Comparative susceptibility of two different genetic types of tilapia to Neobenedenia sp. (Monogenea)

Two different genetic types of tilapia, Mozambique tilapia Oreochromis mossambicus (MT), and Pargo-UNAM (PU; a synthetic hybrid whose genetic composition is 50% Florida red tilapia, 25% Rocky Mountain tilapia, and 25% red variant Oreochromis niloticus), were acclimatized to salinity and exposed to seawater from the Gulf of Mexico off the port of Veracruz, Mexico. Both fish types were infected by the monogenean ectoparasite Neobenedenia sp. and were killed within 2 to 3 wk. A crude worm extract was prepared from whole specimens collected during the original outbreak and used to immunize naive hosts of the same 2 types of tilapia. Immunized fish were then exposed to seawater, which resulted in Neobenedenia sp. infection. Immunization did not confer any protection against Neobenedenia sp. infection. However, the experiment enabled detailed analysis of the dynamics of infection and comparison of the effects of the parasite on the 2 host types. Although both tilapia types exhibited similar resistance to infection (as they harbored similar parasite burdens in the early phase of infection), PU is less tolerant to Neobenedenia sp., as a mean parasite abundance of ca. 50 worms fish-' killed all hosts within a fortnight, while 22% of MT survived up to 3 wk, harboring a mean parasite abundance of ca. 900 worms fish-'. Our results suggest that, as reported elsewhere, Neobenedenia sp. could negatively affect mariculture off the Mexican coast of the Gulf of Mexico.     

Functional relationship between sealworm (Pseudoterranova decipiens, Nematoda, Ascaridoidea) burden and host size in Atlantic cod (Gadus morhua).

A model is proposed to describe the accumulation of sealworm parasites (Pseudoterranova decipiens, Nematoda, Ascaridoidae) in Atlantic cod (Gadus morhua). The model predicts the total amount of food eaten by a fish to reach its size. In young fish growth is linear in time and average parasite burdens are predicted to increase with the cube of the average length of the fish divided by its growth rate. An analysis of worm burdens in cod caught by the commercial fishery in the Firth of Clyde (Strathclyde, Scotland) between 1964 and 1970 illustrates how average parasite abundances at age, length, or fixed time of the year can be compared. It is concluded that exposure did not vary significantly over these years. Extensive apparent inter-annual variations in worm burdens of cod newly recruited to the fishery are well explained by differences in the age composition of cod catches.     

Schistosoma japonicum: protective immunity induced by schistosomulum-derived cells in a mouse model

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.     

Infection of immunosuppressed mice with the abomasal nematode parasite of ruminants, Haemonchus contortus

Infective larvae of Haemonchus contortus established in mice either immunosuppressed with the corticosteroid, dexamethasone, or the cytotoxic drug, cyclophosphamide, or treated with the histamine H2 inhibitor, cimetidine. Infections persisted for as long as the immunosuppressive treatment (7 days) and growth of larvae was similar to that seen in sheep. Virtually no larvae survived in untreated mice. Accordingly, it would appear that adaptive immunity is an important barrier against primary infection by H. contortus in mice and is a determinant of host-range for this parasite. Antibody raised in either sheep or mice against soluble extracts of adult H. contortus precipitated with different but overlapping sets of worm antigens. This suggests that the unique antigens recognized by the mouse compared with the sheep are crucial for the rapid protective responses which prevent primary infection.     

Nippostrongylus brasiliensis: local humoral responses in the lungs and intestines of rats following vaccination with irradiated larvae

Groups of rats were infected with 2000 normal larvae of Nippostrongylus brasiliensis or larvae irradiated with 10 to 120 kR. On Day 10 after infection half the animals from each group were autopsied. The remainder were challenged with 5000 unirradiated larvae on Day 15 and killed ten days later. During the experiment enteric antibody levels were estimated by coproantibody measurement. At autopsy the worm burdens were determined and worm-specific antibodies evaluated in lung extracts and serum. It was found that the levels of coproantibody detected with adult worm metabolites were positively correlated with the number of adult nematodes recovered from the intestine after primary infection. The challenge induced a similar increase of these antibodies in all immunised rats which reflected a high immunity to reinfection of vaccinated animals. Preliminary immunochemical studies suggested that the coproantibodies had SIgA properties. In lung extracts of rats immunised with larvae irradiated at 40, 80, or 120 kR and in all animals after challenge, antibodies reacting with infective larval antigens were found. Their titres were negatively correlated with serum antibody levels. The significance of bronchial and enteric antibodies in conferring protection against challenge remains to be elucidated.     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

