Safety and protective efficacy of live attenuated Salmonella Gallinarum mutants in Rhode Island Red chickens

Vaccine 31 (2013) 1094–1099

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Safety and protective efficacy of live attenuated Salmonella Gallinarum mutants in Rhode Island Red chickens Arindam Mitra a , Amanda Loh a , Amanda Gonzales a , Paweł Łaniewski a , Crystal Willingham a , Roy Curtiss III a,b , Kenneth L. Roland a,∗ a b

The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States

a r t i c l e

i n f o

Article history: Received 8 May 2012 Received in revised form 1 November 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Fowl typhoid Poultry Live attenuated vaccines Regulated delayed attenuation Lipopolysaccharides

a b s t r a c t Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, an important systemic disease of poultry with economic consequences in developing nations. A live attenuated orally applied S. Gallinarum vaccine could provide a low cost method for controlling this disease. We constructed S. Gallinarum strains in which the expression of the crp, rfc and rfaH genes, important for virulence of Salmonella Typhimurium in mice, were under the control of an arabinose-regulated promoter. We evaluated the virulence of these strains compared to wild-type S. Gallinarum and to mutants carrying deletions in these genes. We found that rfc mutants were fully virulent, indicating that, unlike the S. Typhimurium mouse model, the rfc gene is dispensable in S. Gallinarum for virulence in birds. In the case of rfaH, the deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence. The strain exhibiting arabinose-regulated crp expression was attenuated. Its virulence was not affected by the inclusion of 0.2% arabinose in the drinking water. Birds immunized with this strain were protected against a lethal S. Gallinarum challenge and against colonization with the human pathogen Salmonella Enteritidis. This work shows that an arabinose-regulated crp strain provides a basis for further development of a fowl typhoid vaccine. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Fowl typhoid is a systemic disease of poultry caused by Salmonella enterica serovar Gallinarum. Fowl typhoid causes significant morbidity and mortality in poultry in several continents including Africa, Asia, Europe and Latin America [1–4]. Control strategies include well-developed vaccination programs, surveillance and biosecurity practices, all of which can greatly reduce mortality due to fowl typhoid [5–7]. Live attenuated Salmonella vaccines offer greater levels of protection than killed injectable vaccines by eliciting not only humoral immunity, but also mucosal and cell-mediated immunity, important for clearance of Salmonella infections [8,9].

Abbreviations: BSG, buffered saline with gelatin; LD50 , lethal dose,50%; LPS, lipopolysaccharides; OMP, outer membrane protein. ∗ Corresponding author at: The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, PO Box 875401, 1001 S. McAllister Avenue, Tempe, AZ 85287-5401, United States. Tel.: +1 480 727 9992; fax: +1 480 727 0466. E-mail address: [email protected] (K.L. Roland). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.12.021

The currently available live S. Gallinarum vaccine strain, 9R is rough, lacking O-antigen, and is likely to have additional, undefined mutations [10]. The vaccine is administered by multiple injections in older birds [11]. This requirement tends to drive up the cost of using 9R, rendering it an unattractive vaccine for the developing world. In addition, it is virulent in some breeds [11,12]. A number of mutations affecting O-antigen synthesis have been shown to result in attenuation of Salmonella enterica serovar Typhimurium [13–15], and some mutants, e.g. rfc and rfaH, elicit protective responses [16–18]. The rfc gene encodes the O-antigen polymerase that catalyzes the polymerization of O-antigen subunits. Mutants defective in rfc produce LPS with a single O-antigen unit [19]. The transcription antiterminator RfaH is required for efficient transcription of the 18-kb gene cluster encoding genes required for O-antigen synthesis [20]. S. Typhimurium strains carrying an rfaH mutation make vanishingly small amounts of Oantigen [21] and are attenuated for virulence in mice [18]. The crp gene, conserved in diverse bacterial species, encodes the cyclic AMP receptor protein, a global regulator that modulates expression of a number of genes required for virulence and carbohydrate metabolism [22]. Introduction of a crp mutation attenuates many bacterial pathogens for virulence while retaining immunogenicity [23–26]. In particular, cya crp mutants of

