The development and survival of the eggs and larvae of O. circumcincta was studied by spreading the daily faecal output of a sheep over a 2 m square grass plot for 1 week. Observations were carried out for 3 years on material spread at 4-weekly intervals and broadly similar results were obtained in each year. Development time was long in eggs placed outside in January, February, March, November and December. Yields of larvae were small and all were dead within 5 months. During the rest of the year development time was about 2 weeks, yields of larvae were high and larvae survived until July or August of the following year. The significance of these findings in devising systems of grazing management to control parasitic gastroenteritis is discussed.

The genetic control of compatibility between laboratory strains of schistosomes and their snail hosts has been studied intensively since the 1970s. These studies show (1) a bewildering array of genotype-by-genotype interactions – compatibility between one pair of strains rarely predicts compatibility with other strains, and (2) evidence for a variety of (sometimes conflicting) genetic mechanisms. Why do we observe such variable and conflicting results? One possibility is that it is partly an artifact of the use of laboratory strains that have been in culture for many years and are often inbred. Here we show that results of compatibility trials between snails and schistosomes – all derived from the same natural population – depend very much on whether one uses laboratory-cultured or field-collected individuals. Explanations include environmental effects of the lab on either host or parasite, and genetic changes in either host or parasite during laboratory culture. One intriguing possibility is that genetic bottlenecks during laboratory culture cause the random fixation of alleles at highly polymorphic loci that control the matched/mismatched status of hosts and parasites. We show that a simple model of phenotype matching could produce dose response curves that look very similar to empirical observations. Such a model would explain much of the genotype-by-genotype interaction in compatibility observed among strains.

The microscopical examination of Giemsa-stained thin blood smears and a nested PCR were performed to detect avian Plasmodium in 275 passerine birds from small and large fragments of Atlantic Forest, Minas Gerais, Brazil. The 275 blood smears were used both for the microscopical examination and nested PCR providing the DNA template used for the reactions. The sensitivity of the nested PCR assay was higher than that observed for blood smears through microscopical examination. High prevalence (39·6%) of Plasmodium infections was detected by nested PCR while the microscopical examination detected only 16·5% positive birds. Poor agreement was observed between the results of the two different tests. The PCR data obtained were correlated to the forest fragment size of the Atlantic Forest and also correlated to the biological characteristics of the birds (nest type construction, diet, participation in mixed-species flocks, age and sex). Birds captured in the large forest areas were more infected than birds captured in the small areas (51·9% and 28·5%, respectively). Diet and participation in mixed-species flocks were correlated to the Plasmodium parasitism. The insectivorous birds and those that participated in mixed-species flocks were more frequently infected (47% and 41·5%, respectively) than the other groups.

The Calliphoridae include some of the most economically significant myiasis-causing flies in the world – blowflies and screwworm flies – with many being notorious for their parasitism of livestock. However, despite more than 50 years of research, key taxonomic relationships within the family remain unresolved. This study utilizes nucleotide sequence data from the protein-coding genes COX1 (mitochondrial) and EF1α (nuclear), and the 28S rRNA (nuclear) gene, from 57 blowfly taxa to improve resolution of key evolutionary relationships within the family Calliphoridae. Bayesian phylogenetic inference was carried out for each single-gene data set, demonstrating significant topological difference between the three gene trees. Nevertheless, all gene trees supported a Calliphorinae-Luciliinae subfamily sister-lineage, with respect to Chrysomyinae. In addition, this study also elucidates the taxonomic and evolutionary status of several less well-studied groups, including the genus Bengalia (either within Calliphoridae or as a separate sister-family), genus Onesia (as a sister-genera to, or sub-genera within, Calliphora), genus Dyscritomyia and Lucilia bufonivora, a specialised parasite of frogs and toads. The occurrence of cross-species hybridisation within Calliphoridae is also further explored, focusing on the two economically significant species Lucilia cuprina and Lucilia sericata. In summary, this study represents the most comprehensive molecular phylogenetic analysis of family Calliphoridae undertaken to date.

