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Evaluation of nanoparticle-encapsulated outer membrane proteins for the control of Campylobacter jejuni colonization in chickens
Evaluation of nanoparticle-encapsulated outer membrane proteins for the control of Campylobacter jejuni colonization in chickens T. Annamalai,1 R. Pina-Mimbela,1 A. Kumar, B. Binjawadagi, Z. Liu, G. J. Renukaradhya, and G. Rajashekara2 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Wooster 44691 ABSTRACT Numerous vaccination strategies have been evaluated to develop effective vaccines against Campylobacter jejuni colonization in poultry but with limited success. The following experiments were conducted to investigate the effect of biodegradable and biocompatible poly (lactide-co-glycolide) nanoparticle (NP) encapsulated outer membrane proteins (OMP) of C. jejuni. Chickens were vaccinated with different routes [subcutaneous (s/c) or oral] and doses (25, 125, or 250 μg) of candidate nanoparticle vaccine with appropriate control groups. Serum and cloacal fecal samples were taken at regular intervals of time, and the birds were euthanized 7 d postchallenge with C. jejuni. The results were interpreted based on anti-OMP immunoglobulin response in chicken and intestinal colonization of C. jejuni. The C. jejuni colonization in cecal and cloacal
contents at 7 d postchallenge was below the detection limit in the s/c vaccinated groups, but the other groups demonstrated varying degrees of colonization. The serum IgA was higher in the group vaccinated s/c with OMP only compared with the rest of the groups. The serum- and fecal-IgY titers were consistently higher in the s/c vaccinated groups (with or without NP) than the rest of the groups. Elevated levels of OMP specific serum antibodies correlated with below the limit of detection levels of Campylobacter colonization in broiler chickens receiving 125 μg of OMP alone and the OMP+NP vaccine s/c. In conclusion, the s/c route of vaccination with or without NP encapsulated OMP of C. jejuni may serve as a candidate vaccine for control of C. jejuni colonization in chickens.
Key words: Campylobacter jejuni, nanoparticle, vaccine, outer membrane protein, food safety 2013 Poultry Science 92:2201–2211 http://dx.doi.org/10.3382/ps.2012-03004
INTRODUCTION Campylobacter jejuni is a normal intestinal inhabitant of food-producing animals including livestock, domestic birds, and other warm-blooded animals. Frequently, humans become infected with C. jejuni (infectious dose ≥500 bacterial cells) by consumption of contaminated meat, especially poultry (Black et al., 1988). Gastroenteritis is the typical manifestation of C. jejuni infections in humans and is often self-limiting. Infrequently, the convalescent patient may have fatal neurological conditions such as Guillain-Barre syndrome, Miller Fisher syndrome, and reactive arthritis (van Koningsveld et al., 2001). In chickens, C. jejuni harmlessly colonizes, especially in the lower intestinal tract (van de Giessen et al., 1992; ©2013 Poultry Science Association Inc. Received December 28, 2012. Accepted April 14, 2013. 1 These authors contributed equally to the work. 2 Corresponding author: [email protected]
Van Deun et al., 2008), and often chicken carcasses become contaminated from intestinal contents at slaughterhouses (Rice et al., 1997). Broiler chickens are a potential reservoir of the Campylobacter strains pathogenic to humans and can carry Campylobacter as high as 106 to 108 cfu/g of feces and remained colonized until slaughter (Beery et al., 1988; Jacobs-Reitsma et al., 1995; Evans and Sayers, 2000). As a control measure, various methods to reduce Campylobacter colonization in the chicken intestine have been proposed, including biosecurity measures to prevent spread of infection among chickens, increase host resistance by competitive exclusion in the gut, vaccination, or genetic selection of chickens, and finally, by use of antimicrobial compounds (Lin, 2009). The strategies to reduce Campylobacter colonization in chickens are more valued because the intestine of living poultry is a major amplification site for Campylobacter in the food chain. Therefore, reducing the fecal Campylobacter load in poultry during primary production is hypothesized to significantly reduce the incidence of human campylobacteriosis (Lin, 2009).