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S. Typhimurium and Salmonella enterica serovar Choleraesuis are highly attenuated and immunogenic in chicks and pigs respectively [27,28], although a cya crp S. Choleraesuis strain was only partially attenuated in mice [24]. S. Typhimurium cya crp strain, ␹3985, is attenuated in chicks and immunization with ␹3985 protects birds against colonization with groups B, D and E Salmonella [29]. Furthermore, a S. Gallinarum crp mutant is attenuated and protective in six-day-old Lohmann Brown chickens [30]. Recently, a regulated delayed attenuation strategy has been described for S. Typhimurium, whereby wild-type attributes are expressed at the time of immunization and strains become attenuated after a few rounds of replication in the host [31]. In S. Typhimurium, this strategy has been applied to crp, rfc and rfaH by replacing the native promoter of each gene with the araC PBAD promoter, resulting in strains carrying arabinose-regulated crp [31], rfc [16] or rfaH [17] genes. All three S. Typhimurium mutants are attenuated and immunogenic in mice. In this study, we designed and constructed S. Gallinarum strains utilizing regulated delayed attenuation strategies that affect Crp and O-antigen synthesis and screened them for virulence and protective efficacy in Rhode Island Red chickens. Our results showed that oral immunization with an arabinose-regulated crp mutant provides excellent protection against S. Gallinarum and against challenge with the human pathogen Salmonella Enteritidis. 2. Materials and methods 2.1. Animal supplies and housing Straight run Rhode Island Red chicks were purchased from Randall Burkey Co., Inc. (Boerne, Texas, USA). Three-day old Rhode Island Red chicks were housed in isolators in groups of five for two weeks. In some studies, chicks were transferred to open-air cages after the boost. All experiments were carried out in compliance with Institutional Animal Care and Use Committee (IACUC) and Animal Welfare Act at Arizona State University. 2.2. Bacterial strains, plasmids, media, reagents and growth conditions Strains and plasmids used in this study are listed in Table 1. Oligonucleotide primers are listed in Table S1. For administration to birds, strains were grown statically overnight in LB broth [32] with appropriate supplementation at 37 ◦ C. Overnight cultures were diluted 1:100 in 50 ml fresh LB media with appropriate supplements and grown at 37 ◦ C with shaking at 200 rpm until the OD600 reached ∼0.8. Strains were routinely supplemented with either 0.05% arabinose for strains ␹11385 (Prfc174 ::TT araC PBAD rfc, hereafter Prfc174 ) and ␹11386 (PrfaH178 ::TT araC PBAD rfaH, hereafter PrfaH178 ) or with 0.2% arabinose for ␹11387 (Pcrp527 ::TT araC PBAD crp, hereafter Pcrp527 ) Strains ␹11570 (crp-633::cam, hereafter crp-633), ␹11572 (rfc-1224::cam, hereafter rfc-1224) and ␹11571 (rfaH489::cam, hereafter rfaH489) were grown in LB media with 20 ␮g/ml Cm. Cells were harvested by centrifugation at 3629 × g for 15 min at room temperature and resuspended in buffered saline with gelatin (BSG) [33]. For determination of the LPS profile, strains were grown overnight in purple broth (Difco, Sparky, MD, USA), a carbohydratefree medium. Overnight cultures were subcultured (1:100) in fresh purple broth with or without the relevant sugar(s) for a second passage. LPS was prepared and visualized by silver staining in polyacrylamide gels as described previously [34]. LPS isolation was performed three times and representative results were shown.

