Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy M.P. Jiménez de Bagués, C.M. Marin, M. Barberán, J.M. Blasco To cite this version: M.P. Jiménez de Bagués, C.M. Marin, M. Barberán, J.M. Blasco. Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy. Annales de Recherches Vétérinaires, INRA Editions, 1989, 20 (2), pp.205213. <hal00901861> HAL Id: hal00901861 https://hal.archivesouvertes.fr/hal00901861 Submitted on 1 Jan 1989 HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
pregnancy Rev1 subcutaneous Original article B. melitensis Rev1 vaccine routes at Responses of ewes to administered by subcutaneous or conjunctival different stages of pregnancy M.P. Jiménez de Bagués C.M. Marin M. Barberán J.M. Blasco Dpto. P!oducció! Animal, SIAlDGA, Apartado 727, 50.080 Zaragoza, Dpto. A. Patológica, Fac. Veterinaria, Zaragoza, Spain (received 3111987, accepted 891988) Summary ― An experiment to determine the innocuousness and the humoral immune response of B. melitensis Rev1 vaccine was carried out in pregnant ewes. Thirtythree ewes were vaccinated on day 55 of pregnancy : 9 ewes received 2 x 10 9 Rev1 of French origin by the subcutaneous route, 9 ewes were conjunctivally vaccinated with 5 x 108 of the same strain, 7 ewes were subcutaneously vaccinated with 109 Rev1 of Spanish origin and 8 ewes received 1.8 x 109 Spanish strain Rev1 by conjunctival route. Another group (8 animals) were subcutaneously vaccinated with a reduced dose (10 6) of the French strain between days 90 and 120 of pregnancy. Finally, 20 ewes were vaccinated on day 120 of pregnancy with the French strain of which 11 received 10! Rev1 by the subcutaneous route and 9 were conjunctivally vaccinated with the same dose. Rev1 strain was isolated from the vaginal discharge of a significantly higher number of ewes vaccinated subcutaneously than in conjunctivally vaccinated ewes (24/35 versus 9/26 respectively; P < 0.001). No difference between the two vaccine strains was recorded in the number of ewes excreting Revi. Ewes subcutaneously vaccinated on day 55 of pregnancy aborted more frequently than conjunctivally vaccinated ewes (11/16 versus 2/17, respectively), hence a significantly reduced (P < 0.001) length of pregnancy. Although 10 of the 11 animals subcutaneously vaccinated on day 120 of pregnancy excreted Rev1 strain, only one aborted. There was no significant effect of the time of vaccination on both abortions and length of pregnancy in ewes conjunctivally vaccinated with the French strain. No abortions were recorded among the 8 animals vaccinated with the reduced dose on days 90120 of pregnancy. Aborted culture positive fetuses had bronchopneumonia, hepatitis and interstitial nephritis. Humoral immune response to standard smooth antigens was slightly lower in conjunctivally vaccinated ewes than in those vaccinated subcutaneously. ovine ― conjunctival route Brucella melitensis vaccine route des brebis à l administration conjonctivale ou souscutanée du vaccin Résumé ― Réponses Rev1 à différents stades de la gestation. Une expérience a été réalisée chez des brebis gestantes pour déterminer l innocuité du vaccin Rev1 et la réponse sérologique des brebis à ce * Author to whom correspondence should be addressed.
