Comparative tests with formolized and irradiated vaccines against leptospirosis*

Bull. Org. mond. Sante 1973, 48, 587-590 Bull. Wid Hlth Org.J Comparative tests with formolized and irradiated vaccines against leptospirosis* B. BABUDIERIQ M. CASTELLI,1 & F. PISONI 2 The effectiveness ofgamma-irradiated antileptospirosis vaccines and of vaccines killed with formol was compared in guineapigs. Irradiated vaccines proved no more effective than formolized vaccines in preventing either death or persistent kidney infection. Anti-pomona vaccines gave little protection against serotype icterohaemorrhagiae, and there was no significant difference between irradiated and formolized preparations. Vaccine prepared from a virulent strain showed little advantage over vaccine preparedfrom weak strains. Prophylactic killed vaccines have been widely employed against leptospiroses in both man and domestic animals. In the first instance the results obtained have generally been very satisfactory; in the second, users' findings have often differed. It has been stated that even if, in most cases, such a vaccine can protect animals from the disease, it cannot pro- tect them from becoming infected, and therefore carriers of leptospires. We have investigated whether the use of vaccines inactivated by gamma radiation gave better protection and whether this protection extended to strains antigenically different from that employed for the preparation of the vaccine. In recent years Hubbert & Miller (1965) and sub- sequently Stalheim (1967, 1968a, 1968b) have pre- pared an antileptospirosis vaccine by irradiating a suspension of virulent leptospires with appropriate doses of gamma rays. The leptospires are not killed by this treatment, but they become incapable of reproduction and lose their pathogenicity. These investigators found such vaccines to be effective, protecting animals both from the infection and from becoming carriers. In addition to small labora- tory animals, Stalheim (1968a, 1968b) has also used this vaccine to protect a small number of cattle and swine. The dose of ionizing radiation selected by Stalheim was 100 000 rad. * From the Istituto di Microbiologia dell'Universita di Trieste, Trieste, Italy. t Professor Babudieri, former Director of the Istituto di Microbiologia, died on 11 May 1973 after the acceptance of this paper for publication. Assistant. ' Student. We have repeated these investigations, comparing the effectiveness of an irradiated vaccine and of a formolized vaccine prepared by the usual methods. We also attempted to determine whether vaccines prepared by the two techniques using (1) a virulent, recently isolated strain, and (2) an attenuated labora- tory strain, would show a difference in activity. Finally, we investigated whether the two types of vaccine-attenuated live vaccine and formolized vaccine-could protect against leptospires of a dif- ferent serotype. MATERIALS AND METHODS The following Leptospira strains were employed for these tests: (1) strain Policlinico (serotype icterohaemorrha- giae), isolated from man by Babudieri in 1965 and maintained in alternate transfers in guineapigs and in Korthof-Babudieri's medium (Babudieri, 1961). This strain has maintained its virulence and is able to kill guineapigs. (2) strain Dellatte (serotype icterohaemorrhagiae),3 isolated from man by Babudieri in 1940 and main- tained since in culture. It has a low virulence and produces subclinical infection in the guineapig. (3) strain New Jersey (serotype pomona), isolated from cattle in 1946 by Baker and Little. This is the strain most often used in the USA for the prepara- ,MAfter these experiments were completed, Babudieri (Ann. Ist. sup. Sanitd, 1972, 8, 159) reclassified strain Dellatte as belonging to serogroup icterohaemorrhagiae, serotype copenhageni. 3053 -587- B. BABUDIERI ET AL. tion of anti-pomona vaccine for cattle. It is main- tained in culture and its virulence is very limited. These strains were cultivated for 10 days at 30°C in Korthof-Babudieri's medium. The cultures were then centrifuged for one hour at 10 000 rev/min and the sediment was resuspended in the same culture medium, but without rabbit serum and vitamin B12. The numbers of leptospires were counted in a Hawksley counting chamber, and the suspension was diluted to a concentration of 1 x 108 spirochaetes per ml. Part of this suspension was treated with 0.05% formol. Another part was placed in a large test tube and irradiated with a cobalt bomb so as to receive 200 000 rad per ml. We chose this dose, instead of the 100 000 rad suggested by Stalheim (1967), because the lower dose did