A genetically engineered derivative of Salmonella Enteritidis as a novel live vaccine candidate for salmonellosis in chickens

Research in Veterinary Science 93 (2012) 596–603

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc

A genetically engineered derivative of Salmonella Enteritidis as a novel live vaccine candidate for salmonellosis in chickens Rahul M. Nandre, Kiku Matsuda, Atul A. Chaudhari, Bumseok Kim, John Hwa Lee ⇑ College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, South Korea

a r t i c l e

i n f o

Article history: Received 3 August 2011 Accepted 15 November 2011

Keywords: Salmonella Enteritidis Vaccine Attenuation Virulence gene Immune response

a b s t r a c t To construct a novel live Salmonella Enteritidis (SE) vaccine candidate, SE was genetically engineered using the allelic exchange method to delete two virulence genes, lon and cpxR. The lon gene deletion is essential to impair Salmonella replication and avoid overwhelming systemic disease in the host. The cpxR gene deletion is needed to enhance the ability of bacteria to adhere and invade the host cell. Scanning electron microscopy revealed that the derivatives JOL917 (Dlon), JOL918 (DcpxR), and JOL919 (Dlon/ DcpxR) had increased surface fimbrial filamentous structures. Significant elevations of extracellular polysaccharide and FimA expression were observed for the derivatives compared to the parental wild type JOL860, while biochemical properties of the derivatives were not altered. In the safety examination by inoculation of the derivatives in chickens, gross lesion scores of the liver, spleen, kidney, small intestine and caecal tonsils were moderate in the JOL917 and JOL918 groups, and significantly lower in the JOL919 group than those of the JOL860. Bacterial counts from the spleen and caeca of the JOL917 and JOL918 groups were moderate, and significantly reduced in the JOL919 group compared to the JOL860 group. In addition, only the JOL919 group showed significantly lower bacterial counts in the faecal samples than those of the JOL860 group. Significant elevations of IgG and secretory IgA levels observed in the derivative groups, while the JOL919 and JOL860 groups showed a potent lymphocyte proliferation response as compared to those of the control group. In the protection efficacy examination, JOL919 immunized group showed significantly lower depression, lower gross lesion in the liver and spleen, and lower number of the SE positive internal organs than those of the control group against a virulent wild type SE challenge. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Salmonellosis is an important public health problem in many countries and a frequent cause of gastroenteritis and zoonotic infections (Dhanoa and Fatt, 2009; Matheson et al., 2010). The serotype Salmonella Enteritidis (SE) may also cause an asymptomatic infection in poultry, which may result in egg contamination with transmission to humans upon consumption of raw or undercooked eggs and their derivatives (Bäumler et al., 2000; Clavijo et al., 2006). Therefore, Salmonella control within poultry farms is an urgent issue. Vaccination is a potentially effective tool for the prevention of salmonellosis. Whole-cell killed vaccines and subunit vaccines have been used with variable results to prevent Salmonella infection in both humans and animals (Mastroeni et al., 2001). It is thought that live vaccines have advantages over killed vaccines as they stimulate both humoral and cell-mediated immunity (Carvajal et al., 2008). The commercially available live Salmonella vaccines of for poultry are either auxotrophic double-marker mutants derived through chemical mutagenesis (Meyer et al., ⇑ Corresponding author. Tel.: +82 63 270 2553; fax: +82 63 270 3780. E-mail addresses: [email protected], [email protected] (J.H. Lee). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.11.005

1993; Springer et al., 2000), or developed on the basis of the principle of metabolic drift mutations (Vielitz et al., 1992; Linde et al., 1997; Hahn, 2000). However, the efficacy of live vaccines is limited depending upon the residual virulence and host clearance, while multiple studies have been carried out to demonstrate the efficacy of live Salmonella vaccines (Takaya et al., 2002, 2003; Matsui et al., 2003; Kim et al., 2009). Lon has been characterized as a powerful negative regulator of the expression of invasion genes encoded on Salmonella pathogenicity island 1 (SPI-1), and it also effects macrophage survival and is required for systemic infection (Takaya et al., 2002, 2003). The CpxAR two-component signal transduction pathway consists of a sensor kinase (SK), CpxA, and a cognate response regulator (RR), CpxR. The SK, CpxA, is located in the cytoplasmic membrane, where it senses diverse signals, including alkaline pH, altered membrane lipid composition, interaction with hydrophobic surfaces, and misfolded pilin subunits. In response, CpxA autophosphorylates and donates its phosphoryl group to activate CpxR. Activated CpxR regulates part of the envelope stress response system, pilus assembly, type III secretion, motility and chemotaxis, adherence, and biofilm development (Wolfe et al., 2008). Therefore, the derivative strains constructed with deletions of lon and

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

597

2. Materials and methods

of Luria–Bertani (LB, Becton, Dickinson and Company, Sparks, MD, USA) broth and incubating at 37 °C while shaking at 250 rpm. The optical density at 600 nm (OD600) was determined every 1.5 h for 9.5 h. For colony morphology observation, each strain was streaked on LB agar and kept for incubation at 37 °C for 24 h. These colonies, grown on LB agar, were gently collected and fixed in 1.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4). After fixation, they were placed in 1% aqueous osmium tetroxide phosphate buffer and serially dehydrated in acetone. Samples were critical point dried and coated with platinum-palladium alloy for scanning electron microscopy (JSM-6400, JEOL, Japan).

