Combined use of two separate but protective vaccine antigens provides protection against Taenia ovis infection in lambs in the presence of protective maternal antibody.

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.

Immunological responses and potency of the EG95NC- recombinant sheep vaccine against cystic echinococcosis.

Cystic echinococcosis (CE) is a zoonotic disease caused by the cestode parasite Echinococcus granulosus. The disease has an important impact on human health as well as economic costs including the cost of treatment as well as loss of productivity for the livestock industry. In many parts of the world where the disease is endemic, sheep and other livestock play an important role in the parasite's transmission. A vaccine to protect livestock against CE can be effective in reducing transmission and economic costs of the disease. A recombinant antigen vaccine has been developed against infection with E. granulosus (EG95) which could potentially be used to reduce the level of E. granulosus transmission and decrease the incidence of human infections. Further development of the EG95 recombinant vaccine as a combined product with clostridial vaccine antigens is one potential strategy which could improve application of the hydatid vaccine by providing an indirect economic incentive to livestock owners to vaccinate against CE. In this study we investigated the efficacy of the EG95 recombinant vaccine produced in Morocco by vaccination of sheep, including a combined vaccine incorporating EG95 and clostridia antigens. Vaccination with EG95 either as a monovalent vaccine or combined with clostridia antigens, protected sheep against a challenge infection with E. granulosus eggs and induced a strong, long lasting, and specific antibody response against the EG95 antigen.

The first report of hydatid disease (Echinococcus granulosus) in an Australian water buffalo (Bubalus bubalis).

A three year old female water buffalo was slaughtered for human consumption on a dairy buffalo farm in eastern New South Wales, Australia. Gross examination of the offal revealed four small, superficial hydatid cysts in the liver and two larger superficial cysts in one lung. All organs were sliced and no other cysts were found. Histology and PCR confirmed the cysts to be cysts of Echinococcus granulosus senso stricto. None of the cysts contained protoscoleces. The source ofinfection is equivocal, but it is most likely from E. granulosus eggs passed in the faeces of wild dogs (dingoes and dingo-wild dog hybrids). Wild dogs are resident in the bush that abuts the farm boundary and from time to time wild dogs are seen in the buffalo paddocks on the farm. Sylvatic transmission of E. granulosus occurs commonly in eastern Australia through a predator/prey interaction between wild dogs and macropod marsupials.

Echinococcosis: Control and Prevention.

Human cystic echinococcosis (CE) has been eliminated or significantly reduced as a public health problem in several previously highly endemic regions. This has been achieved by the long-term application of prevention and control measures primarily targeted to deworming dogs, health education, meat inspection, and effective surveillance in livestock and human populations. Human CE, however, remains a serious neglected zoonotic disease in many resource-poor pastoral regions. The incidence of human alveolar echinococcosis (AE) has increased in continental Europe and is a major public health problem in parts of Eurasia. Better understanding of wildlife ecology for fox and small mammal hosts has enabled targeted anthelmintic baiting of fox populations and development of spatially explicit models to predict population dynamics for key intermediate host species and human AE risk in endemic landscapes. Challenges that remain for echinococcosis control include effective intervention in resource-poor communities, better availability of surveillance tools, optimal application of livestock vaccination, and management and ecology of dog and wildlife host populations.

Microdiversity of Echinococcus granulosus sensu stricto in Australia.

Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

Monitoring the outcomes of interventions against Taenia solium: options and suggestions.

There is an increasing interest in reducing the incidence of human neurocysticercosis, caused by infection with the larval stage of Taenia solium. Several intervention trials are currently assessing various options for control of T. solium transmission. A critical aspect of these trials will be the evaluation of whether the interventions have been successful. However, there is no consensus about the most appropriate or valuable methods that should be used. Here, we undertake a critical assessment of the diagnostic tests which are currently available for human T. solium taeniasis and human and porcine cysticercosis, as well as their suitability for evaluation of intervention trial outcomes. Suggestions are made about which of the measures that are available for evaluation of T. solium interventions would be most suitable, and which methodologies are the most appropriate given currently available technologies. Suggestions are also made in relation to the most urgent research needs in order to address deficiencies in current diagnostic methods.

