Vaccines against cysticercosis and hydatidosis.

The recombinant vaccines that have been developed against cysticercosis and hydatidosis in sheep and cattle are remarkable for their effectiveness and are prominent as examples of the very few non-living vaccines against parasitic diseases. Their development has been through practical application of molecular parasitology, utilising immunochemical techniques in antigen identification and recombinant DNA methods in antigen production. This brief overview discusses the contribution of molecular techniques to the successful development of recombinant vaccines against Taenia ovis, Taenia saginata and Echinococcus granulosus as well as the immunological and genomic studies that have arisen from their development.     

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.     

Taenia saginata derived synthetic peptides with potential for the diagnosis of bovine cysticercosis

Immunity in Taeniids is predominantly antibody mediated and thus many serological immuno-determinants will have potential in both protection and diagnosis. The antigenicity of six peptides derived from four potentially protective molecules cloned from a Taenia saginata oncospheres cDNA library have been evaluated as targets for the specific diagnosis of bovine cysticercosis. The six peptides consist of: two peptides (HP6-2 and HP6-3) derived from the sequence of the 18 kDa surface/secreted oncospheral adhesion antigen identified by McAb-HP6, two peptides (Ts45W-1 and Ts45W-5) derived from the sequence of the T. saginata homologue of the T. ovis 45W protective gene family, one peptide (TS45S-10) derived from a T. saginata sequence with significant similarity to the T. ovis 45S protective antigen, and one peptide (TEG-1) derived from the sequence of the T. saginata homologue of Echinococcus spp. main surface protein. Longitudinal studies indicate that T. saginata infected cattle respond to all six peptides by 3-4 weeks post-infection and that the antibody levels remain high for at least 12 weeks post-infection. As protection against Taeniid parasites is predominantly antibody mediated, some of these six peptides may be of value as immuno-prophylactic tools and hence also in assays to determine resistance to infection with the parasite. For diagnosis, on the other hand, only three peptides (HP6-2, TEG-1 and Ts45S-10) performed with the necessary sensitivity and specificity to determine exposure to infection with T. saginata, and now merit an exhaustive evaluation prior to employment as routine diagnostic tools.     

OVINE CYSTICERCOSIS IN THE ALBANY REGION OF WESTERN AUSTRALIA

A survey of the cestode infections of 304 dogs from 134 properties in the Albany area of Western Australia was performed. Purgation was induced by oral administration of arecoline and the purge examined for cestodes. The cestodes found and their infection rates were: Dipylidium caninum 16.4%; Taenia spp 36.5%; T. ovis 8.9%; T. hydatigena 15.1%; T. pisiformis 15.1%; T. serialis 2.3%; Echinococcus granulosus 0.7%; Questionnaires completed on 133 of the 134 properties at the time the dogs were purged showed that:– 73.6% of farmers were feeding raw sheep-meat and 24.8% were feeding raw offal to their dogs; dogs were treated on a regular basis with a cestocide on 23.3% of properties; dogs were allowed to roam on 21.8% of the properties and stray dogs were considered prevalent on 23.3% of properties.
Suggestions for control of Cysticercus ovis are made based on an interpretation of the significance of these findings.     

Vaccines against veterinary helminths

The majority of attempts to develop commercial vaccines for veterinary helminths have focussed on identifying protein antigens, which could be formulated as protective vaccines. Notable successes have been achieved for some cestode parasites, where recombinant proteins have been developed into highly effective vaccines. Although effective protection can also be obtained using some nematode proteins in their native forms, it has not yet been possible to formulate commercially successful vaccines for other helminth parasites of veterinary significance. Increasing evidence suggests that parasite glycan moieties may provide an alternative source of vaccine antigens, and increased attention is now being given to this class of compounds. In addition to identifying candidate protective antigen(s), an increased research effort is needed to develop appropriate strategies for the formulation and delivery of helminth vaccines.     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

异源性抗原抗猪囊尾蚴感染的研究

本文报道了泡状带绦虫活化六钩蚴的超声裂解抗原可以诱导猪体产生抗猪带绦虫攻击感染的交叉保护作用。猪囊尾蚴匀浆抗原也使猪体产生了较强的抗猪带绦虫攻击感染的保护作用。泡状带绦虫六钩蚴超裂抗原免疫组与猪囊尾蚴匀浆抗原免疫组的保护情况是相似的,这表明异源免疫也可使猪体产生较好的抗猪囊尾蚴感染的免疫。由于制备异源性抗原的泡状带绦虫能够从狗的体内获得,因此在体外培养猪带绦虫未获成功之前可以解决从人体获取猪带绦虫的困难。     