In this study Australorbis glabratus from Puerto Rico, the Dominican Republic, Venezuela, Surinam and Brazil, Biomphalaria pfeifferi from Liberia, and B. boissyi from Egypt were exposed to strains of Schistosoma mansoni from Puerto Rico, Venezuela, Surinam and Egypt, and to seven cross-strains of S. mansoni. In many instances, incompatibilities between vectors and parasites of different endemic areas were noted. It is suggested that these incompatibilities might be accounted for in two ways: (1) interspecific and intraspecific differences between vectors in respect to the physiological factors that are responsible for the development of the parasite in the snail, and (2) physiological differences between strains of the parasite. Evidence would seem to show that there are interspecific physiological differences between Australorbis glabratus and Biomphalaria boissyi; and that there are intraspecific differences between Australorbis glabratus from Brazil and A. glabratus from other countries here dealt with. In addition, it would appear that the parasite of Egypt differs physiologically from the parasites of Puerto Rico, Venezuela and Surinam; and that the parasite of Brazil differs physiologically from the parasites of the latter countries.

We wish to express our gratitude to the following persons for their co-operation in collecting and shipping snails for this study: Drs M. Abdel Azim, C. H. Barlow, J. O. Coutinho, A. Gabaldon, the late W. A. Hoffman, G. W. Luttermoser, J. Oliver-Gonzalez, H. A. Poindexter, Bichat de Almeida Rodriques and A. E. Wolff. We are also indebted to the late Dr W. A. Hoffman, Dr M. Mayer and Dr J. Alves Meira for sending us animals infected with Schistosoma mansoni.

Orgyia leucostigma haemocytes were able to encapsulate and destroy eggs of Hyposoter species, thereby preventing successful parasitism. When host larvae had previously been parasitized by Cotesia melanoscela, however, this response was not observed, and 3 different Hyposoter species could be reared to maturity in the normally non-permissive host. Appropriate injection experiments established that C. melanoscela virus and venom were both required in order for successful parasitism by Hyposoter to occur. Covalent cross-linking of C. melanoscela viral DNA led to an immune response against Hyposoter larvae, but not eggs, suggesting that an active viral genome may be required for complete development of Hyposoter species in tussock moth larvae.

Autoradiography of compressed mouse tissues has been used to estimate the numbers of Se-labelled schistosomula present in different mouse organs. Day 7 parasites extracted from the lungs of donor mice were delivered by injection to the lungs, systemic organs and liver of recipient mice as a discrete pulse. The numbers detected in various locations with time post-injection were then used to analyse the dynamics of intravascular migration. Approximately 98% of cercaria-associated label was lost during the first 14 days of parasite life, two-thirds of this in the first 7 h post-infection. Nevertheless, 99–113% of schistosomula could be detected 30 min post-injection into the locations chosen. The efficiency of the parasite delivery system was 95%. The time required for the number of foci in the lungs to decline to 50%, after injection of parasites via the femoral vein, was 55 h. Adjustment of this data to allow for parasites returning to the lungs after passage round the systemic vasculature gave a value of 30–35 h for the true mean time of lung transit. The distribution of parasites to systemic organs after their exit from the lungs was proportional to the fractional distribution of cardiac output. The probability (P) of a schistosomulum being distributed to splanchnic beds was estimated at 0·32 and its P of being trapped in the hepatic portal distributaries within the liver as 0·72–0·86. On this basis, the entire hepatic portal population of adult schistosomes would be recruited during 2–3 circuits of parasites around the pulmonary-systemic vasculature. The mean transit time of schistosomula through intestinal capillaries was 6·5 h whilst that through other systemic organs combined (muscles, kidneys, brain, etc) was 16 h, considerably more rapid than lung transit. The time taken for schistosomula to pass between organs, in arterial and venous blood, was shown to be less than 30 min in both cases (probably much less).

Infectious agents have considerable potential to regulate or constrain the population growth of vertebrate hosts in natural habitats. A broad theoretical framework provides many insights into how the biology of the parasite and the demography of the host interact to determine this impact. It may manifest itself as a steady influence over time via stable endemic infection or in a recurrent epidemic fashion, sometimes with unpredictable intervals between epidemics depending on the generation time of the pathogen (time from infection to recovery or host death), its ability to induce lasting immunity and the population growth rate of the host species. Building on these notions, the paper focuses on recent work on the population dynamics of genetically variable pathogen populations and examines the factors that determine the evolution of virulence and the maintenance of genetic diversity in both host and pathogen. Recent research extends conventional theoretical templates to include population genetic elements and the within-host dynamics of the parasite and its interaction with the vertebrate immune system.

