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Aims To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites. Methods and results EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL‐4, IL‐5 and IL‐13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL‐5 and IL‐9 (exsheated L3 and L4) and IFN‐γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL‐4, IL‐5 and IL‐10 while concomitantly enhancing the expression of IFN‐γ. Conclusion The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.

Background Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. Objectives Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). Methods This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. Results We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. Conclusions Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Summary Freeze-thawed extracts of mature schizonts of Plasmodium knowlesi (strain Wl) were electrophoresed in sodium dodecylsulphate (SDS) polyacryl-amide gels, then transferred electrophoretically to diazophenylthioether paper. The transferred antigen was probed using a purified polyvalent, polyclonal immune serum pool isolated from rhesus monkeys (Macaca mulatto). This method for identifying parasite antigens was compared with immunoabsorption, using parasites which had been metabolically labelled in vitro with 35S-methionine. The methods showed general correspondence but some antigens failed to react after transfer because of loss of antigenicity during SDS-polyacramide gel electrophoresis (SDS-PAGE). Some antigens were observed preferentially after the transfer procedure, possibly because of their failure to elute from the immunoabsorbant or because they may have a low methionine content. The transfer method failed to reveal the antigen reactivity of three out of four monoclonal antibodies tested, apparently because the epitopes involved were inactivated by SDS. During P. knowlesi infection in the resistant kra monkey (Macaca fascicularis) the transfer method showed the presence by day 10 of antibodies against two components having an apparent molecular weight by SDS-PAGE similar to those of putative protective antigens described in other studies using monoclonal antibodies.

An attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine is under development, in part, based on studies in mice with P. berghei. We used P. berghei and P. yoelii to study vaccine-induced protection against challenge with a species of parasite different from the immunizing parasite in BALB/c mice. One-hundred percent of mice were protected against homologous challenge. Seventy-nine percent immunized with attenuated P. berghei sporozoite (PbSPZ) (six experiments) were protected against challenge with P. yoelii sporozoite (PySPZ), and 63% immunized with attenuated PySPZ (three experiments) were protected against challenge with PbSPZ. Antibodies in sera of immunized mice only recognized homologous sporozoites and could not have mediated protection against heterologous challenge. Immunization with attenuated PySPZ or PbSPZ induced CD8+ T cell-dependent protection against heterologous challenge. Immunization with attenuated PySPZ induced CD8+ T cell-dependent protection against homologous challenge. However, homologous protection induced by attenuated PbSPZ was not dependent on CD8+ or CD4+ T cells, and depletion of both populations only reduced protection by 36%. Immunization of C57BL/10 mice with PbSPZ induced CD8+ T cell-dependent protection against P. berghei, but no protection against P. yoelii. The cross-protection data in BALB/c mice support testing a human vaccine based on attenuated PfSPZ for its efficacy against P. vivax.

We describe how hookworms interact with their human hosts by comparing lymphocyte phenotyping, proliferative responses, and cytokine and chemokine secretion patterns in adults who are either mono-infected with Necator americanus or egg-negative controls resident in an area of high transmission in Brazil. Cellular immune responses against crude hookworm antigen extracts from different developmental stages were evaluated simultaneously. Principal component analysis (PCA) was used to reduce the standardized immune responses. Random effects multivariate regression was then used to investigate whether principal components (PC) differ between the two groups once potential confounders and effect modifiers have been accounted for. Although hookworm patients had reduced percentages of T and B cells, they had higher levels of activated CD4(+) T and CD19(+) B cells. This state of 'immune activation' coincided with lower proliferative responses, especially to third-stage larval antigen. Cytokine levels in mono-infected adults were also lower and characterized by a mixed Th1/Th2-type profile. Excretory/secretory antigen from adult worms was a potent modulator of the immune response, resulting in diminished TNF-alpha and IL-10 secretion in peripheral blood mononuclear cells (PBMC) from hookworm infected patients. We propose that the longevity of hookworms in their human hosts results from a stage-specific, down-modulation of the immune response.

Aim An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. Materials and results The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4-7 and SDS-PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti-A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver-stained gel and subjected to mass spectrometry in MALDI-TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against A siamensis is discussed in the paper. Conclusion Three of the identified proteins namely, bromodomain-containing protein, anaphase-promoting complex subunit 5 and elongation factor-2 could possibly serve as vaccine candidates against argulosis in carps.

The parasitic ciliate Cryptocaryon irritans, which infects almost all marine fish species occurring in both tropical and subtropical regions throughout the world. The disease, cryptocaryonosis, accounts for significant economic losses to the aquaculture industry. This review attempts to provide a comprehensive overview of the biology of the parasite, host-parasite interactions, and both specific and non-specific host defense mechanisms are responsible for the protection of fish against challenge infections with this ciliate. Also, this paper reflects the current interest in this subject area and the quest to develop an available vaccine against the disease. Due to the high frequency of clinical fish cryptocaryonosis, the study of fish immune responses to C. irritans provides an optimal experimental model for understanding immunity against extracellular protozoa.

