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Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Selection for increased resistance to Salmonella carrier-state (defined as the persistency of the bacteria 4 wk after inoculation) could reduce the risk for the consumer of food toxi-infections. The effects of two genomic regions on chromosomes 7 and 17 harboring two genes, NRAMP1 (SLC11A1) and TLR4, known to be involved in the level of chicken infection 3 d after inoculation by Salmonella were thus tested on a total of 331 hens orally inoculated at the peak of lay with 10(9) bacteria. The animals and their parents were genotyped for a total of 10 microsatellite markers mapped on chromosomes 7 and 17. Using maximum likelihood analysis and interval mapping, it was found that the SLC11A1 region was significantly involved in the control of the probability of spleen contamination 4 wk after inoculation. Single nucleotide polymorphisms (SNP) within the SLC11A1 and TLR4 gene were tested on those animals as well as on a second batch of 279 hens whose resistance was assessed in the same conditions. As the former was significantly associated with the risk of spleen contamination and the number of contaminated organs, SLC11A1 appears to be involved in the control of resistance to Salmonella carrier state. The involvement of the TLR4 gene was also highly suspected as a significant association between SNP within the gene, and the number of contaminated organs was detected.

An experimental population (1216 lambs from 30 sires) of the Inra401 sheep was created in an Inra flock to allow QTL detection for susceptibility to Salmonella infection, wool and carcass traits. The Inra401 is a sheep composite line developed from two breeds: Berrichon du Cher and Romanov. At 113 days of age on average, the lambs were inoculated intravenously with 10(8) Salmonella abortusovis Rv6 (vaccinal strain). They were slaughtered 10 days after the inoculation. Several traits were measured at inoculation and/or slaughtering to estimate the genetic resistance of the lambs to Salmonella infection: specific IgM and IgG1 antibody titres, body weight loss, spleen and pre-scapular node weights and counts of viable Salmonella persisting in these organs. This paper presents a quantitative analysis of the genetic variability of the traits related to salmonellosis susceptibility. The heritabilities of the traits varied between 0.10 and 0.64 (significantly different from zero). Thus, in sheep as well as in other species, the determinism of resistance to Salmonella infection is under genetic control. Moreover, the correlations between the traits are in agreement with the known immune mechanisms. The genetic variability observed should help QTL detection.

373 thirteen-week-old chicks issued from a commercial cross and 312 chickens from the L2 line were intravenously inoculated with 10 (6) Salmonella enteritidis and the numbers of Salmonella in the spleen, liver and genital organs were assessed 3 days later. Heritabilities of the number of Salmonella were estimated at 0.02 +/-0.04 and 0.05 +/-0.05 in the liver; at 0.29 +/-0.07 and 0.10 +/-0.06 in the spleen; and at 0.16 +/- 0.05 and 0.11 +/-0.08 in the genital organs, in the first and second experiments, respectively. The difference between the two experiments could result from sampling variations and from differences in the genetic structure of the two populations possibly including both heterosis and additive effects as well as their interaction in the first experiment. Genetic correlations between the number of bacteria in the genital organs and liver (0.56 +/-0.58 and 0.76 +/- 0.32 in the first and second experiments, respectively) and spleen (0.37 +/-0.24 and 0.79 +/-0.23) were positive. Moreover a significant within-sire effect of VIL1, a marker gene for NRAMP1, was observed in 117 progeny resulting from 25 informative matings. These results indicate that there are genetic differences in the resistance to visceral infection by S. enteritidis in these commercial egg-laying flocks, and suggest that these differences are at least partly due to genetic polymorphism in the NRAMP1 region.

