Abstract

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.

Introduction

Salmonella enterica serovar Abortusovis (S. abortusovis) is the leading cause of abortion in sheep among Salmonella serovars and has been mostly isolated in European countries where sheep herding is common. This serovar naturally infects ovines but not other animals of agricultural importance or humans. Infected animals present no signs of gastroenteritis, i.e., diarrhea, and the presence of the microorganism is generally investigated when several abortions occur in a flock.

Non-typhoid Salmonella serovars, including S. abortusovis, contain virulence plasmids that are required for the extra-intestinal phase of disease [1–3]. A common feature of the virulence plasmids is the presence of the spv genes, designated spvRABCD. The spv genes are involved in infection of extra-intestinal tissues, such as mesenteric lymph nodes, spleen, and liver, by increasing the growth rate of Salmonella in the intracellular compartment [4]. Analysis of 69 wild-type S. abortusovis isolates from different geographic areas showed the presence of a plasmid ranging in size from 50 to 67 kb in all strains [5]. Observations based on Southern blotting analysis of the spv locus suggest that the S. abortusovis plasmid may be considered a virulence plasmid [5], but the function of the plasmid in S. abortusovis pathogenicity has not yet been determined.

Even though naturally occurring S. abortusovis infection appears to be limited to the ovine, infection of mice has been successfully used as a model to evaluate the virulence of wild-type and attenuated S. abortusovis strains [6,7]. Importantly, results from the mouse model have been confirmed in ovine infection [8]. In the present study, our objectives were: (1) to evaluate the invasion of Peyer's patches, mesenteric lymph nodes, and spleen by S. abortusovis as compared to S. typhimurium in a murine model of oral infection; and (2) to examine the effect of S. abortusovis plasmid curing and mutagenesis after oral and intraperitoneal infection of BALB/c mice.

Materials and methods

Bacterial strains and plasmids

The strains and plasmids used in this study are described in Table 1. Wild-type S. abortusovis SS44 [5] was isolated from the fetal tissue of a sheep, mouse-passaged, and stored at −70°C. TT2251 is S. typhimurium carrying a Tn10 insertion in the virulence plasmid at an unknown position that does not affect virulence. Plasmid pLL6 is a temperature-sensitive (42°C) for replication, non-self-transferable, kanamycin-resistant plasmid of 55 kb previously used to construct plasmid-cured derivatives of S. typhimurium LT2 [9].

1

Bacterial strains and plasmids

Strain or plasmid Description Reference or source 
S. abortusovis 
SS44 wild-type [5
SU40 Plasmid-cured derivative of SS44 this study 
SSM56 spvR23::Tn5 from UF006 this study 
SU60 ΔspvRABCD::tet from UF109 this study 
S. typhimurium 
χ3456 SR-11, wild-type [1
TT2251 LT-2, zzc69::Tn10, tetr B.A.D. Stocker 
χ3477 LT-2, galE, rough LPS [15
UF006 spvR23::Tn5, kanr [10
UF109 ΔspvRABCD::tet, tetr P.A. Gulig 
UF110 ΔspvRABCD::tet, tetr [11
Plasmids 
pLL6 temperature-sensitive, kanr [9
pGTRO61 carries S. typhimurium spvRABCD and orfE, ampr [14
Strain or plasmid Description Reference or source 
S. abortusovis 
SS44 wild-type [5
SU40 Plasmid-cured derivative of SS44 this study 
SSM56 spvR23::Tn5 from UF006 this study 
SU60 ΔspvRABCD::tet from UF109 this study 
S. typhimurium 
χ3456 SR-11, wild-type [1
TT2251 LT-2, zzc69::Tn10, tetr B.A.D. Stocker 
χ3477 LT-2, galE, rough LPS [15
UF006 spvR23::Tn5, kanr [10
UF109 ΔspvRABCD::tet, tetr P.A. Gulig 
UF110 ΔspvRABCD::tet, tetr [11
Plasmids 
pLL6 temperature-sensitive, kanr [9
pGTRO61 carries S. typhimurium spvRABCD and orfE, ampr [14

Curing S. abortusovis SS44 of the virulence plasmid and mutagenesis of the spv genes

