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.

Serological survey of Toxoplasma gondii and Campylobacter fetus fetus in sheep from New Zealand.

AIM: To determine the prevalence of antibody titres to Toxoplasma gondii and Campylobacter fetus fetus in sheep from New Zealand. METHODS: As part of a free screening service, unsolicited blood samples were supplied by veterinarians wishing to gauge the exposure of their clients' ewe flocks to T. gondii and C. fetus fetus. Blood samples were submitted from mixed-age ewes throughout New Zealand, from 2006 to 2009, that had not been vaccinated for T. gondii and C. fetus fetus. A total of 2,254 sera were serologically titrated for T. gondii and 3,429 for C. fetus fetus. A latex agglutination kit available commercially was used to quantify antibodies to T. gondii, and an agglutination test developed in-house was used for C. fetus fetus. For T. gondii, titres of ≥1:16 and ≥1:64 were used to define a positive response, and for C. fetus fetus a titre of ≥1:10 was defined as positive. A flock was defined as positive if ≥1 ewe had a positive titre. RESULTS: Of the sera tested for T. gondii, 1,917/2,254 (85%) were positive, using a titre of ≥1:16, and 1,384/2,254 (61%) with a titre of ≥1:64. All 198 ewe flocks tested were seropositive to T. gondii, at a titre of ≥1:16, and all but three were at a titre of ≥1:64. A bimodal distribution was evident in the prevalence of titres to T. gondii suggesting that a percentage of titres ≤1:64 may have been non-specific. Of the sera tested for C. fetus fetus, 1,644/3,429 (48%) were positive to at least one of the four test antigens at titre of ≥1:10. Only 34/298 (11%) flocks tested for C. fetus fetus were completely seronegative. The percentage of seropositive ewes to both T. gondii and C. fetus fetus was significantly higher in the North Island than the South Island. CONCLUSIONS: The study demonstrated that exposure to these two important infectious abortifacients was both considerable and widespread. Minimum titres were postulated to establish a 'cut-off' for a positive result and to allow comparison with past and future studies. The bimodal distribution evident for T. gondii suggested a titre of 1:64 may be an appropriate cut-off. The widespread on-farm exposure probably stimulates the immune response of vaccinated ewes. CLINICAL RELEVANCE: Further studies are required to confirm the clinical significance of flock-based antibody responses, and to validate their use in identifying recently aborted ewes, especially where there are no aborted fetuses for examination.

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants.

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.

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.

Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres.

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.

Parasite vaccine development: large-scale recovery of immunogenic Taenia ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies.

Genetically modified Escherichia coli expressing the Taenia ovis fusion protein GST-45W(B/X) as inclusion bodies were grown in volumes ranging up to 1000 l. Bacteria were inactivated by heat or chemical treatment without affecting immunogenicity. The fusion protein was recovered in a highly immunogenic form from washed inclusion bodies and from urea-solubilised inclusion bodies. The fusion protein was found to be stable in solution after storage at 4 degrees C for up to 2 years. Vaccines formulated with fusion protein from urea-soluble inclusion bodies gave consistently high protection (89-100%) against challenge infection. The methods described enabled the production of sufficient vaccine for large field trials. These trials generated the data required for product registration and manufacture of a vaccine to prevent T. ovis infection in sheep.

Maternal transfer of protection from Echinococcus granulosus infection in sheep.

Although passive immunity against larval metacestodes of the genus Taenia is well established, the transfer of immunity against Echinococcus granulosus infection has not been demonstrated convincingly. The immune status of newborn lambs born to ewes that had been infected or immunised with E granulosus eggs or oncospheres was investigated. The ewes and their six- to eight-day-old lambs were subsequently challenged. Lambs born to triply infected ewes were 80 per cent protected from a challenge infection whereas lambs born to simply infected ewes were only 45 per cent protected. Lambs born to ewes that had been immunised with preparations of sonicated oncospheres had the lowest levels of immunity. The infected ewes were challenged intramuscularly with activated oncospheres and showed some degree of immunity. Ewes which had received oncospheres solubilised in sodium dodecylsulphate or sonicated oncospheres were protected from subsequent oral challenge by 60 to 66 per cent. Radial immunodiffusion revealed that lambs with statistically significantly lower levels of IgG were more susceptible to challenge. However, the degree of protection did not show a simple relationship to the titre of antibody, as determined by an ELISA using a solubilised antigen.

Identification of host-protective antigens of Taenia ovis oncospheres.

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.

Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

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).

Echinococcus granulosus: development of an intermediate host mouse model for use in vaccination studies.

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.

Vaccination against ovine cysticercosis using a defined recombinant antigen.

Cysticercosis caused by larval tapeworms is a major public health problem and a cause of substantial economic losses in the farm-animal industries. Taenia ovis in sheep is a particularly important example. Immunity to reinfection with the larvae has a central role in regulating natural transmission of the parasites, and vaccination with antigens from the early larval oncosphere stage can induce complete protection against infection. As it is impractical to obtain enough oncospheres for a commercial vaccine against these tapeworms, an alternative approach is to use recombinant DNA methods to generate a cheap and plentiful supply of antigens. We report here the expression in Escherichia coli of complementary DNA encoding T. ovis antigens as fusion proteins with the Schistosoma japonicum glutathione S-transferase. Vaccination of sheep with these fusion proteins gave significant, although not complete, immunity against challenge infection with T. ovis eggs. Commercial development of a vaccine is being pursued.

Toxoplasma gondii: purification of zoites from peritoneal exudates by eight methods.

Many methods have been proposed for removing contaminating host cells from mouse peritoneal exudates infected with Toxoplasma gondii tachyzoites. Of these, eight established methods were compared. They were density gradients, sonication and trypsin digestion, differential centrifugation, haemolysin digestion, filtration through glass wool and cellulose columns, and sintered glass and polycarbonate filtration. The methods were assessed for zoite recovery, host cell removal, effect on zoite viability and antigenic integrity, time, cost, and ease. They were almost all capable of removing greater than 90% of the mouse leucocytes, but in some cases this resulted in low zoite recoveries. The sonication and trypsin method produced the best zoite recovery and highest purity, but appeared to affect zoite viability and antigenic integrity. The haemolysin digestion procedure has been adopted by our laboratory because of its high recovery of zoites, and it is inexpensive, quick, and easy to perform.

The production of bacteriocin-like substances by the oral bacterium Streptococcus salivarius.

Inhibitory substances produced by six strains of Streptococcus salivarius were isolated and partially characterized. The six prototype producer strains were selected initially on the basis of their differing spectra of inhibitory activity when tested against a set of nine standard indicator strains. Optimal production conditions were defined for each producer strain and inhibitor-containing extracts were obtained for characterization studies. All of the inhibitors appeared to be proteinaceous substances of molecular weight greater than 3500. When tested against a Streptococcus pyogenes indicator strain, one of the inhibitors was bactericidal, but the other five appeared to be bacteriostatic. Some differences between the inhibitors were observed with respect to heat and enzyme sensitivities.

Hepatic trematodiasis in a Ganges River dolphin.

Hepatic trematodiasis caused by Cyclorchis campula was diagnosed in a juvenile Ganges River dolphin that had been in captivity at an aquarium for approximately 1 year. Histopathologic findings were severe chronic suppurative cholangitis, hyperplasia of the bile duct epithelium, and periductal fibrosis associated with fluke infection of the large bile ducts.