Cross-protection induced by primary infection with Abortusovis and Gallinarum was examined against challenge injection with these Salmonella serotypes as well as with Dublin and Choleraesuis, the other virulent serotypes. Abortusovis induced efficient protection against the other Salmonella. Gallinarum was ineffective against Choleraesuis. Even with low multiplication in mice, the Gallinarum J91 strain induced a weak but significant protection against Dublin (same O group serotype). The antibodies in the blood of mice were tested with ELISA specific for the Salmonella antigens used to prime or to challenge animals. The Gallinarum J91 strain was detected to be more antigenic in ELISA than the other Salmonella antigens. It is difficult to conclude on a correlation between IgM or IgG antibodies and induction of protection, because of the variability in immune response according to the different serotype used. Nevertheless, the negative linkage between a number of bacteria in the spleen of mice challenged with Gallinarum and Dublin, and the level of IgM and IgG antibodies specific for the challenging serotype, showed that humoral immune response could be one element of cross-protection, mainly by the immune response against the same O serotype.

In order to investigate the determinism of the host specificity and to better understand the host resistance mechanisms, infections of sheep were performed with either S. abortusovis, serotype specific for ovine species, or with S. dublin, serotype adapted to cattle and accidentally transmissible to human. Following a subcutaneous challenge, S. dublin disseminated more rapidly towards lymphoid tissues than S. abortusovis. However, S. abortusovis tended to persist in spleen more efficiently than S. dublin. Using a quantitative RT-PCR method, the expression level of ovine cytokines genes was measured in the draining lymph node and in the spleen, in the course of infection. Inflammatory cytokine response was characterised by an early and strong increase of IL-1beta and TNFalpha mRNA in both lymphoid organs following S. dublin infection, while S. abortusovis challenge only induced IL-1beta mRNA increase in the spleen at day 3 post-inoculation. Likewise, S. dublin infection provoked a marked increase of IL-12 mRNA and a slight up-regulation of IFNgamma gene transcription in the local lymphoid site, in contrast to S. abortusovis infection. Elsewhere, both serotypes induced a strong and early IL-10 mRNA production and had no effect on IL-4 gene expression. Finally, taken together, these data suggest that the intensity of inflammatory and anti-infectious cytokine responses, but not the type 2 cytokine response, is serotype-dependent. They also suggest that the host-specific serotype, by limiting the host cytokine-mediated defence, could favour its persistence within lymphoid organs.

The early interferon-gamma (IFN-gamma) response after Salmonella infection was compared in resistant and susceptible congenic mice strains differing by the allele of Nramp1. IFN-gamma gene expression in vivo was studied by quantitative reverse transcription-PCR performed from the spleen of mice challenged by Salmonella either in multiplication or in resting phase. The spleen colonisation by Salmonella in vivo was investigated and was shown to be identical in a genetically defined host, whatever the initial phase of the bacteria. However, the bacterial load of the spleen was significantly reduced in Nramp1(r) (resistant) mice compared to Nramp1(s) (susceptible) mice. The background level of IFN-gamma mRNA was higher in the spleen of resistant mice than in the susceptible mice, before infection. Interestingly, the early upregulation of IFN-gamma gene transcription, which was observed after infection with Salmonella, was reproducibly delayed in susceptible mice compared to resistant mice. Finally, the kinetics of the host IFN-gamma response seems to be Nramp1 dependent. Resistant mice present the advantage of being more prompt to express this anti-infectious cytokine gene than susceptible mice in response to Salmonella infection.

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

Knowledge of the host response, of the resistance process, and of the mediators committed against Salmonella infection is essential to progress towards better means of prophylaxis and eradication. In this context, the present contribution attempts to interconnect, with the pivotal role of the macrophage, the early resistance process under the control of the Nramp1 gene and the cytokine response for resolving infection. IL-12 produced by macrophages is an inducer of IFN-gamma production, which in turn activates the macrophage antibacterial activity and synergizes its effects with TNF-alpha. All three of these cytokines are powerful actors in the first line of anti-Salmonella defence. It can be pointed out that susceptible and resistant individuals do not seem to see the cytokine environment the same way, the former being unresponsive to IL-1 or GM-CSF treatment and deficient in IFN-gamma production. These discrepancies may rely on cell signalling events that could be defective in macrophages of the susceptible phenotype.

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.