2.1. Bacterial strains and genetic manipulation

2.3. Biotype

Genetic manipulation of the derivatives was accomplished by deletion of the genes using the allelic exchange method. The left and right arms of the lon and cpxR genes were amplified by polymerase chain reaction (PCR) with lon and cpxR primers to construct Dlon, DcpxR and Dlon/DcpxR derivatives, which consisted of flanking DNA sequences excluded in the lon and/or cpxR genes, using wild-type SE JOL860 (Table 1). The amplified DNA segments were cloned into a T-vector (Promega, Madison, WI, USA). The cloned left and right arms were directly ligated between the two fragments, and the recombinant connected by two arms was completed. The recombinant DNA fragment was digested with the appropriate restriction enzymes and then cloned into suicide vector pMEG375 (Table 1). The transfer of recombinant suicide plasmids into Salmonella was accomplished by conjugation using pMEG375 as the suicide vector. The electroporation method was used for the plasmid transformation into Salmonella competent cells (Sambrook et al., 1989). The recombinant suicide plasmid pBP294 was obtained by cloning the ligated fragment of lon gene into pMEG375. The plasmid pBP210 was generated by the same method as in the two flanking PCR amplicons of the cpxR gene into pMEG375. To induce the deletion of the lon gene, the plasmid pBP294 was conjugationally transferred into SE JOL860. The selected colonies were confirmed by PCR using the specific primer sets of 50 -CAGGAGTTCTTACAGGTAGA-30 /50 -CCACACTCCGCTGTAGG TGA-30 (lon). By conjugational transfer of pBP210 into JOL860, the deletion of the cpxR gene was introduced. The selected colonies were confirmed by PCR using the specific primer sets of 50 -CATCATCTGCGGGTTGCAGC-30 /50 -GATAATTTACCGTTAACGAC-30 (cpxR).

The biochemical phenotypes of the derivatives were analyzed with the API 20E system (bioMérieux, Rhône, France). Cells were grown for 24 h at 37 °C on LB agar, suspended in 0.85% sodium chloride, and processed as per the manufacturer’s instructions.

cpxR are expected to increase capability for adhesion or invasion, but decreased survival and systemic infection in the host cell, resulting in easy eradication from host cells without causing damage. In this study, the lon and/or cpxR genes were deleted from SE and the potential of the derivatives for use as an attenuated live vaccine candidate against wild-type SE was examined. For this, their morphology, physiological changes, biochemical properties, safety, and immunogenicity were evaluated.

2.4. FimA expression To compare relative expression of FimA between strains, the FimA gene was cloned from a Salmonella Typhimurium (ST) wild isolate to pET28a using a primer set of 50 -GGATCCGCTGATCCTA CTCCGGTGAG-30 , 50 -CTCGAGTTCGTATTTCATGATA AAGG-30 for over-expression of the 6 His-tag attached protein, which was purified using Ni–NTA Agarose (Peptron, South Korea) and inoculated into rabbits to obtain anti-FimA serum. Colonies grown on LB agar for 24 h at 37 °C were suspended in PBS at an OD600 of 0.5, and diluted 1:100. The diluted suspension of each strain was divided into triplicate samples. The concentration of each suspension was determined by the colony count method after spreading of these triplicate samples on LB agar plates for normalization. A piece of ProtranÒ nitrocellulose membrane with a pore size of 0.2 lM (Schleicher and Schuell, Dassel, Germany), was immersed in Trisbuffered saline (TBS, 10 mM Tris, 0.9% sodium chloride, pH 7.4) and set in a 96-well Bio-DotTM apparatus (Bio-Rad Laboratories, USA), according to the manufacturer’s instructions. The result was expressed as mean ratio ± standard error of the mean (SEM) of the normalized value after division by the average of the normalized value for JOL860. This experiment was repeated for four times. 2.5. Extra-cellular polysaccharides (EPS)

2.2. Growth character, colony morphology and scanning electron microscopy Growth character was studied by adding a 1/100 volume of an overnight culture of wild type and derivative strains into 200 mL

Table 1 Bacterial strains and plasmids used in this study. Strains/ plasmid

Description

S. Enteritidis JOL860 S. Enteritidis wild type, originated from chicken salmonellosis JOL917 S. Enteritidis JOL860 derivative Dlon JOL918 S. Enteritidis JOL860 derivative DcpxR JOL919 S. Enteritidis JOL918 derivative Dlon JOL1182 S. Enteritidis virulent strain, originated from chicken salmonellosis Plasmid pMEG375 pBP294 pBP210

Suicide vector to construct derivatives of S. Enteritidis pMEG375 Dlon pMEG375 DcpxR

Reference

Fluorometric quantification of EPS by the Concanavalin A (ConA) binding assay was performed (Robitaille et al., 2006). Briefly, cells grown on LB agar were suspended in PBS at an OD600 of 0.5. Fluorescein isothiocyanate-conjugated Con-A (Sigma–Aldrich, St. Louis, MO, USA) was added at 4 lg/mL, incubated for 30 min, and washed twice with PBS. Then, 200 lL of suspension of each strain was transferred to 5 wells of a 96-well microplate and fluorescence intensity was recorded by TriStarLB941 (Berthold Technologies GmbH and Company, Bad Wildbad, Germany). This experiment was repeated for four times.

This study

2.6. Preparation of mutant and wild strains This This This This

study study study study

(Dozois et al., 2003) This study This study

The strains were maintained as glycerol-frozen cultures in LB broth at 70 °C. The frozen cultures were streaked on LB agars and incubated at 37 °C for 16 h. A colony per strain was inoculated in the LB broth for overnight incubation. For inoculation to chickens, the overnight culture was inoculated in fresh LB broth at 1:20 dilution, incubated at 37 °C to an optical density (OD)600 of 0.6. Cells were harvested by centrifugation at 13,200 rpm for 5 min. The pellets were washed and re-suspended in sterile PBS,

