Vaccinal properties of Salmonella abortusovis mutants
for streptomycin: screening with an ovine model
P Pardon, J Marly, F Lantier, R Sanchis
To cite this version:
P Pardon, J Marly, F Lantier, R Sanchis. Vaccinal properties of Salmonella abortusovis mutants
for streptomycin: screening with an ovine model. Annales de Recherches Vétérinaires, INRA
Editions, 1990, 21 (1), pp.57-67. <hal-00901921>
HAL Id: hal-00901921
https://hal.archives-ouvertes.fr/hal-00901921
Submitted on 1 Jan 1990
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
Vaccinal
properties of Salmonella abortusovis
mutants for streptomycin:
screening with an ovine model
P Pardon
1
J
Marly
F Lantier
R Sanchis
INRA, Station de pathologie de la reproduction, Unité de pathologie infectieuse ovine,
Tours-Nouzilly,
2
37380 Nouzilly
Ministère de l’agriculture, Services vétérinaires, Laboratoire national de pathologie
des petits ruminants, 63, avenue des Arènes, 06051 Nice Cedex, France
(Received 27 October 1988; accepted 7 June 1989)
Summary &horbar; Living attenuated vaccines may help control infection with Salmonella enterica subspecies enterica ser abortusovis in sheep without constraints incompatible with husbandry. Virulence
and immunogenicity of 2 Abortusovis streptomycin-dependent strains and 3 reverse mutants were
compared in sheep, the virulent parental strain Abortusovis 15/5 being used as reference. Reduction
of virulence with conservation of some immunogenicity was demonstrated for the 5 mutant strains after intravenous or subcutaneous inoculation. A 5-day bacteriemia was detectable after intravenous
inoculation with parental strain, whereas none was shown with mutant strains. Mutant reverse
strains survived in loco-regional lymph nodes for at least 12 days. The dependent strain D5 and reverse strain Rv6 were selected for subcutaneous vaccination of 2 groups of 18 ewes 32 days before
fecundation. Ten pregnant ewes per group and 10 unvaccinated ewes were randomly selected
among pregnant animals and were challenged subcutaneously at mid-gestation. Both vaccines conferred protection, but Rv6 was more efficient than D5 and was chosen as the vaccinal strain.
vaccine / Salmonella abortusovis / ovine / mutant strain
Résumé &horbar; Propriétés vaccinales de souches du sérotype Salmonella abortusovis mutantes
vis-à-vis de la streptomycine : criblage avec un modèle ovin. Un vaccin vivant atténué peut faciliter le contrôle de l’infection ovine par Salmonella enterica subspecies enterica ser abortusovis sans
imposer des contraintes incompatibles avec l’élevage. La virulence et l’immunogénicité de 2
souches mutantes streptomycino-dépendantes de ce sérotype et de 3 mutants réverses ont été
comparées sur ovins, la souche sauvage parentale servant de contrôle. La réduction de la virulence
avec conservation d’une immunogénicité a été démontrée pour ces 5 souches mutantes après inoculation intraveineuse ou sous-cutanée. Une bactériémie était détectable pendant les 5 premiers
jours après inoculation intraveineuse de la souche sauvage, tandis qu’aucune bactériémie des
souches mutantes n’était détectable. Les bactéries réverses survivaient au moins 12 jours dans les
ganglions lymphatiques loco-régionaux après injection sous-cutanée. Une souche dépendante (D5)
et une souche réverse (Rv6) ont été retenues pour la vaccination sous-cutanée de 2 lots de 18 brebis 32 jours avant fécondation; 10 brebis par lot et f0 brebis non vaccinées ont été tirées au sort
parmi les brebis présumées gravides et ont été éprouvées par voie sous-cutanée à mi-gestation.
Les 2 souches augmentaient la résistance des brebis à l’épreuve; la souche Rv6 étant plus protectrice, cette souche a été retenue comme souche vaccinale.
vaccin /Salmonella abortusovis / ovin / souche mutante
INTRODUCTION
Abortion and vaginal bacterial excretion
are the main characteristics of endemic infection of sheep due to Salmonella enterica
subsp enterica
ser
abortusovis
(called
Abortusovis; LeMinor and Popoff, 1987).
Collective and repeated antibiotic treatment is expensive and usually unsatisfactory. Inactivated vaccines require 2 or 3 injections per gestation and give irregular
results. Living attenuated vaccines may
help control Abortusovis infection without
constraints hardly compatible with sheep
husbandry.