gestation vaccin voie voie vaccin. Trentetrois brebis ont été vaccinées à 55 jours de gestation : 9 ont reçu 2 x 10 9 Rev1 bactéries d origine française par voie souscutanée, 9 ont été vaccinées avec 5 x 108 bactéries de la Rev1 même souche par voie conjonctivale, 7 brebis ont été vaccinées ar voie souscutanée avec 109 bactéries Rev1 d origine espagnole et 8 brebis ont reçu 1.8 x 1 bactéries Revl espagnole par voie conjonctivale. Huit brebis ont été vaccinées avec f 60 bactéries de souche 90 française entre et 120 jours de gestation. Finalement, 20 brebis ont été vaccinées à 120 jours de gestation avec la souche française : 11 animaux ont reçu 109 bactéries Revl par voie souscutanée et 9 ont été vaccinées par voie conjonctivale avec la même dose. La souche Rev1 a été isolée à partir des pertes vaginales d un nombre significativement plus grand (P < 0.001) de brebis vaccinées par voie souscutanée (24/35) que de brebis vaccinées par voie conjonctivale (9/26). Il ny a pas de différence entre les souches de vaccins, en ce qui concerne le nombre de brebis excrétant Revl. Les brebis vaccinées par voie souscutanée à 55 jours de gestation avortent plus fréquemment (11/16) que celles vaccinées par voie conjonctivale (2/17), d où une durée de significativement réduite gestation (P < 0. 00 1). Chez les brebis vaccinées à 120 jours de gestation par voie souscutanée, la souche Revf a été excrétée par 10 des 11 animaux vaccinés mais seul un avortement s est produit. Il n y a pas eu d effet du moment de la vaccination sur le nombre d avortements et sur la durée de la gestation quand le vaccin français est inoculé par voie conjonctivale. Il n ÿ a pas eu d avortement parmi les 8 animaux vaccinés avec une faible dose 90 entre et 120 jours de gestation. Les foetus de brebis ayant avorté, chez lesquels des bactéries Revf ont été isolées, présentaient des lésions de bronchopneumonie, d hépatite et de interstitielle. La néphrite réponse sérologique a des antigènes lisses standards est légèrement plus faible chez les brebis vaccinées par voie conjonctivale. sous ovin cutanée Rev1 Brucella melitensis conjonctivale Introduction Ovine brucellosis caused by B. melitensis cannot be efficiently erradicated by only testing and slaughtering in heavily infected countries. Vaccination of 36 month old ewes with live strain Rev1 is one of the best methods available for the control of infection. However, in countries with extensively managed sheep, this method can fail because of practical difficulties that result mainly from the failure of flock owners to plan the breeding of female replacements. Therefore, attention has to be given to vaccination of the whole flock as the only way to control this disease under these conditions. Rev1 vaccine can be used in non ewes without pregnant and lactating undesirable effects (Jones and Marly, 1975). When this vaccine has been given to pregnant sheep some contradictory results have been obtained. It has been stated that Rev1 could cause abortions in pregnant ewes (Crowther et al, 1977; Elberg, 1981). However, this effect was not observed in 18 ewes at 56 days of pregnancy that received a revaccination of 2.9 x 10 8 Rev1 conjunctivally 6 months after the first inoculation (Fensterbank e t al, 1982). Similarly, Falade (1983) did not observe abortions in 35 pregnant sheep vaccinated subcutaneously with 10 9 Revl. On the other hand, only 5 abortions were recorded among 600 sheep belonging to a B. melitensisinfected flock that were subcutaneously inoculated with 5 x 10 8 Rev1 late in pregnancy (Blasco et al., 1984). The aim of this study was to obtain additional information on the safety and serological responses of sheep vaccinated during 3 periods of pregnancy with Rev1, administered conjunctivally or subcutaneously.
Materials and Methods Animals and immunization procedure Sixtyone yearling, pregnant Aragonesa ewes were used. All animals were seronegative in rose bengal and complement fixation tests for brucellosis. The animals were vaccinated as described in Table I. The French strain was originally obtained from Dr. S.S. Elberg and kindly provided by Dr. J.M. Verger (Station de Pathologie de la Reproduction, INRA, France). The Spanish strain was a commercial vaccine of unknown origin. Methods described by Alton et al. (1976) were used to assess the count of viable organisms on the day of vaccination and the smoothness of cultures. Examination procedures Rectal temperature was taken before and after vaccination in all animals. Vaginals swabs from all animals were taken weekly from the day of vaccination until 2 months after lambing or abortion. When abortions occurred, vaginal swabs were also taken for bacteriological examination. At lambing, both vaginal swabs of ewes and rectal swabs of newborn lambs were also taken for bacteriological purposes. Aborted fetuses (born dead before day 149 of pregnancy) and dead