Molecular genetic analyses of P. falciparum have led to the cloning and sequencing of a number of antigens that are potential candidates for vaccination against malaria. Seroepidemiological studies in endemic areas have attempted to assess the relative importance of these antigens in protection against malaria. In this paper, we attempt to evaluate the relative contributions of conserved and strain-specific immune responses by modelling their influence of age-specific patterns of infection and disease. The modelling exercises in this paper clearly demonstrate that the observed patterns of age-prevalence are best explained by proposing that the accumulation to a threshold of an immune response against a conserved determinant is required for protection against infection, while 'anti-disease' immunity develops more linearly with exposure. This is compatible with the conjecture that the parasite population is structured into several independently transmitted strains, that each confers some degree of 'anti-disease' immunity, but does not protect against further infection by the same strain. Within this framework, the average duration of parasitaemia increases with age, as previously encountered strains endure for longer periods at a subclinical level. Indirect evidence for the increase in duration of parasitaemia with age may be obtained from a comparison of age-prevalence curves between dry and rainy seasons. By using mathematical methods to structure epidemiological and immunological information, we provide a coherent theoretical framework for the dissection of the important components of naturally acquired immunity to malaria.

SUMMARY Hookworms are bloodfeeding nematodes that reside in the intestinal mucosa. These parasites secrete proteins that induce robust systemic immune responses in humans and experimental animals. By contrast, mucosal immune responses in and around the site of attachment are not described as well. This paper presents data from studies aimed at examining hookworm-specific mucosal antibody responses in a hamster model of Ancylostoma ceylanicum infection. Intestinal flush prepared from infected hamsters was analysed by ELISA and shown to be enriched in IgA-specific for A. ceylanicum excretory–secretory (ES) products. Evaluation of mucosal IgA responses by immunoblot demonstrated that infected hamsters recognized a broad range of ES proteins. Hamsters repeatedly exposed to drug-terminated infections were shown to have enhanced serum IgG and mucosal IgA responses, as well as a high level of protection from challenge infection. Parasite-specific IgA was also detected in the faeces of hamsters undergoing a primary infection, and increasing faecal IgA responses were coincident with significant reductions in intestinal worm burdens and faecal ES output over time. Together these results suggest that secretory IgA may act in concert with other components of the mucosal and systemic immune response to promote protective immunity against hookworm infection and/or disease.

SUMMARY Despite the plethora of evidence on the consequences of host nutrition on their immune responses to gastrointestinal parasites, the identity of molecules and mechanisms that drive the manifestations of immunity under nutrient abundance are not yet known. This is partly due to limitations of the methodologies employed to date that have failed to give comprehensive answers. The great advancements in the technological front over the past few years have opened a window of opportunity to identify these effector molecules, to explore both the mechanisms and cellular pathways and to evaluate their importance in the immune response to parasites. The aim of this paper is to present some of the novel, high-throughput technologies that are currently available in immunology and to explore their use in advancing our knowledge in interactions between nutrition and immunity to nematode parasites, with special reference to sheep. In the first part, we introduce the technologies and we discuss advantages and pitfalls of their use. We bring in successful examples of how their employment advanced knowledge and improved our understanding of the mechanisms that regulate immune responses to pathogens (both micro- and macroparasites). In the second part, we focus on the impact of nutrition on the immune response to parasites, and explore how these technologies can be used to advance our knowledge of immunonutritional interactions. We use as our starting point well-established models that have been successfully used to investigate the consequences of nutrition on the manifestations of immunity to parasites, which we further consider in the context of the novel technologies. We conclude by emphasizing the great potential of the described methodologies in unravelling the complex interactions between nutrition and immunity, but we also recommend caution when interpreting the outcomes.

Nematode parasites cause significant infections in both humans and animals. They are complex, multicellular organisms that present unique challenges for the host, in particular with respect to the recognition of their unusual surface structures by the innate defence system. The innate immune system is now recognized to be a critical component in the development of an adaptive effector response as well as a driver of vaccine-induced immunity. This paper will give an overview of current research on the innate barriers and immune mechanisms, cells, and receptors involved in the innate host response to nematode parasites. It will also review the 'nematode-associated molecular patterns' that may be specifically recognized by the host, in addition to other signals, such as nervous stimulation and tissue damage, that may alert the innate system to parasite invasion.

