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Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

The EG95 vaccine is effective in protecting grazing animals from infection with Echinococcus granulosus. Six male lambs were used in the study, two were each vaccinated subcutaneously with 50μg EG95/1mg Quil-A, two animals were each vaccinated with 50μg EG95/1mg Quil-A in 1% chitosan thermolabile gel subcutaneously, and two animals served as non-vaccinated controls. Two vaccinations were given at a 7 week interval. Two vaccinations induced a significantly higher antibody titre in the chitosan group compared with the Quil-A only group. The chitosan vaccine group also had a significantly higher antibody titre compared with a positive control sera from vaccinated and challenged sheep. Incorporating the EG95/Quil-A vaccine in a thermo-responsive chitosan sol-gel stimulated, after the second injection, a high level of antibody absorbance which remained high for at least one year. This response was significantly greater than the response to vaccine without the gel.

SUMMARY The tapeworm Echinococcus granulosus is the causativeagent of hydatid disease and affects sheep, cattle, dogs andhumans worldwide. It has a two-stage life cycle existing asworms in the gut of infected dogs (definitive host) and ascysts in herbivores and humans (intermediate host). Thedisease is debilitating and can be life threatening where thecysts interfere with organ function. Interruption of the hyda-tid life cycle in the intermediate host by vaccination may bea way to control the disease, and a protective oncosphereantigen EG95 has been shown to protect animals againstchallenge with E. granulosus eggs. We explored the use ofrecombinant vaccinia virus as a delivery vehicle for EG95.Mice and sheep were immunized with the recombinant vec-tor, and the result monitored at the circulating antibodylevel. In addition, sera from immunized mice were assayedfor the ability to kill E. granulosus oncospheres in vitro.Mice immunized once intranasally developed effective onco-sphere-killing antibody by day 42 post-infection. Antibodyresponses and oncosphere killing were correlated and weresignificantly enhanced by boosting mice with either EG95protein or recombinant vector. Sheep antibody responses tothe recombinant vector or to EG95 protein mirrored those inmice.Keywords Echinococcus granulosus, EG95, hydatid, recom-binant poxvirus vaccine, vaccinia virus

The parapoxvirus, orf virus (ORFV) causes superficial skin lesions in infected sheep. Unattenuated ORFV is used globally to vaccinate against orf. Recombinant poxviruses are proven delivery systems and we investigated strategies to express the immunogenic Echinococcus granulosus peptide EG95 from ORFV with the aim of developing a recombinant bivalent vaccine. EG95 is an oncosphere protein of the cestode E. granulosus, a parasite responsible for causing cystic hydatid disease in a wide range of hosts including humans and grazing animals such as sheep. Recombinant viruses were produced in which EG95 was expressed by itself or fused to ORFV envelope-associated structural proteins 10 kDa and F1L. Infection studies in sheep showed that specific antibodies were produced against ORFV and EG95 and that the antibody levels against EG95 were comparable to that of animals immunized with purified EG95 in Quil A adjuvant, an immunization regime that is known to afford protection. A single exposure to the dual vaccine has potential for protecting lambs against orf and for priming against EG95 so as to respond strongly to a later injection of EG95 protein.

An oil-based formulation of the EG95 vaccine to protect grazing animals against infection with Echinococcus granulosus was formulated in Argentina. The efficacy of the vaccine was monitored by serology in sheep and llama (Lama glama) and was compared to the serology in sheep previously published using a QuilA-adjuvanted vaccine. Long-term efficacy was also tested in sheep by challenging with E. granulosus eggs of the G1 strain 4 years after the beginning of the trial. The serological results for both sheep and llama were similar to those described previously, except that there was a more rapid response after the first vaccination. A third vaccination given after 1 year resulted in a transient boost in serology that lasted for about 12 months, which was similar to results previously described. Sheep challenged after 4 years with three vaccinations presented 84·2% reduction of live cysts counts compared with control group, and after a fourth vaccination prior to challenge, this reduction was 94·7%. The oil-based vaccine appeared to be bio-equivalent to the QuilA vaccine.

Although immunity to Echinococcus granulosus in sheep has been shown to be antibody-mediated and complement-dependent and can be passively transferred in colostrum, in animals vaccinated with EG95, the relationship between protection against an oral challenge infection with E. granulosus eggs and anti-EG95 IgG ELISA absorbance values at the time of challenge has not been satisfactorily proven. Using a combination of results from three EG95 vaccination trials, we have found that the IgG ELISA absorbance at the time of challenge infection explains approximately 50% (P ≤ 0·001) of the variability in the percentage protection against an oral challenge with E. granulosus eggs (transformed with arcsin).

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite's natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.

Germline transformation of a parasitic nematode of mammals has proven to be an elusive goal. We report here the heritable germline transformation of Parastrongyloides trichosuri, a nematode parasite whose natural hosts are Australian possums of the genus Trichosurus. This parasite can undergo multiple free-living life cycles and these replicative cycles can be maintained indefinitely in the laboratory. Transformation was achieved by microinjection of DNA into the ovary syncytium of either free-living or parasitic adult females. By selecting for the transgenic progeny of successive free-living life cycles, it was possible to establish and maintain transgenic lines. All three transgenic lines tested were shown capable of establishing patent infections in possums and to transmit the functional transgene to their progeny. The transgene, driven by the Pt hsp-1 promoter, was constitutively expressed in intestinal cells at all stages of both parasitic and free-living life cycles, although gene silencing appears to occur in some transgenic progeny. This is the first report of heritable transgenesis in a parasitic nematode of a mammal and we discuss a variety of previously inaccessible experimental avenues that will now be possible with this powerful model system.