598

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

and were adjusted at appropriate concentrations based on the optical density for inoculations. 2.7. Bacterial inoculation Female day-old layer chickens (Brown Nick), (N = 100) were divided into five groups (n = 20), and provided water and antibioticfree food ad libitum. Each of four groups was inoculated orally with 100 lL of bacterial suspension containing 1  107 colony forming units (cfu) of the wild type or derivative strains. The fifth group was inoculated with PBS as a non-vaccinated control. The animal experiments were conducted under ethics approval (CBU 200801-0019) from the Chonbuk National University Animal Ethics Committee in accordance with the guidelines of the Korean Council on Animal Care. 2.8. Observations of gross lesions and bacterial recovery from organs At the 10th day post inoculation, five randomly selected animals per group were euthanized. Gross lesions of enlarged and necrotic foci in the liver, spleen, kidney, small intestine, and caecal tonsils were scored as 0, 1, 2, or 3. A score of 0 indicated no lesion, 1 – specified necrotic foci, 2 – designated an enlarged and necrotic organ, and 3 – used for more soften, necrotic and enlarged organs. To assess bacterial recovery from the infected organs, splenic and caecal samples were weighed and homogenized in 2 mL buffered peptone water (BPW, Becton, Dickinson and Company, Sparks, MD, USA). 100 lL homogenate was inoculated on Brilliant Green Agar (BGA, Becton, Dickinson and Company, Sparks, MD, USA) for enumeration and incubated overnight at 37 °C for 24 h. The number of SE in the sample was determined and expressed as mean ± SEM log10 cfu/g (Betancor et al., 2005). In addition, the homogenized tissue samples were pre-enriched in 2 mL BPW in shaking incubator at 37 °C for 18 h, followed by 300 lL of the pre-enrichment culture was transferred to 3 mL Rappaport–Vassiliadis R10 (RV) broth (Becton, Dickinson and Company, Sparks, MD, USA) for 48 h at 42 °C. A loop of the enriched broth was streaked onto BGA and Salmonella-type colonies were examined after incubation at 37 °C for 24 h. Samples that were positive only after enrichment were counted as 1 log10 cfu/g, while samples still negative after enrichment were indicated as 0 log10 cfu/g (Betancor et al., 2005). The colonies were confirmed by PCR using SE specific primers (Alvarez et al., 2004). 2.9. Bacterial isolation from faecal shedding The presence of the wild-type and derivative strains in faeces was monitored on the second, third, and fourth weeks post-inoculation. The collected faecal samples of chickens were weighed and diluted 1:10 by weight with BPW, and homogenized. Enumeration and enrichment cultures were performed as described above. The presence of wild-type or derivative strains was confirmed by PCR.

sured using adenosine triphosphate (ATP) bioluminescence as Ò marker of cell viability with a ViaLight Plus Kit (Lonza Rockland, ME, USA), to provide an estimation of mitochondrial activity (Crouch et al., 1993). The emitted light intensity was measured using a luminometer (TriStarLB941, Berthold Technologies GmbH and Company, Bad Wildbad, Germany). The blastogenic response against a soluble antigen was expressed as the mean stimulation index (SI) as previously reported (Rana and Kulshreshtha, 2006). 2.11. Enzyme-linked immunosorbent assay (ELISA) Indirect ELISA was performed with an outer membrane protein fraction (OMP) that was extracted from the SE wild type strain (Kang et al., 2002). ELISA was performed using chicken IgG and IgA ELISA Quantitation Kits (Bethyl laboratories, TX, USA) for determination of IgG and IgA concentrations according to the product information. Microlon ELISA plate wells (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with OMP (0.1 mL) at a concentration of 5 lg/mL. During the ELISA procedure, intestinal wash and plasma samples were diluted 1:5 with PBS. After dilution of intestinal wash and plasma samples, ELISA plate wells were incubated with goat anti-chicken IgG horseradish peroxidase (HRP)conjugate at a 1:100,000 dilution for 1 h. The bound HRP activity was determined using o-phenylenediamine dihydrochloride (Sigma–Aldrich, St. Louis, MO, USA). After ELISA, OD values were measured at 492 nm (TECAN, Austria). 2.12. Protection against challenge Twenty, day-old chickens were divided into two groups (n = 10), provided water and antibiotic-free food ad libitum. The immunized group was inoculated via the oral route with 100 lL of bacterial suspension containing 1  107 cfu of JOL919. The control group was inoculated with PBS. At the fourth week, chickens were orally challenged with the virulent wild type SE JOL1182 at 1  109 cfu per chicken. The birds were observed daily for depression for two weeks. Parameters used for depression evaluation included anorexia, reluctance to move, drowsiness, and lack of response to external stimuli. Each animal was scored on a scale of 0 (no depression) to 3 (severe depression), and the mean depression score was calculated for each group. After two weeks of challenge, all birds were euthanized. Gross lesions of liver and spleen were observed for chickens of the control and JOL919 immunized groups as described above. For bacterial recovery, samples of liver, spleen, and caeca were minced in 2 ml BPW, and an adequate dilution was inoculated on BGA. Enrichment culture was made in RV broth as described above. The challenge strain was confirmed by PCR using SE specific, and lon and cpxR specific primer sets. 2.13. Statistical analysis

2.10. Peripheral lymphocyte proliferation assay The lymphocyte proliferation assay was performed (Rana and Kulshreshtha, 2006; Xie et al., 2008) to evaluate cell-mediated immunity in the immunized groups. Soluble antigen was prepared from the SE wild-type strain JOL860 (Rana and Kulshreshtha, 2006). On the third week post immunization, the peripheral lymphocytes were separated from the blood of five randomly selected chickens per group using the gentle swirl technique (Gogal et al., 1997). After trypan blue dye exclusion testing, 100 lL of viable mononuclear cell suspensions at 1  105 cfu/mL in RPMI-1640 medium were incubated in triplicate in 96-well tissue culture plates with 50 lL of medium alone or medium containing 4 lg/ mL of soluble antigen at 40 °C, in a humidified 5% CO2 atmosphere for 72 h. The proliferation of stimulated lymphocytes was mea-

Analyses were performed with SPSS 16.0 (SPSS Inc., USA). The t test was applied to examine whether there are significant difference in the in vitro experimental data of the mutants comparing to the wild type. Further t-test with Bonferroni correction was applied to examine the data whether or not significant differences are found between the mutants. In the safety examination, the gross lesion scores and bacterial recovery from organs of the derivative groups were compared to those of the JOL860 by the Man Whitney U test. The t test was also used to analyze the immune responses in the control group and the derivative groups. In the protection efficacy experiment, the Chi-square test was used to show significantly lower number of SE positive birds in the immunized group as compared to that of the control group. In addition, the Chisquare test was also used to show significantly lower gross lesion