Plasmid pLL6 encoding kanamycin resistance was introduced into S. abortusovis SS44 by electroporation. An isolated kanamycin-resistant colony was selected at random and subjected to three passages in broth culture with 50 μg ml−1 of kanamycin at 30°C to enable displacement of the resident plasmid (the virulence plasmid and pLL6 are incompatible). Because the sizes of the two plasmids were nearly identical, we could not screen for loss of the resident plasmid by electrophoretic analysis. We therefore proceeded to eliminate pLL6 and examine for loss of both plasmids at the same time. After plating at 30°C on kanamycin, 10 colonies were selected at random and grown in broth without antibiotic at 42°C to cause the loss of the temperature-sensitive pLL6. A dilution was then plated out on non-selective plates at 42°C, and five isolate colonies were randomly chosen to carry out rapid minilysate plasmid extractions. Four of the five colonies were kanamycin-sensitive and contained no plasmid DNA by electrophoretic analysis.

DNA was transferred into smooth salmonellae by transduction with phage P22HTint. spvR::Tn5 S. abortusovis mutant SSM56 was obtained by transduction of the spvR::Tn5 allele from S. typhimurium UF006 [10] to S. abortusovis SS44 and selecting for kanamycin-resistant colonies. Similarly, S. abortusovis SU60 was constructed by transduction of the ΔspvRABCD::tet mutation from S. typhimurium UF109 (Gulig, unpublished results), which is a precursor strain of S. typhimurium UF110 [11], into SS44 using P22 phage. The virulence plasmid of S. typhimurium UF109 contains a deletion of a 6.3-kb ClaI fragment encoding the spvRABCD′ genes replaced by a tetracycline marker (ΔspvRABCD::tet).

Mouse infections

Groups of five female BALB/c mice 6–8 weeks of age (specific-pathogen-free) were inoculated intraperitoneally (i.p.) or orally (p.o.) as previously described [1]. In specific experiments, following p.o. inoculation of mice, Peyer's patches, mesenteric lymph nodes, and spleen were examined for colony-forming units (CFU) as described by Gulig and Doyle [4]. Briefly, mice were inoculated p.o. with ∼1×108 CFU of S. abortusovis, and 5 days later, Peyer's patches, mesenteric lymph nodes, and spleens were removed, homogenized in glass tissue homogenizers with phosphate-buffered saline, and plated to enumerate CFU.

Results and discussion

Tissue tropism of S. abortusovis in infected BALB/c mice

BALB/c mice were infected orally with wild-type S. abortusovis SS44, and the in vivo dissemination and survival of SS44 were compared to those of wild-type S. typhimuriumχ3456. Bacterial recovery from the Peyer's patches and mesenteric lymph nodes of S. abortusovis-infected mice was significantly lower than that observed in mice infected with S. typhimurium (Table 2). In contrast, the splenic infections were very similar between the two serovars. Therefore, it appears that S. abortusovis invades the Peyer's patches and mesenteric lymph nodes before reaching the spleen, but replicates less in Peyer's patches and mesenteric lymph nodes compared to S. typhimurium (Table 2).

2

Gut-associated lymphoid tissue and splenic infections after oral inoculation of S. abortusovis and S. typhimurium

Strain CFU (log10±S.D.) 
 Peyer's patches Mesenteric lymph nodes Spleen 
SS44 2.3±0.3* 3.1±0.5* 5.8±0.3** 
χ3456 4.9±0.2 4.9±0.5 5.6±0.8 
SSM56 2.7±0.9 2.2±0.5∧∧ 1.8±0.5∧∧∧ 
BALB/c mice were inoculated orally and killed 5 days later. Peyer's patches, mesenteric lymph nodes, and spleens were aseptically removed, homogenized, and plated to enumerate CFU. P values for SS44 were calculated vs. χ3456: *P<0.001; **P>0.5.P values for SSM56 were calculated vs. SS44: P>0.5; ∧∧P=0.02; ∧∧∧P<0.001. 
Strain CFU (log10±S.D.) 
 Peyer's patches Mesenteric lymph nodes Spleen 
SS44 2.3±0.3* 3.1±0.5* 5.8±0.3** 
χ3456 4.9±0.2 4.9±0.5 5.6±0.8 
SSM56 2.7±0.9 2.2±0.5∧∧ 1.8±0.5∧∧∧ 
BALB/c mice were inoculated orally and killed 5 days later. Peyer's patches, mesenteric lymph nodes, and spleens were aseptically removed, homogenized, and plated to enumerate CFU. P values for SS44 were calculated vs. χ3456: *P<0.001; **P>0.5.P values for SSM56 were calculated vs. SS44: P>0.5; ∧∧P=0.02; ∧∧∧P<0.001. 