Parastrongyloides trichosuri is a nematode parasite of Australian brushtail possums that has an alternative free-living life cycle which can be readily maintained indefinitely in a laboratory setting. The ability to maintain this parasite in a free-living cycle and induce it to parasitism at the free-living L1 stage makes this an excellent model for the study of genes associated with parasitism. A 70kD protein from infective larvae of P. trichosuri that appears to be immunogenic in infected possums has been identified as a heat shock protein (Hsp)70 homologue. The complete gene for Pt-Hsp70 was cloned and sequenced. The protein encoded by the Pt-Hsp70 gene is the likely orthologue of the Caenorhabditis elegans protein, Hsp70A, also known as hsp-1. Reverse transcriptase-PCR data indicate that Pt-Hsp70 (designated Pt-hsp-1) is expressed at readily detectable levels in all developmental stages of both the parasitic and free-living P. trichosuri life cycles and the promoter is mildly inducible by heat shock. Bioinformatic analysis of expressed sequence tag databases indicates that C. eleganshsp-1 homologues, together with C. eleganshsp-3 homologues, are the predominant members of the Hsp70 superfamily that are normally expressed in parasitic stages of the Strongyloididae family. Promoter fusions to a beta-galactosidase coding sequence were prepared and introduced into wild type C. elegans to produce transgenic nematodes. Reporter gene expression was clearly present within embryonic cells and within intestinal cells of larval and adult stages. Thus, the expression of the Pt-hsp-1 promoter within P. trichosuri and transgenic C. elegans appears similar to the known expression of C. elegans hsp-1. This promoter should be of value in efforts to develop genetic manipulation tools for P. trichosuri.

Despite the importance of spatial processes in host-parasite interactions, parasite dispersal has been the subject of few experimental studies. Introduced marsupial common brushtail possums (Trichosurus vulpecula) are a major environmental and agricultural pest in New Zealand. Parastrongyloides trichosuri, an intestinal rhabdiasoid nematode parasite specific to possums, is being evaluated as a self-disseminating delivery system for engineered fertility control vaccines. This study addressed whether an artificial infection could be established in a naïve, free-living possum population, by measuring the post-release dynamics of possum-parasite interactions at the release site, and by following the spread of the parasite into surrounding possum populations. Infection was established efficiently by applying infective larvae to the skin of possums on a single occasion. All experimentally infected possums recaptured 3 weeks after infection had parasite eggs in their faeces. Over the subsequent 2.5 years, infection spread steadily over an area of about 6000 ha. Infection persisted at the original release site for the 3.5 years of the study and at a nearby site infected by natural spread for more than 3 years. Seasonal changes in faecal egg counts were similar at the two sites. The rapid establishment of the parasite and its spread provide additional support for its ongoing development as a vaccine delivery system.

Introduced Australian brushtail possums (Trichosurus vulpecula) in the North Island, New Zealand, are infected commonly with the possum‐specific nematode, Parastrongyloides trichosuri. In the South Island, however, the parasite was thought to be confined to the Longwood Range, Southland, site of the first successful introduction of possums to New Zealand. Current, more widespread surveys revealed, however, that the parasite was distributed widely but discontinuously across Southland and southern Otago, and restricted to the southern third of the Longwood Range. Repeated sampling of possums on two transects along the north‐west and south‐east of the Range suggested that the parasite may be spreading northwards naturally. We hypothesise, therefore, that P. trichosuri has infected possums in the Longwood Range only relatively recently. A similar recent introduction of the parasite to Otago and Southland could also explain its current limited distribution. The most likely explanation for a recent int...

To assess fully the potential of parasites for biological control of Australian brushtail possums (Trichosurus vulpecula) introduced to New Zealand, factors affecting prevalence and intensity of infection need to be determined. The present study investigated local spatial variation in endoparasite infection in possums at sites along a 24 km forest margin transect, lower North Island, New Zealand, where most of the endoparasite species recorded from possums in New Zealand were present. Sampling sites varied little in the parasite species present in possums, or the prevalence and intensity of infection. Possum sex and age had much stronger effects on prevalence and intensity of infection than sampling site. Most site variation in prevalence and intensity of infection occurred among the Trichos‐trongylus spp., for which possums are not the primary hosts, presumably reflecting variation in contact between possums and the primary hosts.

To assess fully the potential of parasites for the biological control of Australian brushtail possums (Trichosurus vulpecula) in New Zealand, factors affecting the prevalence and intensity of such infections need to be determined. In this study we investigated the effects of season, sex, and age on endoparasite infection in possums from a single forest/farmland site in the lower North Island, New Zealand. Seasonal changes in prevalence and intensity of infection were found for most of the parasites, but the patterns of change varied between species. Parasite prevalences were generally similar in male and female possums, except for the Trichostrongy‐lus spp., which were more prevalent in males. The prevalences of Parastrongyloides trichosuri, Paraustrostrongylus trichosuri and Trichostrongy‐lus retortaeformis were generally lower in young animals (0–1 year) and in animals aged 4 years and older. A body condition index and prevalence of infection showed a significant negative relationship for cocci...

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

Endoparasites may be used for the future biological control of possums (Trichosurus vulpecula) in New Zealand. Because of this we determined the parasite fauna of possums from six areas of the North Island of New Zealand (Hawke's Bay, Castlepoint, Northland, Orongorongo, Para‐paraumu, and Palmerston North). Parastrongyloid.es trichosuri, Trichostrongylus spp., and Eimeria spp. were present in all areas, and Bertiella trichosuri in all but Paraparaumu. Paraustrostrongylus trichosuri was not present in the Northland area. The intensity of infection in high prevalence areas was relatively similar in each region. Paraustrostrongylus trichosuri was the nematode with the highest prevalence and intensity of infection. Comparisons made between our data and records from the South Island, offshore islands of New Zealand, and Wanganui (North Island) show that most of the specific endoparasites of possums, with the exception of Adelonema trichosuri, are present in the lower North Island.