Helminth parasites of vertebrates usually select very specific regions or habitats in their hosts, and this is often preceded by a tortuous migration through various host organs. However, the proximate mechanisms of migration and habitat selection have remained enigmatic despite considerable effort by parasitologists. In this paper, a new approach to studying helminth behaviour in the host is proposed. The core idea is that behaviour strategies must be considered from the perspective of the parasites and their perceptions of their environment. A guiding principle is that the environmental features to which an animal responds, and the actions which are required for responding to the environment, form a fundamental unit of behaviour. Thus, we can deduce an animal's behavioural strategy from the details of its response to environmental signals and from its sensory capabilities. The evidence presented suggests that helminth behaviours in the host often occur as fixed (or modal) action patterns which are usually seen in response to constant, or predictable environmental features. Thus, a working hypothesis is that the mechanisms of physiological and biochemical homeostasis within the host provide an extremely predictable environment for the parasite. Under these conditions, a parasite needs to perceive only small subsets of the total information available from the environment to respond appropriately. Studies on sensory and nervous systems of these organisms are critical to understanding parasite perception, but there are formidable technical obstacles that prevent easy access to parasite nervous systems. Therefore, a multidisciplinary approach, using ideas from parasitology, ecology, evolutionary biology and neuroethology, is considered requisite for reconstructing the parasites' behaviour strategies. It is suggested that future directions should pursue integration of studies on sensory physiology with the behavioural ecology of these organisms.

A new virus disease of tomatoes is described for which the name tomato black-ring is suggested. The host range of the virus is wide but no insect vector has yet been identified. The longevity in vitro of the virus is 7 days or longer, the thermal inactivation point is about 58° C. and the concentration of the virus in the host plant is low. Tomato black-ring is essentially a disease of young plants which rapidly lose their symptoms if they survive the severe initial infection. The virus is carried without symptoms by a large number of miscellaneous plants.

Note added in proof. In a private communication Kassanis has pointed out that the tomato black ring virus produces enations on the undersides of the leaves of cucumber. The writer has confirmed this but finds that the enations develop only in the greenhouse type of cucumber, never on the outdoor or ridge variety. The enations do not seem to develop under winter conditions.

The writer's best thanks are due to Miss Margaret Short for her assistance in this work and to Dr Roy Markham for taking the photographs.

Effective vaccines are available for many protozoal diseases of animals, including vaccines for zoonotic pathogens and for several species of vector-transmitted apicomplexan haemoparasites. In comparison with human diseases, vaccine development for animals has practical advantages such as the ability to perform experiments in the natural host, the option to manufacture some vaccines in vivo, and lower safety requirements. Although it is proper for human vaccines to be held to higher standards, the enduring lack of vaccines for human protozoal diseases is difficult to reconcile with the comparatively immense amount of research funding. Common tactical problems of human protozoal vaccine research include reliance upon adapted rather than natural animal disease models, and an overwhelming emphasis on novel approaches that are usually attempted in replacement of rather than for improvement upon the types of designs used in effective veterinary vaccines. Currently, all effective protozoal vaccines for animals are predicated upon the ability to grow protozoal organisms. Because human protozoal vaccines need to be as effective as animal vaccines, researchers should benefit from a comparison of existing veterinary products and leading experimental vaccine designs. With this in mind, protozoal vaccines are here reviewed.