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2.3. Strain construction All vaccine candidates were derived from S. Gallinarum strain 287/91 [35]. Promoter replacements were introduced by conjugational transfer of suicide plasmids using donor E. coli strain ␹7213 [36]. As S. Typhimurium and S. Gallinarum share high sequence similarity in the flanking region surrounding these promoters (>99%), pre-existing suicide plasmids carrying S. Typhimurium DNA sequences were used [16,17,31,39]. Deletions of crp, rfc and rfaH were constructed via the lambda Red recombination method [40]. Flanking sequences were based on the S. Gallinarum genome using the primers described in Table S1. 2.4. Determination of lethal dose, 50% (LD50 ) in three-day old chicks and one-month old chickens Strains were grown and harvested as described above in Section 2.2. The pellet was resuspended in BSG and adjusted to achieve a dose of 101 to 109 CFU in a volume of 50 ␮l for orally inoculating chicks. The virulence of wild-type strain, 287/91, and its derivatives were assessed in chicks, while the virulence of the challenge strain, ␹4173 was determined in 28-day-old birds by the same method except that the inoculating volume was 500 ␮l. The LD50 was calculated using the Reed and Muench method [41]. Note that for deletion/insertion strains ␹11570 and ␹11571, chicks received 102 , 104 , 106 or 108 CFU and all chicks survived. They were then treated as vaccinated chicks, boosted with 1 × 108 CFU of ␹11570 or ␹11571 and challenged as described in Section 2.5. 2.5. Vaccination, booster and challenge regimen No food or water was provided prior to primary immunization and food was withdrawn for 6 hours before the boost. Groups of 3-day old chicks were inoculated over the tongue with 50 ␮l of BSG or PBS containing ∼1 × 108 CFU of mutant strains, and boosted two weeks later (day 17) with the same dose of the same strain. Thirty minutes after inoculation, food and water were provided ad libitum. In one experiment, 0.2% arabinose was added to the water. At four weeks of age, birds were orally challenged with ∼1 × 107 CFU of virulent S. Gallinarum strain ␹4173 (LD50 in 4 week old birds = 7.7 × 103 CFU) or with ∼1 × 106 CFU of S. Enteritidis strain ␹3700 in 500 ␮l BSG. Deaths were recorded daily. Fifteen days after S. Gallinarum challenge or four days after S. Enteritidis challenge, organs were inspected for lesions, collected and homogenized. Dilutions of the homogenate were made in BSG and plated onto Brilliant Green Agar plates for enumeration of Salmonella present in each tissue. Preenrichment with Tryptic Soy Broth followed by enrichment with Rappaport Vassiliadis broth and subsequent plating onto Brilliant Green Agar plates was carried out for organ samples in which no Salmonella was detected by direct plating. 2.6. Scoring system of birds post immunization or post challenge We developed a ten-point scoring system to evaluate bird health at necropsy. Necropsies were performed three weeks postprimary vaccination and two weeks after challenge. The birds were assigned a score from 0 to 10. Healthy organs with no lesions or signs of pathology were assigned a score of 0. Organs with the following signs of disease were assigned a score of 1: hepatomegaly, splenomegaly, pericarditis, necrotic foci on the liver, splenic lesions, and pale yellow nodules on the myocardium, for a maximum possible pathology score of 6. No individual bird received score of 7, 8 or 9. After challenge, dead birds or sick birds requiring euthanasia received a score of 10 while healthy control birds received an overall score of 0.

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Table 1 Bacterial strains and plasmids used in the study. Strains

Genotype

Source/Parent

␹7213 ␹3796 ␹4173 287/91 ␹11385 ␹11386 ␹11387 ␹11570 ␹11571 ␹11572

thr-1 leuB6 fhuA21 lacY1 glnV44 recA1 asdA4 (zhf-2::Tn10) thi-1 RP4-2-Tc::Mu [␭pir] Wild-type S. Gallinarum Chick passaged isolate of ␹3796 Wild-type vaccine parent S. Gallinarum Prfc174 ::TT araC PBAD rfc PrfaH178 ::TT araC PBAD rfaH Pcrp527 ::TT araC PBAD crp crp-633::cam rfaH489::cam rfc-1224::cam

Roland et al. [36] Case Poppe ␹3796, Susan Porter SGSC4941, [35] 287/91 287/91 287/91 287/91 287/91 287/91

Plasmids pKD46 pYA3832 pYA4298 pYA4304

Lambda red expression plasmid Suicide vector for generating Pcrp527 ::TT araC PBAD crp Suicide vector for generating Prfc174 ::TT araC PBAD rfc Suicide vector for generating PrfaH178 ::TT araC PBAD rfaH

[40] [31] [16] [17]

2.7. Delayed-type hypersensitivity (DTH) response in immunized birds post challenge. To evaluate induction of cell-mediated immunity, we determined the delayed type hypersensitivity (DTH) response of birds following challenge. Outer membrane protein (OMP) samples were prepared as previously described [42] from a galE mutant S. Gallinarum strain grown in LB broth without addition of galactose. Four days after S. Gallinarum challenge, groups of five birds from each treatment group were injected intradermally between the second and third digit of the right foot with 20 ␮g of OMPs in 50 ␮l of phosphate-buffered saline. As a control, 50 ␮l of PBS was injected between the second and third digit of the left foot of the same birds [43]. The difference in thickness between the right and left toe web was measured with a digital caliper 24 h post injection. The difference, if any, between the left and right toe web before injection was set at zero. 2.8. Histopathology of liver and spleen Fifteen-days post S. Gallinarum challenge, sections of liver and spleen were removed from chickens at necropsy. Tissue samples were soaked in PBS after fixing in 10% formalin for 24 h. Samples were cut and stained with Hematoxylin and Eosin (H & E) by the Histology Core at the Mayo Clinic, Scottsdale, Arizona. The H & E stained