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Although, the above-mentioned control measures undoubtedly will help to decrease shedding of Campylobacter by chickens and may reduce the number of positive flocks, vaccination of poultry against Campylobacter will probably be most effective though this remains a major challenge. Outer membrane proteins (OMP) have been exploited for vaccine development because they are easily recognized by the immune system and have been demonstrated as promising candidates for vaccine against gram-negative bacteria (Stern et al., 1990; Lin et al., 2002a; Shoaf-Sweeney et al., 2008). Immunogenic OMP identified in Campylobacter include flagellum (Fla; Guerry, 1997), major outer membrane protein (major OMP; Zhang et al., 2000), cell-binding factor (Peb1; Pei and Blaser, 1993), multidrug efflux pump component (CmeC; Lin et al., 2002b, 2003; Lin et al., 2005), and ferric enterobactin receptor (CfrA; Zeng et al., 2008). In broiler chickens, Campylobacter infection leads to only a mild stimulation of immune system and colonization most likely involves immune tolerance (Hermans et al., 2012). Hence, we hypothesized that the use of C. jejuni crude OMP lysate that may strongly stimulate immune response in chicken could be an attractive vaccine candidate to prevent/ reduce shedding of C. jejuni from the intestinal tract of chickens. To effectively deliver OMP at the site of interest, we encapsulated OMP in the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP). The PLGA NP have been shown to protect encapsulated contents from early degradation and ensure controlled and effective sustained release of the active components. Therefore, the aim of this study was to investigate NP encapsulated OMP as a potential vaccine candidate against C. jejuni colonization of the chicken intestine.
MATERIALS AND METHODS Culture Growth The highly invasive strain C. jejuni 81–176 isolated from an outbreak associated with raw milk was used in the present study (Korlath et al., 1985). Campylobacter jejuni 81–176 strain was grown on Mueller-Hinton agar (MH; Becton Dickinson and Company, Sparks, MD) and incubated for 18 to 24 h at 42°C under microaerobic conditions (85% N2, 10% CO2, and 5% O2) in a DG250 Microaerophilic Workstation (Microbiology International, Frederick, MD).
Preparation of OMP of C. jejuni 81–176 The outer membrane proteins were extracted as described previously (Blaser et al., 1983) with a few modifications. Briefly, the cells grown on MH agar were harvested in MH broth and centrifuged at 6,000 × g for 15 min at 4°C. The pellet was resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride (Pefabloc SC, Boehringer, Mannheim, Germany) as protease in-
hibitor. The cells in suspension were sonicated 15 times with each sonication for 20 s duration and 30 s cooling between the sonications in a sonicator (Vibra-Cell, Sonics and Materials Inc., Newtown, CT) with setting of 3 pulser and 4 amplitude. The sonicated materials were centrifuged at 6,000 × g for 10 min at 4°C, and the supernatant was centrifuged again for at 10,000 × g for 2 h at 4°C and the pellet was freeze-dried and stored. The protein concentration was estimated using Bio-Protein Assay dye reagent kit (Bio-Rad, Hercules, CA) and 5 µg of OMP lysate was resolved on 12% SDS-PAGE and stained with Coomassie blue (Thermo Fisher Scientific, Rockford, IL) to check the quality of the extracted OMP.
Entrapment of OMP in PLGA NP The OMP entrapped PLGA NP were prepared by double-emulsion solvent evaporation method (waterin-oil-in-water, w/o/w) as described previously (Rajapaksa et al., 2010) with a few modifications. Briefly, 5% PLGA (85:15) polymer solution was prepared by dissolving 0.25 g of PLGA in 5 mL of methylene chloride/dichloromethane. Five milligrams of outer membrane protein suspended in 0.5-mL PBS was mixed with 0.25-mL of 2% polyvinyl alcohol solution prepared in 10 mM HEPES buffer (pH 7.5); then 0.05 mL of 20% sucrose and 0.05 mL of 20% magnesium hydroxide were added to the PLGA polymer solution and emulsified using probe sonication for 30 s (Branson-Sonifier 450, Ringwood, NJ) with a duty cycle of 30% and output control of 3. For the ease of sonication, the resulting emulsion (water-in-oil) was divided into 2 tubes and 11.5 mL of 2% polyvinyl alcohol and 1 mL of 12.5% Poloxamer 188 solution were added to each tube and emulsified by sonication for 30 s twice to obtain the final w/o/w emulsion. Contents of both the tubes were combined in a 100-mL beaker and stirred for 20 h at 4°C with a magnetic stirrer at 400 rpm to allow solvent evaporation. The NP were collected by centrifugation at 11,000 × g for 30 min at 4°C (FX6100 rotor, Beckman-Coulter, Brea, CA) for 30 min and resuspended in 45 mL of sterile distilled water; the washing step was repeated 3 times with sterile distilled water by repeated centrifugation and resuspension. The pellet of entrapped NP was finally suspended in 5 mL of 5% sucrose solution, freeze-dried for 16 to 18 h, and then stored at −20°C. Determination of OMP Entrapment Efficiency. The OMP entrapment in the PLGA NP was estimated using a bicinchoninic acid protein assay kit. Approximately 5 mg of freeze-dried NP was added to a 1-mL lysis buffer (5% SDS in 0.1 M NaOH) and incubated with gentle shaking for 1 h at 37°C. The solution was centrifuged at 11,000 × g for 5 min at 4°C, and the supernatant was collected and protein content was estimated using a bicinchoninic acid protein assay kit (Bio-Rad). The protein entrapment efficiency (wt/wt)
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was expressed as the amount of protein entrapped (in percentage) relative to the amount of protein used for entrapment. Scanning Electron Microscopy. The morphology of the protein-loaded NP was visualized using scanning electron microscopy. Briefly, the NP were placed on a double-sided adhesive tape attached to an aluminum stub and sputter-coated with gold/palladium beam for 2 min. The coated sample was imaged using Philips XL30-FEG scanning electron microscopy (Philips, Eindhoven, the Netherlands) at the Molecular and Cellular Imaging Center [Ohio Agricultural Research and Development Center (OARDC), Wooster, OH].