A dihydrofolate reductase has been isolated from free-parasite preparations of the primate malarial parasite, Plasmodium knowlesi. Its properties are similar to those reported for the enzyme from the rodent malarial parasite, P. berghei, even though the base compositions of the DNA of these two species are quite different. The Kms for substrate and cofactor are 3 × 10−6M and 1 × 10−6M respectively and the molecular weight is ˜ 200000. Concentrations of pyrimethamine and trimethoprim as low as 1 × 10−9M and 3 × 10−8M respectively are sufficient to cause 50% inhibition of enzyme activity. The sensitivity to inhibition by pyrimethamine and trimethoprim of growth of cultures of P. knowlesi has also been investigated. Preliminary experiments showed that it was only the schizont stage that was susceptible to the action of the drugs and that in their presence, normal nuclear divisions and segmentation did not occur and subsequently, no reinvasion of fresh red cells took place. The minimum concentrations of drug required to produce these effects were 10−9M for pyrimethamine and 10−7M for trimethoprim. Thus, there is a close correlation between the concentrations of pyrimethamine and trimethoprim required to inhibit the dihydrofolate reductase in a cell-free system and the growth of the parasite in vitro. Pyrimethamine (10−9M) did not, however, affect the incorporation of radioactive precursors into DNA, RNA or protein of schizont stage parasites until after morphological damage could be seen and reinvasion was complete in control cultures. The time courses of incorporation of [14C]algal protein hydrolysate into protein in the presence (10−9M) or absence of pyrimethamine are the same as those described recently with immune serum. The possibility is thus raised as to whether pyrimethamine and immune serum act in the same way.

One of us (P.I.T.) received financial assistance from the World Health Organization. We thank Dr F. Hawking for many helpful discussions, Miss Jane Dunnett and Mr T. Scott-Finnigan for technical assistance and Dr O. D. Standen for samples of pyrimethamine and trimethoprim.

Basilia hispida is the only insect parasite of the flat-headed bats Tylonycteris pachypus and T. robustula in Selangor, Malaysia where this study was conducted between September 1966 and August 1968. The climatic conditions in the laboratory closely approximated to those in the field: seasonal variations in climate were small but diurnal fluctuations occurred. The life cycle of the fly was not affected either by the season or by the sex or species of the host.

A typical life cycle was as follows: adult B. hispida reached sexual maturity within 5 or 6 days of their emergence from the puparium. Copulation largely occurred with newly emerged flies or with females at the time of prepupal deposition; one copulation sufficed for numerous subsequent offspring. The entire larval life of three instars was passed within the female, nourished by maternal secretions. After reaching maturity the female deposited prepupae at 9-day-intervals on the host's roosting quarters, deposition occurring during the day, stimulated by increasing temperature. The length of the pupal stadium depended upon the presence of a host bat; if one was present the pupal period was ca. 25 days. Thus the total life cycle from emergence of the female to the emergence of her first offspring was typically 39 (5 + 9 + 25) days. The sexes were produced in equal numbers. The greater part of postembryonic mortality occurred in the adult instar and was largely due to host-predation. Females were longer lived than males, and it was suggested that they had an average life expectancy of 5–6 weeks in the field.

The life cycle of five other species of Nycteribiidae from two other species of host were briefly examined and no major differences from B. hispida were noted. The results of this and of previous studies indicate that the life cycle is fairly uniform throughout the family.

1. An English anautogenous race and three European strains of the autogenous race of Culex pipiens have been studied comparatively for 3 years under laboratory conditions.

2. Prolonged illumination has an activating influence on females of the anautogenous race, both hibernating and laboratory bred, and greatly stimulates gorging.

3. With the aid of artificial light during the winter months the anautogenous race has been kept breeding in the laboratory throughout the year for eleven generations without the occurrence of cyclical hibernation (asthenobiosis). Consequently it is suggested that the length of daylight may be a factor of importance in controlling the natural hibernation of females of this race.

4. Females of the English anautogenous race, whether of hibernating, or of active generations, laid normally after one blood meal and did not exhibit gonotrophic dissociation. They also oviposited readily after being artificially fed on bird blood.

5. Unfed females lived for a maximum of 19 days and never accumulated fat-body autotrophically.

6. Females, if fed on apple for 5 weeks, accumulated sufficient reserves to enable a small percentage of them to survive 15 weeks of starvation.

7. In the anautogenous race pairing always begins in the air although it may be completed on the ground. The females do not lay without a blood meal, and they show little tendency to bite man but bite birds voraciously.