lambs (died during the first 3 days of life) were autopsied and samples of lungs, liver, spleen and gastric contents were cultured. All cultures were grown on Farrell s medium (Farrell, 1974) and incubated in air at 37 C for 10 days. Suspected colonies were identified by Gram staining, catalase, oxidase and urease tests and by growth in media containing 5 8g/ml of streptomycin with inhibition in media containing 5 IU/ml of penicillin (Alton et al., 1976). Portions of lungs, liver, spleen, kidneys and testicles of aborted or dead lambs were fixed in 10% buffered neutral formalin and embedded in paraffin. Sections 56 11m thick were cut and stained with hematoxylineosin according to conventional procedures for pathological studies. All vaccinated animals were bled 1, 4, 8, 12 and 16 weeks after vaccination for serological studies. Humoral immune responses to standard smooth antigens were studied by rose bengal and complement fixation tests, as described by Alton et al. (1976). Titers of 1/8 or higher in the complement fixation test were considered as positive. Statistical analyses Comparisons between treatments were made by analysis of variance for the duration of pregnancy and by the chi square test for numbers of ewes with vaginal excretion or abortions. Results Fever response to vaccination Mean rectal temperature before vaccination was 38.9 ± 0.1 C. This value was not changed by the conjunctival inoculation of the ewes. However, subcutaneously inoculated ewes showed an increase in rectal temperature to 40.08 ± 0.07 C on the first day, 39.44 ± 0.4 C on the second and returned to normal levels on the third day after vaccination. Vaginal excretion of vaccine strains All reproductive and bacteriological results are summarized in Table I. The vaccinal strain was isolated from the vaginal discharge of a significantly higher number of ewes vaccinated subcutaneously than conjunctivally (24/35 versus 9/26 respectively; P < 0.001). In the ewes vaccinated on day 55 of pregnancy that had vaginal discharges there was a considerably shorter duration of discharge in those vaccinated conjunctivally than in those vaccinated subcutaneously (7.0 versus 13.75 ± 2.4 days respectively; P < 0.05). There was no significant difference between strains with respect to the number of animals excreting Rev1 by the vagina. Most of the ewes which aborted excreted Rev1 by the vagina before the abortion. In animals vaccinated on day 120 of pregnancy with the French strain, there was a significant difference (P < 0.001) between the number of vaginal excretors vaccinated subcutaneously (10/11) and conjunctivally (2/9). However, there was no significant effect of route of vaccination on the duration of excretion. There was one subcutaneously vaccinated ewe that excreted Rev1 for 54 days but most of the animals ceased excretion after 1 or 2 weeks. Abortions and duration of pregnancy Twelve animals aborted among the 35 inoculated subcutaneously, while only 2 abortions were recorded among the 26 ewes vaccinated conjunctivally (P < 0.001 The first abortion was recorded on day 26 after vaccination.
The distribution of the 13 abortions that occurred among the 33 animals vaccinated on day 55 of pregnancy is presented in Fig. 1. Most of the abortions (69.2%) occurred between 4 and 8 weeks after vaccination. In the ewes vaccinated on day 55 of pregnancy, the French strain caused abortion in 38.8% of animals and the Spanish strain in 40% of vaccinated ewes. In these animals, both strains produced a statistically significant (P < 0.001) higher number of abortions when administered subcutaneously than when administered conjunctivally (11/16 versus 2/17 respectively for the 2 strains). Subcutaneous vaccination on day 55 of pregnancy significantly (P < 0.001) reduced the average duration of pregnancy; no significant differences were found between strains. However, the animals conjunctivally vaccinated with the Spanish strain had shorter pregnancies than those vaccinated with the French strain (140 ± 17.6 versus 148.7 ± 1.6 days respectively, P < 0.05). The duration of pregnancy was normal in 8 animals vaccinated with 106 French strain and only 1 ewe excreted Rev1 for a week. Among the 20 ewes vaccinated on day 120 of pregnancy, only 1 subcutaneously vaccinated animal aborted. However, 12 of these animals excreted the vaccinal strain. In the animals vaccinated subcutaneously with the French strain on day 55 or 120 of pregnancy, there were significant differences in the number of abortions produced (7/9 versus 1/11, respectively, P < 0.05) and, on the average length of pregnancy (113.5 ± 28.7 versus 148.5 ± 1.8 days respectively, P < 0.001 There was, however, no significant effect of vaccination time on both abortions and lenght of pregnancy when the same strain was administered conjunctivally. Lambs and examination of fetuses Most newborn lambs (93.75%) were alive 1 of the 3 dead 3 days after lambing. Only lambs had a positive culture at autopsy. Aborted fetuses in which Rev1 was isolated, had purulent bronchopneumonia, hepatitis and interstitial nephritis. Round cell infiltrates were also seen in the testicles and epididymis of aborted male fetuses (Fig. 2). Serological response Subcutaneously vaccinated ewes showed a more rapid increase of positive reactors in the rose bengal test and higher complement fixation titers than conjunctivally i vaccinated ewes (Fig. 3). Both the percentage of reactors in the rose bengal test and the mean complement fixation titers decreased slightly faster in vaccinated ewes than in the conjunctivally subcutaneously vaccinated ewes.