Successful immunization of naturally reared pigs against porcine cysticercosis with a recombinant oncosphere antigen vaccine

Taenia solium causes cysticercosis in pigs and taeniasis and neurocysticercosis in humans. Oncosphere antigens have proven to be effective as vaccines to protect pigs against an experimental infection with T. solium. A pair-matched vaccination trial field, using a combination of two recombinant antigens, TSOL16 and TSOL18, was undertaken in rural villages of Peru to evaluate the efficacy of this vaccine under natural conditions. Pairs of pigs (n=137) comprising one vaccinated and one control animal, were allocated to local villagers. Animals received two vaccinations with 200 μg of each of TSOL16 and TSOL18, plus 5mg Quil-A. Necropsies were performed 7 months after the animals were distributed to the farmers. Vaccination reduced 99.7% and 99.9% (p<0.01) the total number of cysts and the number of viable cysts, respectively. Immunization with the TSOL16-TSOL18 vaccines has the potential to control T. solium transmission in areas where the disease is endemic, reducing the source for tapeworm infections in humans.     

The efficacy of praziquantel against cestodes in cats, dogs and sheep

Praziquantel is a new type of acylated isoquinoline-pyrazine. A single, low oral or subcutaneous dose of the compound is reliably effective against all tested juvenile and adult cestodes in cats, dogs and sheep. Praziquantel is the first cestodicide which is also effective on bile duct cestodes. In cats and dogs, 5 mg praziquantel per kg is completely effective on all stages of Taenia hydatigena, T pisiformis, T ovis, T taeniaeformis, Dipylidium caninum, Mesocestoides corti, Echinococcus multilocularis and E granulosus. Because of its very wide therapeutic index praziquantel is thus particularly suited for eradication programmes, eg, echinococcosis.     

Prevalence and seasonal incidence of larval and adult cestode infections of sheep and goats in eastern Ethiopia

A study on the prevalence and seasonal incidence of cestode parasite infections of sheep and goats was carried out in eastern Ethiopia for 2 years (May 2003-April 2005). During this period, viscera including liver, lungs, heart, kidneys and the gastro-intestinal tract were collected from 655 sheep and 632 goats slaughtered at four abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga. At the abattoirs the abdominal, thoracic and pelvic cavities as well as the muscle surfaces of all animals were visually examined for the presence of larval (cystic) stages of cestode parasites. The viscera were transported within 24 h to the parasitology laboratory of Haramaya University and were examined for larval and adult cestodes following standard procedures. The most prevalent metacestodes (larval cestodes) were Cysticercus ovis (Taenia ovis), Cysticercus tenuicollis (T. hydatigena) and hydatid cysts (Echinococcus granulosus). In sheep, the overall prevalence was 26% for C. ovis, 79% for C. tenuicollis, and 68% for hydatid cysts. Similarly, for goats, the corresponding prevalence was 22%, 53% and 65%, respectively. The difference between sheep and goats in prevalence of C. tenuicollis was significant. The high prevalence of hydatid cysts in both sheep and goats indicates that cystic echinococcosis/hydatidosis is a public health problem in these regions which requires implementation of control measures, including public health education, strict meat inspection and control of stray dogs. The results of the survey also implies that infections of small ruminants with these metacestodes are responsible for condemnation of substantial quantities of affected organs and muscles and therefore of direct economic importance. Intestinal infections with adult tapeworms of Moniezia expansa, Avitellina centripunctata and Stilesia globipunctata, and bile duct infections with Stilesia hepatica were also common in both sheep and goats. In sheep, the overall prevalence of these tapeworms were 61%, 20%, 24% and 39%, respectively. Similarly, the overall prevalence of these parasites in goats was 53%, 21%, 27% and 36%, respectively.     

Evaluation of the results of intradermal tests in spontaneous cysticercosis in cattle

The diagnostic value of intradermal tests was verified on 126 bulls (out of this number 32 autopsically positive) subject to intravital diagnostics of bovine cysticercosis. As allergen were used homologous antigens from adult larvae of Taenia saginata and heterologous antigens from larvae of Taenia crassiceps, as well as hydatid fluid from Echinococcus granulosis. With the antigen T. saginata the sensitivity reached 53.1% and specificity 90.4%, with the antigen T. crassiceps 53.1% and 80.9% and with the antigen E. granulosus 16.7% and 89.5%.     