slides were visualized by using a Zeiss CP-ACHROMAT 100×/1.25 oil objective in a Zeiss Axioskop 40 microscope and representative sections were reported. 2.9. Statistical analysis Differences in S. Gallinarum colonization between the vaccinated and control birds after challenge were determined using the Student’s two-tailed t-test. One-way ANOVA followed by a Dunnett’s multiple comparison test was used for analysis of protective efficacy and lesion scoring. A p value less than 0.05 was considered significant. All statistical analyses were performed using Graphpad prism. 3. Results 3.1. Sugar dependent O-antigen synthesis and crp expression The rfc mutant, ␹11572, produced a single O-antigen unit as expected, while O-antigen synthesis in strain ␹11385 (Prfc174 ) was arabinose-dependent (Fig. 1). Strain ␹11385 produced low levels of O-antigen in the absence of arabinose, similar to that reported for this construct in S. Typhimurium [16]. O-antigen was essentially undetectable in the rfaH mutant ␹11571, while strain ␹11386 (PrfaH178 ) exhibited arabinose-dependent O-antigen

Fig. 1. Silver-stained polyacrylamide gels of reversibly rough S. Gallinarum mutants grown in purple broth. Arabinose-dependent O-antigen synthesis in ␹11385 (Prfc174 ::TT araC PBAD rfc) and ␹11386 (PrfaH178 ::TT araC PBAD rfaH). ␹11572 (rfc-1224::cam) exhibited a semi-rough LPS with single O-antigen unit while ␹11571 (rfaH489::cam) exhibited a fully rough LPS phenotype. The wild-type parent of mutant strains, 287/91 was also grown with or without arabinose as indicated.

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Table 2 Attenuation and protective efficacy of Salmonella Gallinarum mutants in Rhode Island Red chickens. Strain 287/91 BSG ␹11386 ␹11385 ␹11572 ␹11570 ␹11571 ␹11387

Genotype

Lethal dose, 50% (CFU)

Alive/totala

Wild-type – PrfaH178 ::TT araC PBAD rfaH Prfc174 ::TT araC PBAD rfc rfc-1224::cam crp-633::cam rfaH489::cam Pcrp527 ::TT araC PBAD crp

6 × 10 – 3 × 104 5.7 × 105 1 × 106 >108 CFU >108 CFU >108 CFU

b

5

2/20 b b b

11/20c 18/20c 19/20d

Survival after challenge on day 28 with 1 × 107 CFU of wild-type S. Gallinarum strain ␹4173. Experiment not done. c These chicks were survivors of the virulence study. Thus, 4 groups consisting of 5 chicks each were immunized with 102 , 104 , 106 or 108 CFU of the indicated strains. All chicks were boosted with 108 CFU of the same strain two weeks later. p < 0.01 compared to BSG group. d Birds were immunized with approximately 1 × 108 CFU of ␹11387 on days 3 and 14. Shown are combined data from 3 independent experiments. p < 0.01 compared to BSG group. a

b

synthesis (Fig. 1). O-antigen synthesis by parent strain 287/91 was not substantially influenced by the addition of arabinose (Fig. 1). Maltose fermentation by strain ␹11387 (Pcrp527 ), as judged by colony color on MacConkey agar plates, was dependent on the addition of arabinose (data not shown) as expected for a crp mutant [23]. S. Typhimurium crp mutants grow slower than their wildtype parent strains [23]. To evaluate this parameter, growth of ␹11387 in LB broth was compared to parent strain 287/91 and crp mutant strain, ␹11570. Strains ␹11387 and ␹11570 grew more slowly than strain 287/91 (Fig. 2). Addition of 0.2% arabinose restored the growth of ␹11387 to near wild-type levels.

3.2. Evaluation of the virulence of S. Gallinarum mutants in three-day old Rhode Island Red chicks The virulence of parent and mutant strains was evaluated in three-day old Rhode Island Red chicks. The wild-type vaccine parent strain, 287/91, had an LD50 of 6 × 105 CFU (Table 2). Strains ␹11385 (Prfc174 ) (LD50 ∼ 5.7 × 105 CFU) and ␹11572, (rfc-1224) (LD50 ∼ 1 × 106 CFU) retained full virulence, as did strain ␹11386 (PrfaH178 ) (LD50 ∼ 3 × 104 CFU). Strains ␹11387 (Pcrp527 ), ␹11570 (crp-633) and ␹11571 (rfaH489) were fully attenuated (LD50 > 108 CFU).