Vaccination and Challenge of Chickens One-day-old chickens were obtained from a local hatching facility at the Food Animal Health Research Program (OARDC, Wooster, OH). All chickens were confirmed to be Campylobacter negative by culturing cloacal swabs before vaccination. Animal experiments were performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. A total of 80 one-week-old chicks were divided randomly into 10 groups with 8 chicks in each group, and the chicks were vaccinated on d 7 followed by a booster vaccination at d 21. The vaccination schedule and the different treatment groups are listed in Table 1. Chickens were immunized either orally or subcutaneously (s/c) with OMP alone or with NP encapsulated OMP reconstituted in PBS. Because our main purpose was to optimize an oral vaccine for C. jejuni, only one dose (125 μg), representing a dose level between the high (250 μg) and low (25 μg) dose used for oral inoculation, was used for s/c administration. Because purified proteins may undergo degradation in the gastrointestinal tract and possibly fail to induce any antibody response when administered orally, the s/c injection also served as a quality control for our approach. The birds in all groups were orally challenged with 1 × 108 cfu/mL of C. jejuni 81–176 in 0.2-mL of PBS 14 d after booster vaccination and the control group received only 0.2 mL of PBS.
Collection and Processing of Chicken Samples The blood samples were collected and the sera were separated on the day of vaccine (d 7), 21, 35, and 42 d, and stored at −20°C until further use. Cloacal fecal samples were taken on d 0, 7, 14, 21, 28, 35, and 42, and in addition, cecal contents were taken on d 42 following euthanasia. The cloacal fecal samples from d 35 (day of challenge) were pooled for each treatment group. The cloacal fecal samples taken on other days were not pooled. For antibody response, cloacal fecal samples were suspended in 1 mL of PBS, suspension was centrifuged at 3,000 × g for 5 min at 4°C, and supernatants were stored at −20°C until further used for antibody detection. For cfu determination, the cecal and cloacal samples were weighed (weight range: ceca, 2 to 3 g; cloacal samples 1 to 2 g) and suspended in 3 and 1 mL of buffered peptone water, respectively, and homogenized. One hundred microliters of the homogenized suspension was serially (10-fold) diluted in buffered peptone water and plated on MH agar containing Campylobacter selective supplement (SR117E, Oxoid, Hampshire, UK) and incubated microaerobically at 42°C for 48 h. The Campylobacter colonies were counted and representative colonies were confirmed by PCR using Campylobacter genus specific 16s rRNA primers (Sanad et al., 2011). The cfu per gram of fecal sample was calculated considering respective weights of samples.
ELISA The ELISA was performed as described previously (Cawthraw et al., 1994), with few modifications. Briefly, to determine the level of C. jejuni specific IgY and IgA antibodies in serum and pooled fecal samples from different groups, ELISA plates (Nunc Maxisorp, Rochester, NY) were coated overnight at 4°C with 50 µL of pretitrated amounts of OMP (4 µg/mL for IgY; 6 µg/ mL for IgA) diluted in coating buffer (0.05 M carbonate buffer, pH 9.6). The plates were washed in PBS containing 0.1% Tween 20 (PBS-T), and 200 µL of
Table 1. Vaccination details for different treatment groups used in the study1 Treatment
n
Route of vaccination/volume (mL)
125 µg of OMP + NP 125 µg of OMP only NP only PBS only (control) 125 µg of OMP + NP 125 µg of OMP only 25 µg of OMP + NP 250 µg of OMP + NP 25 µg of OMP only 250 µg of OMP only
8 8 7 8 8 8 8 8 8 8
Oral/0.5 Oral/0.5 Oral/0.5 Oral/0.5 Subcutaneous/0.5 Subcutaneous/0.5 Oral/0.5 Oral/0.5 Oral/0.5 Oral/0.5
1OMP
Age at first vaccine (d)/booster (d)
= outer membrane protein of Campylobacter jejuni; NP = nanoparticle; n = sample size.