The cellular and humoral immune response to Schistosoma mansoni infections in inbred rats: I. Mechanisms during initial exposure

Fischer rats were infected with Schistosoma mansoni by exposure to cercariae. The effects of animal age at exposure and the size of that exposure were determined by quantitation of subsequent worm burdens. Optimal conditions for assay of protection mediated by cells or serum transferred from exposed donors were ascertained. The major initial protective immune response to infection was mediated by thymusdependent lymphocytes as demonstrated by discrete subpopulation cell transfer experiments. Simultaneously harvested serum enhanced worm survival and obviated the protective effects of transferred cells when serum and cells were given concomitantly. Subsquently, host immunity became dependent on antibody formation. In the chronic phase of infection, a small number of surviving parasites coexisted with antibody which specifically interacted with schistosomal antigens but did not protect in transfer experiments. These studies suggest a form of immunologic enhancement with antibody modulation of cellular immunity as possible components of the immune response to both acute and chronic schistosomiasis.     

Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes

BALB/c mice can be protected against a fatal Leishmania major infection by immunization with whole radio-attenuated promastigotes; however, neither the antigens responsible for protection nor the protective immunologic mechanisms have been defined. In this study, the ability of promastigote fractions to elicit similar immunity to that obtained with whole organisms, and the immune responses associated with such protection were analyzed. Intraperitoneal immunization with a soluble, membrane-free parasite extract was found to induce protection against L. major challenge equal to that obtained with whole organisms. Induction of immunity (89% protection in seven experiments) was most effective with 100 micrograms of the soluble leishmanial antigen (SLA) and required concomitant injection of the bacterial adjuvant, Corynebacterium parvum (CP), followed by an i.p. boost of SLA alone 1 wk later. Vaccinated animals exhibited Leishmania-specific cell-mediated immunity, as assessed both by lymphocyte transformation and the production of macrophage-activating factors (MAF). In addition, although SLA + CP-immunized mice failed to exhibit delayed-type hypersensitivity (DTH) before challenge, splenic lymphocytes from these mice could transfer a local DTH reaction to naive recipients. Immunization also induced the production of antibodies against two major metabolically labeled proteins of m.w. 30,000 and 53,000, but failed to stimulate a detectable humoral response against promastigote surface antigens. Thus, these experiments demonstrate that vaccine-induced immunity against cutaneous leishmaniasis is strongly associated with the induction of cell-mediated immunity, but does not require the development of an antibody response to promastigote surface antigens. In addition, these studies establish the feasibility of employing soluble, nonmembrane-derived parasite material as a source of protective immunogens.     

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.     

Schistosoma mansoni: reduced worm burdens in mice immunized with isolated Fasciola hepatica antigens.

Mice immunized with Fasciola hepatica antigens are protected to a challenge exposure with Schistosoma mansoni cercariae. This protection is manifested in a 28–54% reduction in worm burdens of the immunized mice over controls. The protective antigens could be isolated by antibody affinity chromatography and react with an antiserum to S. mansoni. These antigens, when used to immunize mice, result in 50–60% reduction in worm burdens over controls. One protective antigen has been isolated which when used alone or in combination with a B-cell adjuvant such as polyadenylic-polyuridylic acid (poly (AU)) results in 56–81% reduction in worm burdens over controls. The complexity of the F. hepatica adult worm antigens was demonstrated by Laurell crossed immunoelectrophoresis. Crossreactivity with antisera to S. mansoni and S. japonicum and the presence of one common antigen between the two genera have been demonstrated.     

Schistosoma mansoni-New Zealand rabbit model: resistance induced by infection followed by active immunization with protective antigens

In previous studies, rabbits immunized with adult worm antigens released from fresh adult schistosomes incubated in saline media showed a significant level of protection against challenge parasites. Focusing on the rabbit-Schistosoma mansoni model, concomitant immunity was investigated. A peculiar form of response to cercarial infection was observed: rabbits subjected to percutaneous infection and similar reinfections at different times after primary infection killed schistosomula from the challenge infection as well as established parasites from the primary infection. In this study the challenge infection stimulus was replaced by active immunization with an adult worm-derived protective antigenic mixture. The results show that immunization of New Zealand rabbits with an adult worm antigenic extract is capable of inducing a response that results in a significant reduction of the mean worm burden of the primary infection earlier than did homologous infection, as compared to worm reduction due to a second infection.     

