Conjunctival vaccination of pregnant ewes and goats with Brucella melitensis Rev 1 vaccine: safety and serological responses E Zundel, Jm Verger, M Grayon, R Michel To cite this version: E Zundel, Jm Verger, M Grayon, R Michel. Conjunctival vaccination of pregnant ewes and goats with Brucella melitensis Rev 1 vaccine: safety and serological responses. Annales de Recherches Vétérinaires, INRA Editions, 1992, 23 (2), pp.177-188. <hal-00902061> HAL Id: hal-00902061 https://hal.archives-ouvertes.fr/hal-00902061 Submitted on 1 Jan 1992 HAL is a multi-disciplinary 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.
Original article Conjunctival vaccination of pregnant ewes and goats with Brucella melitensis Rev 1 vaccine: safety and serological responses E Zundel JM Verger M Grayon R Michel 1 Institut National de la Recherche Agronomique, Centre de Tours, Pathologie Infectieuse et Immunologie, 37380 Nouzilly; 2 Vétoquinol, 70204 Lure Cedex, France (Received 14 January 1992; accepted 10 February 1992) Summary ― When Brucella melitensis strain Rev 1 vaccine (Rev 1) is administered by the standard method (1-2 x 109 viable bacteria injected subcutaneously), it may induce long-lasting serological responses and/or cause abortion in pregnant animals. The conjunctival route considerably reduces these drawbacks. In the present experiment a 1 x 108 CFU dose for both ewes and goats conjunctivally vaccinated at mid-pregnancy was tested for innocuousness (outcome of pregnancy, contamination of unvaccinated contact animals, duration of serological responses) in comparison with 3 x 108 CFU (ewes and goats), 19 x 10 and 39 x 10 CFU (ewes) doses. No reaction was observed at the time of vaccination, and the risk of environmental contamination with Rev 1, due to the conjunctival administration of the vaccine, is negligible. Abortions occurred later at surprisingly severe rates (over 60% of pregnant vaccinated animals), except in the 1 g x 10 CFU ewes group (20%). Moreover, the serological reactions of the 1 xl 80 CFU ewes which normally lambed were negative again as early as 12 weeks after vaccination. Although the dose of 1 x 108 CFU Rev 1 was safer for pregnancy than the standard dose mainly in ewes as compared to goats, the innocuousness was not yet sufficient to propose the former dose to indiscriminately vaccinate sheep and goats by the conjunctival route, whatever the age or physiological status. brucellosis I vaccine I Brucella melitensis Rev 1 I conjunctival route I safety Résumé ― Vaccination conjonctivale de chèvres et de brebis gestantes avec le vaccin Brucella melitensis Rev 1 : Innocuité et réponses sérologiques. La souche Rev 1 de Brucella melitensis (Rev 1) est un vaccin efficace contre la brucellose des petits ruminants. Son emploi par la méthode standard (1-2 x f09 bactéries viables injectées par voie sous-cutanée) peut induire des réponses sérologiques de longue durée, et des avortements chez les animaux gestants. La voie conjonctivale réduit considérablement ces inconvénients. La présente expérimentation a utilisé 38 brebis et 23 chèvres vaccinées à mi-gestation avec Rev 1, par voie conjonctivale, pour évaluer l innocuité (issue de la gestation, contamination des animaux contacts non-vaccinés, durée des réponses sérologiques) d une dose 1 x 10 6 UFC comparée à des doses de 3 x 108 UFC (brebis et chèvres), 1 x 910 et 3 x 109 UFC (brebis). Aucune réaction n a été observée au moment de la vaccination, et le risque de contamination de l environnement par Rev 1, inhérent à l administration conjonctivale, est négligeable. Les taux d avortements ont été étonnamment élevés (plus de 60% des animaux gestants vaccinés), sauf pour le groupe des brebis vaccinées avec 1 x 810 UFC, dont * Correspondence and reprints