The heart can be seriously affected in human trichinellosis, and cardiac involvement can cause death. Experimental infections in rats have suggested the possible participation of immunopathological processes. The aim of the present paper was to investigate the possible presence in trichinellosis patient sera of antibodies recognizing host tissues and particularly the myocardium. Nineteen sera from late period trichinellosis patients, who acquired infection in the Poznan region (Poland), were tested by immunoblot on extracts from normal rat or human heart ventricle wall, spleen, placenta, kidney and skeletal muscle. Patients' sera recognized several antigens that were not recognized by normal sera. On rat and human heart ventricle wall, a high proportion of sera (42%) reacted with a protein of 68 kDa (P < 0.05 compared to normal sera). The reactivity with this antigen, however, was not significantly different in patients with or without cardiac involvement. When sera were tested on skeletal muscle we found that 47% reacted with a protein of 27 kDa and 53% reacted with a protein of 41 kDa (P < 0.05 for both proteins, compared with normal sera). The reactivity against the 68 kDa antigen and against the 27 and 41 kDa skeletal muscle antigens was not observed on kidney, placenta and spleen extracts. Moreover, very few bands were observed on these tissues as compared to heart and skeletal muscle tissues, thus suggesting a high tissue specificity of the reactivity of trichinellosis sera. In conclusion, this study identifies organ-specific autoantibodies in trichinellosis patient sera, their role in the pathogenesis of cardiac involvement being still unclear.

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

SUMMARY Since the publication of the mouse genome, attention has focused on the strains that were selected for sequencing. In this paper we report the results of experiments that characterized the response to infection with the murine gastrointestinal nematode Heligmosomoides polygyrus of eight new strains (A/J, C57BL/6, C3H, DBA/2, BALB/c, NIH, SJL and 129/J), in addition to the well-characterized CBA (poor responder) and SWR (strong responder) as our controls. We employed the repeated infection protocol (consisting of 7 superimposed doses of 125L3 each administered at weekly intervals, faecal egg counts in weeks 2, 4 and 6 and assessment of worm burdens in week 6) that was used successfully to identify quantitative trait loci for genes involved in resistance to H. polygyrus . SWR, SJL and NIH mice performed indistinguishably and are confirmed as strong responder strains to H. polygyrus . CBA, C3H and A/J mice all tolerated heavy infections and are assessed as poor responders. In contrast, DBA/2, 129/J and BALB/c mice performed variably between experiments, some tolerating heavy worm burdens comparable to those in poor responders, and some showing evidence of resistance, although only in one experiment with female 129/J females and one with female BALB/c was the pattern and extent of worm loss much like that in SWR mice. Because the genetic relationships between six of the strains exploited in this study are now well-understood, our results should enable analysis through single nucleotide polymorphisms and thereby provide more insight into the role of the genes that control resistance to H. polygyrus.

SUMMARY CD4 T cells play a central role in the immune response to malaria. They are required to help B cells produce the antibody that is essential for parasite clearance. They also produce cytokines that amplify the phagocytic and parasitocidal response of the innate immune system, as well as dampening this response later on to limit immunopathology. Therefore, understanding the mechanisms by which T helper cells are activated and the requirements for development of specific, and effective, T cell memory and immunity is essential in the quest for a malaria vaccine. In this paper on the CD4 session of the Immunology of Malaria Infections meeting, we summa- rize discussions of CD4 cell priming and memory in malaria and in vaccination and outline critical future lines of investi- gation. B. Stockinger and M.K. Jenkins proposed cutting edge experimental systems to study basic T cell biology in malaria. Critical parameters in T cell activation include the cell types involved, the route of infection and the timing and location and cell types involved in antigen presentation. A new generation of vaccines that induce CD4 T cell activation and memory are being developed with new adjuvants. Studies of T cell memory focus on differentiation and factors involved in maintenance of antigen specific T cells and control of the size of that popula- tion. To improve detection of T cell memory in the field, efforts will have to be made to distinguish antigen-specific responses from cytokine driven responses.

This paper reports the results of a genome-wide search for quantitative trait loci (QTL) influencing immunological responses to infection with the gastro-intestinal nematode parasite Heligmosomoides polygyrus in an F2 population created by crossing the resistant SWR and the susceptible CBA inbred mouse strains. Following infections, intestinal granuloma score at post mortem, mucosal mast cell protease 1, and IgE and IgG1 titres were recorded. The susceptible CBA mice had significantly higher IgG1, but significantly lower IgE, mucosal mast cell protease 1 and granuloma scores than SWR mice. Significant QTL were mapped to chromosomes 4, 11, 13 and 17 for granuloma score; chromosomes 12 and 17 for IgE; chromosome 10, 17 and 18 for IgG1 and chromosomes 1, 9, 10, 11, 17 and 18 for mucosal mast cell protease 1. Chromosomes 10, 11, 17 and 18 had QTL affecting more than one trait, and these are most likely to represent single QTL with multiple effects rather than multiple QTL. Some of these QTL map to regions known to harbour genes responsible for the induction of immunological responses to intestinal worms.