Immunization of Atlantic salmon against the salmon louse: identification of antigens and effects on louse fecundity

Injection of extracts derived from adult caligid copepods induced a partial immunity to Lepeophtheirus salmonis in Atlantic salmon. Antigens were derived from the supernatant of adult lice extracts and were partially purified by Con A affinity chromatography. The antigens were also present in the pellet derived from adult lice extracts, Immunohistochemistry showed that antibodies present in the serum of rats immunized with the Con A purified extract bound predominantly to the gut of L. salmonis. Components of apparent sizes > 205 000, 165 000, 133 000, 130 000, 125 000, 114 000, 110 000, 96 000, 82 000, 78 000, 65 000, 46 000, 35 000, 31 000 and < 29 000 were present in the extract. Although the numbers of attached lice were initially higher on those fish in the immunized group, throughout the course of the challenge experiment the total numbers of lice did not vary greatly between the immunized or control groups of fish. Initially, an overall average of about 17 attached stages were recorded from each fish sampled and by the end of the experiment this figure had fallen to about five adult lice per fish. However, compared with control fish fewer gravid female lice were present on immunized fish and furthermore these lice possessed fewer eggs (P<0.01). No major differences in egg hatching success were recorded. Further exploitation of this would require the isolation and purification of the antigens responsible for the observed effects. The possibilities for the development of a salmon louse vaccine are promising.     

Natural Regulatory T Cells in Malaria: Host or Parasite Allies?

Plasmodium falciparum malaria causes 500 million clinical cases with approximately one million deaths each year. After many years of exposure, individuals living in endemic areas develop a form of clinical immunity to disease known as premunition, which is characterised by low parasite burdens rather than sterilising immunity. The reason why malaria parasites persist under a state of premunition is unknown but it has been suggested that suppression of protective immunity might be a mechanism leading to parasite persistence. Although acquired immunity limits the clinical impact of infection and provides protection against parasite replication, experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to the aetiology of severe disease. Thus, an appropriate regulatory balance between protective immune responses and immune-mediated pathology is required for a favourable outcome of infection. As natural regulatory T (T_reg) cells are identified as an immunosuppressive lineage able to modulate the magnitude of effector responses, several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during malaria. The main findings to date are summarised in this review and the implication for the induction of pathogenesis and immunity to malaria is discussed.     

Epizootiology of spirorchiid infection in green turtles (Chelonia mydas) in Hawaii

We describe the epizootiology of spirorchiid trematode infections in Hawaiian green turtles (Chelonia mydas) by quantifying tissue egg burdens in turtles submitted for necropsy and by assessing antibody response to crude adult worm and egg antigens among a variety of age groups. Hapalotrema sp. and Laeredius sp. predominated in turtles infected with spirorchiids. Tissue egg burdens decreased with increasing size and increased with deteriorating body condition of turtles. No relationship was found between tissue egg burdens and sex or fibropapillomatosis status. Tissue egg burdens increased in turtles from southeast to northwest in the main Hawaiian Islands (Hawaii to Kauai). Hatchling and captive-reared turtles had significantly lower levels of antibodies against crude worm and egg antigens. Based on tissue egg burdens and antibody status, we hypothesize that immature turtles become infected with spirorchiids shortly after recruiting into coastal foraging pastures from the pelagic environment, that infection levels decrease with age, and that spirorchiids detrimentally affect the body condition of sea turtles independent of tumor burden. The low intensity of infection in turtles with the endemic trematode Carettacola hawaiiensis suggests either that turtles are less susceptible to infection with this parasite or that the parasite is outcompeted by species of Hapalotrema and Laeredius. Given that the 2 latter species are found in the Pacific and other oceans, they are not likely endemic and were probably introduced into Hawaii through an undetermined route.     