7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21
Age at challenge (d)/status (yes/no) 35/yes 35/yes 35/yes 35/no 35/yes 35/yes 35/yes 35/yes 35/yes 35/yes
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Figure 1. Scanning electron microscopy image of outer membrane protein (OMP) of Campylobacter jejuni entrapped in poly(lactide-coglycolide) (PLGA) nanoparticles (NP). Nanoparticles were placed on a double-sided adhesive tape attached to an aluminum stub and sputtercoated with gold/palladium beam for 2 min. The coated sample was imaged using a Philips XL30-FEG scanning electron microscope. a and b: Representative images at resolution levels 5,000× and 13,000×, respectively, of selected field of OMP entrapped in PLGA NP.
blocking buffer (5% skim milk suspension in PBS-T) was added to the wells and incubated at 37°C for 2 h. The plates were subsequently washed in PBS-T, and the test samples were diluted in blocking solution and then 50 µL was added to each well in duplicate and incubated at 37°C for 2 h. Serum samples were diluted 1:10 for IgA assay and in 4-fold dilutions starting from 1 in 64 for the IgY assay. Fecal supernatants were diluted 1:2 for both IgY and IgA assays. After 2 h of incubation, the plates were washed in PBS-T and incubated for 30 min at 37°C in 50 µL of peroxidase conjugated anti-IgA (1: 2,500) or anti-IgY (1:20,000) antibodies (Novus Biologicals, Littleton, CO) diluted in blocking solution. The plates were then washed in PBS-T and 50 µL of peroxidase substrate solution (TMB) was added to the wells (KPL, Gaithersburg, MD). The reaction was stopped with 1 N HCl solution after 10 min of color development. The plates were read at 450 nm using a Spectramax 340PC microplate reader (Molecular Devices, Sunnyvale, CA). Serum OMP-specific IgY antibody responses were measured by titrating serum samples by serial dilution, and antibody titers were determined using a cutoff of mean + 3 SD of the negative control group. Higher magnitude of serum IgY antibody responses allowed us to use the titration method. However, due to low magnitude of responses, the IgA titers were presented as OD values.
Statistical Analysis The ELISA results were analyzed with the JMP software (JMP, SAS Institute Inc., Cary, NC) using 1-way ANOVA followed by Tukey’s honestly significant difference test. For the chicken experiment, because sufficient replicates (n = 8) in each group were used to obtain statistically accurate data, the experiment was not repeated. Counts of Campylobacter cfu per gram were presented as means ± SE. The mean was determined by averaging the cfu counts from samples (n = 7 or 8);
for samples with below the level of detection of Campylobacter, detection limit was used as the minimum value. Data were log10 transformed and analyzed using 2-way ANOVA (GraphPad Prism v.5 Software Inc., La Jolla, CA). Correlation coefficient between antibody levels and cfu per gram were performed using GraphPad Prism v.5 software. A P-value of <0.05 was used to indicate statistically significant differences.
RESULTS Entrapment of OMP in PLGA NP Using the scanning electron microscopy, size of the OMP entrapped NP was determined and found to be ranging from 200 to 600 nm in diameter (Figure 1). The entrapment efficiency of OMP in PLGA NP was approximately 54%.
Campylobacter cfu Enumeration in Cecal and Cloacal Fecal Samples The vaccine trials were designed to check how effectively potential candidate vaccines under trial could reduce C. jejuni colonization (cfu/g) in intestinal contents when infected experimentally. Hence, we enumerated the cfu from cecal and cloacal contents from different vaccine trial groups. Our result showed that the groups were significantly different in the number of cfu per gram of tissues. The groups receiving the s/c vaccine had significantly lower cfu (below the detection limit) in cecal contents than all the groups receiving the oral vaccine (P < 0.05). The cfu per gram of cecal content was below the detection limit (<10 cfu) in the s/c groups that received either encapsulated OMP or OMP alone (Figure 2). The cecal cfu of the group given NP only orally was significantly higher than the other orally vaccinated groups except the group given 125 µg of OMP encapsulated in NP. The percentage of
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Figure 2. Campylobacter cfu per gram in cecal and cloacal fecal samples. The birds were orally challenged with 1 × 108 cfu of Campylobacter jejuni at 28 d post-first vaccination. The cecal contents and cloacal fecal samples were taken 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) indicate the groups differ significantly within sample from each organ (P < 0.05). OMP = outer membrane proteins; NP = nanoparticle; s/c = subcutaneous.