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

599

scores and bacterial recovery in the immunized group than those of the control group. 3. Results 3.1. Constructions of the derivatives Deletions of the lon and cpxR genes from SE JOL860 by the allelic exchange method resulted in constructions of Dlon, DcpxR, and Dlon/DcpxR derivatives. Successful deletions of the lon gene in JOL917, of the cpxR gene in JOL918, and of both genes in JOL919 were confirmed by PCR with expected amplicon sizes of 3.88 kb and 1.54 kb for the intact wild and derivative of the Dlon gene, respectively, and 2.71 kb and 2.04 kb for the intact wild and derivative of the DcpxR gene, respectively. 3.2. Morphological, physiological and biochemical properties of the derivatives The derivatives were examined to observe whether the gene deletions had an effect on morphological structures, physiological characteristics and biochemical phenotypes. The growth curves of all derivatives in the LB broth under high-oxygen conditions were fundamentally similar to that of the wild type. The colony morphologies of JOL917, JOL918, and JOL919 were mucoid, while the wild type strain was non-mucoid and slightly larger in size. SEM revealed that the wild type had smooth surfaces with no apparent fimbrial structures (Fig. 1A); JOL917 (Dlon) appeared elongated and corrugated with fimbriae (Fig. 1B); JOL918 (DcpxR) had a smooth surface with long fimbrial structures (Fig. 1C); and JOL919 (Dlon, DcpxR) also appeared elongated with corrugated morphology and filamentous structures (Fig. 1D). Furthermore, the FimA expression of JOL917, JOL918, and JOL919 was detected 19.2, 8.9, and 28.0 times, respectively, compared to that of the wild type (P < 0.01, Fig. 2A). In addition, the FimA expression of JOL919 was also significantly higher than those of JOL917 and JOL918 (P < 0.05, Fig. 2A) based on the t-test with Bonferroni correction. The EPS per cell of JOL917, JOL918, and JOL919 were 4.5, 2.2, and

Fig. 2. Phenotype examinations of wild type SE (JOL860), Dlon (JOL917), DcpxR (JOL918), and DlonDcpxR (JOL919) derivative strains: (A) relative FimA expression of 24-h culture on Luria–Bertani (LB) agar by dot-blot analysis; (B) relative extracellular polysaccharide (EPS) of 24-h culture on LB agar examined by ConcanavalinA (CON-A) binding fluorometric assay. Error bars indicate the standard error of the mean (SEM). ⁄Significant difference of derivatives from the parental strain JOL860 (P < 0.01). ⁄⁄Significant difference of JOL919 compared to other derivatives, JOL917 and JOL918 (P < 0.05).

21.5 times increased, respectively, compared to that of the wild type (P < 0.01, Fig. 2B). In addition, the EPS per cell of JOL919 was also significantly higher than those of the JOL917 and JOL918 (P < 0.05, Fig. 2B) based on the t-test with Bonferroni correction.

Fig. 1. Scanning electron microscope observations (Magnification, 10 k): (A) wild type Salmonella Enteritidis (SE) (JOL860) revealed non-fimbriated smooth surface; (B) 4lon derivative (JOL917) revealed elongated structures with fimbriae appendage; (C) 4cpxR derivative (JOL918) exhibited a smooth surface with long fimbriae; and (D) 4lon4cpxR derivative (JOL919) appeared corrugated with abundant filamentous structures.

600

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

Table 2 Gross lesions from internal organs of chickens inoculated with SE derivatives. Strain

JOL860 JOL917 JOL918 JOL919

Dose (CFU)

n

107 107 107 107

5 5 5 5

Gross lesionsa Liver

Spleen

Kidney

Small intestine

Caecal tonsils

2.0 ± 0.6b 0.2 ± 0.4c 0.4 ± 0.5c 0.0 ± 0.0c

1.0 ± 0.6 0.8 ± 0.4 0.4 ± 0.5 0.0 ± 0.0c

1.0 ± 0.0 0.6 ± 0.5 0.2 ± 0.4c 0.0 ± 0.0c

0.6 ± 0.5 0.2 ± 0.4 0.6 ± 0.5 0.0 ± 0.0c

0.6 ± 0.5 0.8 ± 0.4 0.4 ± 0.5 0.0 ± 0.0c

n, Numbers of the birds used. a Gross lesions were observed after euthanasia of five randomly selected animals per group on the 10th day post inoculation. b Group lesion score (mean ± SEM). c Gross lesion score is significantly different from the JOL860 group (P < 0.05).

Table 3 Bacterial recovery from organs and faeces of chickens inoculated with SE derivatives. Strain

JOL860 JOL917 JOL918 JOL919

Dose (CFU)

107 107 107 107

n

Bacterial recoverya Spleen

5 5 5 5

4.1 ± 0.6 1.3 ± 1.1b 2.2 ± 1.8 0.8 ± 1.2b

Caeca

7.0 ± 0.6 3.1 ± 2.5 3.6 ± 3.0 2.0 ± 1.7b

Faecal sample 2nd Week

3rd Week

4th Week

2.0 ± 1.5 0.0 ± 0.0c 3.3 ± 2.0 0.0 ± 0.0c

2.3 ± 1.4 1.9 ± 1.5 1.9 ± 1.6 0.0 ± 0.0c

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

n, Numbers of the birds used. a Mean ± SEM log10 cfu/g. b The bacterial recovery for spleen and caeca is significantly different from the JOL860 group (P < 0.05), observed on the 10th day post inoculation. c The bacterial recovery from faeces is significantly different from the JOL860 group (P < 0.05), examined on the 2nd, 3rd and 4th week post inoculation.

Fig. 3. The immune responses to Salmonella antigen in chickens inoculated with the SE wild type and derivative strains on the fourth week post inoculation: (A) the mucosal sIgA immune response; (B) the systemic IgG immune response. Error bars indicate SEM. ⁄Significant differences in the values of wild type and derivative groups compared to the PBS inoculated control group (P < 0.05).