Neospora caninum is recognized as a major cause of infectious abortion in cattle. Very little is known about immunity to Neospora. Cell mediated responses have previously been shown to be important in the development of protective immunity to the closely related parasite Toxoplasma gondii, and may therefore be an important component in the immune response to Neospora. In this paper we report that a group of low molecular weight NCI strain tachyzoite antigens (< or = 30 kDa) separated by SDS PAGE and bound to nitrocellulose membrane stimulated proliferation in vitro of CD4+ T cells from calves experimentally infected with N. caninum. Proliferation was accompanied by production of high concentrations of IFN-gamma. Several of these antigens were also recognized by antibody produced in these animals. As the most effective vaccines require the stimulation of both humoral and cell mediated immune responses, these antigens may be important in the development of a vaccine against neosporosis.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) have been shown to differ in inflammatory response to intradermal administration of blowfly (Lucilia cuprina) antigens and artificial challenge. The current paper describes analysis of antibody responses to L. cuprina antigens in the R and S animals. Serum antibody titres and specificities to larval antigens were examined and the specificity of wound exudate antibodies was also investigated in animals artificially challenged with L. cuprina. Titres of L. cuprina specific serum IgA, IgM, IgG2 and IgG1 were measured by ELISA, while specificities were examined on two-dimensional immunoblots of larval homogenates. Exposure to L. cuprina stimulated the production of specific antibody in both R and S animals, however antibody titres did not differ between the R and S animals. There was large variation in antibody specificity between individual animals and some L. cuprina proteins appear to be more frequently recognized by sera from either resistant or susceptible animals, however the recognition of a specific protein could not be solely attributed to the resistance status of the animal. It appears that resistance in these animals may be independent of serum antibody and is likely to be an innate response. Despite high levels of IgG in wound exudates, this antibody recognized few antigens in comparison with serum from the same animal, suggesting that exudate contains little functional antibody in comparison to serum.

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

This paper describes the evaluation of the protective antibody response of sheep to vaccination against Taenia ovis infection with a defined recombinant antigen (45W). Sera from 181 vaccinated sheep, collected prior to experimental challenge with T.ovis, were assessed for 45W specific IgA, IgG, IgG1, IgG2 and IgM levels and these results correlated with protection data. There were significant relationships (P < 0.001) between IgG, IgG1 and IgG2 titres and protection. Serum IgA levels did not correlate with protection and there were no significant levels of 45W specific IgM detected. Killing of several other taeniid cestodes has been shown to be complement mediated and the findings in this study are consistent with the involvement of this immune mechanism in 45W vaccinated sheep. A comparison of the adjuvants used in this study (saponin and oil in water) demonstrated that whereas both adjuvants stimulated the production of similar levels of 45W specific IgG1, the IgG2 response was significantly higher in sheep vaccinated with oil adjuvant.

In this paper we describe the relationship between antibodies to Necator americanus stage-specific antigens and parasite burden in an endemically-infected population in Papua New Guinea. Using an age-structured analysis, we show that the correlation coefficient between levels of IgG against adult worm excretory-secretory (ES) antigen and parasite burden declined significantly with host age from positive in younger hosts to significantly negative in older hosts. A trend towards similar patterns was present for anti-larval IgG both pretreatment and after reinfection, and for anti-ES IgM and anti-ES IgE pretreatment. These patterns are consistent with a role for these isotypes in a protective immune response, although parasite-induced immunosuppression may provide an alternate explanation. This is another demonstration of possibly protective responses to N. americanus infection.

Field studies of parasitic helminths in endemically infected human communities have provided quantitative information on the relationships between parasite burdens, immune responses and age. There are considerable difficulties in the interpretation of these immunoepidemiological data due to the complexities of the biological processes generating the observed patterns. In this paper simple mathematical models are used to explore the expected patterns of variation with host age in parasite burdens, the aggregation of parasites among hosts, levels of immune response, and the correlation between parasite burdens and immune responses. These relationships reflect rates of infection, rates of parasite mortality, the strength of the immune response, and the duration of immunological memory. The models generate some complex and counterintuitive patterns. The analysis suggests that some of these patterns might serve to (i) distinguish effects due to acquired immunity from effects due to age-dependent exposure, (ii) identify potentially protective immune responses, and (iii) identify the parasite stages important in the development of acquired immunity. The results imply that previous analyses of immunoepidemiological data may have been overly simplistic and, especially, that patterns believed to be inconsistent with protective immunity may have been incorrectly interpreted.