Discussion From the results presented in this report, it can be stated that Rev1 causes a high proportion of abortions when injected by both standard routes and doses into pregnant sheep. This side effect was seen in goats vaccinated with standard doses of Rev1 (Alton, 1970) and also in cattle vaccinated with standard doses of B19 (Corner and Alton, 1981 ).
Living avirulent vaccine strains can contain mutants with different degrees of residual virulence. The isolation of mutants of strain 19 in erythritolutilizing aborting animals that were vaccinated during pregnancy (Corner and Alton, 1981; Beckett and McDiarmid, 1985) is in agreement with this hypothesis. Similarly, variations have been noted in both the persistence after vaccination and the immunogenicity between 4 Rev1 strains of different origins, in a mouse model (Bosseray, 1985). In this trial, no significant differences were observed between strains of different origins with respect to vaginal excretion, number of abortions produced and the length of pregnancy in ewes subcutaneously vaccinated on day 55 of pregnancy. There were significant differences (P < 0.05) in both the number of abortions recorded and the length of pregnancy among conjunctivally vaccinated ewes, however the animals inoculated with the Spanish strain had received a 4fold higher dose than those vaccinated with the French strain. Unfortunately, these differences between strains in sheep vaccinated on day 120 of pregnancy could not be tested. In spite of effects of both strain and day of pregnancy at inoculation, most of the significant differences observed were due to the route of vaccine administration. A greater number of subcutaneously inoculated ewes aborted, excreted Rev1 strain and had a shorter pregnancy, compared with conjunctivally inoculated ewes. It has been stated that Rev1 infection the second week after generalizes in subcutaneous vaccination of normal sheep. Rates of infection then decrease being restricted to lymph nodes in the head and the prescapular region (Lantier and Fensterbank, 1985). Therefore, it is likely that this generalization leads to colonization of the uterus and subsequent abortion in vaccinated pregnant ewes. Most of the abortions recorded occurred between 28 and 56 days after vaccination. Therefore, vaccination of sheep late in pregnancy (> 120 days) leads to only a few abortions in spite of the high proportion of Rev1 vaginal excretors. This agrees with previous late results from sheep vaccinated in pregnancy and belonging to B. melitensis infected flocks (Blasco et al., 1984). The administration of standard doses of Rev1 by the conjunctival route appears to be a good alternative to subcutaneous vaccination in young ewes (Fensterbank et al., 1985). This method limits the generalization of Rev1 infection to lymph nodes in the head (Alton et al., 1984). In this trial, the side effects of Rev1 vaccination were minimized by this method of inoculation. Only 2 ewes receiving 1.8 x 109 Spanish strain aborted among the 26 conjunctivally inoculated animals. Although the mouse model (Bosseray et al., 1984) predicted only small differences of immunogenicity between doses of 10 3 and 10 7 Rev1, the side effects observed when vaccinating pregnant animals appear to be dose related (Alton, 1970; Crowther et al., 1977). Therefore, low doses of Rev1 should minimize the risk of abortion in pregnant animals. The subcutaneous inoculation with 4 Rev1 reduced doses at a level of 5 x 10 did not cause abortion in pregnant goats (Alton, 1970), while it conferred a level of protection similar to that produced by standard doses. In contrast, greatly reduced doses (10 810 9) of B. abortus strain 19 injected subcutaneously produced good levels of protection in pregnant cows (Nicoletti et al., 1978;