Sensitivity of partial carcass dissection for assessment of porcine cysticercosis at necropsy.

Many interventions against Taenia solium are evaluated by assessing changes in the prevalence of porcine cysticercosis ascertained by carcass dissection. Financial and logistical difficulties often prohibit dissection of entire pig carcasses. We assessed 209 pigs from rural areas of Cameroon and Peru for the presence of T. solium cysticerci and determined the distribution of parasites within the musculature of infected animals. Considering the presence of cysts in the tongue, masticatory muscles and heart, 31 of the 38 (81%) naturally infected animals were identified as having cysts. Dissection of only the tongue, masticatory muscles and heart provides a relatively sensitive and highly specific method for diagnosis of porcine cysticercosis.

Cysticercosis and echinococcosis.

Cysticercosis and cystic echinococcosis are zoonotic parasitic diseases commonly transmitted by livestock animals. Past and future efforts to reduce transmission of these diseases adopt a One Health approach where control measures are implemented largely in the parasites' animal hosts in order to bring about, indirectly, a reduction in human disease. New and highly effective vaccines have been produced which are capable of preventing infections with Echinococcus granulosus (cystic echinococcosis) and Taenia solium (cysticercosis) in their animal intermediate hosts. Application of vaccines, together with taeniacides in the parasites' definitive hosts, provides new opportunities for control of these diseases and a reduction in the global burden of human cysticercosis and cystic echinococcosis.

Antigenic differences between the EG95-related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination.

Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95-1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95-related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95-1G1 vaccine. The proteins (EG95-1G6 and EG95-5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95-1G1. Differences in the amino acid sequence of EG95-related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.

Oncospheral penetration glands are the source of the EG95 vaccine antigen against cystic hydatid disease.

Immunohistochemistry and immunogold labelling techniques were used to localize the EG95 vaccine antigen in Echinococcus granulosus oncospheres. In non-activated oncospheres, the cytoplasm of 2 pairs of bilateral cells exhibited specific positive labelling for the presence of EG95. No surface localization was seen in non-activated or recently activated oncospheres. Besides the staining of 2 pairs of bilateral cells, there was also a generalized distribution of specific staining for EG95 throughout the parenchyma of activated oncospheres. Immunogold labelling of non-activated oncosphere revealed specific reactivity for EG95 involving 2 pairs of bilateral cells and the ultrastructural characteristics of these cells were consistent with them being penetration gland cells. No other oncospheral structures stained specifically for the presence of EG95. The absence of surface location of EG95 in oncospheres suggests that the parasite may not be susceptible to vaccine-induced antibody and complement mediated attack until some post-oncospheral development has occurred. Further studies would be required to determine when the EG95 antigen associates with the parasite's surface, thus making them susceptible to immune attack.

Comparative pathology of pulmonary hydatid cysts in macropods and sheep.

The development and appearance of hydatid cysts of Echinococcus granulosus in experimentally infected tammar wallabies (Macropus eugenii) and sheep during the period 9-17 months post-infection (mpi) were studied. Cysts of unknown age were also examined from mature, naturally infected sheep. The cysts grew more rapidly and became fertile within a shorter period in wallabies compared with sheep. Cysts from the wallabies were larger in absolute size and were larger relative to the size of the lungs. Microscopical examination revealed that wallaby hydatid cysts developed in small bronchioles. Hydatid cysts in the wallabies had a thicker germinal membrane, with more nuclei and a thicker laminated layer (LL), than hydatid cysts of similar age found in sheep. In contrast, the adventitial layer was thicker in the ovine cysts, comprising a hyalinized layer of degenerate collagen and necrotic cellular debris surrounded by a layer of granulation tissue that was largely absent from lesions in the wallabies. Multilocular cysts were present in sheep, but not in wallabies. The greater thickness of the germinal membrane in wallaby cysts suggests greater parasite activity, which may explain the more rapid growth rate in this host, whereas the thicker adventitial layer in sheep cysts may be restrictive to growth while simultaneously protecting the hydatid from the host immune response. These differences in the parasite-host relationship between macropods and sheep may reflect the relatively recent introduction of the parasite into Australia.