猪带绦虫不同阶段及羊囊尾蚴蛋白质分析

利用浓度梯度SDS－聚丙烯酰胺凝胶电泳对猪囊尾蚴的囊液和虫体，猪带绦虫成虫的头颈节、成节、孕节，猪带绦虫的虫卵以及羊囊尾蚴的蛋白成分进行了对比分析。结果发现：囊液、虫体、头颈节、成虫、孕节、虫卵、羊囊尾蚴分别有35条（15．0－150．4kd）、44条（15．0－133．7kd)、22条（13．3－163．9kd)、19条（13．3－90．1kd)、19条（13．3－90．1kd)、30条（13．3－99．4）和38条（15．0－133．4kd)蛋白带；其中15．0kd和35．5kd两条共同抗原蛋白带，在猪带绦虫各阶段和羊囊尾蚴中含量最大的蛋白带同时也是阶段性的共同抗原蛋白带。这些结果为下一步的保护性抗原筛选奠定了基础。     

猪囊虫病基因工程疫苗的研制

从猪带绦虫六钩蚴ｃＤＮＡ文库中筛选目的基因并进行克隆表达,重组抗原用血清学方法和猪体免疫进行鉴定。钭具有免疫保护作用的重组抗原纯化,与免疫刺激复合物疫苗佐剂混合,制备成猪囊虫病基因工程疫苗,免疫仔猪并攻虫。该疫苗安全,无毒副作用,免疫猪减虫率９２．２％,完全保护率５５．５％,且免疫组发现的囊虫多数已死亡。     

AN EPIDEMIOLOGICAL ANALYSIS OF FACTORS INFLUENCING THE INCREASE IN TAENIA OVIS PREVALENCE DURING THE NEW ZEALAND ECHINOCOCCUS GRANULOSUS CONTROL PROGRAM

There was an 8-fold increase in the national prevalence of Taenia ovis in dogs during the first decade of the New Zealand hydatid disease control program. The temporal patterns in the prevalence of T. ovis were similar in both rural working dogs and urban pet dogs, with the highest prevalence found in the sheep farming areas. The dramatic increase in the prevalence of T. ovis was a reflection of the change in source of food for many working dogs, from the traditional raw viscera to carcase meat of sheep, a change necessitated by hydatid disease control. Other factors influencing the rise in prevalence of T. ovis in rural dogs were investigated using stepwise multiple regression techniques. The results of the regression analyses suggested that the rate of increase in the prevalence of T. ovis was influenced by 4 major factors: Taenia hydatigena variables (initial prevalence and rate of progress in control), an ethnic variable (Maori proportion in local population), canine variables (numbers of dogs per farm and per owner), and animal husbandry variables (pasture type, sheep density and numbers of sheep per farm). The results were discussed in the light of present knowledge of the epidemiology and immunology of the taeniid cestodes.     

Evaluation of tongue inspection and serology for diagnosis of Taenia solium cysticercosis in swine: usefulness of ELISA using purified glycoproteins and recombinant antigen

Evaluation of serology using glycoproteins (GPs) purified by preparative isoelectric focusing (pH 8.8) and recombinant chimeric antigen (RecTs) of Taenia solium was carried out using (1) blood samples on filter papers from pigs infected with different doses of eggs of T. solium in Mexico, (2) serum samples from pigs found infected naturally in Vietnam and Ecuador and (3) serum samples from pigs suspected to be infected with T. solium by tongue inspection in Tanzania. Antibody responses (IgG) were detectable in experimentally infected pigs confirmed harbouring 16 or more cysts at necropsy from 30 days after egg inoculation. One of three pigs naturally infected and harbouring 2.5 cysts/kg muscle and most of pigs harbouring=5.0 cysts/kg were also seropositive by ELISA. Although pigs may be infected with other taeniid species such as Taenia hydatigena, pigs harbouring this parasite were negative in ELISA. Approximately, 76 and 78% of sera from pigs having nodule(s) in the tongue (positive tongue inspection) were serologically positive by both ELISA and immunoblot, respectively. Furthermore, approximately 34 and 18% of sera from pigs having no nodules in the tongue (negative tongue inspection) were also seropositive by ELISA and immunoblot, respectively. ELISA using the two antigens was more sensitive than immunoblot and reliable for differentiation of pigs infected with cysticerci of T. solium from those either uninfected or infected with other taeniid species. Pigs without nodule by tongue inspection should be checked serologically in endemic areas.     