birds positive for Campylobacter in the cecal contents were also zero in groups vaccinated s/c with the encapsulated OMP or OMP alone (Table 2). The orally vaccinated groups showed presence of C. jejuni in the cecal contents: encapsulated OMP (88%), OMP alone (63%), and the group given NP alone (57%). The groups that received 25 or 250 µg of OMP alone were 67 and 50% positive for Campylobacter, respectively, in cecal contents. The groups that received 25 or 250 µg of encapsulated OMP were 63 and 38% positive for Campylobacter, respectively, in cecal contents. The group vaccinated with 125 µg of encapsulated OMP s/c had the lowest cfu in the cloacal fecal sample (below the detection limit) and was significantly dif-
ferent from all the other challenged groups. The group vaccinated with 125 µg OMP alone s/c was also different from the other orally vaccinated groups. The group given 250 µg OMP alone orally did not differ from the other orally vaccinated groups. The percentage of birds’ cloacal samples positive for C. jejuni was different in the oral groups and s/c groups. Sixty-three percent of chickens in groups vaccinated orally with 125 μg of OMP with or without encapsulation and 86% of chickens in group given NP alone were positive for C. jejuni. The cloacal samples from the group vaccinated s/c with encapsulated OMP (125 μg) had no detectable Campylobacter, whereas only 13% was positive in groups given OMP alone s/c. The groups that received
Table 2. Cecal and cloacal samples positive for Campylobacter expressed in percentage (n = 7 or 8 chicks/group)1 Cecum OMP + NP Amount of OMP (µg) 25 125 250
Cloaca OMP alone
s/c
Oral
s/c
Oral
NT BDL NT
63 88 38
NT BDL NT
67 63 50
OMP + NP
OMP alone
s/c
Oral
s/c
Oral
NT BDL NT
50 63 50
NT 13 NT
67 63 50
1OMP = outer membrane protein of Campylobacter jejuni; NP = nanoparticle; s/c = subcutaneous; BDL = below detection limit; NT = 25- and 250-µg doses were not tested by s/c route.
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25 or 250 µg of OMP alone were 67 and 50% positive for Campylobacter, respectively, in cloacal contents. The groups that received 25 or 250 µg of encapsulated OMP were both 50% positive for Campylobacter in cloacal contents (Table 2). The control group cecal and cloacal samples were negative for the Campylobacter. Because oral vaccination of birds with OMP (with and without encapsulation) using a maximum dose of 250 µg per bird did not induce any protection against C. jejuni colonization, only the serum and fecal samples from birds in groups vaccinated 125 μg (orally or s/c) were analyzed for antibody response.
ELISA Results Serum IgA. The serum optical density (OD) values due to secretory IgA response in the groups differed significantly at 14 d postvaccination (dpv; 21 d of age, P < 0.0001, Figure 3). The secretory IgA response of groups vaccinated with OMP by the s/c route with or without encapsulation was higher than the rest of the groups. However, the group vaccinated s/c with encapsulated OMP was different from the group vaccinated s/c with OMP alone. The rest of the groups’ serum OD values did not differ from one another. At 28 dpv (day of challenge, 35 d of age), the serum OD values of the group receiving OMP only by the s/c route had a higher OD value than the rest of the groups (P < 0.0001). The other groups did not differ from one another. At 7 d postchallenge (dpc, 42 d of age), the OD values of different groups did not differ from one another. Sig-
nificant negative correlation (r = −0.50, P < 0.0001) was observed between serum IgA antibody titers and cloacal cfu levels. Serum IgY. The serum IgY titers of the groups differed significantly at 14 dpv (21 d of age, P < 0.0001, Figure 4). The titers of groups vaccinated s/c with OMP or encapsulated OMP were higher than the rest of the groups. However, the groups s/c vaccinated with encapsulated OMP and OMP alone did not differ. The groups vaccinated with encapsulated OMP orally had higher titers than the control and lower titers than the s/c groups but did not differ from the groups given OMP or NP alone orally. At 28 dpv (day of challenge, 35 d of age), the serum titers of the different groups differed significantly (P < 0.0001). The differences between groups were similar to 14 dpv, and the s/c groups had higher titers than the rest of the groups. At 7 dpc (42 d of age), the serum titers of the groups were significantly different (P < 0.0001). The serum titers of s/c groups with encapsulated OMP or OMP alone were higher than the rest of the groups. However, the groups vaccinated s/c with encapsulated OMP or OMP alone did not differ. In addition, a significant negative correlation (r = −0.58, P < 0.001) was observed between serum IgY antibody titers and cloacal cfu levels. The oral groups given encapsulated OMP or OMP/NP alone did not differ from the control group. Fecal IgA. The OD values of the different groups at 7 dpv (14 d of age) differed significantly (<0.0001; Figure 5). The s/c group with encapsulated OMP had higher OD than the group administered encapsulated
Figure 3. Serum IgA in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The serum samples were taken at 14 and 28 d postvaccination and 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
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Figure 4. Serum IgY titers in birds vaccinated subcutaneously (s/c) and orally with outer membrane protein (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The serum samples were collected at 14 and 28 d postvaccination and 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05).