Similar findings were obtained by Pascopella et al. [12] for S. gallinarum, the causative agent of the fowl typhoid syndrome, a disseminated infection that is also characterized by the absence of enteritis. These authors, while investigating the early steps of infection by S. gallinarum in the murine model, demonstrated that S. gallinarum has low tropism for the murine Peyer's patch epithelium.

Association of the S. abortusovis virulence plasmid with virulence in BALB/c mice

Wild-type S. abortusovis SS44 was cured of its resident virulence plasmid by using an incompatible antibiotic-resistant plasmid, and the virulence of plasmid-cured mutant SU40 was tested by means of two routes of inoculation. Preliminary assays of LD50 determinations showed that high doses of S. abortusovis are required to kill BALB/c mice both p.o. (LD50∼5×107) and i.p. (LD50∼1×105) (data not shown). Similar high values were previously found to be required for the subcutaneous and intravenous route of infection [6,7].

Mice infected p.o. with wild-type SS44 (1.5×108) presented signs of disease after 5 days, whereas those infected with plasmid-cured SU40 (5.0×108) appeared healthy. The SU40 splenic counts were significantly lower than those from SS44-infected mice (Table 3). As has been reported for other serovars, these results show that the S. abortusovis virulence plasmid is necessary for systemic infection of the reticuloendothelial organs in orally inoculated mice.

3

Virulence in BALB/c mice of wild-type S. abortusovis SS44 and its plasmid-cured derivative SU40

Strain Log10 splenic CFU (mean±S.D.) n=5 
 p.o. infection i.p. infection 
SS44 (w.t.) 5.8±0.07 6.3±1.2 
SU40 (plasmid-cured) 1.8±0.39* 6.0±1.0 
SU40/pGTRO61 4.1±0.38** N.T. 
Mice were inoculated p.o. with 1.5×108 CFU of SS44 or 5.0×108 CFU of SU40 and killed after 5 days. In a second set of experiments, 3.1×105 CFU of SS44 or 5.4×105 CFU of SU40 were injected i.p. into mice. Animals were killed after 3 days. *P<0.001 vs. p.o. SS44; **P=0.004 vs. p.o. SS44 and P<0.001 vs. p.o. SU40. P=0.840 vs. i.p. SS44. N.T.=not tested. 
Strain Log10 splenic CFU (mean±S.D.) n=5 
 p.o. infection i.p. infection 
SS44 (w.t.) 5.8±0.07 6.3±1.2 
SU40 (plasmid-cured) 1.8±0.39* 6.0±1.0 
SU40/pGTRO61 4.1±0.38** N.T. 
Mice were inoculated p.o. with 1.5×108 CFU of SS44 or 5.0×108 CFU of SU40 and killed after 5 days. In a second set of experiments, 3.1×105 CFU of SS44 or 5.4×105 CFU of SU40 were injected i.p. into mice. Animals were killed after 3 days. *P<0.001 vs. p.o. SS44; **P=0.004 vs. p.o. SS44 and P<0.001 vs. p.o. SU40. P=0.840 vs. i.p. SS44. N.T.=not tested. 

To evaluate the significance of the S. abortusovis virulence plasmid on the bacterial replication and survival following dissemination to the systemic sites, the virulence of S. abortusovis strain SS44 (3.1×105) and its cured derivative, SU40 (5.4×105), was also assessed by i.p. inoculation of BALB/c mice. Mice in both groups showed signs of disease 1 day post infection and were near death at 3 days when they were killed. The spleen counts obtained from SS44- and SU40-challenged animals did not differ significantly (Table 3). Similar results were observed by others with a plasmid-cured S. pullorum strain, which demonstrated a low, although significant, attenuation after parenteral inoculation, whereas orally inoculated cured salmonellae were completely avirulent [13]. It is possible that the i.p. route of infection initially results in less intracellular infection than oral inoculation. Because the plasmid-encoded spv genes increase the replication rate of salmonellae within host cells [4], there could be less dependence on the virulence plasmid for pathogenesis after i.p. inoculation of mice with S. abortusovis.