8. Strains of the autogenous race of C. pipiens were obtained from rural areas in Greece, Malta and Hungary and have been maintained in the laboratory for over 3 years.

9. Continuous breeding under autogenous conditions (no blood meals) for several years and through forty-five to forty-nine generations has had no deleterious eflect on these autogenous strains.

10. Spanogyny, or the gradual decrease in the numbers of females produced in succeeding generations, does not necessarily follow prolonged autogeny if the cultures are kept under favourable conditions.

11. Males of the autogenous race pair with resting females, so that pairing can take place in very small areas.

12. Cross-mating was easily obtained between the two races, autogenous and anautogenous, in both directions: male autogenous + female anautogenous; and male anautogenous + female autogenous. Stenogamy and autogeny are hereditary characters. Stenogarny always appears in the F1 generation, but autogeny sometimes appears in the F1 generation and sometimes not until the F2 generation.

13. Eggs are quickly killed by temperatures below freezing-point; and young larvae die within 24 hours at 0° C. Adults, even hibernating females, die within 4 days at −16° C.

We investigated changes in the behaviour of Stagnicola elodes associated with the emergence of Plagiorchis elegans cercariae. Within 15 min of the reduction in light intensity, which triggered the onset of cercarial emergence, infected snails moved to the top of the water column and remained there for 2–3 h. Seventy-nine percent of all cercariae that emerged from the snail did so during this period. Uninfected snails showed no such behavioural changes following the change in light intensity. Cercariae were released in a dense cloud around the snail at the water surface and dispersed passively. Within 3–4 h more than 80% of all cercariae had settled in the bottom 5 cm of the water column. The infectivity of cercariae increased from less than 20% upon emergence from the snail to greater than 75% 4–6 h post-emergence, and then declined steadily to below 5% by 24 h post-emergence. Cercarial longevity was greater than 30 h and exceeded the period of infectivity. This may be related to steadily falling endogenous glycogen levels. A delay in attaining maximum infectivity may represent an adaptive mechanism allowing time for cercarial dissemination, thus reducing superinfection, and subsequent parasite-associated mortality, of second intermediate hosts.

The immune-dependent elimination of a challenge infection in rats vaccinated with highly-irradiated cercarriae of Schistosoma mansoni was analysed by passive transfer of serum, recovery of the challenge from the lungs and liver and by transferring lung-stage schistosomula. Recipients of serum from rats immunized with either unirradiated, 20 or 40 krad.irradiated cercariae, most of which die in the liver, lungs and skin, respectively, were equally resistant if the serum was injected on the day of infection or 5–7 days after infection. In addition, vaccinated rat serum transferred to mice and vaccinated rabbit serum transferred to rats conferred comparable protection when injected on day 0 or 5 days after infection of the recipents. This apparent susceptibility of the lung schistosomula to immune attack was confirmed by challenging 20 or 40 krad.-irradiated cercariae vaccinated rats with lung-stage schistosomula derived either from mice or rats. All the detectable attrition of a cercarial challenge in vaccinated rats occurred between 7 and 10 days post-challenge, before the parasites reached the liver. Since there was no evidence of damage or attrition in the skin or lungs before day 7 it was concluted that immune-dependent elimination occurred rapidly following a ‘window of sensitivity’ coinciding with the migration of the parasites from the lungs to the liver.

Eleven gamma-emitting radionuclides (49Sc, 54Mn, 59Fe ( + 2 and + 3), 60Co, 65Zn, 75Se (as selenomethionine, selenocystine, selenite and selenate), 109Cd, 125Sb, 133Ba, 137Cs and 203Hg) were screened as labelling agents for Schistosoma mansoni cercariae by incubation of infected Biomphalaria glabrata snails in radioactive solution according to the technique of Christensen (1977). Only [75Se]methionine yielded satisfactorily labelled cercariae. Multiple regression analysis of volume, number of cercariae and radioactivity from a series of 10 aliquots of unwashed cercarial suspensions yielded estimates of unbound and cercarial-bound radioactivity that were equivalent or superior to estimates based on assay of washed cercariae and eliminated loss of cercariae. Washing of cercarial suspensions over 8 μm pore diameter Millipore filters was found to result in entanglement of 60–90% of the cercariae on the filter disc. Differential external radioassay, a new technique employing partial body shielding within a total body counter, permitted separate estimation of tail and body radioactivity of conscious mice previously exposed by tail immersion to 75Se-labelled cercariae, with measurements repeated as often as desired. Approximately 39% of the 75Se present in emergent cercariae was retained by schistosomula transformed in vitro but this was subject to considerable variation, especially in schistosomula transformed in vivo. Secreted or catabolized label from penetrant cercariae and schistosomula was rapidly removed from the skin by the bloodstream. Numbers of schistosomula in tail skin were directly proportional to the number of reduced silver foci counted on tail autoradiograms; only a very small fraction of tail radioactivity represented unbound (‘spurious’) label. Migration of schistosomula away from skin was 50% complete at 3·8–4·3 days, as determined by probit analysis of autoradiographic data.