The biochemical phenotypes of all derivative strains examined by the API 20E system were consistent with the wild type. 3.3. Safety and immunogenicity of the derivative strains in chickens 3.3.1. Observations of gross lesions Safety and immunogenicity of the derivative strains were examined using 100, day-old chickens, divided into five groups (n = 20) for the wild type, JOL917, JOL918, JOL919 and non-inoculated control groups. To evaluate the safety of the derivatives, five randomly selected chickens from each group were euthanized for postmortem examination on the 10th day post oral inoculation. Findings of enlarged white spots on the liver, spleen, kidney, small intestine, and caecal tonsils were scored from 0 to 3. Means of the organ lesion scores for the derivative groups were compared to the wild type group. The wild type group showed the highest score compared to the other derivative groups (Table 2). The JOL917 group scored significantly low in the liver and small intestine (P < 0.05), while JOL918 group scored significantly low in the liver, spleen and kidney compared to those of the wild type (P < 0.05). The JOL919 group scored significantly low in all the five organs compared to those of the wild type group (P < 0.05). 3.3.2. Bacterial recovery from organs and faeces By both direct colony count of homogenized samples on BGA and sample enriched RV cultures from the JOL860, JOL917, JOL918, and JOL919 groups, recovery of bacteria from the spleen and caeca was attempted to evaluate safety. As shown in Table 3, the wild type JOL860 showed bacterial counts 4.1 ± 0.6 and 7.0 ± 0.6 cfu/g for the spleen and caeca, respectively, while those for JOL917 were 1.3 ± 1.1 and 3.1 ± 2.5 cfu/g, and JOL918 were 2.2 ± 1.8 and 3.6 ± 3.0 cfu/g, respectively. The JOL919 group showed lowest bacterial counts for spleen, 0.8 ± 1.2 cfu/g and caeca, 2.0 ± 1.7 cfu/g (P < 0.05, Table 3).

Fig. 4. The stimulation index of lymphocyte proliferation assay for the chickens inoculated with the SE wild type and derivative strains on the third week post inoculation. Error bars indicate SEM. ⁄Significant differences of values of wild type and derivative groups compared to the phosphate buffered saline (PBS) inoculated control group (P < 0.05).

Furthermore, we also attempted to isolate the wild type and derivative strains from faecal samples collected on the second, third, and fourth week post inoculation. JOL860, JOL917 and JOL918 were isolated from faecal samples up until the third week post inoculation. As shown in Table 3, JOL860 showed bacterial counts as 2.0 ± 1.5 and 2.3 ± 1.4 on the second and third week post inoculation, respectively. In addition, bacterial counts of JOL917 were 0.0 ± 0.0 and 1.9 ± 1.5, while those of JOL918 were 3.3 ± 2.0 and 1.9 ± 1.6, on the second and third week post inoculation, respectively. However, JOL919 was not isolated from either direct colony count on BGA or from enrichment culture of the group faecal samples (Table 3). 3.3.3. Humoral and cellular immune responses Systemic IgG and mucosal secretory IgA (sIgA) levels were examined on the fourth week post inoculation to investigate the humoral immune responses induced by the derivatives. The reaction was

601

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

analyzed by determining specific IgG in plasma and sIgA in intestinal wash fluid by indirect ELISA using the specific antigen of SE. As shown in Fig. 3, the mucosal sIgA titres showed approximately 1.3-, 3.3- (P < 0.05), 3.5- (P < 0.05) and 1.9-fold (P < 0.05) increases for JOL860, JOL917, JOL918 and JOL919, respectively, compared to the PBS control. Furthermore, the plasma IgG titers showed approximately 2.9, 2.4, 2.5, 3.7-fold increases for JOL860, JOL917, JOL918 and JOL919, respectively, compared to the PBS control (P < 0.05). In addition, cellular immune responses induced by the derivative strain were examined using the peripheral lymphocyte proliferation assay. On the third week post inoculation, the SI values of 2.28 ± 0.58 (P < 0.05), 1.54 ± 0.15, 1.11 ± 0.06, and 2.25 ± 0.58 (P < 0.05) were observed in the JOL860, JOL917, JOL918 and JOL919 inoculated groups, respectively; while the SI value was 1.15 ± 0.21 for the PBS-control group (Fig. 4).

3.4. Protection against virulent challenge To determine the protective efficacy of JOL919, the chickens were challenged with the virulent wild type SE strain, and were evaluated by the depression score, gross lesion score and bacterial recovery (Table 4). In the immunized group, two birds showed slight and temporal depression followed by recovery, while in the unimmunized control group, all chickens showed signs of depression. The depression score of the immunized group (0.2 ± 0.4) was significantly lower than the control group (1.2 ± 0.8). The liver and spleen in the immunized group revealed no or very little gross lesions, while the control group showed enlargement with necrotic lesions in both organs (Fig. 5). Gross lesion scores of the liver (0.0 ± 0.0) and spleen (0.5 ± 0.5) in the immunized group were significantly lower than those in the control group (P < 0.05). In

Table 4 Depression, gross lesions and bacterial recovery post challenge. Group

Control JOL919

n/Na

7/10 2/10b

Depression score

1.2 ± 0.8 0.2 ± 0.4c

Gross lesion score

Bacterial recovery

Liver

Spleen

Liver

No. of positive

Spleen

No. of positive

Caeca

No. of positive

0.6 ± 0.5 0.0 ± 0.0d

1.5 ± 0.8 0.5 ± 0.5d

1.0 ± 1.3 0.0 ± 0.0e

4/10 0/10f

1.8 ± 1.6 0.3 ± 0.7e

7/10 2/10f

0.6 ± 0.8 0.2 ± 0.6

6/10 1/10f

a

n/N, Number of SE-positive birds per total number of birds observed by direct and enrichment culture methods. The number of SE positive birds of the JOL919 immunized group is significantly lower than that of the control group (P < 0.05). c The depression score (mean ± SEM) of the JOL919 immunized group is significantly lower than that of the control group (P < 0.05), observed for 14 days post-challenge. d Gross lesion scores (mean ± SEM) of the liver and spleen of the JOL919 immunized group are significantly lower than those of the control group (P < 0.05), observed on the 14th day post challenge. e Bacterial counts (mean ± SEM log10 cfu/g) from the liver, spleen, and caeca of the JOL919 immunized group are significantly lower than those of the control group (P < 0.05), examined on the 14th day post challenge. f Significantly lower number of SE positive organs from the JOL919 immunized group compared to the control group (P < 0.05). b