Effect of spv mutagenesis on S. abortusovis virulence in BALB/c mice

To assess the capability of the spv genes to restore virulence of SU40 in mice, we introduced the recombinant plasmid pGTRO61 [14] carrying the S. typhimurium spvRABCD genes into SU40. The cloned S. typhimurium spv genes only partially complemented the lack of the entire virulence plasmid in SU40 for virulence (Table 3) since splenic CFU for SU40/pGTR061 were significantly higher than for SU40 alone, yet significantly lower than for SS44. This result is in contrast to the ability of pGTRO61 to fully restore virulence to plasmid-cured S. typhimurium[14]. The observation that the cloned S. typhimurium spv genes on pGTRO61 could not fully complement virulence of SU40 raises the possibilities that either the expression of S. typhimurium spv genes was not correctly regulated from pGTRO61 in S. abortusovis or that other plasmid-encoded genes were involved in virulence of S. abortusovis. To examine these possibilities and to evaluate the role of spv genes in S. abortusovis, we constructed spvR23::Tn5 (SSM56) and ΔspvRABCD::tet (SU60) S. abortusovis derivatives. The SSM56 and SU60 strains were then tested for virulence in mice by oral inoculation. Both strains were avirulent (Table 4). Recovery of both spv mutant strains from spleens was as low as that obtained with the plasmid-cured SU40 strain. In an attempt to complement the ΔspvRABCD::tet mutation, we introduced the spv+ plasmid pGTRO61 into SU60 and orally inoculated mice. Five days later, the spleen counts showed that pGTRO61 was not able to fully restore virulence (Table 4). Interestingly, when pGTRO61 was placed into wild-type SS44 the splenic CFU detected 5 days after infection were 10-fold lower than those of the wild-type parent, but the difference was not statistically significant (Table 4). These results are consistent with inappropriate expression of the S. typhimurium spv genes in S. abortusovis and are not consistent with other plasmid-encoded genes being involved with virulence in S. abortusovis.

4

Virulence of SpvS. abortusovis derivatives in BALB/c mice

Strain Log10 splenic CFU (mean±S.D.) 
SS44 (w.t.) 5.8±0.11 
SSM56 (spvR1.9±0.60 
SU60 (spvRABCD2.2±0.40 
SU60/pGTRO61 4.0±0.85* 
SS44/pGTRO61 4.8±1.0** 
Mice were inoculated orally; three to five mice were used per group. Inocula were 2.1×108 CFU for SS44, 3.0×108 CFU for SSM56, 1.1×108 CFU for SU60, 2.4×108 CFU for SSM56/pGTRO61, 5.1×108 CFU for SU60/pGTRO61, and 3.3×108 CFU for SS44/pGTRO61. P values were calculated vs. SS44: *P=0.009; **P=0.116; P<0.001. 
Strain Log10 splenic CFU (mean±S.D.) 
SS44 (w.t.) 5.8±0.11 
SSM56 (spvR1.9±0.60 
SU60 (spvRABCD2.2±0.40 
SU60/pGTRO61 4.0±0.85* 
SS44/pGTRO61 4.8±1.0** 
Mice were inoculated orally; three to five mice were used per group. Inocula were 2.1×108 CFU for SS44, 3.0×108 CFU for SSM56, 1.1×108 CFU for SU60, 2.4×108 CFU for SSM56/pGTRO61, 5.1×108 CFU for SU60/pGTRO61, and 3.3×108 CFU for SS44/pGTRO61. P values were calculated vs. SS44: *P=0.009; **P=0.116; P<0.001. 

To examine in more detail the differences in virulence between the wild-type strain and the Spv derivatives, mice were infected orally with wild-type SS44 and spvR::Tn5 SSM56, and after 5 days Peyer's patches, mesenteric lymph nodes, and spleens were examined for CFU. SSM56 showed a 104-fold decrease in splenic CFU compared with SS44 (Table 2). Infection of mesenteric lymph nodes by SSM56 was also significantly reduced, although to a lesser extent, whereas infection of Peyer's patches by the two strains was not significantly different (Table 2).