As part of a search for pathogens that might be useful agents for biological control of possums, the three largest offshore islands of New Zealand that still have possums were surveyed to determine the pathogens present in these isolated populations. Brushtail possums from Kawau Island (n = 158), Chatham Island (n = 214) and Stewart Island (n = 194) were examined for internal parasites. Possums from Kawau Island were infected with Eimeria spp. (16.7%), Bertiella trichosuri (5.2%) and Purustrongyloides trichosuri (15.5%). No Paraustrostrongylus trichosuri or Trichostrongylus spp. were found. Possums from Chatham Island were infected with Eimeria spp. (10.9%). Bertiella trichosuri (3.6%), T colubriformis (6.6%), T retortaeformis (1%) and T. vitrinus (0.5%). No Parastrongyloides or Paraustrostrongylus spp. were found. Possums from Stewart Island were infected only with Eimeria spp. (4.6%). Because of their paucity of some parasites, the opportunity exists to use these offshore islands to study the introduction and spread of a parasite into a possum population, and what technology would be required to bring it to hyperendemicity.

The ectoparasites of the brushtail possum Trichosurus vulpecula were examined at 15 original release sites in New Zealand. Three species of fur mite—Atellana papilio Domrow, 1958, Petrogalochirus dycei (Domrow, 1960), and Murichirus anabiotus Domrow, 1992—were present in all population samples from the North, South, Stewart, and Kawau Islands. M. anabiotus was absent from the Chatham Islands. A. papilio was isolated from 82 (98%) of the 84 digested pelts, P. dycei from 78 (93%) of 84, and M. anabiotus was found in 67 (93%) of the 72 pelts digested from populations where the mites were present. The follicle mite Marsupiopus trichosuri Fain, 1968 was detected in two (7%) and one (3%) of pelts examined from Kawau Island and the Orongorongo Valley, respectively. Trichosurolaelaps crassipes Womersley, 1956 was present on 99% of possums and absent only from Kawau Island. Nymphs and larvae of the cattle tick Haemaphysalis longicornis were isolated from four (33%) of the 12 Northland pelts. The loss of e...

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.

The methodology and results of a parasite survey of 209 possums from Wanganui, western North Island, in the spring of 1994 are described. No somatic protozoan parasites were detected, but 23% of possums had coccidia, Eimeria sp. Oocysts in their faeces. Prevalences of intestinal nematodes were: Parastrongyloides trichosuri (55%), Trichos‐trongylus spp. (59%), and Paraustrostrongylus trichosuri (2.4%). Only one animal was found infected with the cestode Bertiella trichosuri. It was demonstrated that prevalence of Parastrongyloides trichosuri was affected by age and habitat and Trichostrongylus spp. by habitat. Four species of external parasites were found: the mites Tricho‐surolaelaps crassipes (49%), Petrogalochirus dycei (65%), Atellana papilio (81%), and Murichirus anabiotus (32%). The parasite fauna of possums in New Zealand appears to be less diverse than that in their native Australia.

SummarySeveral years have elapsed since the publication by Johnson et al. (1989) of the cloning of a recombinant antigen from the cestode parasite Taenia ovis which stimulated high levels of protective immunity in sheep. A great deal of subsequent research and development was necessary to bring the fledgling vaccine to the point of being a registered commercial product. The results of these subsequent studies are dealt with briefly in this paper, including the results of field trials. The T. ovis vaccine was registered by the New Zealand Animal Remedies Board in February 1994. Where then is the commercial product ? This paper gives a background to market problems which have emerged through the politics (and realities) of the NZ T. ovis control campaign. It serves as notice that the best science dedicated to producing vaccines or products for parasitic, or other, diseases often faces significant hurdles in the real world of commerce and politics.

Cattle may act as hosts for the transmission of the cestode parasite Echinococcus granulosus and play a role in transmission of the parasite leading to human cystic echinococcosis (CE). The recombinant EG95 vaccine has been shown to be able to protect cattle and other intermediate host species against CE. Ideally the immunisation of bovines against E. granulosus, using EG95 vaccine, should occur early in life so as to provide maximum protection against the establishment of hydatid cysts. Maternally derived antibody from vaccinated cows may provide some protection for the neonate, but may also interfere with the active response to vaccination. Experiments were undertaken to determine the optimal regime for protection of young cattle against CE. One group of pregnant cattle received 2 vaccinations of EG95 antigen+Quil A adjuvant two months and one month prior to calving. The control group of pregnant cattle were not vaccinated. Calves were either challenged with E. granulosus eggs at 4, 9, 13 or 17 weeks post-birth or were given their first vaccination at 8, 12 or 16 weeks post-birth. Sera obtained at regular intervals were tested by ELISA to assess the immunological response. All calves were experimentally challenged with E. granulosus eggs and subsequent necropsy confirmed the levels of protection. Maternal antibody was shown to protect calves to some extent for at least 17 weeks. Calves from vaccinated cows responded well serologically if the first vaccination was given at 8 or 12 weeks, but full protection against a challenge infection was achieved only if the first vaccination was delayed until 16 weeks after birth. Calves from non-vaccinated cattle also were not fully protected if the first vaccination was at 8 or 12 weeks, but were fully protected if the first vaccination was given when they were 16 weeks old. This suggests that immunological maturity is not acquired in calves until 4 or 5 months of age. No safety problems were observed following two vaccinations of 40 pregnant cows or 30 suckling calves.