Fig. 5. The gross lesions of the liver and spleen were observed in the unimmunized control group and the JOL919 immunized group on the 14th day post challenge with a virulent wild type JOL1182 SE (Size bar, 10 mm). Arrows indicate gross lesions on liver and spleen. (A) liver and (B) spleen of the unimmunized control chicken; (C) liver and (D) spleen of the JOL919 immunized chicken.

602

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

addition, in bacterial recovery attempts from the liver, spleen, and caeca, two birds were positive for SE in the immunized group, while seven birds were positive in the control group (Table 4). In the immunized group, the bacterial counts for liver, spleen and caeca were 0.0 ± 0.0, 0.3 ± 0.7 and 0.2 ± 0.6 cfu/g on log10 scale, respectively, while those of the control group were 1.0 ± 1.3, 1.8 ± 1.6 and 0.6 ± 0.8 cfu/g. Bacterial cells isolated from the recovery attempts were significantly lower in the liver and spleen of the immunized group compared to those of the control group (P < 0.05). In addition, the number of SE positive liver, spleen and caeca were significantly lower in the immunized group than those of the control group (P < 0.05).

4. Discussion In this study, the effect of the deletion of lon and/or cpxR genes from SE was evaluated. Considerable involvement of these genes in pathogenic mechanisms has been suggested recently and the possibility of utilizing target genes for the construction of live vaccines was proposed (Kim et al., 2009). Lon protein negatively regulates the ability of the bacterium to invade epithelial cells and the expression of invasive genes carried on SPI through degradation of HilC and HilD (Takaya et al., 2005). Furthermore, Lon participates in controlling many pathways: cell division (Schoemaker et al., 1984), capsule synthesis (Torres-Cabassa and Gottesman, 1987), sporulation (Schmidt et al., 1994), and cell cycle progression (Wright et al., 1996). Lon is also essential for systemic infection by Salmonella (Takaya et al., 2003). Therefore, disruption of the lon gene would impair its replication in the host cell and its ability to cause overwhelming systemic disease (Takaya et al., 2003). CpxR is associated with the controlling part of the envelope stress response system, and is also associated with pilus assembly, which can be related to both adhesion and invasion of epithelial cells (Wolfe et al., 2008). The CpxR protein reduces adhesion and invasion due to its excessive expression (Humphreys et al., 2004). Therefore, strains constructed by deletion of the lon and/or cpxR genes are expected to possess increased capability of adhesion and invasion but reduced replication in the host cell without causing any side effects and systemic disease. Furthermore, the possibility of reversion to the wild-type pathotype is very unlikely due to the complete deletion of two virulence-associated genes (Boyen et al., 2008). After deletion of the lon and/or cpxR genes from SE, the growth character and biochemical properties of the derivatives rarely changed in the present study. These findings indicate that these genes are not involved in the regulation of growth and the biochemical reactivity of SE. However, physiological structures, such as FimA production and EPS secretion were significantly changed by the lon and/or cpxR gene deletions. The FimA structural genes are involved in production of type 1 fimbriae of SE (Müller et al., 1989). For SE, type 1 fimbriae have been shown to contribute to adherence in gut epithelia, including Peyer’s patches, and pathogenesis in the mouse (Aslanzadeh and Paulissen, 1992). The in vitro experiments showed that the FimA production was elevated in JOL917, JOL918, and JOL919 derivatives compared to the wild type. The JOL919 group also showed significantly higher FimA expression as compared to those of the JOL917 and JOL918 groups (Fig. 2A). Electron microscopic results also indicated that JOL917, JOL918, and JOL919 derivatives produced more fimbriae than the wild-type (Fig. 1). These results might be due to the fact that CpxR and Lon proteins are involved in the expression and production of Salmonella fimbriae (Kim et al., 2009). With regards to the fact that the JOL917 and JOL919 strains produced more filamentous structures by electron microscopic observation, deficiency of the lon gene has been reported to induce filamentous