Echinococcus granulosus,Taenia hydatigena,T. ovis: Evaluation of cyst fluids as antigen for serodiagnosis of larval cestodes in sheep

In order to monitor the progress of New Zealand's hydatids eradication campaign, a specific, serological, diagnostic test is required to identify infected sheep. An indirect haemagglutination test, using pyruvic aldehyde-stabilized sheep erythrocytes as the antigen carrier, was developed for the serodiagnosis of larval cestode infections of sheep. Using cyst fluid from Echinococcus granulosus, Taenia hydatigena and T. ovis as the antigens in this test, it was shown that the larval cestode species, responsible for an infection in sheep harbouring a single specific infection, could be identified by the higher titre given with the homologous antigen, in comparison to that given with the heterologous antigens. Sera of sheep infected with two or more species were also tested by this method, and the only specific infections to be diagnosed by differential titres were those due to the presence of live E. granulosus cysts. These antigens cannot be used for the diagnosis of specific larval cestode infections in the field because of the cross-reactivity between cyst fluids. However, the test did show that infection with larval cestodes could be diagnosed on a non-specific basis.     

Pilot field trial of a recombinant Taenia ovis vaccine in lambs exposed to natural infection

Previous trials of an experimental Taenia ovis vaccine using the recombinant antigen GST--45W(B/X) established that it was possible to achieve >90% 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.     

猪带绦虫45W-4B基因在家蚕细胞中的表达

猪带绦虫45W基因已被认为是预防猪囊虫病的基因工程疫苗候选基因之一。其中4B基因是45W基因家族中高度保守的一个成员（以下称45W-4B）。本试验将重组于pGEM-3Z载体上的4B基因转载到pVL1393转移载体中，通过鉴定获得重组质粒pVL1393-4B，然后将重组质粒与BmNPV病毒共转染，筛选出重组BmNPV-4B重组病毒，用该重组病毒感染Bm细胞，收集细胞上清，SDS-PAGE电泳鉴定表达蛋白，并经Western blot检测表明该蛋白能识别囊虫病人（猪）阳性血清，45W-4B蛋白的成功表达对进一步研究45W蛋白的结构和功能以及开发高效猪囊虫基因工程疫苗打下了基础。     

A comparison of DNA vaccines expressing the 45W, 18k and 16k host-protective antigens of Taenia ovis in mice and sheep

The immunogenicity of DNA vaccines encoding three different Taenia ovis host-protective antigens was compared in mice and sheep. DNA vaccines encoding the 45W, 18k and 16k antigens of T. ovis were constructed. The ability of DNA vaccines encoding the 45W and 18k genes to express antigen was confirmed by Western blotting of transfected Cos-7 cells. BALB/c mice were vaccinated intramuscularly with 45W, 18k or 16k DNA vaccines and the humoral immune response analysed by ELISA. DNA vaccines expressing 45W, 18k or 16k antigen were immunogenic in mice and generated significant titres of antigen-specific antibody. Intramuscular vaccination of outbred sheep with the T. ovis DNA vaccines generated significantly lower titres of 45W-specific antibody and failed to generate 18k or 16k-specific antibody. The findings of this study show that each of the three T. ovis host-protective antigens are amenable to delivery via DNA vaccines, and that the parameters governing the efficacy of DNA vaccines in sheep require further investigation.     

Parasite vaccines--a reality?

Over the last decade, the anti-parasitics market has been the fastest growing sector of the overall $18 billion animal health market. While drugs for the treatment of parasites of livestock still dominate this sector and will continue to be developed or re-formulated, because of consumer demands for chemical-free food and of concerns regarding the environment and animal welfare there is a growing interest in the development of safe and effective vaccines. There is also a call for vaccines in the lucrative $3 billion-plus companion animal market. These demands for vaccines will add a greater impetus to an area that has seen tremendous success in the last 15 years. A number of anti-parasite vaccines have been developed, e.g. the recombinant 45w and EG95 oncosphere proteins against Taenia ovis and Echinococcus granulosis, respectively, and the Bm86 vaccine against Boophilus microplus. In addition, the cathepsin L vaccines against the liver fluke, Fasciola hepatica, and the H11 vaccine against Haemonchus contortus are progressing well. There are also many additional vaccine candidates for H. contortus and for other nematodes such as Ostertagia and Trichostrongylus spp. that may ultimately lead to broad-spectrum gastrointestinal worm vaccines. Live or attenuated-live vaccines are available for the control of avian coccidiosis, toxplasmosis in sheep and anaplasmosis in cattle, although molecular vaccines against protozoans are still proving elusive. The wealth of information in genomics, proteomics and immunology that has been forthcoming together will new methods of vaccine production and delivery should see many new vaccines reach the marketplace in the near future.