Analogy with the Brucella melitensis
strain Rev 1 vaccine (Elberg, 1981) has
directed research to a living vaccine (Pardon et al, 1980). Eight streptomycindependent and 6 reverse mutants were
selected (Lantier et al, 1981Screening of
the vaccinal strain among these 14 mutant
strains was carried out in vivo, in mice and
then in sheep. Virulence and immunogenicity studies in a murine model (Pardon
and Marly, 1979) allowed us first to exclude 9 out of the 14 mutant strains
(Lantier et al, 1981).The final choice
among the 5 remaining strains was made
at the end of 3 successive experiments in
sheep (Pardon et al, 1983; Sanchis and
Pardon, 1984). The first 2 experiments led
to only 2 strains being retained, 1 per category of mutants, according to virulence
and immunogenicity in non-gravid sheep.
The last experiment, a vaccinationchallenge test in gravid ewes, led to the
choice of the vaccinal strain Rv6.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Abortusovis 15/5
Plommet from the
was originally isolated by
placenta of an aborting ewe
(Pardon and Marly, 1979). This streptomycinsensitive (Str
) strain was used to obtain the res
sistant mutant, 15/5 Str
, the dependent mur
tants, Dl and D5 and the reverse mutants, Rvl,
Rv4 and Rv6 (Lantier et al, 1981 Conditions of
strain preservation, diluent and preparation of
the inoculum have been described elsewhere
(Pardon and Marly, 1979). Strains were plated
Trypticase Soy Agar (TSA; BioM6rieux,
Lyon) or Salmonella-Shigella medium (SS; BioMérieux, Lyon). Mutant strains differed from the
s by their growth caparent wild strain 15/5 Str
pacity. In the presence of streptomycin (500 pg/
ml; Specia, Paris) added to the culture medium,
growth was observed only with the resistant mur (colonies of at least 1 mm in diamtant 15/5 Str
eter in less than 24 h of incubation) and the reverse mutants Rvl, Rv4 and Rv6 (colonies of at
least 1 mm in diameter in over 24 h). The parent
on
s was used as a referstrain Abortusovis 15/5 Str
ence for virulence, and the Abortusovis strain
15/5 Str
r as a challenge strain.
Animals
of both sexes or male lambs (7 to 8
months old), Préalpes-Lacaune reared in an
isolated flock free of contagious abortions, were
distributed randomly in experimental groups and
placed in a specialized building 1 week before
inoculation. Ewes were fertilized in a controlled
covering after hormonal heat synchronization.
Three successive experiments with different
aims were made between August and October
1978.
Sheep
Multiplication and persistency
Nine adult sheep (7 ewes, 2 rams) were distributed in 3 experimental groups. Each group received in the jugular vein 5 ml of bacterial suspension containing 6 x 10! to 7 x 10! CFU of 1
of these strains: wild 15/5 Str
, dependent D5 or
s
reverse Rv6. Blood samples for serological tests
were taken each day for 2 weeks, then twice
during the 3rd week, and once a week until
slaughter. Blood samples for bacteriological examination were taken at 30 min and 6 h after inoculation, then twice a day for 4 days, and each
morning for the next 4 days. Rectal tempera-
tures were recorded at the time of inoculation
and then each morning for 6 days. At slaughter,
42 days after inoculation, samples were harvested: supramammary, precrural, prescapular, medial retropharyngeal, submaxillary, parotid, hepatic, mediastinal and distal jejunal lymph
nodes; portions of liver, spleen, lung, kidneys,
udder or testis; gall-bladder; contents of rumen
and intestine at different levels; bile and heart
clot.
lected with a vaginal swab. All aborted foetuses
and dead lambs were necropsied, and cultures
were made from colostrum and from spleen,
lung, stomach contents and brain. The 28 proven gravid ewes in challenged groups were
slaughtered and necropsied between 2.5 and 3
months after abortion or normal lambing. At
slaughter, samples for Salmonella detection
were: prescapular, hepatic, ileo-caecal and distal jejunal lymph nodes; gall-blader and uterus.