Summary In the present paper, we have characterized the specificity of a series of monoclonal antibodies (MoAbs) against Plasmodium berghei sporozoites, selected for their lack of reactivity with the repeat domain of the circumsporozoite (CS) protein. We found that these MoAbs recognize Pb44, the mature membrane form of the CS protein, but they do not react with Pb54, its precursor. Furthermore, these MoAbs do not react with any of the synthetic peptides representing the linear sequence of the P. berghei CS protein nor do they react with a recombinant CS (rCS) protein. These observations indicate that the epitope recognized by these antibodies is expressed only after the processing of the CS protein has occurred. This ‘processing dependent epitope’ is present in sporozoites of P. berghei, and also in those of P.falciparum, P. yoelii and P. brasilianum. It is not present in sporozoites of the P. cynomolgi-P. vivax complex and of P. gallinaceum. These anti-sporozoite MoAbs strongly inhibit sporozoite invasion of hepatoma cells, in vitro, however, they displayed no protective effect in an in vivo assay.

Despite the plethora of evidence on the consequences of host nutrition on their immune responses to gastrointestinal parasites, the identity of molecules and mechanisms that drive the manifestations of immunity under nutrient abundance are not yet known. This is partly due to limitations of the methodologies employed to date that have failed to give comprehensive answers. The great advancements in the technological front over the past few years have opened a window of opportunity to identify these effector molecules, to explore both the mechanisms and cellular pathways and to evaluate their importance in the immune response to parasites. The aim of this paper is to present some of the novel, high-throughput technologies that are currently available in immunology and to explore their use in advancing our knowledge in interactions between nutrition and immunity to nematode parasites, with special reference to sheep. In the first part, we introduce the technologies and we discuss advantages and pitfalls of their use. We bring in successful examples of how their employment advanced knowledge and improved our understanding of the mechanisms that regulate immune responses to pathogens (both micro- and macroparasites). In the second part, we focus on the impact of nutrition on the immune response to parasites, and explore how these technologies can be used to advance our knowledge of immunonutritional interactions. We use as our starting point well-established models that have been successfully used to investigate the consequences of nutrition on the manifestations of immunity to parasites, which we further consider in the context of the novel technologies. We conclude by emphasizing the great potential of the described methodologies in unravelling the complex interactions between nutrition and immunity, but we also recommend caution when interpreting the outcomes.

The apicomplexan parasite Toxoplasma gondii is remarkable in several aspects, since it is a protozoan that infects most nucleated cells in many warm-blooded animals, worldwide. Although the cellular immune response against T. gondii is critical for infection control, antibodies may either enhance or block protective mechanisms, and even mediate immunological damage, directly or indirectly. Since cytokines regulate the class/subclass switch, antibodies may also be the biomarkers of protective or pathological cellular immune events. There is a scientific and clinical interest in the presence of natural and autoreactive antibodies, as well as in the 'chronic' immunoglobulin M (IgM) response and the post-treatment 'rebound'. Another interesting aspect is compartmentalization; certain immunoglobulins may uniquely be found in specific host fluids. Local synthesis has been demonstrated, but antibodies may also traverse several cell layers, like the blood-brain and haemato-ocular barriers, and the placenta. In some instances, Fc receptors (FcRs) facilitate transport and may even have a concentrator effect, which can be related to resistance or pathology. These aspects of the humoral response against T. gondii are reviewed in the present paper.

The present paper reviews the available literature on the development of immunity to animal Schistosoma infections. The majority of the studies on animal schistosomiasis were performed in cattle and pigs and only Schistosoma mattheei, S. bovis and S. japonicum received particular attention, mainly because of their recognized veterinary significance or zoonotic aspect. Although it is an accepted fact that acquired resistance to Schistosoma is of major importance in the regulation of infection intensity in the field, almost nothing is yet known of either the nature of the antigens or of the immune mechanisms involved. The recent studies on immunity development focus in particular on the occurrence of maternal to foetal transfer of immunological substances related to animal Schistosoma infections and possible effects of these transfers on the immunity development of the foetus/newborn. Since congenital infections for Schistosoma species other than S. japonicum are extremely rare, the most plausible route for foetal contact is the transplacental or postnatal transfer of immunological substances. Prenatal transfers of specific antibodies and antigens via placental lesions and postnatal transfers via the colostrum were observed in cattle and pigs, and subsequent modifications of the immune response of the newborn were observed. Placental lesions induced by Schistosoma eggs could allow other pathogens to cross the placenta.

SUMMARY The baboon model of schistosomiasis has been used extensively to study parasite biology, immune responses and pathological manifestations after natural and experimental infections. The body of knowledge accumulated so far has placed this animal model at the pinnacle in the continuing search for new interventions and might hold the key to the development of new anti-schistosome vaccines. In this review paper, we highlight previous and recent studies that have elevated the baboon to be the model of choice for schistosomiasis research. In particular, the long-term studies of re-infection after chemotherapy as well as the interaction between vaccination, chemotherapy and infection are highlighted.