Cystic echinococcosis is endemic in the Rio Negro province of Argentina. After 30 years of control using praziquantel in dogs the transmission rate to humans and sheep has decreased significantly, however transmission persists. The objective of the study is to assess the impact of the inclusion of the EG95 vaccine for sheep in the control programme, including analysis of the vaccine's operative feasibility in field conditions. The vaccine was applied in an area comprising four communities of native people including 79 farms with 3146 lambs and 311 dogs in total. Seventy one farms were designated as control areas where no vaccinations were undertaken while vaccinations of lambs undertaken on 91 farms. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection when the animals were 1-1.5 years of age. Farm locations were defined using GPS coordinates for the houses. Evidence for Echinococcus granulosus transmission was monitored by coproantigen ELISA on samples of dog faeces, by E. granulosus-specific PCR using soil samples, and anti-E. granulosus antibody assessments in sera from 2 to 4 teeth lambs, purgation of dogs to detect E. granulosus worms and necropsy on adult sheep. Before the vaccine was introduced, 26.2% of sheep with 2-4 teeth were positive using ELISA/WB, the prevalence decreased to 7.8% at the third year following use of the vaccine. Necropsy of animals older than 6 years (not vaccinated) showed that 66.1% of animals were infected with E. granulosus. In dogs, 4% was found positive for E. granulosus using arecoline purgation and 24.7% of the farms were infected using coproELISA/WB. During the first year of vaccination 2721 lambs received the first vaccine dose and 2448 received a booster. In the second year 2138 lambs were initially vaccinated and 1745 received a booster, and 1308 animals received the third dose. During the third year 1110 lambs received the first dose from which 539 received a booster and 723 animals received the third dose. An analysis of advantages and limitations of the diagnostic techniques used and the ability of the geospatial analysis to detect risk area are included. Based in the immunodiagnostic techniques, the EG95 vaccine has been able to prevent the infection in animals up to 3 years old. Also, the difficulties in the field for the correct vaccine administration and the social features and habits that may impact on echinococcosis control are included in the analysis.

Hydatid disease is an important human zoonosis. Humans become infected from carnivores that are infected with the Echinococcus granulosus tapeworm. Carnivores become infected after consuming hydatid cysts from grazing animals, which are generally sheep, goats and cattle. A vaccine, known as EG95, can protect sheep and goats against cystic echinococcosis. This paper describes the adaptation of the EG95 vaccine for use in cattle. The monitoring of results used serology and also infection with E. granulosus eggs, followed by necropsy. Immunisation with living E. granulosus oncospheres showed that cattle could be immunised against E. granulosus. Immunisation of cattle with EG95 plus QuilA was also successful. A dose-response and adjuvant trial showed best results were achieved with 250 μg of antigen and 5mg of the adjuvant QuilA, which was 5 times the recommended sheep dose. After two vaccinations given one month apart, 90% protection was maintained for 12 months. At 12 months a third vaccination boosted protection to 99% which was maintained for a further 11 months.

7 páginas. -- The definitive version is available at http://www.journals.elsevier.com/acta-tropica/#description, This paper is based on the experience of the authors, with the aim to define the challenges for Echinococcus granulosus (E.g./CE) diagnosis and control for those countries that may now or in the future be contemplating control of hydatid disease. A variety of methods are available for diagnosis in humans but a universal gold standard is lacking. Diagnosis in definitive hosts can avoid necropsy by the use of methods such as coproantigen detection but test performance is variable between populations. A sylvatic cycle adds challenges in some countries and the epidemiology of the parasite in these hosts is poorly understood. Control by solely administering praziquantel to dogs is not effective in developing countries where the disease is endemic. Additional avenues to pursue include the instigation of participatory planning, use of an existing vaccination for intermediate hosts and development of a vaccine and long-acting anthelmitic implants for definitive hosts. Promoting public acceptance of control of the dog population by humane euthanasia and reduced reproduction is also essential.

A heat-labile component of normal sheep serum (56 degrees C for 30 min but not 50 degrees C for 30 min) was able to lyse oncospheres in vitro. The degree of effect, and the proportion of oncospheres lysed, was related to the concentration of normal unheated sheep serum complement, or other sources of complement (rabbit, mouse) in the culture. Lower concentrations were required for lysis if the culture serum was obtained from sheep immune to E. granulosus infection. Heat inactivation of normal or immune sheep serum removed any lytic ability. No lysis occurred in any concentration of unheated foetal lamb serum. However, unheated foetal lamb serum was able to restore the lytic effect to heated normal or immune serum. This suggests that lysis in both immune and normal serum is antibody-dependent and complement-mediated. The lysis in normal serum would appear to be due to natural cross-reacting antibodies that can fix complement at the oncosphere surface. The complement lesion resulted in damage to the plasma membrane. This then peeled back, predisposing the oncosphere to osmotic destruction. The use of bleach to dissolve the embryophore caused damage to the plasma membrane similar to that caused by complement. Developing metacestodes at 3 days of age in vitro in immune serum were susceptible to the addition of complement at that time.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).

Background Human cystic echinococcosis (CE) is highly endemic in the Tibetan regions of Sichuan where most families keep guard dogs and where there are considerable numbers of ownerless/stray dogs. Strong Buddhist beliefs do not allow for elimination of stray dogs, and many strays are actually fed and adopted by households or monasteries. On account of the high altitude (3900–5000 m), pasturage is the major agricultural activity in this area. The harsh mountainous climate often leads to many grazing animals dying on the pasture at the end of a hard winter. The skin and some meat are taken, and the rest of the animal is left for scavenging birds and animals. The poor sanitation and hygiene, the Buddhist doctrine of allowing old livestock to die naturally, plus the unrestricted disposal of animal viscera post-slaughter may be responsible for the high prevalence of human CE in this setting. Methods and Findings As part of a large collaborative control program for CE in Ganzi County, situated in the west of Sichuan Province, surveillance for Echinococcus infection in domestic dogs using a coproantigen method and necropsy of unwanted dogs was carried out prior to (in 2000) and after (in 2005) dog anthelminthic treatment (5 mg/kg oral praziquantal at 6 month intervals) to determine the efficacy of the treatment for control. The prevalence of E. granulosus only in dogs by necropsy was 27% and 22%, and prevalence of both Echinococcus spp. by necropsy was 63% and 38%; prevalence of both Echinococcus spp. by coproantigen analysis was 50% and 17%. Necropsy of sheep/goats (age, Author Summary Human cystic echinococcosis (CE) is highly endemic in Tibetan regions of Sichuan. As part of a control program for CE in Datangma district, Ganzi County, necropsy of strays and coproantigen-ELISA of all dogs was carried out prior to and post-drug treatment to determine the efficacy of the treatment for control. Examination of sheep/goats and yaks was undertaken to determine the baseline transmission pressure to dogs. The necropsy results indicated no apparent significant change in the overall prevalence of E. granulosus in unwanted dogs after 5 years of 6-month treatment. In contrast, there was a highly significant decrease in Echinococcus prevalence in domestic/stray dogs after the 5-year treatment program shown by coproantigen-ELISA. This indicated a decreasing but continuing risk for re-infection of dogs resulting from high infection pressure from the numerous infected domestic animals. DNA genotyping indicated the presence only of the sheep strain (G1) of E. granulosus in the study area. A combination of livestock vaccination with the highly effective Eg95 vaccine and dog drug treatment, targeting two critical points of the parasite life-cycle, would avoid the conflicts of religion or local culture and achieve the goal of hydatid control in the long term in the area.