Fact or hypothesis: Taenia crassiceps as a model for Taenia solium, and the S3Pvac vaccine.

Research undertaken over the past 40 years has established many of the general principals concerning immunity to taeniid cestodes. Although much is well understood about the host-protective mechanisms against taeniids and this knowledge has been exploited in studies on vaccine development, many aspects require further investigation or confirmation. Some phenomena have come to be regarded as being well established, while careful analysis of the published data would suggest that they may be better regarded as hypotheses rather than established facts. This review considers one selected issue pertaining to immunity to cestode infections and examines carefully the nature of the evidence that is available to support conclusions that have been made in this area. The issue examined is the use of Taenia crassiceps as a model for cysticercosis in pigs caused by Taenia solium, together with the S3Pvac vaccine, which has been developed based on this model. Strong evidence is found to support the conclusion that defined T. crassiceps antigens can limit intraperitoneal proliferation of the ORF strain of T. crassiceps in mice; however, the potential for these antigens to affect T. solium infection in pigs requires further confirmation.

Variation in the cellular localization of host-protective oncospheral antigens in Taenia saginata and Taenia solium.

Immunohistochemistry and immunofluorescence with confocal microscopy were used to localize the host-protective antigens of Taenia saginata (TSA9 and TSA18) and Taenia solium (TSOL16, TSOL18 and TSOL45). In nonactivated oncospheres, TSA9 and TSOL45 antigens were found primarily in the cytoplasm of the penetration gland type one (PG1) cell. A similar pattern of staining was seen for TSOL45 in oncospheres of T. solium that remained within the oncospheral membrane. In addition, there was less intense staining of TSA9 and TSOL45 in the quadri-nucleate penetration gland type 2 (PG2) cell. TSA18, TSOL16 and TSOL18 were predominantly found in the PG2 cell. In activated oncospheres that had escaped the oncospheral membrane, the antigens (other than TSA9) were seen both in the penetration gland cell locations and throughout the oncospheral parenchyma. Co-localization analyses revealed that only TSOL16 and TSOL18 antigens were co-localized in the PG2 cell of oncospheres that had not escaped the oncospheral membrane. However, in activated oncospheres that escaped the oncospheral membrane, all three antigens of T. solium were co-localized as they were present throughout the parenchyma. No positive staining was observed on the surface of nonactivated or recently activated oncospheres of T. saginata or T. solium.

Fact or hypothesis: concomitant immunity in taeniid cestode infections.

Sustained research efforts over the last 50 years have revealed a considerable amount of information about immunity to taeniid cestode infections in the parasites' intermediate hosts. As a product of this research, a series of effective recombinant vaccines have been developed which have no parallel in any other group of parasitic organisms. There are, however, many important aspects relating to immunity that remain to be elucidated. Some concepts have come to be firmly held as facts and yet the supportive data are either conflicting or unconfirmed. This review considers the phenomenon of immunity to re-infection with taeniid cestodes in their intermediate hosts, examining carefully the nature of the evidence that is available to support conclusions that have been drawn in this area.

Antibody responses to the host-protective Taenia solium oncosphere protein TSOL18 in pigs are directed against conformational epitopes.