(à l exception d une brebis qui avait avorté) les réactions sérologiques étaient négatives dès la 12B semaine après vaccination. L innocuité de la dose 1 x f08 UFC est meilleure que celle de la dose stan- 8 dard, principalement chez la brebis. Elle n est cependant pas suffisante pour recommander 1 x 10 UFC comme dose unique pour la vaccination conjonctivale par Rev 1 de tous les petits ruminants, quels que soient leur âge et leur état de gestation. brucellose / vaccin / Brucella melltensis Rev 1 / voie conjonctlvale / Innocuité INTRODUCTION Brucella melitensis strain Rev 1 (Rev 1) has proved to be a very effective vaccine against ovine and caprine brucellosis (Alton and Elberg, 1967; Elberg, 1981 However Rev 1 has 2 drawbacks when injected subcutaneously at full (standard) dose, ie 1-2 x 19 O viable bacteria enumerated as colony forming units (CFU): it may induce long-lasting serological responses (Fensterbank et al, 1982) which do not allow discrimination between infected and vaccinated animals, and, if injected during pregnancy, it may cause abortion in pregnant ewes and goats (Alton and Elberg, 1967; Elberg, 1981). For these reasons, vaccination is restricted to young animals between 3 and 8 months of age. However, in many areas, the lambing (kidding) season is extended and the prevalence of brucellosis is high. Control programs in these areas need a method to vaccinate both young and adult animals, including those that may be pregnant (Alton et al, 1972; Alton, 1990). Attempts to avoid previously quoted drawbacks have focused on subcutaneous vaccination with reduced doses or on conjunctival vaccination. Greatly reduced doses (10 4 CFU Rev 1) were safe (Alton, 1970; Crowther et al, 1977) but protection against challenge at mid-pregnancy has not been demonstrated. A log CFU Rev 1 dose administered by the conjunctival route at 4 months of age induced protection which was almost as good as that given by the subcutaneous route, with serological titres disappearing in all animals by 4 months after vaccination (Fensterbank et al, 1985). Conjunctival doses of 10 8and 10! CFU Rev 1 were also found to be effective for ewes (Fensterbank et al, 1985) and goats (Fensterbank et al, 1987) respectively; a dose of 1 x 8 10 CFU Rev 1 should therefore be effective for both sheep and goats. It has been shown that the innocuousness of Rev 1 for pregnant ewes was improved by the conjunctival administration of the vaccine, and that it also depended on the dose and time of vaccination (Jiménez de Bagues et al, 1989). The present experiment was devised to test whether a 1 x 10 8 CFU Rev 1 dose for both ewes and goats conjunctivally vaccinated at mid-pregnancy compared with 3 x 108 (ewes and goats), 1 x 10 9 and 3 x 109 (ewes) CFU Rev 1 doses could have satisfactory safety levels with regard to the following: i), the outcome of pregnancy in vaccinated animals (innocuousness); and ii), the contamination of unvaccinated control animals kept in close contact with the vaccinated animals. Both vaccinated and contact animals were also examined for the presence and persistence of vaccinal antibodies. The results showed that although the dose of 1 x 10 8 CFU Rev 1 was safer for
pregnancy than the standard dose (1 x 109) mainly in ewes as compared to goats, the innocuousness was not yet sufficient to propose the former dose for the universal conjunctival vaccination of sheep and goats. MATERIALS AND METHODS Animals and experimental design Ewes Thirty-eight 3-5-year-old pregnant ewes of the Préalpes-Lacaune breed and born in the brucellosis-free flock at the INRA-Nouzilly station were used in this experiment. The ewes were randomly allotted to 6 groups, as shown in table I. The animals in 4 of the groups were placed in 4 separate pens, then vaccinated by the conjunctival route: 5 animals with 1 x 10 8, 5 with 3 x 10 8, 11 with 1 x 109 and 6 with 3 x 109 CFU Rev 1. Eleven unvaccinated animals in the 2 remaining groups were kept as controls, 6 in close contact with the 1 x 109 group and 5 with the 3 x 109 group. Goats Twenty-three 3-5-year-old supposedly pregnant goats of the Alpine breed were purchased from a brucellosis-free flock. The goats were randomly allotted to 4 groups, as shown in table 11. The animals (all pregnant) in 2 groups were vaccinated by the conjunctival route: 6 with 1 Bx 10 and 5 with 3 x 108 CFU Rev 1. Twelve unvaccinated animals were kept as controls: 6 (3 pregnant) with the 1 x 108 vaccinated group and 6 (2 pregnant) with the 3 x 10 8one. culture of B melitensis strain Rev 1 maintained at Nouzilly (France). Briefly, smooth colonies were grown on suitable agar slants after control for purity and colony morphology. The culture was diluted to the required concentration according to an absorbance reference curve. Actual concentrations used were measured by viable counts. Animals were vaccinated by dropping one 33-pl drop of the vaccinal suspension onto the conjunctiva. Examination procedures Clinical examination