It has been shown that vaccination with three doses of the Leishmania infantum poly-protein Q containing five genetically fused antigenic determinants from the Lip2a, Lip2b, H2A and P0 proteins, mixed with BCG induces clearance of parasites in 9 out of 10 Leishmania infantum-infected Beagle dogs, in addition to clinical protection. In the present paper we analysed the immunogenic potential of the poly-protein Q and the specificity and polarization of the response against the antigenic determinants of Q when mixed with various adjuvants. The data showed that the Q protein had high intrinsic immunogenic potential and that it was able to induce a long-lasting IgG response. The IgM immunogenic potential of the poly-protein was mainly due to the LiP2a and LiP2b determinants, whereas the IgG immunogenic potential was mainly due to the LiP2a component. It was observed that the protein itself elicited a mixed IgG2a/IgG1 response and that the determinants of Q were endowed with different IgG2a/IgG1 potential. It was also observed that the adjuvants did not influence the intensity or specificity of the IgM response but that they modulated the intensity, the specificity and the polarization of the IgG response against the determinants of Q. CpG-ODN motifs or double-stranded DNA plasmids containing CpG motifs when mixed with Q induced a predominant IgG2a response mainly observed at early stages post-immunization. The data showed that a CpG + Q mix induced significant protection against L. infantum infection in Balb/c mice.

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.

The development of a malaria vaccine is a priority for improved and sustained malaria control. Optimal use of a vaccine in Africa will only be achieved if it can be delivered through the Expanded Programme of Immunization (EPI). We have completed a trial of the peptide vaccine SPf66 in Tanzanian infants, given alongside the EPI vaccines. This paper describes the humoral responses to SPf66 and the EPI vaccines. A total of 1207 infants were recruited into a two-arm, double-blind, individually randomized placebo-controlled trial of SPf66. The objectives of the trial were to determine the safety, immunogenicity and efficacy of SPf66 and to assess interactions with EPI vaccines when three doses of SPf66 were delivered alongside the EPI vaccines. Cross-sectional surveys were carried out to asses seroconversion rates to the EPI vaccines and the antibody response to SPf66 (NANP)50 and Plasmodium falciparum lysates. Seroconversion rates to EPI vaccines were high and no statistically significant differences in prevalence or titres were found between SPf66 and placebo recipients. IgG antibodies against SPf66 (NANP)50 and whole P. falciparum lysate were present in high titres in mothers of recruited children at the time of birth. Vaccination with SPf66 stimulated a good anti-SPf66 IgG response which declined to preimmunization levels by 2 years of age and which was not associated with protection against clinical malaria. The vaccine induced no IgG antibodies against (NANP)50 or P. falciparum lysates. SPf66 stimulated a humoral immune response when given to very young infants and did not interfere with seroconversion to EPI vaccines. The response to SPf66 was qualitatively different from that seen in older children, in whom SPf66 has been shown to be moderately efficacious. This difference raises concerns about the difficulties of immunizing very young infants who need to be targeted by antimalarial vaccination programs.

SUMMARY The control of tick populations by using conventional strategies poses several problems, including the appearance of organophosphate resistant strains, among others. The possibility of using alternative strategies such as vaccination with tick antigens has been suggested by several authors. One particular antigen (Bm86) has been described and shown to be able to induce a protective immunity against the cattle tick Boophilus microplus. In this paper we demonstrate by means of immunohistochemical staining that this antigen is conserved among several strains of this species. These results correlate with those showing that animals vaccinated with a preparation of recombinant Bm86 were protected against challenge with the four different strains tested, including one resistant to organophosphates. These results favour the immunization with recombinant Bm86 for the control of the cattle tick B. microplus.

SUMMARY Epidemiological evidence is widely cited in support of the hypothesis that certain antibody responses to human helminth, especially schistosome, infection exhibit blocking activity. This evidence includes positive correlations between antibody levels and the rate of re-establishment of infection following chemotherapy, antibody levels which peak in younger and more susceptible age classes, lower ratios of blocking antibodies to others in older and less susceptible age classes. In this paper simple mathematical models are used to explore expected age-specific relationships between antibody levels, parasite burdens and re-establishment rates for different combinations of protective, neutral and blocking immune responses. In general, all the above cited patterns may be generated without invoking blocking activity, especially if the abilities to produce different antibody responses have different immunological memories, i.e. persist for different lengths of time in the absence of continued exposure to antigen. None of these patterns, including a positive correlation between antibody levels and rates of re-establishment following chemotherapy, offers unambiguous evidence for blocking activity. Blocking activity is also predicted to affect the shape of the age-intensity curve and the relationship between susceptibility to infection and age in ways which are not necessarily consistent with the epidemiological evidence. The importance of the blocking activity, which has been convincingly demonstrated in vitro, to population level immunoepidemiological patterns in the field therefore remains uncertain.