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.

To the Editor: The People’s Republic of China accounts for >500,000 cases of echinococcosis and more disability-associated life years (DALYs) lost because of this disease than any other world region (1,2). Hydatid cysts of Echinococcus granulosus (cystic echinococcosis [CE]), or the more pathogenic lesions with multiple vesicles caused by E. multilocularis infection (alveolar echinococcosis [AE]), usually grow slowly in the liver, so that severe illness and death may eventually occur in a high proportion of those with untreated infections (3,4). Apart from surgery, long-term anthelminthic therapy (>6 months) with the benzimidazole compound albendazole, although parasitostatic only, has a beneficial outcome in >50% of cases (5). To control the transmission of this zoonosis, veterinary public health measures must be emphasized (6). In 2004 the Chinese Ministry of Health (MoH) undertook a nationwide assessment of 8 parasitic diseases, including malaria, schistosomiasis, and echinococcosis. To identify echinococcosis, 7 provincial MoHs carried out a mass abdominal screening of 34,500 persons using portable ultrasound scanners. The overall prevalence (2.5%) was highest in Tibetan communities in the Tibet Autonomous Region and in northwestern Sichuan and Qinghai Provinces (these latter regions form part of the eastern Tibetan Plateau). Collaborative studies involving the Sichuan Center for Disease Control and Prevention (based in Chengdu) and an international consortium of research institutes partly funded by the US National Institutes of Health (Bethesda, MD, USA) have shown an increasingly serious public health problem at the village, township, and county levels. In Shiqu County of Ganze Tibetan Autonomous Prefecture, 414 (12.9%) of nearly 3,199 persons surveyed by ultrasound (with serologic confirmation) exhibited CE or AE, including 19% in this category (7). The effects of human echinococcosis are substantial, with >50,000 DALYs lost in a population of 63,000 in Shiqu County (8). Despite increased urbanization in China, >70% of Tibetans still live as seminomadic pastoralists on the high grasslands at an altitude >3,500 m. Most Tibetan herdsmen families keep at least 1 dog, and large numbers of ownerless stray dogs are tolerated by pastoralists and Buddhist monks. Risk factors for human echinococcosis (both CE and AE) in Tibetan communities usually include occupation, age (older persons are at higher risk), gender (higher risk for female), environment (pastoral landscapes), livestock ownership, and a history of dog ownership, as well as indicators of low socioeconomic status, including poor water quality and illiteracy (7,9) The prevalence levels of human AE in Ganze Tibetan Autonomous Prefecture (Sichuan Province) are among the highest recorded anywhere in the world. This situation presents a formidable challenge for early diagnosis, optimal affordable treatment, and prevention and control. Markham Hospital in Aba Tibetan Autonomous Prefecture (Sichuan) performed 1,200 operations for echinococcosis from 1992 through 2005, 20% for AE disease. For remote, high-altitude, pastoral Tibetan communities, however, long-term albendazole therapy is the only realistic treatment option, but regular follow-up of patients is difficult in these poorly accessible communities. To address the public health concerns and consider options for controlling hydatidosis/echinococcosis in the eastern Tibetan Plateau, an International Workshop on Treatment, Prevention and Control of Echinococcosis was held in Chengdu in May 2006 with support from the Sichuan Center for Disease Control and Prevention, MoH Beijing, the New Zealand International Aid and Development Agency [NZIAD]), Fogarty-National Institutes of Health (USA), Xinjiang Medical University, and the Boulder-Lhasa Sister City Project. Recommendations stressed the following public health needs: improved treatment centers within the known disease-endemic counties or prefectures for long-term follow-up of patients after surgery and chemotherapy for both CE and AE disease, a better understanding of the epidemiology and ecology of transmission, and planning for pilot control interventions against both CE and AE transmission. NZAID made a detailed report of the implementation and effects of a pilot echinococcosis control program (2000–2006) in Datangma County, Ganze Tibetan Autonomous Prefecture (Sichuan Province). Problems occurred chiefly because of poor intersectoral cooperation, difficult logistics, cultural antagonism, lack of participatory planning, difficult access, treatment of dogs (with praziquantel), vaccination of livestock with the new EG95 vaccine (6), and lack of adequate surveillance of dog and livestock infection levels. The report indicated how many of these difficulties could be overcome. Consequently, the People’s Republic of China MoH and provincial disease control networks approved funding in July 2006 to initiate pilot intervention programs against echinococcosis in 17 Tibetan autonomous counties of northwest Sichuan. Control options initially focused on regular supervised dosing of owned dogs and stray dogs (with praziquantel) by local operatives from district disease control centers and on improving health education at primary healthcare levels. Surveillance relies on measuring regularly the degree of Echinococcus infection in dogs by using a coproantigen test. Also, a specific age cohort of schoolchildren is monitored by ultrasound and serologic testing each year to determine changes in the prevalence of the 2 diseases. Albendazole is provided free, and the cost of surgery for hydatid disease is also subsidized through the new National Rural Cooperative Medical Insurance System. In addition to the major public health problem now being recognized for echinococcosis in Tibetan communities, their general health indices are low (higher prevalence of tuberculosis, bone diseases such as arthritis, and poorer health in general) because of living and working at altitudes >4,000 m, compared with those in most other areas of China. Access and outreach should be improved (in conjunction with animal health initiatives) (10) for effective delivery of treatment, vaccination, and health education packages to these largely scattered and marginalized pastoral communities.