Local and general reactions were observed, rectal temperature was taken daily in all animals (10 8 vaccinated ewes groups excepted) for 4 days before and 7 days after vaccination. Abortions and full-term deliveries were recorded. Abortion was defined as the delivery of 1 (or several) stillborn kid(s) or lamb(s) or newborn that died within 48 h of birth. Bacteriology All cultures were carried out on Farrell s selective medium (Farrell, 1974), and plates were read after 4 and 7 days of incubation at 37 C. Detection limits were 3-5 CFU per g or ml of solid or liquid specimens respectively. The number of Rev 1 colonies per plate was recorded as: 0, no Brucella; 1, 1-5 colonies; 2, 6-25; 3, 26-125; 4, 126-625; 5, > 625. Ocular, nasal and buccal swabs from all animals were taken daily for a week and on the l4th day after vaccination. The local persistence index (LPI), expressed as a percentage, was calculated for each group of animals as follows: Vaccination The batch of Rev 1 vaccine used was freshly prepared as previously described (Fensterbank et al, 1987) from a vial of a freeze-dried seed where M = sum of the marks recorded as previously mentioned for all animals for 7 days after vaccination; n = number of animals;
105 = 3 (daily swabs) x 7 d (duration of the sampling period taken as LPI basis) x 5 (maximum mark per cultured swab). Vaginal swabs were taken weekly between vaccination and parturition or abortion, daily for a week thereafter, then every 2 weeks for 3 months. Swabs were directly smeared onto the surface of the Farrell s medium. Colostrum (or milk) samples were taken daily after parturition (or abortion) for a week, then fortnightly for 3 months. These samples were centrifuged, and cream and sediment were separately plated with a sterile loop. Aborted foetuses or dead kids or lambs were necropsied and samples of lungs, liver, spleen and gastric content were cultured. Serology All animals were bled before vaccination (week No 0), fortnightly to week 26, then monthly to week 33 (goats), 37 (10 8 ewes), 61 (10 9 contact and vaccinated ewes). Sera were subjected to Rose Bengal plate test (RBPT) (Benga-test, lffa-m6rieux, Lyon, France), and to complement fixation test (CFT) (Antifix,
Rh6ne-M6rieux, Lyon, France), as described by Alton et al (1988). Sera were considered as positive when showing any degree of agglutination (RBP T), or 50% fixation at a dilution of 1/4 or higher (CF!. RESULTS Clinical results Statistics Durations of pregnancy were compared by the non-parametric Mann and Whitney ll-test. Numbers of animals with positive ocular, nasal and buccal swabs in the 7 days after vaccination, numbers of abortions and numbers of viable kids or lambs were compared by the X2-test. Local and general responses No local reaction was observed in vaccinated animals, whatever the species or the dose. Mean rectal temperatures were 39.0 ± 0.2 C in ewes, 38.8 ± 0.3 C in goats before vaccination, and remained un-
changed after conjunctival inoculation of Rev 1. Abortions and duration of pregnancy The number of abortions, viable lambs and viable kids is given in tables I (ewes) and 11 (goats). The number of abortions and the number of viable lambs were significantly different between the 1 x 10 8 and 19 x 10 ewe groups (P < 0.05 and P < 0.001 respectively). There was no significant difference between the vaccinated goats groups. No abortion occurred among the non-vaccinated controls kept in close contact with their respective vaccinated groups. The mean durations of pregnancy for the different groups are given in tables I and II. The results on the 11 control ewes were not sta- were pooled because they tistically different (table 1). A significant difference between the 1 x 108 and 1 x 109 vaccinated groups was observed in ewes (P < 0.05). Durations of pregnancy were significantly shorter for the 4 aborted goats in the 3 x 108 group (107.5 days) than for the 1 x 108 group (121.5 days) (P < 0.05). Bacteriological results Ocular, nasal and vaginal secretions after vaccination Brucella were recovered during 5 (1 x 10 8, ewes) to 14 (3 x 109, ewes; 3 x 10 8, goats) days after vaccination mainly from ocular and nasal swabs as compared to buccal swabs of vaccinated animals. They were never recovered from swabs of contact animals. Positive cultures were always very slight, as shown by LPI, which expresses the