structures due to repression of cell division and may lead to an increase in the production of capsular polysaccharide, which is a major antigenic component (Torres-Cabassa and Gottesman, 1987). The EPS production was also significantly elevated in all the derivative groups, while the JOL919 group showed more prominent elevation in EPS production (Fig. 2B). These results indicated that EPS might have been produced by the deletion of the lon and/or cpxR genes. Since Salmonella has been reported to have the ability to adhere to form a biofilm on chicken intestinal epithelium in a type I fimbriae-dependent manner, followed by EPS secretion, suggesting involvement of both type I fimbriae and EPS on attachment and invasion of Salmonella in chicken cells (Ledeboer and Jones, 2005), these strains may have increased adherence and invasiveness into host cells. To evaluate the safety of the SE derivatives as live vaccines, the faecal shedding bacteriology, gross lesions, and bacterial persistence were measured after inoculation of these strains into susceptible chickens. The faecal shedding of genetically manipulated microorganisms through faeces is considered undesirable from a health and environmental safety point of view (Ghany et al., 2007). Faeces can also serve as a nutritional reservoir of Salmonella (Gantois et al., 2009). Previously reported Salmonella live vaccines have shown faecal excretion (Tan et al., 1997). In this study, the derivative strains were excreted into faeces in all chicken groups inoculated with a dosage of 107 except the JOL919 group (Table 3). Furthermore, the JOL917 group showed significantly lower gross lesion scores in the liver and small intestine, while the JOL918 group showed significantly lower gross lesion scores in the liver, spleen and kidney. The gross lesions of all five examined internal organs in the JOL919 group were scored significantly lower, which indicated satisfactory attenuation of virulence induced by deletion of both genes (Table 2). The mean number of bacteria recovered from organs of the JOL917 and JOL918 groups was moderate as compared to the wild type group for spleen and caeca. The JOL919 group showed the lowest bacterial count in the spleen and caeca as compared to the wild type group (P < 0.05). These data also suggest that JOL919 has less bacterial persistence in the organs compared to the wild type and other derivatives (Table 3), indicating that this strain may be free from undesirable complications caused by bacteria with longer persistence. Cell-mediated immune mechanisms are important for recovery from Salmonella infections, presumably because Salmonella survive and replicate within macrophages (Mastroeni et al., 1993; Beal et al., 2006). In this study, ATP bioluminescence was used as a marker of cell viability, and significant responses were detected at the third week post immunization in the JOL919 derivative group (Fig. 4), while the JOL917 and JOL918 groups did not show significant cellular immune response. In addition, mucosal sIgA and systemic IgG responses were induced significantly in chickens of the JOL917, JOL918 and JOL919 groups (Fig. 3A and B). The high inductions of sIgA and IgG levels may be related to the deletion of lon and/or cpxR genes as the lon and/or cpxR gene deletions lead to increase the synthesis of capsular polysaccharides and the expression of fimbriae, which are important antigenic components required to induce an effective immune response. Our collective data revealed that JOL919 was the safest and most immunogenic candidate among the derivatives. Thus, JOL919 was further evaluated for protection efficacy against a virulent wild type SE challenge. In the examination of protection, the higher bacterial counts in the livers and spleens of the unimmunized control group might be correlated with the failure of innate immunity to control Salmonella replication in the spleen and liver, leading to pronounced hepatosplenomegaly, with lesions in these organs (Fig. 5; Chappell et al., 2009). On the other hand, the majority of Salmonella appear to be cleared successfully by the adaptive immunity with only small numbers persisting within the spleen

R.M. Nandre et al. / Research in Veterinary Science 93 (2012) 596–603

and caeca of the JOL919 immunized group, which indicates that immunization with JOL919 can provide an efficient protection against SE infection. 5. Conclusion In conclusion, the Dlon derivative JOL917 and the DcpxR derivative JOL918 showed significantly higher EPS production, FimA expression, mucosal sIgA and systemic IgG responses but not satisfactory attenuation. In contrast, the derivative with both lon and cpxR genes deleted, JOL919, induced significant EPS production and FimA expression, and cellular, mucosal and systemic immune responses with satisfactory attenuation, which can provide the safety and effective protection against salmonellosis. 6. Conflict of interest statement Authors declare no conflict of interest. Acknowledgment This work was supported by Mid-career Researcher Program through NRF grant funded by the MEST (No. 2011-0000076). References Alvarez, J., Sota, M., Vivanco, A.B., Perales, I., Cisterna, R., Rementeria, A., Garaizar, J., 2004. Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples. Journal of Clinical Microbiology 42, 1734–1738. Aslanzadeh, J., Paulissen, L.J., 1992. Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella Enteritidis in mice. Microbiology and Immunology 36, 351–359. Bäumler, A.J., Hargis, B.M., Tsolis, R.M., 2000. Tracing the origins of Salmonella outbreaks. Science 287, 50–52. Beal, R.K., Powers, C., Davison, T.F., Barrow, P.A., Smith, A.L., 2006. Clearance of enteric Salmonella enterica serovar Typhimurium in chickens is independent of B-cell function. Infection and Immunity 74, 1442–1444. Betancor, L., Schelotto, F., Fernandez, M., Pereira, M., Rial, A., Chabalgoity, J.A., 2005. An attenuated Salmonella Enteritidis strain derivative of the main genotype circulating in Uruguay is an effective vaccine for chickens. Veterinary Microbiology 107, 81–89. Boyen, F., Haesebrouck, F., Maes, D., Immerseel, F.V., Ducatelle, R., Pasmans, F., 2008. Non-typhoidal Salmonella infections in pigs: A closer look at epidemiology, pathogenesis and control. Veterinary Microbiology 130, 1–19. Carvajal, B.G., Methner, U., Pieper, J., Berndt, A., 2008. Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chicken. Vaccine 26, 5423–5433. Chappell, L., Kaiser, P., Barrow, P., Jones, M.A., Johnston, C., Wigley, P., 2009. The immunobiology of avian systemic salmonellosis. Veterinary Immunology and Immuno-pathology 128, 53–59. Clavijo, R.I., Loui, C., Andersen, G.L., Riley, L.W., Lu, S., 2006. Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumen. Applied and Environmental Microbiology 72, 1055–1064. Crouch, S.P., Kozlowski, R., Slater, K.J., Fletcher, J., 1993. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods 160, 81–88. Dhanoa, A., Fatt, Q.K., 2009. Non-typhoidal Salmonella bacteraemia: epidemiology, clinical characteristics and its’ association with severe immunosuppression. Annals of Clinical Microbiology Antimicrobials 8, 15. Dozois, C.M., Daigle, F., Curtiss, R.I.I.I., 2003. Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proceedings of the National Academic of Sciences of the United States of America 100, 247–252. Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Gast, R., Humphrey, T.J., Van Immerseel, F., 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews 33, 718–738. Ghany, M.A.E., Jansen, A., Clare, S., Hall, L., Pickard, D., Kingsley, R.A., Dougan, G., 2007. Candidate live, attenuated Salmonella enterica serotype Typhimurium vaccines with reduced fecal shedding are immunogenic and effective oral vaccines. Infection and Immunity 75, 1835–1842. Gogal Jr., R.M., Ahmed, S.A., Larsen, C.T., 1997. Analysis of avian lymphocyte proliferation by a new, simple, nonradioactive assay (lympho-pro). Avian Diseases 41, 714–725. Hahn, I., 2000. A contribution to consumer protection: TAD Salmonella vac1 E – a new live vaccine for chickens against Salmonella Enteritidis. Lohmann Information 23, 29–32.