Many field studies of human helminth infections have reported a positive correlation between parasite burdens and the rate of re-establishment of infection following chemotherapy, i.e., predisposition. Some studies have also reported the relationships between re-establishment and exposure and immune responses. The interpretation of these data is made difficult by the complexity of the underlying immunoepidemiological processes. In this paper, simple mathematical models are used to explore expected patterns, especially in relation to host age. These patterns are determined by rates of infection, parasite life expectancy, the level of immunological responsiveness, the duration of immunological memory, and may be greatly affected by the immunogenic roles of different parasite stages. In general, acquired immunity is predicted to reduce the degree of predisposition. This reduction is age-dependent and may generate 'negative predisposition' in some age classes. Age-dependent reductions in the correlation between re-establishment and exposure are also predicted. The correlation between re-establishment and protective immune responses is also predicted to be age-dependent, but may remain positive for all ages despite significant acquired immunity. The results suggest that great care is needed in the interpretation of immunoepidemiological data from treatment-reinfection studies.

Summary Experiments described in this paper were aimed at determining whether subcutaneous inoculation of live, avirulent Leishmania major would protect mice against infection by the virulent parasite. To this effect, promastigotes or amastigotes of a highly virulent strain of L. major (MRHO/IR/76), used in human trials of leishmanization, and which induces non-healing skin lesions in both CBA and BALB/c mice, were rendered non-pathogenic by gamma irradiation. A dose of 150 krad was required to abrogate the virulence of the parasite as tested on BALB/c mice. Strikingly, however, not all leishmanias were completely inactivated by this procedure since live parasites were detected in the footpads and/or the inguinal lymph nodes as long as 28 days (CBA) or 18 weeks (BALB/c) after injection. Furthermore, 150 krad-irradiated promastigotes retained the capacity to transform into amastigotes intracellularly in vitro. Subcutaneous inoculation of this irradiated ‘vaccine’ confered onto CBA mice a high degree of protection against challenge by both the homologous and a heterologous (MRHO/SU/59/P) strains of L. major. Lymph node cells from protected animals acquired the capacity to activate infected macrophages in vitro to kill intracellular L. major. To allow for maximum development of immunoprotection, the irradiated promastigotes had to remain viable, perhaps reflecting a requirement for transformation into amastigotes in the vaccinated host.

The KEK (Lysine-Glutamic acid-Lysine) motif is frequently found in the primary structure of certain malaria proteins involved in invasion, and plays an important role in the interaction of these proteins with the erythrocyte. This motif is contained in a peptide which forms part of the polymeric synthetic malaria vaccine SPf 66, currently undergoing extensive human trials. Analysis of the antibody titres against the subunit peptides that comprise this vaccine has shown that protection is associated with high titres to the KEK-containing peptide. In this paper we examine the fine recognition of this motif by polyclonal sera from protected vaccinated individuals, demonstrating the critical role played by the interacting ion pair formed between the amino terminal lysine (K) and glutamic acid (E), which act as contact residues for an important proportion of the antibody population directed against this vaccine. This ion pair in the KEK motif constitutes perhaps one of the most important malaria epitopes involved in protection, and could explain the mechanism through which protective immunity is acquired.

Summary Two mouse strains maintained in this laboratory (WEHI) are variably resistant to infection with Schistosoma japonicum and S. mansoni in that worms cannot be found in the liver and portal system in a high proportion (WEHI 129/J mice) or low proportion (C57BL/6 mice) some weeks after exposure to cercariae. Resistance can be as high as 100% in WEHI 129/J mice and is usually around 20% in C57BL/6 mice. The proportion of resistant mice closely parallels the proportion of mice that demonstrate a shunting of microbeads, injected into a mesenteric vein, from liver to lungs. This applies to F1 x WEHI 129/J backcross mice in which the data suggest oligogenic genetic effects although no evidence for a participation of MHC-linked genes in the phenomenon has emerged. 129/J mice derived from the Jackson Laboratory do not show a shunting of beads from the portal system to the lungs but their progeny bred at WEHI do. Germ-free WEHI 129/J mice resemble conventionally-maintained, SPF-derived WEHI 129/J mice in their variable resistance to schistosome infection. No satisfactory explanation for hepato-portal system peculiarities in WEHI 129/J and C57BL/6 mice can be advanced as yet and a possibility raised in this paper is a contribution from nutritional factors such as hypervitaminosis A superimposed on a genetic predisposition in these two related mouse strains.

Vaccines currently being evaluated against malaria are based on proteins derived from the blood, sporozoite and sexual stages. Antigens from the liver stage, which is now recognized as the major target of protective sporozoite induced immunity, have received comparatively little attention. This paper describes the generation of a monoclonal antibody (MoAb), which recognizes an antigen specific to the liver stage of the rodent malaria Plasmodium berghei. The antigen is expressed throughout liver stage development and appears to be localized to the parasitophorous vacuole membrane. The MoAb did not affect the growth of liver stages cultured in vitro nor could protection be demonstrated in vivo following passive transfer of the antibody.