OMP orally, and both groups had higher OD than the rest of the groups; the OD values of the rest of the groups did not differ. At 14 dpv (21 d of age), the s/c group given OMP alone had higher OD than the rest of the groups (P < 0.001), but the rest of the groups did not differ from each other. At 21 dpv (28 d of age), the groups differed from each other significantly (P < 0.001). The s/c group given encapsulated OMP had higher OD than the rest of the groups. The oral groups administered with encapsulated OMP or NP alone had significantly lower OD values than the rest of the groups. The groups given OMP s/c or orally did not differ significantly from the control group. At 7 dpc (42 d of age), the OD values of the groups differed significantly (P < 0.01). The s/c group with OMP alone had higher OD than the control group and the groups administered encapsulated OMP or OMP alone orally. However, the s/c group given OMP alone did not differ significantly from the group given NP alone orally. A significant negative correlation coefficient (r = −0.56, P = 0.0002) was observed between fecal IgA antibody titers and cloacal cfu levels. Fecal IgY. At 7 dpv (14 d of age), the s/c group with encapsulated OMP had a significantly higher OD value than the rest of the groups (P < 0.001; Figure 6). At 14 dpv (21 d of age), there was no significant differ-
ence between the groups. At 21 dpv (28 d of age), the group given OMP alone s/c differed from the rest of the groups (P < 0.01). At 7 dpc (42 d of age), the OD values of the groups differed significantly (P < 0.0001). The s/c group given OMP alone had higher OD value than the s/c group given encapsulated OMP, and both these groups had higher OD values than the rest of the groups.
DISCUSSION The commensal nature of C. jejuni in avian species is still a debated topic, but it is beyond doubt that the experimental C. jejuni infection triggers both systemic and mucosal immune responses in chickens (de Zoete et al., 2007). Moreover, the elevated levels of Campylobacter-specific serum antibodies correlated with the reduced colonization level of Campylobacter, suggesting a protective role of antibodies in Campylobacter infection in chickens. The high levels of Campylobacter maternal antibodies in younger chickens may partly contribute to the lack of Campylobacter infection in young chickens in natural environments during the first 2 wk of life, which was also supported by experimental challenge studies (Sahin et al., 2001, 2003b). Our findings on the protective nature of Campylobacter-specific antibodies further
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Figure 5. Fecal IgA in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The fecal samples were taken at 7-d intervals postvaccination and 7 d postchallenge. The cloacal fecal samples within each treatment group were pooled for 28 d postvaccination (35 d of age) collection and have no error bars. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
Figure 6. Fecal IgY in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The fecal samples were taken at 7-d intervals postvaccination and 7 d postchallenge. The cloacal fecal samples within each treatment group were pooled for 28 d postvaccination (35 d of age) collection and have no error bars. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
CAMPYLOBACTER JEJUNI OUTER MEMBRANE PROTEIN VACCINE
support the practicability of developing immunizationbased approaches to curtail Campylobacter infection in chickens. To date, there is no vaccine available to control Campylobacter colonization in poultry. In previous research studies, attempts were made to develop an effective vaccine against C. jejuni using immunogenic flagellar proteins administered intranasally (Lee et al., 1999), formalin inactivated C. jejuni administered orally (Rice et al., 1997), and inner membrane antigen of C. jejuni with Salmonella carrier administered orally (Wyszyńska et al., 2004, 2008). Widders et al. (1996) demonstrated that the intraperitoneal route of vaccination with killed C. jejuni was more effective than the oral or s/c route in reducing Campylobacter intestinal colonization in chickens. In addition, Noor et al. (1995) reported that an in ovo vaccine was capable of eliciting early antibody response in chicken. However, none of these vaccine trials were successful in completely preventing the Campylobacter infection in chickens. Here we designed a vaccine trial using potent immunogenic OMP of C. jejuni entrapped in NP and administered through oral and s/c routes (Islam et al., 2010, Zeng et al., 2010). To increase the mucosal immune response, encapsulation of the OMP in NP was attempted in this study. Previous studies have demonstrated that vaccine particles encapsulated in PLGA NP are efficient in eliciting potent immune response (Sarti et al., 2011; Dwivedi et al., 2012). The NP are used in mucosal vaccines because mucosal candidate vaccines on their own are not very effective due to degradation in the gastrointestinal tract. The NP-encased vaccines efficiently elicit immune response if administered by the s/c, intranasal, or oral route (Dwivedi et al., 2012). The PLGA NP are readily taken up by the Peyer’s patches in the ileum and can stimulate antigen presenting cells and subsequently T cells (Igartua et al., 1998; Chong et al., 2005; Waeckerle-Men and Groettrup, 2005). The NP-encased vaccines administered orally in mice induced gut-specific IgA response as well as IgG response (Sarti et al., 2011). Intranasal administration of Chitosan-DNA NP carrying flagellar protein genes of C. jejuni in chickens increased the immune response, but was not very effective in reducing C. jejuni infection (Huang et al., 2010). The present study highlighted the importance of administration of the OMP in controlling intestinal colonization of C. jejuni, and it correlated with the induction of Campylobacter-specific immunoglobulins when delivered s/c but not orally. Oral vaccines that contain only protein may not be effective due to protein degradation in the gut (Garinot et al., 2007). Therefore, attempts have been made to encapsulate the proteins in NP so that they can be released slowly and taken up by the intestinal microfold cells, resulting in induction of effector and memory immune responses (Brayden et al., 2005; des Rieux et al., 2006). Therefore, we encapsulated the OMP in PLGA NP, but this approach failed to elicit the desired immunity when the vaccine