not appear sufficiently sure. One of the guineapigs inoculated by Hubbert & Miller (1965) with a vaccine irradiated with 50000 rad developed a persistent kidney infection; we therefore considered that only a four-fold dose would be safe. The absence of contamination in each vaccine batch was checked by means of transfers in the customary media and in Korthof-Babudieri's me- dium, and by darkfield examination. The irradiated leptospires were motile under the microscope, but were unable to multiply in Korthof-Babudieri's medium. With each type of vaccine we inoculated a group of 20 guineapigs each weighing 175-280 g. The vac- cine was inoculated in a single dose of 1 ml per guineapig. A group of 20 unvaccinated guineapigs was used as a control. On the 21st day all the guinea- pigs, including controls, received single intraperito- neal injections of 1 ml of a rich 10-day-old culture of the virulent strain Policlinico. After vaccination, the guineapigs were kept under observation for 21 days in order to determine if they developed morbid symptoms due to the vaccine. Guineapigs that became infected were kept under observation for 23 more days. Post-mortem examina- tions were carried out on those that died; we sought the characteristic signs of Leptospira infection, and made haemocultures and cultures of liver and kidney tissue in Korthof-Babudieri's medium. All surviving guineapigs were sacrificed after the period of obser- vation and subjected to post-mortem examination. Samples ofkidney tissue were transferred to Korthof- Babudieri's medium, and the sera were examined for the presence of agglutinins. In order to ascertain that the irradiated vaccine was harmless, 21 days after inoculation we sacrificed 4 guineapigs of each of the two groups that had received irradiated vaccine prepared with leptospires of the icterohaemorrhagiae serotype (strains Poli- clinico and Dellatte). With these guineapigs, too, we carried out autopsies and culture tests, and exam- ined the serum for agglutinating antibodies. The results were evaluated statistically by com- paring the percentages obtained by the x2 test, using 2 x 2 frequency tables. RESULTS The irradiated vaccines used in the tests proved completely safe. They caused no evident pathological changes in guineapigs, and even the 8 animals sacri- ficed on the 21st day showed no anatomical or pathological alteration. The cultures prepared from kidney tissues proved negative. Agglutinins were not detected in the serum, even at a titre of 1 : 10. This is not surprising, as it is well known that the limited amount of antigen contained in one dose of killed Leptospira vaccine is usually unable to provoke an evident rise in antibody in guineapigs. The antigenic activity of live but irradiated leptospires, unable to multiply, corresponds to that ofan equivalent amount of a killed antigen. Seven of the vaccinated guinea- pigs and 1 control animal died before challenge. Post-mortem examinations and cultures in no way indicated that their death could be attributed to inoculation with the vaccine. The sera of vaccinated guineapigs that survived until challenge agglutinated the two icterohaemorrhagiae strains at titres varying from 1: 200 to 1: 600. The results obtained in the different tests are sum- marized in Table 1. DISCUSSION In evaluating the efficacy of an antileptospirosis vaccine, a distinction must first be made between protection against disease (and death) and protection against infection. In the first instance the lack of any symptom of disease does not exclude the possibility that the vaccinated person or animal might become a carrier and spreader of leptospires. In man the carrier state is of no practical impor- tance because it lasts for a very short time and usually does not constitute a source of infection for other people. In cattle and other domestic animals, vaccinated individuals that nevertheless become car- riers spread leptospires in limited quantities and for a short period of time. In this paper, in the statistical 588 VACCINES AGAINST LEPTOSPIROSIS Table 1. Results of vaccination of guineapigs with gamma-irradiated and formolized vaccines and subsequent challenge with strain Policlinico Vaccination Challenge Positive Positive Vaccines kidney Animals PosaititiveyAnimals Mortality Animals cultures infected from Surviving cultures in group in group sacrificed from by leptospirosis animals from sacrificed injection survivors animals Irradiated Policlinico 20 1 4 0 15 3 12 (80%) 2 Dellatte 20 1 4 0 15 4 11 (73%) 0 NewJersey 20 3 0 - 17 11 6 (35%) 1 Formolized Policlinico 20 1 0 - 19 2 17 (89%) 2 Dellatte 