In the present study, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was developed to identify 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory–secretory (E–S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the HinfI or DdeI endonuclease restriction of the 2800 bp fragment; T. nativa by the RsaI restriction of the 2800 bp fragment, or by the AluI restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the AluI restriction of the 1250 bp fragments, and can be discriminated between them by the SspI restriction of the 2800 bp fragment; T. pseudospiralis by the MspI restriction of the 372 bp fragment; T. nelsoni by the HhaI or AluI restriction of the 2800 bp fragment; Trichinella T5 by the HhaI restriction of the 2800 bp fragment; Trichinella T6 by the AluI restriction of the 1250 bp fragment; and Trichinella T8 by the SspI or RsaI restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi, Trichinella T5 and T6.

Lactacystin, a specific inhibitor of proteasomes in eukaryotic cells, did not block parasite entry or the establishment of the parasitophorous vacuole, but did inhibit parasite growth and daughter cell budding, as well as DNA synthesis. Two other proteasome inhibitors, MG-132 and proteasome inhibitor 1, also blocked parasite growth and intracellular development. Adding lactacystin to established, dividing parasites, rapidly blocked parasite growth and daughter cell budding at all stages in the process. Pre-treating host cells with lactacystin did not block parasite entry or development. Moreover, under the conditions used, the host cells appeared not to be adversely affected indicating that host cell proteasome activity was not essential for parasite entry or development. Concomitant with these effects on parasite growth and division were morphological changes in the parasite including the appearance of whorls of ER-derived membranes presumably related to the failure to breakdown misfolded proteins. These changes were specific to lactacystin and were not seen in parasites treated with other protease inhibitors. Although the ER-derived structures resembled autophagic bodies, similar structures could not be induced by serum starvation nor did the membranous whorls acidify or undergo morphological changes consistent with autophagosomal maturation. These results highlight the possible role of proteasome activity in Toxoplasma in intracellular development and the regulation of parasite replication. However, how the dividing parasite recycles its organelles and the functional relationship between any lysosomal–autophagic pathway and proteasomes in the parasite remains unresolved.

Neospora caninum, the causative agent of neosporosis, is a recently identified apicomplexan parasite which is structurally and biologically closely related to, but antigenically distinct from, Toxoplasma gondii. Molecules associated with the surfaces of N. caninum tachyzoites are likely to participate in the host cell entry process, could be involved in the interaction of the parasite with the immune system, and they could influence the pathogenesis of neosporosis. Isolated N. caninum tachyzoites were extracted with the non-ionic detergent Triton X–114 and were further analysed using a polyclonal anti-N. caninum antiserum. Immunoblots revealed several reactive bands, 1 of which represented a glycoprotein of approximately 36 kDa (Nc-p36). This molecule was present in 2 isolates of Neospora (NC-1 and Liverpool), but was absent in Toxoplasma (RH-strain) tachyzoites. Immunofluorescence and pre-embedding immunogold transmission electron microscopy employing affinity-purified anti-Nc-p36 antibodies showed that the Nc-p36 is a cell surface-associated protein. Immunogold on-section labelling of LR-White-embedded parasites, fixed prior and at defined time-points after host cell entry, demonstrated the presence of this molecule on the surface as well as within the dense granules of N. caninum tachyzoites.