TSOL18 is a recombinant protein that has been shown in repeated experimental trials to be capable of protecting pigs against challenge infection with the cestode parasite Taenia solium. Antibodies raised by the vaccine are capable of killing the parasite in an in vitro culture and it is believed that antibody and complement-mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. Investigations were undertaken to characterize whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N(-)) and the two truncations (TSOL18N(-)-1 and TSOL18N(-)-2) were used in inhibition ELISA. TSOL18N(-) was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N(-) antibodies to TSOL18N(-) in ELISA. However, neither TSOL18N(-)-1 nor TSOL18N(-)-2, either alone or when combined together, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N(-) antibodies with TSOL18N(-). It is concluded that the dominant antibody specificities, and probably the host-protective specificities, of TSOL18 are conformational epitopes.

Pig-farming systems and porcine cysticercosis in the north of Cameroon.

A survey was conducted in 150 households owning 1756 pigs in the rural areas of Mayo-Danay division in the north of Cameroon. A questionnaire survey was carried out to collect information on the pig-farming system and to identify potential risk factors for Taenia solium cysticercosis infection in pigs. Blood samples were collected from 398 pigs with the aim of estimating the seroprevalence of T. solium cysticercosis. The results showed that 90.7% of the pigs are free roaming during the dry season and that 42.7% of households keeping pigs in the rural areas have no latrine facility. Seventy-six per cent of the interviewed pig owners confirmed that members of the household used open-field defecation. Enzyme-linked immunosorbent assay (ELISA) for antigen and antibody detection showed an apparent prevalence of cysticercosis of 24.6% and 32.2%, respectively. A Bayesian approach, using the conditional dependence between the two diagnostic tests, indicated that the true seroprevalence of cysticercosis in Mayo-Danay was 26.6%. Binary logistic regression analysis indicated that a lack of knowledge of the taeniasis-cysticercosis complex and the absence of a pig pen in the household were associated with pig cysticercosis.

The ultrastructure of taeniid cestode oncospheres and localization of host-protective antigens.

Taeniid eggs contain an infective larval form of the parasite, known as the oncosphere, which has been found to be highly susceptible to attack by the host's immune system and this fact has been exploited in the development of highly effective vaccines. Relatively little is known about the structure of taeniid oncospheres and the localization of host-protective antigens within or on the oncosphere. Here, we briefly review the current state of knowledge of the structure of the oncosphere and present preliminary data on the localization of a host-protective antigen within the oncospheres of Taenia ovis. The precise localization of the antigens, in the context of a detailed knowledge of the ultrastructure of the parasite, may reveal the immune mechanisms by which the taeniid parasites are killed by vaccine-induced immune responses, which, in turn, may provide clues about how vaccines could be developed against other parasitic helminths.

Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: a study using the hydatid vaccine EG95.

The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.

Preliminary field trial of a vaccine against coenurosis caused by Taenia multiceps.

Taenia multiceps is a taeniid cestode that in its adult stage lives in the small intestine of dogs and other canids. In the intermediate hosts, the larval stage of T. multiceps causes coenurosis, a common disease in the CNS of ruminants, which typically leads to the death of the infected animals. Recent research into new methods for control of coenurosis and other taeniid cestode infections such as hydatidosis has identified vaccination as a potentially valuable new tool. In order to test the applicability of vaccination as an approach for control of T. multiceps infection in sheep, a field trial was carried out against natural infection in Sardinian farms (Italy) with recombinant proteins of T. multiceps. The recombinant proteins with Quil A as adjuvant were injected subcutaneously, the first administered to lambs at 10-12 weeks of age and a booster dose given after 2-4 weeks. A total of 632 sheep were selected, belonging to the "replacement quota" of six different farms, of which 424 were used as controls (unvaccinated) and 208 were vaccinated. After a period of more than 40 months from the beginning of the field trial, 33 episodes of cerebral coenurosis occurred in the monitored farms, including 32 cases in control sheep and l case in a vaccinated animal. Statistical analysis revealed a significant reduction in the number of coenurosis cases in the vaccinated animals (chi(2)=14.08, P<0.001). This is the first successful field test of a practical vaccine against T. multiceps and, considering the high degree of effectiveness achieved, could be a prelude to routine application in field situations of particular risk, such as Sardinia.