20 0 0 - 20 11 9 (45%) 2 NewJersey 20 1 0 - 19 13 6 (31 %) 2 Unvaccinated controls 20 1 0 - 19 17 2 (11 %) 2 evaluation of the results, we took into consideration both partial protection (protection against illness and death) and complete protection (protection against illness and death and against the carrier state). An analysis of the data in Table 1 indicates that there was no statistically significant difference be- tween the protection afforded by icterohaemorrhagiae irradiated and formolized vaccines, when they were compared (partial protection, x2= 0.4; complete pro- tection, X2 = 0.8). The irradiated vaccine prepared with strain Policlinico was practically as effective as the corresponding formolized vaccine. The irradiated vaccine prepared with strain Dellatte gave protection to more animals than the corresponding formolized vaccine. However, the difference was not statistically significant (partial protection, x2= 1.8; complete pro- tection, x2=3.6). Both in the groups that received irradiated vac- cine, and in those treated with formolized vaccine, some of the surviving guineapigs became renal car- riers of leptospirosis. The irradiated vaccines did not afford better protection against this condition than the formolized ones. Statistical comparison showed that vaccine pre- pared with the virulent strain Policlinico may give greater protection than vaccine prepared with strains of low virulence, but only if we consider the criterion of partial protection (x2=5.3), and not complete protection (X2=2.7). Apart from the question of virulence, it should be borne in mind that strain Policlinico was also used for the challenge, and the identity of the antigenic pattern could be a factor in the better result achieved by the Policlinico vaccines. The two vaccines prepared with serotype pomona, although less effective than the icterohaemorrhagiae vaccines (partial protection, x2 = 12.3; complete pro- tection, X2 = 11.6), clearly give protection against serotype icterohaemorrhagiae, at least as regards complete protection (partial protection, X2 = 2.3; complete protection, x2 = 4). In this case again, the irradiated vaccines did not prove more effective than the formolized vaccine. This conclusion may be of some interest if related to the experiments by Kemenes (1964), who found that a formolized anti-pomona vaccine did not give any appreciable protection against infection by sero- type icterohaemorrhagiae, while the protection was very reliable in animals that had overcome an infec- tion by serotype pomona. This finding might have suggested that a live vaccine, although irradiated, could give a cross-immunity far superior to that given by a killed vaccine. However, this did not take place in our experiments, and the capacity to immunize of irradiated leptospires, which are incapable of reproduction, is a phenomenon quite different from the greater capacity of virulent and aggressive lepto- spires. 589 590 B. BABUDIERI ET AL. To conclude, it does not appear that gamma- irradiated antileptospirosis vaccine is more effective than the common formolized vaccines, at least in the guineapig, even for conferring protection against becoming a carrier. As the irradiated vaccine cannot be kept for a long time, but must be employed at once after its preparation, it is evident that while irradiated vaccine can be used as a substitute for formolized vaccine, there are practical disadvantages. RE2SUME ESSAIS COMPARATIFS DE VACCINS FORMOLES ET DE VACCINS IRRADIES CONTRE LA LEPTOSPIROSE On a compar6 chez le cobaye l'efficacite de vaccins anti-leptospirose vivants, irradi6s par rayonnement gam- ma A la dose de 200 000 rad/ml, et de vaccins tues par le formol. Les vaccins irradi6s ne se sont pas montr6s plus actifs que les vaccins formoles correspondants, meme en ce qui conceme la pr6vention de l'infection r6nale et de l'6tat de porteur de germes. Les vaccins anti-pomona n'ont suscit6 qu'une faible protection contre l'infection par le serotype icterohaemorrhagiae. Ici encore, les vaccins irradi6s n'ont pas donne de meilleurs r6sultats que les vaccins formoles. Les vaccins obtenus a partir de souches virulentes ne presentent apparemment que peu d'avantages par rap- port aux vaccins pr6pares a I'aide de souches faiblement pathogenes. REFERENCES Babudieri, B. C. (1961) Bull. Wld. Hlth Org., 24, 45 Hubbert, W. T. & Miller, J. N. (1965) J. Immunol., 95,759 Kemenes, F. (1964) Z. Immun.- Allergie-Forsch., 127, 209 Stalheim, 0. H. V. (1967) Amer. J. vet. Res., 28, 1671 Stalheim, 0. H. V. (1968a) Amer. J. vet. Res., 29, 473 Stalheim, 0. H. V. (1968b) Amer. J. vet. Res., 29, 1463