Early growth of Salmonella typhimurium in spleen and liver of mice is controlled by the mouse chromosome 1 locus Ity/Nramp 1. Genetic control of resistance to the attenuated vaccine strain Rv6 of Salmonella abortusovis was studied in mice infected by the intravenous route. Comparison of kinetics of bacterial colonization of spleen and liver in two congenic BALB/c-susceptible (Itys) and -resistant (Ityr) mouse lines showed that BALB/c mice (Itys) were significantly more susceptible to infection than C.D2 mice (Ityr) suggesting that infection by this vaccine strain is controlled by a gene which is close or identical to Ity/Nramp 1. Congenic mice also differed in their anti-Salmonella antibody response, measured by ELISA: susceptible mice had a significantly higher antibody level than resistant mice, whatever the immunoglobulin isotype (IgM, IgG1, IgG2a, IgG3, IgA, and total immunoglobulins). The two congenic BALB/c mouse lines had equal serum C3c levels in response to infection. However, we observed a highly significant difference according to the sex of mice, suggesting a role of sex hormones in the regulation of the level of some complement factors. These results, obtained with congenic mice, strongly suggest that the Ity/Nramp 1 locus controls susceptibility to infection by the S. abortusovis vaccine strain Rv6 and influences the antibody response., (Copyright 1998 Academic Press Limited.)

The efferent lymph duct of the ovine prescapular lymph node was cannulated, and Salmonella abortusovis (SAO), a specific pathogen for sheep inducing abortion and mortality of newborn lambs, was inoculated by the subcutaneous route in this lymph node drained area. While the prescapular lymph node draining the inoculation site represented an efficient barrier for the vaccinal SAO Rv6 strain spreading, SAO 15/5 virulent bacteria were steadily detected in efferent lymph of infected sheep. The inoculation of the virulent strain of SAO induced a greater increase of the cell output than did the attenuated vaccinal strain, but proportions of blast cells appearing in the efferent lymph were similar in both cases. Flow cytometry analysis showed that B and T cell outputs were both increased during SAO infections, but while T cell subset proportions slightly decreased, B cell percentages significantly rose, and, at the peak response, almost all of the lymphoblast cells were activated B cells. Typical antibody profiles characteristic of a primary immune response were observed, and antibody titres were greater in the efferent lymph of animals inoculated with the virulent strain of SAO. Many of the cytokine mRNAs we investigated were steadily detected by RT-PCR in efferent lymph cells of control sheep, but frequencies of detection of IL-2, IFN gamma, IL-1 beta and TNF alpha mRNAs were augmented in efferent lymph cells following inoculation of both SAO virulent or vaccinal strains. IL-10 and IL-8 mRNAs could only be detected after a SAO inoculation, while detection of IL-4 mRNAs was increased only in efferent lymph cells from SAO virulent strain-infected sheep. The efferent lymph cannulation technique thus appeared a very powerful way to study the in vivo development of the immune response to SAO, in its natural host, the sheep.

Of the 302 monoclonal antibodies included in the Third Workshop on Ruminant Leukocyte Antigens, 167 have been tested for their reactivity on uninfected and Salmonella-infected sheep lymph node leukocytes using FACS analysis. Only 47 of them showed specificities which could be related to those of some control monoclonal antibodies, such as the anti-CD5, anti-Ig light chain and anti-MHC Class II monoclonal antibodies.

1. Four groups of hens, each of a different line, were inoculated at peak of lay, per os in the crop with 1 ml of a suspension containing 10(9) cfu/ml Salmonella enteritidis PT4 (SE). The kinetics of SE contamination in the environment, egg shell and yolk were studied during the first 28 d post inoculation. On the day of slaughter, intestines, caeca, spleen, liver, ovary, oviduct and content were investigated for SE contamination. 2. The commercial egg-type line L2 was found to be the most susceptible to SE. It laid many SE-positive yolks (13.8%) and internal and faecal organs were frequently infected. 3. Certain lines are found to exhibit a degree of resistance to SE; the cause of which is unknown and might be attributed to major genes.