Invasiveness and immunogenicity
Clinical examination
Young rams (18; 7-8 months old) were distributed in 6 experimental groups. Each group received subcutaneously (sc) behind the right
shoulder 2 ml of bacterial suspension containing
between 5 x 10
8 to 1 x 10! viable bacteria of 1
of these strains: wild 15/5 Str
, dependent D1 or
s
D5, reverse Rv1, Rv4 or Rv6. Rectal temperatures were recorded twice a day for 3 days after
inoculation and each morning for the following 7
days. Blood samples for serological tests were
collected at the time of inoculation and at days
2, 5, 7, 9 and 12 after inoculation. Skin thickness at the site of inoculation was measured
each morning from the day of inoculation to day
9 after inoculation. At slaughter, 12 days after inoculation, samples were harvested: prescapular,
distal jejunal and hepatic lymph nodes, spleen,
Animals were observed once or twice daily. Skin
thickness at the site of sc injection was measured with a caliper.
liver, gall-bladder, kidneys, testis, epididymis
Serological tests
Venous blood samples were placed in tubes
without anticoagulant (Vacutainer, Becton Dickinson, Meylan). Sera were subjected to agglutination tests with a microtechnique described
previously (Pardon et al, 1983; Sanchis et al,
1985).
Bacteriologic tests
and seminal vesicles.
Protection against abortion
Venous blood samples were placed in tubes
with ethylene diaminetetraacetic acid (Vacutainer, EDTA). Bile and gall-bladder, fcetal stomach
contents and intestinal contents were individually and directly placed in sterile pots. Other sam-
Ewes (55) were distributed in 3 experimental
groups. Two out of the 3 groups received as a
ples, entirely
vaccination an sc injection behind the left shoulder of 2 ml with 7 x 10
8 Abortusovis strain D5 or
Rv6. Thirty-four days later, ewes were fertilized
in a controlled covering after hormonal heat synchronization. About 2 months after fecundation,
each initial group was reduced to 10 ewes presumed to be gravid. From each group, the 8 remaining ewes were isolated as unchallenged unvaccinated controls. The 3 groups of 10 ewes
received as challenge a sc injection behind the
9 Abortusovis
right shoulder of 2 ml with 8 x 10
15/5 Str
. At each lambing, cultures were made
r
from colostrum and from vaginal discharge col-
and swabs were directly plated
SS medium. Blood was also enriched in
Trypticase Soy Broth (BioM6rieux, Lyon) before
plating on SS medium. Uteruses were ground
separately in a homogenizer (Kenwood, Woking, UK) with a small volume of diluent. Other organs were fragmented with a stomacher (Col-
or partially removed, were dissected, flamed and individually placed in sterile pots.
Liquid samples
on
worth Stomacher, Cofralab, Bordeaux, France)
in a sterile plastic bag after addition of a small
volume of sterile diluent. Intestinal contents
were diluted with a small volume of diluent and
crushed with a stomacher. One aliquot of each
ground or crushed sample was seeded onto 2
1 of SS medium and the other of SS medium with added streptomycin (500 ¡1g/ml). For
the numeration, tissues were weighed, diluted
plates,
(1/5 weight/volume), crushed and aliquots were
plated. After 48 h at 37 °C, the identity of dubious colonies was checked by agglutination with
sera against 0 or H Salmonella antigens (Diagnostics Pasteur,
Marnes-La-Coquette, France).
Definitions
A lambing was noted as an abortion when it
took place before 140 days of gestation. A sample was noted as infected when at least 1 colony of Abortusovis was isolated. A lambing was
noted as infected when Abortusovis was isolated from at least 1 of the samples taken from the
ewe or from products of this lambing.
Presentation of results and statistics
Mean (m) and standard errors (SE) were calculated from CFU of all animals per experimental
group. Samples from which no Abortusovis was
isolated were noted as containing 1 viable bacteria. The level of infection of a sample was also
noted semi-quantitatively, the score being
scaled from 0 to 4 (1, 1 to 5 colonies; 2, 6 to 25;
3, 26 to 125; 4, >125). Titres of sera in aggluti2 of renating antibodies were expressed in log
ciprocal of dilutions. Comparisons between experimental groups were statistically evaluated
using a t-test or an F-test with the appropriate
number of degrees of freedom of error (df), the
level of significance between means being established by comparison with the calculated value of the least significant differences (isd).
RESULTS
Systemic multiplication
and persistency
The intravenous route was used to explore
capacity for survival and systemic multiplication of the bacterial strains. The group
inoculated with the parent wild strain presented irregularly detectable bacteremic
phases (table I); all positive blood samples
were taken during the first 5 days after inoculation. The number of Abortusovis in
the blood was low: on 11 positive blood
samples, direct plating without enrichment
was successful once. Samples taken at
slaughter, 42 days after injection, were not
infected, including those from the group inoculated with the wild strain.