603

Humphreys, S., Rowley, G., Stevenson, A., Anjum, M.F., Woodward, M.J., Gilbert, S., Kormanec, J., Roberts, M., 2004. Role of the two-component regulator CpxAR in the virulence of Salmonella enterica serotype Typhimurium. Infection and Immunity 72, 4654–4661. Kang, H.Y., Srinivasan, J., Curtiss, R.I.I.I., 2002. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine. Infection and Immunity 70, 1739–1749. Kim, S.W., Moon, K.H., Baik, H.S., Kang, H.Y., Kim, S.K., Bahk, J.D., Hur, J., Lee, J.H., 2009. Changes of physiological and biochemical properties of Salmonella enterica serovar Typhimurium by deletion of cpxR and lon genes using allelic exchange method. Journal of Microbiological Methods 79, 314–320. Ledeboer, N.A., Jones, B.D., 2005. Exopolysaccharide sugars contribute to biofilm formation by Salmonella enterica serovar Typhimurium on HEp-2 cells and chicken intestinal epithelium. Journal of Bacteriology 187, 3214–3226. Linde, K., Hahn, I., Vielitz, E., 1997. Development of live Salmonella vaccines optimally attenuated for chickens. Lohmann Information 20, 23–31. Mastroeni, P., Chabalgoity, J.A., Dunstan, S.J., Maskell, D.J., Dougan, G., 2001. Salmonella: immune responses and vaccines. Veterinary Journal 161, 132– 164. Mastroeni, P., Villarreal-Ramos, B., Hormaeche, C.E., 1993. Adoptive transfer of immunity to oral challenge with virulent Salmonella in innately susceptible BALB/c mice requires both immune serum and T cells. Infection and Immunity 61, 3981–3984. Matheson, N., Kingsley, R.A., Sturgess, K., Aliyu, S.H., Wain, J., Dougan, G., Cooke, F.J., 2010. Ten years experience of Salmonella infections in Cambridge, UK. Journal of Infection 60, 21–25. Matsui, H., Suzuki, M., Isshiki, Y., Kodama, C., Eguchi, M., Kikuchi, Y., Motokawa, K., Takaya, A., Tomoyasu, T., Yamamoto, T., 2003. Oral immunization with ATPdependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar Typhimurium. Infection and Immunity 71, 30–39. Meyer, H., Koch, H., Methner, U., Steinbach, G., 1993. Vaccines in salmonellosis control in animals. Zentralblatt Bakteriologie 278, 407–415. Müller, K.H., Trust, T.J., Kay, W.W., 1989. Fimbriation genes of Salmonella Enteritidis. Journal of Bacteriology 171, 4648–4654. Rana, N., Kulshreshtha, R.C., 2006. Cell-mediated and humoral immune responses to a virulent plasmid-cured mutant strain of Salmonella enterica serotype Gallinarum in broiler chickens. Veterinary Microbiology 115, 156–162. Robitaille, G., Moineau, S., St-Gelais, D., Vadeboncoeur, C., Britten, M., 2006. Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes. Journal of Dairy Science 89, 4156–4162. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning, a laboratory manual, in: Cold Spring Harbor Laboratory. Cold Spring Harbor, New York, Second ed.. Schmidt, R., Decatur, A.L., Rather, P.N., Moran Jr., C.P., Losick, R., 1994. Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G. Journal of Bacteriology 176, 6528–6537. Schoemaker, J.M., Gayda, R.C., Markovitz, A., 1984. Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death. Journal of Bacteriology 158, 551–561. Springer, S., Lehmann, J., Lindner, T., Thielebein, J., Alber, G., Selbitz, H.J., 2000. A new live Salmonella Enteritidis vaccine for chicken-experimental evidence of its safety and efficacy. Berliner und Münchener tierärztliche Wochenschrift 113, 246–252. Takaya, A., Kubota, Y., Isogai, E., Yamamoto, T., 2005. Degradation of the HilC and HilD regulator proteins by ATP dependent Lon protease leads to downregulation of Salmonella pathogenicity island 1 gene expression. Molecular Microbiology 55, 839–852. Takaya, A., Suzuki, M., Matsui, H., Tomoyasu, T., Sashinami, H., Nakane, A., Yamamoto, T., 2003. Lon, a stress-induced ATP-dependent protease, is critically important for systemic Salmonella enterica serovar Typhimurium infection of mice. Infection and Immunity 71, 690–696. Takaya, A., Tomoyasu, T., Tokumitsu, A., Morioka, M., Yamamoto, T., 2002. The ATPdependent lon protease of Salmonella enterica serovar Typhimurium regulates invasion and expression of genes carried on Salmonella pathogenicity island 1. Journal of Bacteriology 184, 224–232. Tan, S., Gyles, C.L., Wilkie, B.N., 1997. Evaluation of an aroA mutant Salmonella Typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor fecal shedding. Veterinary Microbiology 54, 247–254. Torres-Cabassa, A.S., Gottesman, S., 1987. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. Journal of Bacteriology 169, 981–989. Vielitz, E., Conrad, C., Voss, M., Löhren, U., Bachmeier, J., Hahn, I., 1992. Immunization against Salmonella-infections using live and inactivated vaccine preparations. Deutsche Tierärztliche Wochenschrift 99, 483–485. Wolfe, A.J., Parikh, N., Lima, B.P., Zemaitaitis, B., 2008. Signal integration by the twocompetent signal transduction response regulator CpxR. Journal of Bacteriology 190, 2314–2322. Wright, R., Stephens, C., Zweiger, G., Shapiro, L., Alley, M.R., 1996. Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. Genes and Development 10, 1532–1542. Xie, D., Wang, Z.X., Dong, Y.L., Cao, J., Wang, J.F., Chen, J.L., Chen, Y.X., 2008. Effects of monochromatic light on immune response of broilers. Poultry Science 87, 1535–1539.