virus. Furthermore it is desirable to obtain at least sufficient data to enable one to assess both size and shape of the particles rather than to assume some shape or some density value which may be incorrect.

Malaria and filaria infection rates were determined for anopheline mosquitoes collected whilst biting and resting in village houses in Papua New Guinea. The number of anophelines infected with both parasites was greater than expected from the infection rates of each parasite and this difference was significant in resting collections. The excess of multiply infected mosquitoes is probably a result of a vector population composed of individuals with differing numbers of opportunities to become infected. Malaria-positive, Anopheles punctulatus from resting catches had a significantly greater number of Stage 3 Wuchereria bancroftilarvae than malaria-negative mosquitoes. However, multiply infected mosquitoes appear to suffer greater mortality than non-infected or singly infected mosquitoes when the filarial worm reaches the third stage. Any potential increase in transmission resulting from multiple infections is thereby offset by a greater mortality rate in these mosquitoes.

The proteolytic activities present in adult Necator americanus excretory–secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6·6, while fibrinogenolytic activity was shown to be greater at pH 3·5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.

Two strains each of Eimeria acervulina and Eimeria mivati were examined for their ability to develop in the chorioallantoic membrane (CAM) of chicken embryos and the caeca of chickens. It was found that between 100 and 1000 times more sporozoites of E. acervulina were needed to produce infections in the caeca and the embryo CAM respectively. The reproduction of both species in infections of chickens was similar and a high degree of cross-protection occurred.

It was found that oocysts of E. mivati differed in size and shape depending upon whether they were produced from infections of embryos or chickens. When maintained by passage in chickens they assumed size and shape characteristics of E. acervulina.

These findings, with the known similarity of the life-cycle stages, pathogenicity and pathology, indicate that E. mivati is not sufficiently different from E. acervulina to be a distinct species. It is suggested that it is best referred to as E. acervulina var. mivati.

This work started in May 1950 and is part of a wider study of parasite populations on lowland pastures.

An account of observations made on ewes has already been given (Crofton, 1954), and the present paper deals with observations made on lambs from the same flocks. It is realized that a proper approach to parasite problems in sheep entails consideration of the flock as a whole, but for convenience, the observations made on different age groups have been described separately. It is for convenience, also, that pasture contamination is only considered briefly here. Later an attempt will be made to consider parasite populations on a lowland farm as a dynamic system of interacting factors.

As mice recover from infection with Babesia microti, abnormal forms of the parasite are present in some red cells. These forms are non-infective, indistinguishable by light microscopy from those present after treatment with amicarbalide, a babesicidal drug, and persist in splenectomized mice.

Electron microscopy confirmed that these abnormal forms are degenerating intra-erythrocytic parasites. In the absence of evidence for death of B. microti elsewhere in the body, it appears that infections of mice with this parasite are normally terminated by their death within circulating red cells. This has important implications for the mechanism of immunity to both this parasite and those Plasmodium spp. with which it cross-protects in this host.

The action of pyrimethamine, sulphadiazine, proguanil and its active metabolite CPT, and 2:4-diaminopteridines against infections of Plasmodium berghei in mice was antagonized by P–aminobenzoic acid and by pteroylglutamic acid. Antagonism was in some instances detected only when P–aminobenzoic acid was given in solution in the drinking water as well as being injected subcutaneously. No antagonism was detected with a number of amino acids and nucleic acid derivatives.

As all of the above group of drugs can be antagonized by P–aminobenzoic acid and by pteroylglutamic acid, it would seem that they are alike in their mode of action. There must, however, be some differences between the mode of action or absorption of these drugs because species of Plasmodium that are very sensitive to the action of one of these drugs are frequently not very sensitive to the action of others.

As P. berghei is dependent on p–aminobenzoic acid, it is suggested that it can utilize this compound in the synthesis of pteroylglutamic acid to a greater extent than can P. gallinaceum, and that it resembles P. knowlesi more than other species of Plasmodium.