Differences between evening and morning rectal temperatures did not contribute
significantly to the observed variations (results not presented); a mean between the
2 daily individual records was used to calculate the evolution of rectal temperatures
in each group. The moments of rise in rectal temperature and those later in serologic
response were not different according to
the groups, but the peaks were lower and
the febrile phase shorter in groups injected with the mutant strains (table I). Animal
No 9 exhibited the lowest hyperthermic response and did not react serologically to
injection of the Rv6 strain; the 2 other animals in this group presented a serological
reaction similar to that of animals injected
with the D5 strain. A local reaction at the
injection site was noted but not measured.
The
serological response was
injection of the mutant strains.
lower after
a
Beside the short in vivo survival of wild
mutant strains, this first experiment
demonstrated a reduction in immunogenicity and in the systemic dissemination capacity of the 2 mutant strains compared
with the parent wild strain.
or
Invaslveness and immunogenicity
In the second
injected
experiment,
bacteria
were
to screen strains
according to
their invasiveness and their capacity for
stimulation of an immunologic response after an sc vaccination. Screening was exsc
of 3 rams were inoculated sc with
dose about 15 times higher than that
used for intravenous inoculation.
Groups
tended to all 5 mutant strains, the parent
wild strain being used as reference.
Twelve days after injection, the 9 examined samples other than the sites of local
reactions and prescapular lymph nodes of
the inoculated side were uninfected in all
groups. The Rv6 strain was first of the mutant strains, in frequence and level of local
and regional persistence of bacteria. The
serologic response was higher after injection of reverse strains than after injection
of dependent strains (table II).
All selected criteria evidenced minimal
reactions after injection of the dependent
mutant strains (table II). The intensity of
cutaneous reactions to Rv4 or Rv6 injections was of the same order as that to the
parent strain injection, but regional lymphadenitis and fever remained lower. Local
by injection of
undetectable at
cutaneous reactions caused
dependent
strains
were
the time of slaughter and consequently no
examination of local infection was carried
out.
The low capacity for systemic colonization of all strains, and the reduced virulence of mutant strains were confirmed.
One strain per category of mutants was retained: D5 because of higher local and regional reactions, and Rv6 because of higher scores of local and regional infection
(table II).
Protection against abortion
Ewes
were
challenged
to test and compare
mid-gestation
protections induced
sc at
by vaccination before gestation with about
7 x 10
8 Abortusovis strain D5 or Rv6. As in
the previous experiment, local postvaccinal
reaction measured by increase in skin
thickness was higher and more persistent
after Rv6 injection than after D5 injection:
at day 7 after vaccination, skin thickness
was 23.0 + 1.0, 11.2 + 0.8 or 2.5 ±0.1 mm
(mean ± SE, n 18) in Rv6, D5 or the unvaccinated group respectively. One abscess discharching pus through the skin
was observed in 6 out of 18 ewes vaccinated with the Rv6 strain. In 4 weeks, local reaction was not longer visible and hardly
palpable except in animals with transiently
discharging abscesses, skin thickness
being 6.1 ± 1.0, 2.8 ± 0.1 or 2.5 ± 0.1 mm
(mean ± SE, n 18) in Rv6, D5 or unvacci=
=
nated group
respectively.
Anti-O agglutinating titers measured at
7 after vaccination were higher in animals vaccinated with the Rv6 strain (Rv6
group: 7.9 ± 0.2; D5 group: 6.6 ± 0.2; unvaccinated group: 2.0 ± 0.2; mean ± SE, n
18). Then the titers in the vaccinated
groups decreased. At the time of challenge, 102 days after vaccination, agglutinating titers remained slightly higher after
Rv6 vaccination (Rv6 group: 4.7 ± 0.16; D5
group: 4.1 ± 0.13; unvaccinated group: 2.3
± 0.18; mean ± SE, n
18). In unchallenged groups of 8 ewes, no vaccinal Sal-
day
=
=
monella was isolated at lambing or from
samples taken at slaughter.