Recently an immunological test for Echinococcus spp. antigens in dog faeces has been developed. The antigens appear to be carbohydrates, which survive proteolytic digestion and environmental degradation. For ELISA a capture antibody is used to capture the antigens, followed by a detection antibody. This paper describes a modification of the test whereby capture and detection antibodies are generated exclusively to the carbohydrate portion of the parasite tegument. Faecal extracts were heated to 70 °C overnight and the addition of foetal calf serum to the extracts was not necessary. The use of this modification as a surveillance tool in an extensive field trial of hydatid control in Western Sichuan is described. From 2003 onwards all dogs received a treatment with praziquantel pills in the spring and the autumn of each year. On each of six occasions 580 faecal samples were collected from 29 villages and analysed. Prevalence of Echinococcus spp. coproantigen-positive samples was 50% in year 2000, and decreased from 35% to 17% through 2003–2005. This coproantigen technique is now being used as part of the Chinese National Hydatid Disease Control Program, initially in 10 counties in Sichuan Province.

Control of hydatidosis is less effective without the support of dog-owners, and this support can only be obtained if the people have a clear understanding of the life-cycle of the hydatid parasite(s) and what risk factors contribute to human infections. Dissemination of this information is the biggest challenge for hydatid control. Participatory planning between dog-owners and community leaders should evaluate the possible control technologies, and should enable a choice of those aspects that suit the sociology and economic status of the particular community. Collection of baseline data is essential, as is on-going surveillance. Hydatid control should be mainly self-funded, which again requires the support of the dog-owner. A pilot hydatid control program for Tibetan herdsmen is described.

As part of a study to assess whether endoparasites could assist in the biological control of brushtail possums in New Zealand, we investigated the composition and distribution of possum endoparasites in the South Island. Possums were collected near five of the original release areas in the South Island : Banks Peninsula, Hokitika, Nelson, Dunedin and Invercargill. Among the nematodes, those most frequently encountered were Trichostrongylus spp., which were present in possums from all five study areas. Trichostrongylus species from possums in the Invercargill area comprised 4.5% T. colubriformis, 0.9% T. vitrinus and 11.3% T. retortaeformis. Paraustrostnmgylus trichosuri and Parastrongyloides trichosuri were found only in the Invercargill area, where they infected 1.4% and 14% of possums respectively. The cestode Bertiella trichosuri was present in possums from all locations except Dunedin. The protozoan Eimeria spp. occurred in all areas. These are the first records of Parastrongyloides trichosuri, Paraustrostrongylus trichosuri, T. vitrinus, T. retortaeformis and Eimeria spp. in South Island possums. The prevalence of endoparasites and the intensity of infection was very low compared to the lower North Island of New Zealand. Endoparasites at the existing levels in the South Island probably have very little effect on possum populations.

To determine the efficacy of eprinomectin, doramectin and a combination of albendazole and levamisole in suppressing or eliminating nematode infections or faecal egg counts (FEC) in possums naturally or experimentally infected with Parastrongyloides trichosuri, Paraustrostrongylus trichosuri and Trichostrongylus colubriformis.To establish an effective dose of eprinomectin, groups of naturally infected possums were treated with 0, 0.5, 2.5, 5.0 or 7.5 mg/kg liveweight (LW) eprinomectin pour-on (n=6 possums/group) and changes in FEC and nematode worm counts at necropsy determined, 18 days later. Efficacy of the 7.5 mg/kg dose was re-examined in a second group of naturally infected possums (n=12) by monitoring FEC weekly for 28 days post-treatment. Persistence of the anthelmintic effect of doramectin injection was tested using nematode-free possums treated with 0, 0.2, 0.4, 0.6 or 0.8 mg/kg LW (n=3 possums/ group), which were experimentally infected 14 days later with T. colubriformis, Paraustrostrongylus trichosuri and Parastrongyloides trichosuri infective larvae. Response to treatment was assessed by FEC and nematode worm counts at necropsy, 42 days posttreatment. Efficacy of a 1.0 mg/kg dose of doramectin was subsequently examined using naturally infected possums (n=11) by monitoring FEC weekly for 28 days post-treatment. To determine the efficacy of a levamisole-albendazole combination drench, possums with naturally acquired nematode infections (n=6) were treated orally with 37.5 mg/kg LW levamisole plus 23.75 mg/kg LW albendazole on 2 occasions, 7 days apart, and response to treatment was assessed by monitoring FEC for 57 days.Eprinomectin 7.5 mg/kg LW reduced Paraustrostrongylus trichosuri worm counts by 98 % (p0.05). Doramectin 0.6 mg/kg LW reduced Parastrongyloides trichosuri and Trichostrongylus spp worm counts by 99% (p0.05) and 0.8 mg/kg LW reduced Paraustrostrongylus trichosuri by 100% (p0.05), in possums challenged with larvae 14 days after treatment. Treating possums with a levamisole-albendazole combination orally, twice, 7-days apart, reduced FEC by 99%.The doses of anthelmintics required to effectively control nematodes in possums were higher than those recommended for animals for which they are currently registered. Possums tolerate the high dose rates of anthelmintics used in this study without apparent adverse effects.

Hydatid disease is a zoonotic parasitic disease that is distributed widely around the world and causes substantial human morbidity and mortality, particularly in developing countries. Reduction of human hydatid disease using anthelmintics, together with changes in human lifestyle and animal management practices, have been unsuccessful in some developing countries where the disease still persists. Substantial progress has been made towards developing a practical, recombinant vaccine in sheep, to interrupt the lifecycle of Echinococcus granulosus and to prevent subsequent transmission from dogs to humans. This review focuses on the scientific advances in the development of a recombinant vaccine for hydatid disease and the remaining challenges facing the widespread use of the vaccine for control of hydatid disease in endemic areas.