3.3. Protective efficacy of S. Gallinarum mutants Strains ␹11385 (Prfc174 ) and ␹11386 (PrfaH178 ) were too virulent to be useful as vaccines (Table 2) and were not tested further. Therefore, of the three arabinose-regulated mutants, only strain ␹11387 (Pcrp527 ) was evaluated for efficacy against challenge with virulent S. Gallinarum strain ␹4173. We performed three separate experiments using treatment groups of 5 or 10 birds. Similar results were obtained in all three experiments and the results combined (Table 2). Immunization with strain ␹11387 (Pcrp527 ) provided significant protection against challenge with ␹4173 compared to nonimmunized control birds (p < 0.05) (Table 2) and resulted in a significant reduction in the number of challenge organisms isolated from internal organs (p < 0.05) (Table 3). The safety of strain ␹11387 was reflected in the low pre-challenge mean health scores in immunized birds (Table S2). Post challenge, vaccinated birds received significantly lower mean health scores than controls (p < 0.05) (Table S2). The vaccinated birds also displayed a DTH response (Table S2), indicating induction of strong cellular responses. In addition, after challenge, vaccinated birds displayed less focal degeneration and necrosis as compared to non-vaccinated challenged birds (Fig. S1). We also examined the ability of ␹11387 to protect against challenge with the human pathogen S. Enteritidis. We were unable to detect S. Enteritidis from systemic organs such as liver and spleen in vaccinated birds 4 days after challenge, a result significantly lower than non-vaccinated controls (p < 0.05) (Table 3). To evaluate protection conferred by deletion strains ␹11571 (rfaH489) and ␹11570 (crp-633), we took the birds that survived the virulence assay and boosted them two weeks later with 1 × 108 CFU of the same strain they received initially and challenged them two weeks later with ␹4173. The rfaH strain ␹11571 conferred a significant protection (p < 0.05; Table 2). Because these birds were originally part of the virulence study, the two birds that died after challenge were primed with a dose of only 1 × 102 CFU. Strain ␹11570 was partially protective, though the observed protection was significant (p < 0.05). The lower survival in this group is likely to be due to the fact that these birds were also part of the

Table 3 Colonization of vaccinated and nonvaccinated Rhode Island Reds by S. Gallinarum (␹4173) or S. Enteritidis (␹3700). Treatment

Fig. 2. Effect on growth of deleting crp gene or replacing its promoter with an arabinose-regulated promoter in S. Gallinarum. Growth of strains ␹11387 (Pcrp527 ::TT araC PBAD crp) with and without addition of 0.2% arabinose, ␹11570 (crp-633::cam) and 287/91 (wild-type) in LB broth.

BSG ␹11387 a b

S. Gallinarum (CFU/g)

S. Enteritidis (CFU/g)

Liver

Spleen

Liver

Spleen

6.05 ± 0.19 0.20 ± 0.18b

5.70 ± 0.19 1.06 ± 0.4b

2.71 ± 0.35 –a,b

3.06 ± 0.49 –b

No SE detected. p < 0.05 compared to BSG group.

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Table 4 Effect of 0.2% arabinose in the drinking water on virulence and efficacy of S. Gallinarum strains ␹11387 (Pcrp527 ) and ␹11570 (crp-633) in Rhode Island Red chicks. Dosea

Combined totalsc

Alive/total (percent survival)

Strain

Prime (CFU)

Boost (CFU)

␹11387 ␹11570 PBS

1.00 × 10 1.26 × 108 –

1.25 × 10 1.18 × 108 –

8

8

+arabinoseb

No arabinose

d

7/10 (70%)d 10/11 (91%)d 0/5 (0%)

9/12 (75%) 6/10 (60%)d 2/5 (40%)

16/22 (73%) 16/21 (76%) 2/10 (20%)

a Birds received the indicated vaccine strain at 3 days of age and boosted at 14 days of age. All chickens were challenged with 1.1 × 107 CFU of S. Gallinarum strain ␹4173 at 30 days of age. Two chicks died prior to challenge. One inoculated with ␹11387 in the no arabinose group died 4 days after the primary immunization and one chick vaccinated with ␹11570 in the + arabinose group died 3 days after the boost. Both chicks that died were runts. b Birds in this group received water containing 0.2% arabinose for the duration of the experiment. None of the birds immunized with ␹11387 that received arabinose in the water died prior to challenge. c Birds from the +arabinose and no arabinose groups were combined. d p < 0.05 compared to PBS group.