A study was undertaken to determine the susceptibility or resistance of 9 outbred experimental or commercial poultry lines to Salmonella enteritidis PT4. Young chicks were inoculated either intramuscularly or orally just after hatching. After intramuscular challenge the lines could be divided into susceptible lines (LD 50% < or = 10(2) Salmonella per animal), intermediate lines (LD 50% about 10(4) Salmonella) and resistant lines (LD 50% > 10(5) Salmonella). The results obtained after oral challenge confirmed these 3 groups for both mortality rates and the probability of the presence of salmonellae in the spleen and liver. There was no difference between lines concerning caecal carriage.

The role of T cells in granulomatous responses and in acquired immunity against Salmonella abortusovis (SAO) infection was studied in a murine model. Mice were subcutaneously (s.c.) vaccinated with a live attenuated strain of SAO. One month after vaccination, the transfer of primed spleen cells (1 x 10(8) cells per mouse) to syngeneic recipient mice conferred a significant protection of 3 log10, measured by spleen colonization on day 6 after s.c. challenge. In vitro treatment of spleen cells, before the transfer, with anti-Lyt-2 monoclonal antibody (IgG2b isotype MAb) and complement significantly impaired the protective activity. Treatment with anti-L3T4 MAb also diminished transferred protection, but to a lesser degree. Depletion of both L3T4+ and Lyt-2+ T cells completely abrogated protection. MAb treatment of spleen cells in vitro did not seem to have any effect on antibody response in recipient mice. Six days after the challenge protected recipient mice showed organized granulomas in the liver containing Mac-1+ macrophages and L3T4+ T cells. In non-protected mice at 6 days post-challenge, large infiltrates of T lymphocytes and macrophages were observed, but as numerous lesions with necrosis of hepatocytes; no granuloma were seen. In our experimental conditions, Lyt-2+ and L3T4+ T cells appeared to play, alone and in synergy, a role in vaccine-induced immunity against SAO and hepatic granulomas may contribute to the control of the infection.

The aim of the present study was to determine whether the Ity gene, which controls the resistance to S. typhimurium infection in mice, also governs the resistance to S. abortusovis, a serotype specific for goat and sheep. During either i.v. or i.p. infection, BALB/c mice (Itys) were not able to control the growth of S. abortusovis and eventually died from infection. In contrast CBA (Ityr) or (C.CB)F1 (Ityr/s) mice were able to control the growth of these bacteria. Using congenic C.D2 Ityr mice, we found that the gene controlling resistance to S. abortusovis was tightly linked to the Ity gene on chromosome 1. Furthermore, in the spleen and the liver of backcross BALB/c x (CBA x BALB/c) mice, the S. abortusovis resistance phenotype cosegregated with the two alleles of the Len-1 gene, a gene tightly linked to the Ity gene. By contrast, in these backcross mice, the level of infection of the peritoneal cavity, the site of inoculation, did not correlated with the Len-1 phenotype of the animal. These results provide evidence that after i.p. inoculation the control of S. abortusovis growth in the spleen and the liver is controlled by the Ity gene, but also suggest that additional gene(s) regulate the number of bacteria at the site of inoculation.

Salmonella enterica subsp. enterica ser. Abortusovis, a sheep-adapted serotype, causes a contagious disease. Abortion is the major symptom and the main source of contamination. Research on this ovine disease may aid farmers, but may also contribute to comparative biological knowledge. Innate resistance partly controlled by the Ity locus, increased resistance to reinfection and humoral and T-cell-mediated immunity were observations gained with a murine model. In ewes, abortion regularly occurs following subcutaneous challenge carried out from the third month of gestation onwards. This ovine model was used to evaluate prevention methods for Salmonella Abortusovis infection. One subcutaneous injection of a live attenuated lyophilized vaccine containing a selected streptomycin-independent reverse mutant was shown to protect ewes against abortion and excretion of Salmonella Abortusovis. This vaccine could be administered simultaneously with other commercial live vaccines such as Brucella melitensis Rev. 1 vaccine. In sheep, application of the vaccine to the conjunctiva (an easy, individual and hygienic route of mucosal vaccination) was followed by lymph node bacterial colonization and a serological response without local or general clinical reactions. The early events of natural infection remain to be explored, as do the mechanisms underlying the host specificity of Salmonella Abortusovis.