Alton et al., 1980). The side effects of this method were minimal (Nicoletti et al., 1978), although strain 19 has been responsible for some abortions in vaccinated cattle (Corner and Alton, 1981; Beckett and McDiarmid, 1985). Information on these aspects is scarce in sheep. Although the work of Crowther et al (1977) did not produce a very clearcut result, it demonstrated the unfavorable side effects of Rev1 in pregnant ewes. Doses of 10 4 Rev1 appeared to be safe but experimental animals were not challenged and the level of protection could not be evaluated. At this dose level, protection is effective in goats (Alton, 1970) but in sheep no protection was obtained when they were conjunctivally vaccinated with two doses of 10 6 Rev1 (Fensterbank et al., 1982). No abortions were recorded in the 8 animals vaccinated with 10 6 Rev1 of French origin. However, at this dose level, the Spanish strain was responsible for a very large number of abortions in sheep vaccinated in midpregnancy in field campaigns (unpublished results). Therefore, it is possible that some differences may exist in residual virulence between Rev1 strains at a dose level. Another important factor that should be considered is the breed of the animals vaccinated, since differences in susceptibility occur (Alton, 1985). From the results presented here, it can be concluded that conjunctival vaccination minimizes the risk of abortion for pregnant ewes and that the risk increases when the animals are vaccinated in the first 2 or 3 months of pregnancy. Due to the small number of animals used, we could not properly evaluate the differences at a dose level between the two strains inoculated. Additional work should be carried out to determine exactly the safest method of vaccination of pregnant ewes which will require challenge experiments with virulent B. melitensis to assess the level of immunity obtained. Acknowledgements This work has been supported by grant CICYT 376 and fellowship CONAI (DGA). We thank S. Lazaro and F. Lahoz for their excellent technical assistance. References Alton G.G. (1970) Vaccination of goats with reduced doses of Rev1 Brucella melitensis vaccine. Res. Vet. Sci. 2, 5459 Alton G.G. (1985) Rev1 and H38 Brucella melitensis vaccines. In : Brucella melitensis (Plommet M. & Verger J.M., eds.), Martinus Nijhoff. Dordrecht, pp. 229236 Alton G.G., Jones L.M. & Pietz D.E. (1976) Las tecnicas de laboratorio en la brucelosis. O.M.S. Monograph. Ser. no. 55, Geneva Alton G.G., Corner L.A. & Plackett P. (1980) Vaccination of pregnant cows with low doses of Brucella abortus strain 19 vaccine. Aust Vet. J. 56, 369372 Alton G.G., Fensterbank R., Plommet M. & Verger J.M. (1984) La brucellose de la chbvre. In : Les maladies de la chèvre. Les Colloques de I INRA, n 28, INRA, Paris, pp. 6991 Beckett F. & McDiarmid S.C. (1985) The effect of reduceddose Brucella abortus strain 19 vaccination in accredited dairy herds. Br. Vet. J. 141, 507514 Blasco J.M., Estrada A. & Mercadal M. (1984) A note on adult sheep vaccination with reduced dose of Brucella melitensis Revl. Ann. Rech.!.15,553556 Bosseray N. (1985) Quality control of four Rev1 antibrucella vaccines. In : Brucella melitensis. (Plommet M. & Verger J.M., eds.), Martinus Nijhoff, Dordrecht, pp. 229236 Bosseray N., Plommet A.M. & Plommet M. (1984) Theoretical, practical and statistical basis for a general control method of activity for
antibrucella vaccines. Dev. Biol. Stand. 56, 257270 Corner L.A. & Alton G.G. (1981) Persistence of Brucella abortus strain 19 infection in adult cattle vaccinated with reduced doses. Res. Vet. Sci. 31, 342344 Crowther R.W., Orphanides A. & Polydorou K. (1977) Vaccination of adult sheep with reduced doses of Brucella melitensis strain Rev1 : Safety and serological responses. Trop. Anim. Health Prod. 9, 8591 Elberg S.S. (1981) Rev1 Brucella melitensis vaccine. Part. II (19681980). Vet. Bull. 51, 67 73 Falade S. (1983) Serological response of sheep to Brucella melitensis Rev1 vaccine. Zentralbl. Veterinaermed. B, 30, 546551 Farrell LD. (1974) The development of a new selective medium for the isolation of Brucella abortus from contaminated sources. Res. Vet. Sci. 16, 280286 Fensterbank R., Pardon P. & Marly J. (1982) Comparison between subcutaneous and conjunctival route of vaccination with Rev1 strain against Brucella melitensis infection in ewes. Ann. Rech. Vét. 13, 295301 Fensterbank R., Pardon P. & Marly J. (1985) Vaccination of ewes by a single conjunctival administration of Brucella melitensis Rev1 vaccine. Ann. Rech. Vet 16, 351356 Jones L.M. & Marly J. (1975) Serological and bacteriological studies of ewes vaccinated with Brucella melitensis strain Rev1 during lactation. Ann. Rech. V6t. 6, 6771 Lantier F. & Fensterbank R. (1985) Kinetics of Rev1 infection in sheep. In : Brucella melitensis. (Plommet M. & Verger J.M., eds.), Martinus Nijhoff, Pub[., Dordrecht, pp. 247251 Nicoletti P., Jones L.M. & Berman D.T (1978) Adult vaccination with standard and reduced doses of Brucella abortus strain 19 vaccine in a dairy herd infected with brucellosis. J. Am. Ve t. Med. Assoc. 173, 14451449