virulence study and some of the birds received low priming doses (see Section 2 and below). 3.4. Effect of dietary arabinose on virulence and efficacy of strain ␹11387 Because crp expression is controlled by arabinose, which may be present in the diet, we evaluated the effect of dietary arabinose on virulence and immunogenicity of ␹11387. We inoculated groups of 20 birds with either ␹11387 or the crp deletion mutant ␹11570 using the same immunization and challenge schedule described above for ␹11387. The birds were split into groups of 10/strain and received either plain water or water containing 0.2% arabinose. Mock-vaccinated control groups of 5 birds each were also included. After vaccination, no birds in any group died from fowl typhoid (Table 4). In this experiment, both groups of birds received similar priming doses and we observed similar efficacy against challenge in birds vaccinated with either ␹11387 or ␹11570. 4. Discussion In this work, we adapted regulated delayed gene expression strategies to S. Gallinarum, targeting rfc, rfaH and crp. Strain ␹11387 (Pcrp527) showed the most promise for further development as a fowl typhoid vaccine. The strain was avirulent (Table 2), caused only modest internal lesions (Table S2), protected against a lethal S. Gallinarum challenge (Tables 2, 4) and protected against colonization by S. Enteritidis (Table 3). The addition of extra dietary arabinose did not affect virulence or immunogenicity (Table 4), although it is interesting that more of the control birds survived when arabinose was present in the diet. However, due to the low number of birds used in this study, a firm conclusion regarding the beneficial effects of dietary arabinose could not be reached. It is unlikely that the arabinose present in feed would trigger the arabinose-regulated promoter in our system, as the arabinose in plants is typically present as poorly digested polymers [46]. In previous work, we showed that the araC PBAD promoter does not turn on when Salmonella is grown in minimal broth supplemented with 1% chicken feed [48]. Our results with the rfc strains were unanticipated, since S. Typhimurium rfc mutants are completely attenuated in mice [13], as is an S. Typhimurium strain with a similar Prfc174 mutation [16]. These results indicate that a single O-antigen subunit is sufficient for S. Gallinarum virulence, at least in Rhode Island Reds. The increase in virulence observed for PrfaH178 mutant strain, ␹11386 was also unexpected, as a similar S. Typhimurium construct was partially attenuated in mice [17]. The lack of attenuation observed with ␹11386 may be due to the up-regulation of other virulence genes in this strain during in vitro growth prior to inoculation. Salmonella pathogenicity island 1 (SPI-1) genes,

including SPI-1 regulators hilC and hilD, and a number of inv genes are down-regulated when rfaH is deleted [21]. SPI-4 genes are also affected [21]. Thus, it is possible that arabinose-regulated synthesis of RfaH in ␹11386, just prior to inoculation, may have resulted in higher than normal levels of RfaH leading to overproduction of one or more virulence factor(s) that resulted in the observed increase in virulence. The importance of SPI-1 in S. Gallinarum virulence is not clear and appears to be influenced by the breed and age of the bird [44,45]. This will be a topic for future studies. We did not observe any difference in protection between crp strain ␹11570 and the arabinose-regulated Pcrp strain ␹11387 (Table 4), indicating that these two strains induced similar levels of immunogenicity. It will be interesting to make a similar comparison in a more resistant bird line, such as White Leghorns, to determine whether the regulated delayed attenuation strategy provides an advantage in those birds. Our ultimate goal is to develop a strain with more than one attenuating mutation, at least one of which does not respond to dietary components, to provide a safe, immunogenic vaccine. Once a final vaccine strain has been identified, it will be evaluated in different breeds and ages of birds for virulence and immunogenicity. Competing interests The authors declare that they have no competing interests. Authors’ contributions RC, KLR and AM designed experiments. AM, AL, CW, AG, PL and KLR performed the experiments. AM and PL performed statistical analyses. AM, RC and KLR wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Jacquelyn Kilbourne and Marta Matulova for their excellent technical assistance during this study. Many thanks go to Timothy Corsi and Julie Pugsley for taking outstanding care of the animals used in this work. We thank Javier Santander for his stimulating discussions and constructive feedback and Shifeng Wang for providing plasmids. This work was funded by BREAD grant IOS0965511 from the National Science Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2012.12.021.

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