Lambings of 28 among the 30 challenged ewes were observed. Eight out of 9
unvaccinated ewes and 7 out of 19 vaccinated ewes aborted after challenge; 8 out
of 9 unvaccinated ewes and 9 out of 199
vaccinated ewes excreted the challenge
strain (table III; fig 2). Local cutaneous reaction to the challenge injection resulted in
an abscess discharging pus through the
skin in 4 or 5 animals depending on the
group; the cutaneous reaction decreased
more rapidly in Rv6-vaccinated animals
(fig 2a). Febrile reaction was shortened in
vaccinated groups (fig 2b). In D5- and
Rv6-vaccinated groups, gestation lengths
were not directly related to maximal values
of antibody titers or of local skin reactions
after vaccinal injections. Individual serological titers remained at the same level during the 2 successive weekly examinations
the day of a normal or infected lambing.
According to clinical and serological results, the protective effect of the Rv6 strain
was higher than that of the D5 strain (table
IV).
Bacteriological examination at lambing
detected on the whole 16 vaginal excretions associated with an infected foetus,
but 1 infected foetus in the Rv6-vaccinated
group which was expelled with uncontaminated lochia. Sampling of mammary secretions was possible from ewes lambing at
term or near term; 1 challenge bacteria excretion was detected in colostrum from 1
out of the 2 infected lambings at normal
term in the Rv6-vaccinated group. From
samples taken at slaughter, bacteria of the
challenge strain were detected only from
right prescapular lymph node of 1 animal in the unvaccinated and challenged
the
group; no vaccinal bacteria
from any challenge animals.
were
isolated
DISCUSSION
Attenuation of virulence in these 5 dependent or reverse mutant strains has been
established in the animal species naturally
susceptible to the infectious disease. The
Rv6 strain was finally chosen as the vaccinal strain because of its immunogenicity
and protective activity. The absence of
bacteriemia detectable during the days following intravenous inoculation of the mutant strains was a first indication of their
low virulence, inasmuch as bacteriemia is
a necessary pathogenic step towards abortion and vaginal excretion of bacteria.
However, in view of results obtained in
mice (Lantier et al, 1981) as well as in
sheep in the first 2 experiments, it was
feared that residual virulence was too low
considering the pursued objective: ie to
find
a vaccine efficient with 1 injection over
reproduction period if not during the economical life of a ewe. Considering that the
in vivo persistence of bacteria and the du-
a
ration of host reactions to infection are related to the duration of immunity, the
strains inducing relatively high biological
reactions and long local persistence of
bacteria were selected after the first 2 experiments (tablesI and II).
However, the small differences between
strains with the same type of mutation
would have required more animals per
group and smaller variations between inoculated doses for a choice based on statistical grounds in the 2 first experiments. Evident pragmatic reasons account for the
small size of groups, as well as for the use
of non-gravid animals and the substitution
of kinetics of numbers of viable bacteria in
organs (Pardon and Marly, 1979; Lantier,
1987) for kinetics of bacteriemia and timepoint observation of bacterial colonization.
In the 3rd experiment, the use of gravid
ewes permitted a better simulation of the
natural disease. Beside measures of indirect effects of infection such as fever, local
reaction and serological response, 2 criteria essential to test a vaccine against a
contagious abortive disease were taken
into account: clinical issue of gestation and
bacterial excretion. Housing in a closed
ventilated building with a metallic slippery
floor, with 1 small compartment for each
group, may be factors explaining the low
number of viable lambs (table III).
The sc route of vaccination was chosen
in view of a polyvalent vaccine (Plommet
et al, 1987) including the Brucella melitensis strain Rev 1 vaccine, which is at
present injected sc (Alton and Elberg,
1967; Elberg, 1981An sc challenge and
a high dose of virulent bacteria injected at
mid-gestation regularly induce abortion in
non-immunized animals, allowing a reduction of the number of animals in experi-
mental groups (Pardon et al, 1983; Sanchis et al, 1985). But the sc route of challenge by-passes initial phases of the natural infectious process, and the particular
site of sc injection is exposed to the risk of
non-uniform behavior of different cutaneous regions in terms of immunological reaction and of bacterial implantation or dissemination.