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.

The multiplication and dissemination of Salmonella abortus ovis, a Salmonella serotype which is specific for small ruminants, were studied in 17 culled non pregnant ewes. The virulent strain 15/5, that has previously been used either for the development of mouse models or as an abortion inducing agent in sheep, was injected by the subcutaneous route at a sublethal dose of 10(9) salmonella per animal. In most animals, it induced only transient hyperthermia and pronounced local reactions. Groups of four to five ewes were killed on days 3, 6, 10, and 14 for bacterial counts of their lymph nodes and organs. Maximal level of infection was on day 6, at which time the lymph node draining the site of inoculation (right prescapular lymph node), spleen, liver and lungs were regularly infected. On day fourteen, S abortus ovis were regularly reisolated only from right prescapular lymph nodes and local reactions.

Salmonella enteritidis subsp. enteritidis ser. abortus ovis, a sheep-adapted serotype, causes a contagious infectious disease with abortion as the main symptom, sometimes accompanied with mortality of lambs. This auxotrophic gram-negative bacterium ranks among the main causes of ovine abortions in some countries of Europe and of western Asia. The disease tends toward an endemic pattern with a multi-annual rhythm in the frequency of abortions. Bacteremia leads to bacterial colonization of the feto-placental unit, the principal site of salmonella multiplication. Peripartum vaginal excretion, coming with abortion or sometimes with lambing of living lamb at term, is massive. Fecal excretion remains generally undetectable, except during septicemic complication of placental retention. Rams develop a serological response to contamination but remain clinically normal. Frequency of carriers of abortus ovis is reported as low-to-relatively-high. Diagnosis rests on laboratory findings. A sero-agglutination test may offer a strong presumption at the flock level. Isolation of abortus ovis confirms the diagnosis; vaginal discharges, placentas or organs of aborted fetus or of dead animals are plated directly on selective media. Differential diagnosis requires consideration of other salmonelloses and other abortifacient diseases predominating in the area. Introduction of sheep from an infected area to a clean area should be avoided. Isolation of aborting ewes and destruction of contaminated bedding and of all products of abortion reduce contamination. Antibiotic treatment during an outbreak is sometimes disappointing. Annual vaccination with dead or living vaccines is advisable in endemic areas.

Strain Rv6, a streptomycin-independent reverse mutant of Salmonella abortus-ovis, multiplied and disseminated in vivo to a lesser extent than the virulent parental strain 15/5, as determined by subcutaneous inoculation of two outbred strains of mice with different susceptibilities to salmonella infection. This lower virulence of strain Rv6 was not modified by 10 passages in susceptible mice. Vaccination with strain Rv6 induced a dose-dependent capacity of mice to control subsequent infection with the virulent parental strain. Induced resistance began as early as 3 days after vaccination and lasted at least 11 months. Its efficiency was controlled by different routes and doses of challenge. Strain Rv6 induced a greater protection than that conferred by heat-killed bacteria or by tested commercial killed vaccines.

Resistance of outbred mice to subcutaneous challenge with S. abortus ovis was increased when previously or simultaneously parasitized with Eimeria Falciformis. This was measured by mortality rates and levels of splenic infection induced by Salmonella in parasitized and control NMRI or CD-1 (CR) mice. No effect was observed when E. falciformis was inoculated after S. abortus ovis.

Streptomycin-dependent and streptomycin-independent reverse mutants of Salmonella abortus ovis were selected on medium with and without added streptomycin. Their in vivo properties were tested with a murine model. Reduced capacities of dissemination and multiplication of all mutant strains were evidenced by means of subcutaneous, intraperitoneal, and intravenous inoculations of two outbred strains of mice with different susceptibilities to experimental S. abortus ovis infection. A factorial immunization experiment demonstrated the effects of vaccinal strains and doses. It showed large differences among the mutants. First-time infection with the fully virulent parent strain excepted, the best protection was given by independent reverse mutants which had kept residual capacity of in vivo dissemination and multiplication. This screening partly determined the selection of strain Rv6 as a potential live vaccine.