Previous trials of an experimental Taenia ovis vaccine using the recombinant antigen GST--45W(B/X) established that it was possible to achieve90% protection against a single artificial challenge of T. ovis eggs. This trial was undertaken to assess vaccine efficacy against artificial challenge and natural infection acquired by lambs grazing contaminated pasture. Two hundred Romney lambs were vaccinated at 6 and 12 weeks of age. One hundred control lambs were not vaccinated but were allowed to run with the vaccinated mob. At 15 weeks of age, 10 controls and 18 vaccinated lambs were artificially challenged with 2000 T. ovis eggs. The remaining control and vaccinated lambs were allowed to graze contaminated pasture for 3 weeks and were then moved to clean pasture for 5 months. The artificially challenged lambs plus 24 of the field-infected lambs were slaughtered and the carcasses dissected to obtain cyst counts. The remaining field-infected lambs were slaughtered at a commercial processing plant and the carcasses examined by conventional meat inspection. The results showed that the vaccine provided a high level of protection against artificial challenge (92%) and natural infection (98%) when assessed by carcass dissection. The data from commercial meat inspection showed that vaccination provided 89% efficacy against downgrading or condemnation compared to non-vaccinated control lambs. The average difference in carcass values between vaccinated and non-vaccinated groups was 4.36 dollars, representing a 35% loss in value due to T.ovis infection in non-vaccinated lambs.

Cystic echinococcosis, caused by the larval stage of Echinococcus granulosus, is a global public health problem. Whilst in a few localities, such as New Zealand, the parasite has been effectively controlled or even eradicated, in most endemic regions it remains a persistent problem. In some areas, such as the former Soviet Union, the disease incidence in humans has increased rapidly in recent years. It is important to have an understanding of the transmission dynamics, both between dogs and domestic livestock where the parasite maintains itself and from dogs to people. It is from this knowledge that effective control measures can be devised to reduce the prevalence of the parasite in animals and hence reduce the incidence of human disease. Mathematical models to describe the transmission of the parasite and the effects of different control strategies were first proposed over twenty years ago. Since then further information has been acquired, new technology has been developed and better computing technology has become available. In this review, we summarise these developments and put together a theoretical framework on the interpretation of surveillance information, how this affects transmission and how this information can be exploited to develop new intervention strategies for the control of the parasite. In particular, the parasite remains a persistent or re-emerging problem in countries of low economic output where resources for an intensive control programme, that has been successful in rich countries, are not available. By understanding of the transmission biology, including mathematical modelling, alternative and cost-effective means of control can be developed.

Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.

A vaccine to protect sheep, goats, and bovines against hydatid disease caused by the cysts of Echinococcus granulosus is prepared as a recombinant fusion protein expressed in Escherichia coli. Solubilised inclusion bodies are injected, together with Quil A, subcutaneously on two occasions 1 month or more apart, and induce protection against infection which lasts for at least 12 months. A third injection given 6-12 months after the second injection induces a high and long-lasting protection against artificial or natural challenge infections. This review describes work carried out on the formulation, safety and efficacy of the vaccine under laboratory and field conditions, using artificial or natural challenges with E. granulosus eggs, followed by necropsy. Hydatid control programmes based on regular treatment of all dogs with the correct dose of a highly-efficient anthelmintic have sometimes not been successful in Continental environments. Access to dogs is difficult in summer because of the distances to summer pastures, and is often impossible in winter because of snow. A control program using strategic twice-yearly anthelmintic treatment of dogs is likely to be successful provided grazing animals are vaccinated as well. Vaccination as a control tool only requires the veterinarians to visit two times a year, and while the veterinarian is present, the dogs can be treated with anthelmintic for little additional cost. One visit should take place after the autumn kill of animals for winter consumption, and this is a good time to vaccinate animals born in the summer, and also all other animals while they are healthy and immunologically responsive. The other visit should take place in the spring, at which time animals born during winter can be vaccinated. Although a single immunization has been shown to induce a useful degree of protection, where possible it is best to give two initial injections, 1 month apart. If it is possible for veterinarians to stay in the field for 2 months in November/December and March/April, in order to give the two injections, a more rapid onset of full protective immunity will initially be achieved than if the injections are given 6 months apart. A large-scale safety and efficacy trial involving 50,000 and 100,000 lambs in Qinghai and Xinjiang Provinces of China has taken place. Results have confirmed safety and efficacy. In most countries, prevalence of infection increases with age. The vaccine has no effect on established cysts, and therefore, in order to prevent the biomass of Echinococcus spp. from increasing, it might be an effective strategy to begin a control programme by vaccinating all animals. Because many of the older stock will already be infected, they will remain a source of infection for dogs for the average lifetime of the stock. Dogs will still be able to be infected from the older stock, and will continue to infect humans. We advocate that a vaccination programme be accompanied by education about hydatid disease, and anthelmintic treatment of dogs in late autumn and early spring.

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.

Infections with the larval stages of taeniid cestode parasites cause substantial human morbidity as well as economic losses in domestic livestock species. Despite ongoing efforts around the world, few countries have been able substantially to reduce or eradicate these infections through the use of anthelmintics and lifestyle changes. Vaccines offer an additional potential tool to assist with the control of parasite transmission. Here, Marshall Lightowlers and colleagues review the substantial progress that has been made towards developing practical vaccines against hydatid disease in sheep and cysticercosis in sheep and cattle. Recombinant antigens have been used to induce more than 90% protection against challenge infections. Such success in animals encourages investigation of the potential use of vaccines in humans to prevent hydatid disease arising from infection with Echinococcus granulosus and cysticercosis from infection with Taenia solium.

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.

Experimental vaccine trials against hydatid disease have been undertaken in sheep using the EG95 recombinant vaccine. Challenge infection was with viable Echinococcus granulosus eggs obtained from a New Zealand isolate (dog/sheep cycle), an Australian isolate (dingo/wallaby cycle) and an Argentine isolate (dog/sheep cycle). Vaccination with EG95 conferred a high degree of protection against challenge with all three parasite isolates (protection range 96-100%). Taken together, the trials demonstrated that 86% of vaccinated sheep were completely free of viable hydatid cysts when examined approximately 1 year after challenge infection. Vaccination reduced the number of viable cysts by 99.3% compared with unvaccinated controls. These results suggest that the EG95 vaccine could have wide applicability as a new tool for use in hydatid control campaigns.