Twelve days after a virulent challenge
regularly inducing placental colonization in
gravid ewes, the wild strain was not detectable in samples other than at the site of
challenge injection and the prescapular
lymph node draining this site. This absence or undetectable level of systemic
colonization is explainable by the low level
and short duration of bacteriemia and by a
maximum of systemic colonization observed early, about 1 week after inoculation (Lantier, 1987). Evolution of rectal
temperature indicates systemic effects
transient after inoculation of
strains. Among the dependent
strains, the D5 strain was preferred to the
D1 strain because of its greater biological
effects (table II). Bacterial colonization of
the inoculated region and serological response are generally more intense with reverse strains than with dependent strains
(table II). Injection of the Rv6 strain caused
relatively high local and regional bacterial
scores (table II), so this strain was retained
as the best choice among the reverse
strains.
In comparing Rv6- and D5-vaccination
regarding protection against abortion, the
higher number of animals observed during
the period preceding the challenge revealed the regularity of the postvaccinal
serological response and confirmed its level as slightly more elevated (about 1 dilution) in the Rv6-vaccinated ewes. In this
group, the local reaction was also more intense. The vaccination-challenge test demonstrated the protective activity of the 2
even
more
mutant
mutant strains against a severe challenge.
The Rv6 strain appears to be more protective in sheep and in mice (table IV; Lantier
et al, 1981Parameters measured before
challenge do not provide information allowing replacement of the virulent inoculation
to test the increased capacity of resistance
to the infection. The low persistence of virulent bacteria in tissues precludes a comparison based on the localization and intensity of colonization of samples at
slaughter and necropsy of ewes a few
weeks after lambing; such parameters
may be utilized in Brucellosis (Fenster-
bank et al, 1982).
genetic and physiological
the dependent and reverse
mutations for streptomycin (Hancock,
1981) indicates that phenotypic variability
Available
knowledge
on
observed between mutants of the
same
The Rv6 strain was further tested with a
reduced vaccinal dose inducing a lower local reaction (Pardon and Marly, unpublished results). Present knowledge on the
epidemiology of the disease does not allow
a precise forecast of an abortive outbreak
in flocks located in endemic areas; experiments aimed at the demonstration of protection against natural contamination
should cover a large number of flocks over
several years (Sanchis and Pardon, 1984).
Protection was confirmed with the reduced
vaccinal dose in a vaccination-challenge
experiment with pregnant ewes maintained
in other environmental conditions, and was
favourably compared with 2 injections of
commercial dead vaccines (Sanchis and
Pardon, 1981).).
ACKNOWLEDGMENTS
type corresponds to several possible sites
of mutation. Reverse mutants originate
from at least 1 mutation added to 1 of
those leading to dependent strains from
streptomycin-susceptible strains. In order
to vaccinate man or various animal species, dependent mutant strains were obtained from several bacterial species (Reitman, 1967; DuPont et al, 1970; Anon,
1972; Meyer et al, 1973; Vladoianu et al,
1975; Wei and Carter, 1978; Chengappa
et al, 1979, 1980; De Alwis et al, 1980;
Kucera et al, 1981). In mice orally infected
by a Typhimurium serotype, Vladoianu
and Dubini (1975) confirmed the stability
of the reduction of virulence in dependent
strains. But among reverse mutants that
can be obtained in vitro from dependent
strains of Enteritidis or Typhimurium serotypes, about 1 % presents a reversion to
virulence of the wild parental strain; the
frequency of this type of reversion from a
9 to 10-1! (Vladoiadependent strain is 10nu and Dubini, 1975). Such a risk of reversion should be very low if a reverse strain
is chosen directly as a vaccinal strain.
This work was supported by a contract between
INRA and the Federation Nationale des Groupements de Defense Sanitaire du B6tail and by a
grant from the EEC programme of research on
Mediterranean agriculture. We thank M Plommet as coordinator of the project of multivalent
living vaccine against sheep abortion, and C Le
Louedec, B Poutrel and M Pépin for helpful discussions. We are indebted to P L6chopier and
G Bourgy for organization of experimental facilities and to R Delaunay, D Musset and W Piemont for the care and maintenance of the animals.
REFERENCES
Alton GG, Elberg SS (1967) Rev. 1. Brucella
melitensis vaccine. A review of ten years of
study. Vet Bull 37, 793-800
Anon. (1972) Les vaccins buccaux contre les
entérobactéries. Rapport technique n° 500.