local persistence of Rev 1 for each group of animals after conjunctival instillation, and ranged from 3.2 to 9.9% (tables I and II). Percentages of positive ewes were significantly lower between the 1 x 108 group and each of the other 3 groups 3 x 8, 10 1 x 9, 1 O 3 x 910 (P < 0.05). No difference was observed between the 2 vaccinated goats groups. was ob- No vaginal excretion of Rev 1 served in pregnant animals between vaccination and abortion or parturition, except in one ewe (3 x 109) which excreted slightly at weeks 3, 4 and 5 but stopped until abortion 3 weeks later. Milk and aborted materials All the ewes or goats which aborted were excretors of Rev 1 strain, and their foetuses had positive cultures at necropsy. Vaginal excretion was heavy during the first week, then decreased and disappeared within 2 to 3 weeks. Rev 1 excretion in milk was weak, irregular, and usually lasted 2 to 3 weeks with 3 long-lasting exceptions (1 ewe and 2 goats). The vaccinated ewes which lambed normally did not excrete Rev 1. No contact animal except one goat excreted Rev 1. This animal kidded normally on day 163 of pregnancy, then excreted Rev 1 weakly for 8 days in vaginal discharges, but excretion was still found in milk 40 weeks later. Serological results Ewes (figs 1 and 2) All vaccinated ewes were positive to RBPT from 2 to 4 weeks (1 x 10 8), 8 (3 x 108, 1 x 10 9), 61 (3 x 109) and from 29 to 57 weeks (1 x 109) after vaccination. They were all positive to CFT from 2 to 10
weeks (3 x 108, 11Q9), 49 weeks (3 x 109) and from 29 to 57 weeks (1 x 10 9) after vaccination. In the 1 x 10 $ group, the maximum proportion of vaccinated pregnant ewes positive to CFT was 4 out of 5 at weeks 2 and 4. One of them aborted
7 weeks after vaccination, and was still positive 37 weeks later. The 4 other animals were negative again at week 12 (CFT) and 16 (RBPT) (figs 1 and 2). Among the &dquo;contact&dquo; ewes, there were a few weakly positive reactions after the abortions: from 10 to 18 weeks and at the 22nd week after vaccination to RBPT (fig 1); only one animal was weakly positive to CFT at weeks 14 and 16 after vaccination (fig 2). Goats (figs 3 and 4) All vaccinated goats were positive to RBPT from 2 to 22 (3 x 10 8) or 33 weeks (1 x 108) after vaccination. They were all positive to CFT from 4 (1 x 10 8) or 6 (3 x 108) to 10 (3 x 108) or 29 weeks (1 x 10 8) after vaccination. Nearly all the non-vaccinated &dquo;contact&dquo; goats gave long-lasting strongly positive serological responses after the onset of abortions, contrary to ewes (figs 3 and 4). DISCUSSION No local (ocular) or general (temperature) reaction to conjunctival vaccination with Rev 1 was observed in this study or by others (Jiménez de Bagubs et al, 1989), contrary to standard subcutaneous vaccination (Lantier and Fensterbank, 1985; Jim6nez de Bagues et al, 1989). The situation was quite different regarding the outcome of pregnancy, which was dramatically affected by Rev 1 conjunctival vaccination of small ruminants at mid-term. Doses of 3 x 10 8 (sheep and goats), 1 x 109 and 3 x 10 9 (sheep) caused very high percentages of abortion (60 and more), similar to those expected when pregnant ewes and goats are subcutaneously vaccinated with the standard dose (1-2 x 109) of Rev 1 (Alton and Elberg, 1967; Alton et al, 1967; Entessar et al, 1967; Elberg, with the virulent strain 1981) or challenged B melitensis H38 (Fensterbank et al, 1982, 1985, 1987). Surprisingly, these abortion
rates were far more severe than those previously reported for ewes conjunctivally vaccinated with similar doses (5 x 10 8 to 1.8 x 109) of Rev 1 but in earlier (55 days) or later (120 days) stages of pregnancy, ie 12% and 0% respectively (Jiménez de Bagues et al, 1989). Other results for both ewes (Entessar et al, 1967; Jiménez de Bagues et al, 1989) and goats (Alton and Elberg, 1967; Alton et al, 1967) corroborate the hypothesis that the stage of pregnancy at the time of vaccination is probably responsible for these significant differences as a consequence of a higher susceptibility of the placenta to Rev 1 infection at the middle than at other stages of pregnancy. The lowest of the 4 doses tested (1 x 108) was the safest for pregnancy, mainly in ewes. The mean duration of pregnancy (145.8 days), and the proportion of abortions (20%), and viable lambs (72.7%) were significantly different