In summary, S. abortusovis is capable of causing lethal systemic infection in BALB/c mice but, in contrast to S. typhimurium, does not rely on proliferation in the intestine as an initial step in the disease process. Furthermore, the S. abortusovis plasmid is not necessary for colonization of the intestine, but is essential for efficient infection of mesenteric lymph nodes before systemic dissemination. The possibility that systemic infection of the spleen mimics transplacental infection of fetuses in pregnant ewes suggests that the S. abortusovis virulence plasmid and spv genes could be essential in ewes for survival in the draining lymph nodes of the intestines and reaching the blood stream as early steps in infection of fetuses.

Acknowledgements

We thank M.P. Satta and P. Nicolussi for excellent technical assistance. This work was supported by NATO Grant 920838; by Regione Autonoma della Sardegna, Progetto Biotecnologie; by EU Grant AIR3-CT96-1743 to S.R.; and by NIH Grant AI24821 to P.A.G., who was an American Heart Association Established Investigator with funds contributed in part by the American Heart Association–Florida Affiliate.

References

[1]
Gulig
P.A.
Curtiss
R.
III
(
1987
)
Plasmid-associated virulence of Salmonella typhimurium
.
Infect. Immun.
 
55
,
2891
2901
.
[2]
Hovi
M.
Sukupolvi
S.
Edwards
M.F.
Rhen
M.
(
1988
)
Plasmid-associated virulence of Salmonella enteritidis
.
Microb. Pathogen.
 
4
,
385
391
.
[3]
Heffernan
E.J.
Fierer
J.
Chikami
G.
Guiney
D.
(
1987
)
Natural history of oral Salmonella dublin infection in BALB/c mice: effect of an 80-kilobase-pair plasmid on virulence
.
J. Infect. Dis.
 
155
,
1254
1259
.
[4]
Gulig
P.A.
Doyle
T.J.
(
1993
)
The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice
.
Infect. Immun.
 
61
,
504
511
.
[5]
Colombo
M.M.
Leori
G.
Rubino
S.
Barbato
A.
Cappuccinelli
P.
(
1992
)
Phenotypic features and molecular characterization of plasmids in Salmonella abortusovis
.
J. Gen. Microbiol.
 
138
,
725
731
.
[6]
Pardon
P.
Marly
J.
(
1979
)
Experimental Salmonella abortus ovis infection of normal or primo-infected CD1 mice
.
Ann. Microbiol. (Paris)
 
130B
,
21
28
.
[7]
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
.
[8]
Pardon
P.
Sanchis
R.
Marly
J.
Lantier
F.
Guilloteau
L.
Buzoni-Gatel
D.
Oswald
I.P.
Pepin
M.
Kaeffer
B.
Berthon
P.
Popoff
M.Y.
(
1990
)
Experimental ovine salmonellosis (Salmonella abortusovis): pathogenesis and vaccination
.
Res. Microbiol.
 
141
,
945
953
.
[9]
Kelln
R.A.
Lintott
L.G.
(
1990
)
Construction of plasmid-free derivatives of Salmonella typhimurium LT2 using temperature-sensitive mutants of pKZ1 for displacement of the resident plasmid, pSLT
.
Mol. Gen. Genet.
 
222
,
438
440
.
[10]
Caldwell
A.L.
Gulig
P.A.
(
1991
)
The Salmonella typhimurium virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene
.
J. Bacteriol.
 
173
,
7176
7185
.
[11]
Gulig
P.A.
Doyle
T.J.
Clare-Salzler
M.J.
Maiese
R.L.
Matsui
H.
(
1997
)
Systemic infection of mice by wild-type but not Spv−Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha
.
Infect. Immun.
 
65
,
5191
5197
.
[12]
Pascopella
L.
Raupach
B.
Ghori
N.
Monack
D.
Falkow
S.
Small
P.L.
(
1995
)
Host restriction phenotypes of Salmonella typhi and Salmonella gallinarum
.
Infect. Immun.
 
63
,
4329
4335
.
[13]
Barrow
P.A.
Lovell
M.A.
(
1988
)
The association between a large molecular mass plasmid and virulence in a strain of Salmonella pullorum
.
J. Gen. Microbiol.
 
134
(
Pt. 8
),
2307
2316
.
[14]
Gulig
P.A.
Caldwell
A.L.
Chiodo
V.A.
(
1992
)
Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid
.
Mol. Microbiol.
 
6
,
1395
1411
.
[15]
Gulig
P.A.
Curtiss
R.
III
(
1988
)
Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium
.
Infect. Immun.
 
56
,
3262
3271
.