Protective efficacy of a cloned Brugia malayi antigen in a mouse model of microfilaremia

Immunization of mice with irradiated Brugia larvae or parasite extracts has been shown to induce partial resistance to microfilaremia and enhance clearance of infective larvae. We recently reported the cloning of a 548 amino acid 62-kDa Brugia malayi Ag identified on the basis of reactivity with antisera to a subset of protective microfilarial Ag. Our study describes the protective efficacy against microfilaremia in mice, immunogenicity, and parasite stage-specificity of this candidate vaccine molecule. Immunization of Swiss or BALB/c mice with 1 to 3 micrograms of a 92-kDa trpE fusion protein encoding amino acids 1-479 reduced the intensity of microfilaremia by 40 to 60% compared to control animals given buffer or bacterial trpE (p less than 0.01 to 0.001). Mice immunized with the 92-kDa fusion protein developed delayed-type hypersensitivity reactivity to B. malayi as assessed by enhanced footpad swelling 24 and 48 h after intradermal injection of adult worm extract and in vitro lymph node mononuclear cell proliferation (3H-thymidine uptake) in response to the fusion protein (mean +/- SD stimulation index 4.7 +/- 0.8 vs 2.0 +/- 1.4 for trpE, p less than 0.05). Proliferative responses of lymph node cells coincubated with three other fusion proteins corresponding to the filarial protein truncated from its carboxyl-terminus suggest that dominant T cell epitopes of the 62-kDa Ag are encompassed by amino acids 437-479. Rabbit antibody to the 92-kDa trpE fusion protein immunoprecipitated a 62-kDa polypeptide from [35S] methionine biosynthetically labeled B. malayi microfilariae, adult female, and adult male worms. These data indicate that a recombinant Ag expressed in several developmental stages of B. malayi is capable of inducing partial resistance against microfilariae and Ag-specific T cell responses in mice.     

Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis

To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccina- tions, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protec- tive immunity against schistosomiasis.     

Expression of Plasmodium falciparum 3D7 STEVOR proteins for evaluation of antibody responses following malaria infections in naïve infants

Clinical immunity to Plasmodium falciparum malaria develops after repeated exposure to the parasite. At least 2 P. falciparum variant antigens encoded by multicopy gene families (var and rif) are targets of this adaptive antibody-mediated immunity. A third multigene family of variant antigens comprises the stevor genes. Here, 4 different stevor sequences were selected for cloning and expression in Escherichia coli and His6-tagged fusion proteins were used for assessing the development of immunity. In a cross-sectional analysis of clinically immune adults living in a malaria endemic area in Ghana, high levels of anti-STEVOR IgG antibody titres were determined in ELISA. A cross-sectional study of 90 nine-month-old Ghanaian infants using 1 recombinant STEVOR showed that the antibody responses correlated positively with the number of parasitaemia episodes. In a longitudinal investigation of 17 immunologically na?ve 9-month-old infants, 3 different patterns of anti-STEVOR antibody responses could be distinguished (high, transient and low). Children with high anti-STEVOR-antibody levels exhibited an elevated risk for developing parasitaemia episodes. Overall, a protective effect could not be attributed to antibodies against the STEVOR proteins chosen for the study presented here.     

Antibodies against Discocotyle sagittata (Monogenea) in farmed trout

The relationship between Discocotyle sagittata intensities and host length, weight and specific anti-parasite antibody titres was studied in 3 year-classes of farmed rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta at the end of the annual transmission cycle. Antibody titres were significantly higher in infected farmed fish than in naive controls, indicating that infection elicits immunoglobulin production. No correlation was found between host size and parasite burdens, nor between infection intensities and antibody titres.     

Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

A preliminary evaluation of factors affecting an experimental system for vaccination-and-challenge with Haemonchus contortus in sheep

A preliminary evaluation of factors affecting an experimental system for vaccination-and-challenge with Haemonchus contortus in sheep. International Journal for Parasitology 19: 169-175. Studies were made with Haemonchus contortus in sheep to ascertain the influence of a range of factors in the domain of the host, the parasite or the vaccine on the formulation of protocols for vaccination-and-challenge to be used in identifying protective immunogens. The results corroborate earlier findings that protective immunity can follow vaccination with homogenates of parasites and show that initial processing of parasites for a vaccine leaves protective immunogen in a functional state. Sonicates of adult worms produced protective immunity and were identified as raw stock in which to prospect for candidate immunogens. By contrast, sonicates of infective larvae and exsheathing fluid invoked no significant protection and were not accredited for the same purpose. In an experiment unaccompanied by protective immunity, ewes contained lower worm burdens than castrate males indicating that vaccination experiments should be made with hosts of one sex only. Again in an experiment unaccompanied by protective immunity, Freund's complete adjuvant increased susceptibility to infection compared with Freund's incomplete adjuvant or no adjuvant implying a profound and persistent interference from killed mycobacteria on resistance against H. contortus.