Antibody isotype and epitope specificities were examined in sheep immunized with EG95, a protective recombinant vaccine against hydatid disease. All sheep immunized with EG95 as a fusion protein with glutathione S-transferase (GST) produced prominent IgG antibodies against the EG95 portion of the protein. Linear, antibody-binding epitope specificities of EG95 were mapped using a series of 25 overlapping synthetic peptides. Three immunodominant regions were identified which generated specific IgG1 and IgG2 antibodies in the majority of vaccinated sheep. These regions corresponded to the EG95-derived sequences SLKAVNPSDPLVYKRQTAKF, DIETPRAGKKESTVMTSGSA and SALTSAIAGFVFSC. An additional immunogenic region was identified which induced almost exclusively IgG2 antibody. This epitope was located within the sequence TETPLRKHFNLTPV. The anti-parasitic, protective effects of the EG95 vaccine correlated with the detection of specific antibody to two or more of the four linear immunogenic regions. The identification of these immunogenic peptides of EG95 maybe useful in the development of a synthetic peptide vaccine as a derivative of the EG95 recombinant.

Vaccination of intermediate hosts will effectively reduce the prevalence of human echinococcosis. The rate of progress can be enhanced by concurrent tactical treatment of definitive hosts. The oncosphere of Echinococcus granulosus contains a specific protein which when expressed on the oncosphere surface will combine with specific antibody In the presence of complement, damage is caused to the plasma membrane, resulting in failure of the oncosphere to develop into a metacestode. Immunisation of sheep with oncosphere secretions or with an oncosphere homogenate results in 99% resistance to a challenge infection of the sheep with E. granulosus eggs. Immunisation of sheep with the specific purified protein results in 92% resistance. The gene coding for the protein has been isolated from an E. granulosus oncosphere cDNA library and expressed in E.coli as a fusion protein with glutathione-S-transferase. A monoclonal antibody to an epitope on the E. granulosus portion of the molecule is able to bind to the surface of oncospheres and to prevent their development to metacestodes in vitro. Immunisation of sheep with the fusion protein has given 97 and 98% resistance to a challenge infection in two separate trials. The shelf-life of formulated vaccine is at least 12 months, and the immunity generated by two injections given 1 month apart persists for at least 12 months. A homologue of the gene has been identified in E. multilocularis. The vaccine has the potential to be used as a tool for control of transmission of E. granulosus through its natural intermediate hosts (particularly domestic ungulates) as part of hydatid control programmes. Traditional methods of hydatid control (anthelmintic treatment of dogs) require regular treatment within the prepatent period for prolonged periods (20-30 years) to achieve satisfactory control, and often sociological factors to do with dog-feeding, dog ownership and dog-control prevent such an achievement. By controlling the disease in both the definitive host and the intermediate host, the shortcomings of either procedure can be overcome, resulting in a cost-effective desired result within a practical time period.

The paper points out the essential aspects of host/parasite relationship during the development of oncospheres (ONC) of Echinococcus granulosus into an early cyst. Secretions of the penetration glands in the hatched ONC causes lysis of host tissue during penetration of the activated ONC. It also protects the parasite against the host's immune response while developing the laminated layer. The microvilli, compressed under the plasma membrane in the hatched ONC, are uplifted in the activated larvae; they increase in number and size and are substituted by short and truncated microtriches in metacestodes 3 days old. At that time appears the first lamination of the laminated layer that surrounds the metacestodes as an electron-dense matrix composed of fine microfibrillated material and remnants of sloughed microvilli. The second lamination appears by day 6–8 and is more electron-dense than the first one. The laminated layer is of parasite origin, formed of a series that emanates from the germinal membrane. It is suggested that Vg secretory vesicles, that are elaborated in the perikaryon of the germinal zone and continuously carried to the syncytium via the microtubular cytoskeleton, are responsible for the laminated layer formation and are involved in initial evasion of the immune response of the host. The cyclical production of laminations could be necessary to create layers that can ultimately be sloughed off as the cyst grows and serve to divert the host cellular response to the parasite.

Hydatid disease (echinococcosis) has a two-host cycle involving the domestic dog and grazing animals. Humans are also infected by the dog. Both unilocular (Echinococcus granulosus in yaks, sheep, and goats) and multilocular (alveolar) (E. multilocularis in hares and rodents) hydatids are common in western Sichuan in the People's Republic of China. Humans and dogs are equally infected with both species. Many yaks (Bos grunniens) were found with multilocular cysts that visually were deemed to be E. multilocularis. However, a histologic and molecular study showed that they were actually E. granulosus. No infective cysts were found in 125 necropsied yaks. We conclude that the yak is an inadequate and dead-end host for the sheep dog (G1) strain of Echinococcus granulosus and also for E. multilocularis.

Immunoblotting and in vitro oncosphere-killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus, and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non-denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity-purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18–12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Sheep were fully protected against challenge infection following immunization with a homogenate of T. ovis oncospheres. Ultracentrifugation of sonicated oncospheres either alone or in the presence of a range of detergents did not reduce the immunogenicity of the extracts. Solubilization of oncosphere extracts in non-ionic detergents or sodium dodecyl sulphate (SDS) enabled analysis of host-protective antigens by isoelectric focusing (IEF) and electrophoresis in polyacrylamide gels (SDS-PAGE), respectively. Immunoblotting analysis of oncosphere antigens with immune sheep sera identified predominantly two groups of antigens with relative mobilities of 31–34 kDa and 47–52 kDa with a common isoelectric point of 5.8. The immunogenicity of these antigens was confirmed in vaccination trials using appropriate fractions cut from SDS-PAGE gels and agarose IEF gels. Affinity-purified antibodies prepared against the candidate antigens were used to select the corresponding recombinant DNA-derived polypeptides, one of which was subsequently found to be host-protective.