Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection.

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.

In vitro potency testing of inactivated biologics: current situation in the E.E.C.

A whole series of regulations are currently being implemented in the European Economic Community in the field of biologics that are complementing the European Pharmacopoeia and widening its field of application. The object of these new regulations is to ensure a very high level of safety and efficacy, but unfortunately, they tend towards the increased use of animals for testing purposes. However, for ethical, economic and practical reasons, the number of animals used must be reduced which makes the development of in vitro tests an important issue. In vitro testing is complicated since inactivated products are often adsorbed onto aluminum hydroxide or presented as an emulsion, and tests are easier to carry out prior to final blending operations. It is suggested that in-process controls can replace finished product controls when batch-to-batch consistency has been formerly validated, a quality assurance system has been adopted, and that system is regularly subjected to a suitable inspection procedure. The new inspection system applied in the United Kingdom should facilitate progress in this field and lead to a parametric release system. To avoid future problems, it is particularly important that efforts be made now for the harmonization of new techniques between Europe and the United States.

Spirochetes: vaccines, animal models and diagnostics.

The detailed characterization of various proteins from spirochetes using molecular biology techniques has made possible new approaches to vaccine and diagnostic development that are described in this session. The importance of animal models was emphasized and illustrated.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.

Efficacy of long-acting oxytetracycline alone or combined with streptomycin in the treatment of bovine brucellosis.

Twenty-nine Brucella abortus culture-positive cows were treated with a long-acting oxytetracycline (20 mg/kg of body weight, IM) alone or combined with streptomycin (25 mg/kg, IM or IV) or were re-treated with the same product. There appeared to be a synergism by the 2 drugs. Of 21 courses of treatment with the combined antibiotics, 14 (67%) were considered successful. Only 3 of 14 (21%) were successful using oxytetracycline alone. The period from onset of therapy to cessation of shedding in udder secretions was variable. Four cows that ceased shedding were culture-positive in tissues taken at slaughter. The titers on tube agglutination and complement-fixation tests were of limited value in short-term evaluations of therapeutic regimens.

Effectiveness of various therapeutic regimens for bovine brucellosis.

Various chemotherapeutic regimens were evaluated in 48 culture-positive dairy cows. Cessation of shedding of Brucella abortus from udder secretions and absence in selected tissues at necropsy were criteria of success. A combination of a long-acting oxytetracycline and streptomycin eliminated Brucella in 10 of 14 (71.4%) cows. Two cows that were retreated with the same regimen also became culture-negative. Other treatment regimens, including the use of liposome-encapsulated antibiotics, were less successful. Serotests were a poor criterion of effectiveness.

Protection by oral administration of brucella abortus strain 19 against an oral challenge exposure with a pathogenic strain of Brucella.

Twenty heifers were vaccinated orally with Brucella abortus strain 19. These heifers and 21 control heifers were challenge exposed in midgestation with strain 2308 by the oral route. Ten of 19 pregnant control heifers aborted and 14 were culture positive. Two additional heifers were seropositive at slaughter. Strain 2308 was recovered from 4 vaccinates at slaughter; none of the vaccinates aborted. Titers after oral vaccination persisted for less than 82 days.