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was delivered by the oral route. Previous studies with mucosal vaccines against Campylobacter have shown mixed results in eliciting mucosal antibody production (Huang et al., 2010; Zeng et al., 2010). However, NP encapsulated OMP vaccine delivered by the s/c route resulted in higher IgY and IgA titers in cloacal feces. The group given encapsulated OMP s/c had higher and earlier fecal IgA response (7 and 14 dpv) and postchallenge. The fecal IgY was higher in both the s/c groups (encapsulated and nonencapsulated OMP). The serum IgA and IgY responses also appeared earlier and at higher levels in the s/c groups. The fecal and cecal C. jejuni cultures were negative (below the detection limit) for the group injected s/c with encapsulated OMP, whereas other vaccinated groups were positive for C. jejuni (Figure 2, Table 2). Therefore, incorporation of Campylobacter immunogenic antigens in a biodegradable NP administered parenterally may have promise in providing better protection from C. jejuni colonization in chickens. Control of Campylobacter infection would reduce the likelihood of contamination of chicken carcasses with Campylobacter during processing and transportation thus will have a significant impact on zoonosis. Our data suggest that the s/c route of vaccination with encapsulated OMP or OMP alone is highly effective in inducing protective antibody responses and preventing C. jejuni colonization in the chicken. The s/c vaccination with OMP provided significant protection against Campylobacter infection by the virtue of higher IgY and IgA responses in both serum and feces. These results point toward superiority of the s/c vaccine over oral vaccine in the control of intestinal infection with Campylobacter. Although the s/c administered vaccines may not be practical for large-scale application in broilers, the vaccine can still be of use in layer and breeder hens. Campylobacter can persist in laying hens for a prolonged period; therefore, if layers/breeders are free of the organism, the possibility of zoonotic threats from the organism transmitted through contaminated eggs or environment or management practices can be minimized. Although, vertical egg transmission of the organism is extremely rare (Sahin et al., 2003a), C. jejuni has been isolated from reproductive organs of chickens (Camarda et al., 2000) and chickens can be infected by ingestion of the bacteria from contaminated shells (Cox et al., 2012). Reducing the Campylobacter colonization in the layer and breeder hen population, therefore, will reduce the environmental contamination and possibility of cross contamination of eggs, thereby minimizing the risk of ingestion of the organism by chicks and subsequent colonization. In summary, our findings suggest the usefulness of NP-based delivery of C. jejuni antigens to confer protection against C. jejuni colonization in chickens; however, further research is needed to optimize the oral delivery of modified NP-based vaccines for cost-effective and practical use in the current broiler production system.
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ACKNOWLEDGMENTS We thank Juliette Hanson and Kingsly Berlin (both from Food Animal Health Research Program, Wooster OH) for their technical assistance with chicken experiment. G. Rajashekara’s laboratory is supported by the OARDC, The Ohio State University, and the Agriculture and Food Research Initiative grant #2012-6800319679, USDA.
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CAMPYLOBACTER JEJUNI OUTER MEMBRANE PROTEIN VACCINE ing the immunostimulant monophosphoryl lipid A. Biomaterials 32:4052–4057. Shoaf-Sweeney, K. D., C. L. Larson, X. Tang, and M. E. Konkel. 2008. Identification of Campylobacter jejuni proteins recognized by maternal antibodies of chickens. Appl. Environ. Microbiol. 74:6867–6875. Stern, N. J., R. J. Meinersmann, and H. W. Dickerson. 1990. Influence of antibody treatment of Campylobacter jejuni on the dose required to colonize chicks. Avian Dis. 34:595–601. van de Giessen, A., S. I. Mazurier, W. Jacobs-Reitsma, W. Jansen, P. Berkers, W. Ritmeester, and K. Wernars. 1992. Study on the epidemiology and control of Campylobacter jejuni in poultry broiler flocks. Appl. Environ. Microbiol. 58:1913–1917. Van Deun, K., F. Pasmans, R. Ducatelle, B. Flahou, K. Vissenberg, A. Martel, W. Van den Broeck, F. Van Immerseel, and F. Haesebrouck. 2008. Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut. Vet. Microbiol. 130:285–297. van Koningsveld, R., R. Rico, I. Gerstenbluth, P. I. Schmitz, C. W. Ang, I. S. Merkies, B. C. Jacobs, Y. Halabi, H. P. Endtz, F. G. van der Meche, and P. A. van Doorn. 2001. Gastroenteritisassociated Guillain-Barre syndrome on the Caribbean island Curacao. Neurology 56:1467–1472. Waeckerle-Men, Y., and M. Groettrup. 2005. PLGA microspheres for improved antigen delivery to dendritic cells as cellular vaccines. Adv. Drug Deliv. Rev. 57:475–482.