World Health Organisation, Geneva
Chengappa MM, Carter GR, Chang TS (1979) A
streptomycin-dependent live Pasteurella multocida type-3 vaccine for the prevention of
fowl cholera in turkeys. Avian Dis 23, 57-61
Chengappa MM, Myers RC, Carter GR (1980) A
streptomycin-dependent live Pasteurella multocida vaccine for the prevention of rabbit
pasteurellosis. Lab Anim Sci 30, 515-5188
de Alwis MCL, Carter GR, Chengappa MM
(1980) Production and characterization of
streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic
septicaemia. Can J Comp Med 44, 418-422
DuPont HL, Hornick RB, Snyder MJ, Libonati
JP, Woodward TE (1970) Immunity in typhoid
fever: evaluation of live streptomycindependent vaccine. Antimicrob Ag Chemother 236-239
Elberg SS (1981) Rev 1 Brucella melitensis vaccine. Part 11. 1968-1980. Vet Bull 51, 67-73
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 Vet 13, 295-301
Hancock REW (1981) Aminoglycoside uptake
and mode of action
with special reference
to streptomycin and gentamycin. I. Antagonists and mutants. J Antimicrob Chemother
-
8, 249-276
Kucera CJ, Wong JCS, Eis RL (1981) Development of
a chemically altered Pasteurella multocida vaccinal strain. Am J Vet Res 42,
1389-1394
Lantier F (1987) Kinetics of experimental Salmonella abortus ovis infection in ewes. Ann
Rech Vet 18, 393-396
Lantier F, Pardon P, Marly J (1981) Vaccinal
properties of Salmonella abortus ovis mutants for streptomycin: screening with a murine model. Infect Immun 34, 492-497
Lantier F, Pardon P, Marly J (1983) Immunogenicity of a low virulence vaccinal strain
against Salmonella abortus ovis infection in
mice. Infect Immun 40, 601-607
LeMinor L, Popoff MV (1987) Designation of Salmonella enterica sp. nov, nom. rev., as the
type and only species of the genus Salmonella. Request for an opinion. lnt J Syst Bacte-
riol37, 465-468
Meyer H, Hartmann H, Steinbach G, Koch H,
Kittlick M, Stelzner A, Linde K (1973) Unter-
suchungen zur Salmonellose des Kalbes. 2.
Mitteilung: lmmunisierungversuche mit atte-
nuierten S.-dublin-Keimen. Archi
Exp
Vete-
rinaermed 27, 301-320
Pardon P, Lantier F, Marly J, Sanchis R (1980)
Mise au point d’un vaccin contre la Salmonellose abortive ovine. Bull Soc Vét Prat Fr 64,
465-469
Pardon P, Marly J (1979) Experimental Salmonella abortus ovis infection of normal or
primo-infected CD1 mice. Ann Immunol
130B, 21-28
Pardon P,
Marly J, Sanchis R, Fensterbank R
(1983) Influence des voies et doses d’inocu-
lation avec Salmonella abortus ovis sur 1’effet
abortif et la réponse sérologique des brebis.
Ann Rech Vét 14, 129-139
Plommet M, Bosseray N, Lantier F, Bernard F,
Pardon P, Rodolakis A (1987) Simultaneous
vaccination by three living attenuated strains
of Brucella Salmonella and Chlamydia in
mice. Vaccine 5, 27-32
Reitman M (1967) Infectivity and antigenicity of
streptomycin-dependent Salmonella typhosa.
J Infect Dis 117, 101-107
Sanchis R, Pardon P (1981) Comparaison de
I’activit6 de trois vaccins par une methode
expérimentale d’avortementSalmonella
abortus ovis chez la brebis. Recl Méd Vdt
157, 501-505
Sanchis R, Pardon P (1984) Essai en milieu
contamin6 d’un vaccin atténué [yophilis6
contre I’avortement de la brebis dO S abortus ovis. Ann Rech Vét 15, 381-386
Sanchis R, Polveroni G, Pardon P (1985) S6rodiagnostic de la Salmonellose abortive des
brebis. Bull Lab V6t (19/20), 45-51
Vladoianu IR, Dubini F (1975) Experimental
model of oral antityphoid vaccination with live
streptomycin-dependent Salmonella typhimurium in C57BL/6 mice. J Hyg 75, 215-218
8
Vladoianu IR, Dubini F, Bolloli A (1975) Contribution to the study of live streptomycindependent Salmonella vaccines: the problem
of reversion to a virulent form. J Hyg 75, 203214
4
Wei BD, Carter GR (1978) Live streptomycindependent Pasteurella multocida vaccine for
the prevention of hemorrhagic septicemia.
Am J Vet Res 391, 534-1537