from their counterparts, ie respectively 131.8 days (P < 0.05), 90.9% (P < 0.05) and 5.6% (P < 0.001 for ewes vaccinated with the 1 x 10 9 dose (table I). In goats the improvement of innocuousness was not as evident, since all animals vaccinated with 1 x 10 $ Rev 1 aborted (table II). However, a dose-effect was observed in the duration of pregnancy, which was significantly (P < 0.05) longer for aborted goats in the 1 x 10 8 group (121.5 days) than for the aborted ones in the 3 x 10 8 group (107.5 days). The clinical response to the 1 x 10 8 Rev 1 dose, which was more severe in goats than in ewes, is in agreement with the well-known higher susceptibility of the former to Brucella infection. This difference is usually turned to account in experiments on brucellosis vaccines to fit the dose of the challenge strain to the animal species, eg for the
strain B melitensis H38, 5 x 10! CFU for ewes (Fensterbank et al, 1982, 1985) and 5 x 10 6 CFU for goats (Fensberbank et al, 1987). Rev 1 conjunctival vaccination of sheep in field conditions did not lead to similar severe clinical results. No abortion was recorded among 1 039 pregnant ewes from 3 field flocks, conjunctivally vaccinated with a single Rev 1 standard dose (1 x 10 9) (Michel, personal communication). Similar differences in susceptibility to a Brucella conjunctival challenge have already been reported between pregnant cows from an experimental station and others purchased from field herds (Goode et al, 1957). Although there is no satisfactory explanation for these differences, it may be assumed that in experimental and well-controlled conditions, animals are more &dquo;naive&dquo; towards bacterial infections and therefore a priori more susceptible to a virulent challenge or a living vaccine. This variability could be added to the breed-related variations in susceptibility to Brucella infection (Crowther et al, 1977; Alton, 1985). In the few days following vaccination, Rev 1 was isolated from ocular, nasal and buccal swabs in 25-55% of the vaccinated animals. However, LPI values were always very low whatever the dose (tables I and II). Rev 1 was never recovered from corresponding swabs in unvaccinated contact ewes and goats which also remained serologically negative until the abortion period of the vaccinated animals. The risk of environmental contamination with Rev 1 due to the conjunctival administration of the vaccine could therefore be considered as negligible. After vaccination at mid-pregnancy Rev 1 was not recovered (except in 1 ewe) from vaginal swabs collected before abortion. It has however been reported that in ewes conjunctivally vaccinated with Rev 1 during early or late pregnancy, most of the animals which aborted excreted the vaccinal strain via the vagina before abortion (Jiménez de Bagu6s et al, 1989), as observed after a virulent challenge with B melitensis strain H38 (Fensterbank et al, 1982). All animals which aborted (and 1 goat which kidded at term) excreted Rev 1 organisms from the vagina and/or in milk for 2-3 weeks after delivery. This is in agreement with previously reported durations of Rev 1 excretions (14-33 days) for both sheep (Entessar et al, 1967) and goats (Alton et al, 1967; Alton, 1970). However, 2 goats out of 11 (18%) still excreted Rev 1 in milk 44 and 49 weeks after abortion, and one ewe out of 19 (5%) for 6 months after abortion. During the abortion period, nonvaccinated ewes were weakly contaminated in spite of the very close contact with vaccinated animals: no abortion occurred, no Rev 1 was recovered from the vagina or the milk, and only 1 ewe was found to be weakly CFT-positive. Contamination was much greater among unvaccinated control goats raised in the same conditions. Although no abortion occurred, one goat excreted Rev 1 from both udder and vagina. All nonvaccinated animals became strongly positive to serological tests after the vaccinated goats began to abort. Furthermore, Rev 1 was recovered during the abortion period from ocular and/or nasal and/or buccal swabs of many more contact goats (and in a much greater amount) than contact ewes (data not shown). These differences between ewes and goats could be connected with the behavioural peculiarities of those species; the latter are known to be both curious and active. However, it is well known that animals can be experimentally contaminated with vaccinal (Alton et al, 1967) or virulent (En-