Summary We have previously reported that Fab fragments of a monoclonal antibody to the 66 kD-related antigens of Plasmodium knowlesi inhibit merozoite invasion of erythrocytes, strongly suggesting a role for these antigens as merozoite receptors for red cells. In this paper we have examined the distribution of these antigens on the surface of free merozoites. Rapid immunofluorescence microscopy on free, unfixed cells demonstrated a preferential association of antigen with a polar region of merozoites. This was confirmed, and localized essentially to the apical region, by immunoelectron microscopy under a variety of fixation regimes. Metabolic inhibitors did not affect the distribution, suggesting that apical localization is not due to capping induced by cross-linking antibodies. These observations suggest that 66 kD-related antigens may play a role in the orientation of the apical prominence of merozoites towards the red cell, and/or in the junction formation that occurs subsequent to orientation.

Summary This paper reports the results of in vitro experiments which attempt to elucidate the mechanisms whereby Gambian children control acute infections of Plasmodium falciparum. It was shown initially that mononuclear cells from children with acute malaria, in the presence of specific antibody, caused a marked reduction in in vitro parasite growth. IgM antibodies appeared to be considerably more effective than IgG. T or B lymphocytes were ineffective in the system; adherent cells alone had some effect, but much less than the unfractionated cell population. Adherent cells were however fully effective after exposure to supernatants from T cells activated either non-specifically by phytohaemaggluti-nin (PHA), or specifically by P. falciparum antigens. Depression of parasite growth was also observed, independent of anti-malarial antibody. This was achieved when adherent cells from healthy Europeans, as well as those from infected children, were exposed to the supernatants from previously stimulated T cells before adding to the culture. Furthermore, intra-erythrocytic parasite death occurred after a short exposure to the supernatants of ‘activated’ adherent cells from both infected children and Europeans.

This paper describes experiments which demonstrated that the survival of Nematospiroides dubius was severely impaired in mice treated with immune serum. CFLP donor mice were given a series of infections ranging from 25 to 200 infective larvae, at weekly intervals for 6 weeks. The mice were treated with anthelmintic on day 21 and/or day 28 to prevent the accumulation of lethal numbers of parasites in the intestine, and were bled between day 42 and day 49. Female NIH recipient mice were given a total of 2.0-2.5 ml of immune serum i/p., in several separate smaller doses at various times in relation to the day of infection. Between the administration of immune serum begun during the first 4 days of infection and the animals being killed within the next 3 weeks, the mice harboured fewer worms than control animals, the worms were stunted and their fecundity was greatly reduced. Furthermore, these worms were subsequently lost from the intestines of treated mice, during and after the fourth week of infecton. These effects on N. dubius were not observed when mice were given normal serum nor when immune serum was administered after day 6 of the infection. The delayed rejection of adult worms from mice treated with immune serum is of particular significance and suggests that immune serum contained factors which facilitated the expression of a second component in worm expulsion not nornally effective in a primary infection. The possible immunologcal mechanisms underlying these findings are discussed and related to the immunosuppression which N. dubius is known to induce in the host.

In this paper, in-vitro experiments are reported which indicate that in dogs infected with the hookworm Ancylostoma caninum, there exists a cooperation between specific IgG antibodies and fixed complement components (C3). This relation is important in the adherence of peripheral blood leucocytes to the surface of the larvae. Fixation of complement occurs via the alternative pathway.

Summary The protozoan parasite Theileria annulata causes a severe disease of cattle in tropical countries; one stage in the parasite life cycle involves the transformation of bovine lymphocytes leading to rapid lymphoproliferation. Immunity to this disease is largely cell mediated and directed against the infected lymphocyte. In this paper we report the identification of three classes of infection specific antigen (using monoclonal antibodies) one of which is found on the surface of the lymphocyte. Such antigens can be used in parasite strain typing, in providing an understanding of the molecules involved in immunity and in providing the basis for a vaccine.

A simple method for producing monospecific rabbit antisera, applied originally to the constituents of human serum (Goudie et al. 1966), has been adapted for use with Schistosoma mansoni egg antigens. Replicate isolated immunoprecipitin arcs resulting from the immunoelectrophoretic reaction between an egg antigen and a polyspecific antiserum were excised, washed extensively to remove non-precipitated contaminants, homogenized, and emulsified with complete Freund's adjuvant. Rabbits were given weekly subcutaneous injections of the emulsion in multiple sites, and a monospecific precipitating antibody against the respective immunizing antigen generally resulted within 6 weeks of the commencement of immunization. Antisera raised in this manner against S. mansoni egg antigens omega 1, alpha 1 and kappa 5 have been used to characterize the antigen with respect to their stage- and species-specificity. After immunoabsorption to remove background activity, the sera could be used to detect unequivocally the respective antigens in crude egg homogenate that had been subjected to SDS-PAGE and electrotransfer to nitrocellulose paper.