tessar et al, 1967; Davis et al, 1990) Brucella strains by natural contact with aborting donor animals. The serological results with both RBPT and CFT were very similar. Most of vaccinated pregnant animals reacted to RBPT and CFT from weeks 2 or 3 after vaccination; immediately after the titres began to decrease (data not shown). Each abortion then looked like a vaccination booster, and this generalized multiplication of bacteria produced a long-lasting serological response. The 1 x 10 8 vaccinated ewes, except one animal which aborted, were again CFT negative as early as 12 weeks after vaccination. In this study the dose of 1 x 10$ CFU Rev 1 was found to be safer for pregnancy than the standard dose (1 x 10 9), mainly in ewes as compared to goats. It is therefore assumed that this dose could be used without risk under field conditions in animals that are usually more resistant than experimental animals, confined in wellcontrolled conditions. However, innocuousness as evaluated under the latter conditions was not yet sufficient to propose the dose of 1 x 10$ CFU Rev 1 to indiscriminatly vaccinate sheep and goats what-ever the age or physiological status. Further trials should be carried out to determine whether 1 x 10! CFU B melitensis Rev 1 is the highest dose which could be both effective and safe. ACKNOWLEDGMENTS We are grateful to P Lechopier for aiding with technical problems and to G Bourgy, P Bernardet, P Calahorra, JR Chardron, R Delaunay, A Dixneuf, D Musset and W Pi6mont for care and maintenance of the animals. This study was supported by Vétoquinol. REFERENCES Alton GG (1970) Vaccination of goats with reduced doses of Rev 1 Brucella melitensis vaccine. Res Vet Sci 11, 54-59 Alton GG (1985) Rev 1 and H38 Brucella melitensis vaccines. In: Brucella melitensis (Verger JM, Plommet M, eds) Martinus Nijhoff, Dordrecht, 215-227 Alton GG (1990) Brucella melitensis. In: Animal Brucellosis (Nielsen K, Duncan JR, eds) CRC Press, Boca Raton, FL (USA), 383-409 Alton GG, Elberg SS (1967) Rev 1 Brucella melitensis vaccine. A review of ten years of study. Vet Bull 37, 793-800 Alton GG, Elberg SS, Crouch D (1967) Rev 1 Brucella melitensis vaccine. The stability of the degree of attenuation. J Comp Pathol77, 293-300 Alton GG, Jones LM, Garcia-Carrillo C, Trenchi A (1972) Brucella melitensis Rev 1 and Brucella abortus 45/20 vaccines in goats: immunity. Am J Vet Res 33, 1747-1751 Alton GG, Jones LM, Angus RD, Verger JM (1988) Techniques for the Brucellosis Laboratory. INRA, Paris Crowther RW, Orphanides A, Polydorou K (1977) Vaccination of adult sheep with reduced doses of Brucella melitensis strain Rev 1: safety and serological responses. Trop Anim Health Prod 9, 85-91 Davis DS, Templeton JW, Ficht TA, Williams JD, Kopec JD, Adams LG (1990) Brucella abortus in captive bison. 1. Serology, bacteriology, pathogenesis, and transmission to cattle. J Wild Dis 20, 360-371 Elberg SS (1981) Rev 1 Brucella melitensis vaccine. Part I I. 1968-1980. Vet Bull 51, 67-73 Entessar F, Ardalan A, Ebadi A, Jones LM (1967) Effect of living Rev 1 vaccine in producing long-term immunity against Brucella melitensis infection in sheep in Iran. J Comp Pathol77, 367-376 Farrell ID (1974) The development of a new selective medium for the isolation of Brucella abortus from contaminated sources. Res Vet Sci 16, 280-286
Fensterbank R, Pardon P, Marly J (1982) Comparison between subcutaneous and conjunctival route of vaccination with Rev 1 strain against Brucella melitensis infection in ewes. Ann Rech Vét 13, 295-301 Fensterbank R, Pardon P, Marly J (1985) Vaccination of ewes by a single conjunctival administration of Brucella melitensis Rev 1 vaccine. Ann Rech Vét 16, 351-356 Fensterbank R, Verger JM, Grayon M (1987) Conjunctival vaccination of young goats with Brucella melitensis strain Rev 1. Ann Rech Vét 18, 397-403 Goode ER, Manthei CA, Amerault TE (1957) Relationship of age to susceptibility of nonvaccinated cattle to Brucella abortus. Am J Vet Res 18, 279-282 Jim6nez de Baguos MP, Marin CM, Barberan M, Blasco JM (1989) Responses of ewes to B melitensis Rev 1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy. Ann Rech Vét 20, 205-213 Lantier F, Fensterbank R (1985) Kinetics of Rev 1 infection in sheep. In: Brucella melitensis (Verger JM, Plommet M, eds) Martinus Nijhoff, Dordrecht, 247-251