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Widders, P. R., R. Perry, W. I. Muir, A. J. Husband, and K. A. Long. 1996. Immunisation of chickens to reduce intestinal colonisation with Campylobacter jejuni. Br. Poult. Sci. 37:765–778. Wyszyńska, A., A. Raczko, M. Lis, and E. K. Jagusztyn-Krynicka. 2004. Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 22:1379–1389. Wyszyńska, A., J. Zycka, R. Godlewska, and E. K. JagusztynKrynicka. 2008. The Campylobacter jejuni/coli cjaA (cj0982c) gene encodes an N-glycosylated lipoprotein localized in the inner membrane. Curr. Microbiol. 57:181–188. Zeng, X., F. Xu, and J. Lin. 2008. Ferric enterobactin receptor CfrA is an attractive vaccine candidate against Campylobacter jejuni. Abstract E-014, 108th General Meeting of American Society for Microbiology, Boston, MA. Zeng, X., F. Xu, and J. Lin. 2010. Development and evaluation of CmeC subunit vaccine against Campylobacter jejuni. J Vaccines Vaccin. 1:112. Zhang, Q., J. C. Meitzler, S. Huang, and T. Morishita. 2000. Sequence polymorphism, predicted secondary structures, and surface-exposed conformational epitopes of Campylobacter major outer membrane protein. Infect. Immun. 68:5679–5689.
contents at 7 d postchallenge was below the detection limit in the s/c vaccinated groups, but the other groups demonstrated varying degrees of colonization. The serum IgA was higher in the group vaccinated s/c with OMP only compared with the rest of the groups. The serum- and fecal-IgY titers were consistently higher in the s/c vaccinated groups (with or without NP) than the rest of the groups. Elevated levels of OMP specific serum antibodies correlated with below the limit of detection levels of Campylobacter colonization in broiler chickens receiving 125 μg of OMP alone and the OMP+NP vaccine s/c. In conclusion, the s/c route of vaccination with or without NP encapsulated OMP of C. jejuni may serve as a candidate vaccine for control of C. jejuni colonization in chickens.
Key words: Campylobacter jejuni, nanoparticle, vaccine, outer membrane protein, food safety 2013 Poultry Science 92:2201–2211 http://dx.doi.org/10.3382/ps.2012-03004
INTRODUCTION Campylobacter jejuni is a normal intestinal inhabitant of food-producing animals including livestock, domestic birds, and other warm-blooded animals. Frequently, humans become infected with C. jejuni (infectious dose ≥500 bacterial cells) by consumption of contaminated meat, especially poultry (Black et al., 1988). Gastroenteritis is the typical manifestation of C. jejuni infections in humans and is often self-limiting. Infrequently, the convalescent patient may have fatal neurological conditions such as Guillain-Barre syndrome, Miller Fisher syndrome, and reactive arthritis (van Koningsveld et al., 2001). In chickens, C. jejuni harmlessly colonizes, especially in the lower intestinal tract (van de Giessen et al., 1992; ©2013 Poultry Science Association Inc. Received December 28, 2012. Accepted April 14, 2013. 1 These authors contributed equally to the work. 2 Corresponding author: [email protected]
Van Deun et al., 2008), and often chicken carcasses become contaminated from intestinal contents at slaughterhouses (Rice et al., 1997). Broiler chickens are a potential reservoir of the Campylobacter strains pathogenic to humans and can carry Campylobacter as high as 106 to 108 cfu/g of feces and remained colonized until slaughter (Beery et al., 1988; Jacobs-Reitsma et al., 1995; Evans and Sayers, 2000). As a control measure, various methods to reduce Campylobacter colonization in the chicken intestine have been proposed, including biosecurity measures to prevent spread of infection among chickens, increase host resistance by competitive exclusion in the gut, vaccination, or genetic selection of chickens, and finally, by use of antimicrobial compounds (Lin, 2009). The strategies to reduce Campylobacter colonization in chickens are more valued because the intestine of living poultry is a major amplification site for Campylobacter in the food chain. Therefore, reducing the fecal Campylobacter load in poultry during primary production is hypothesized to significantly reduce the incidence of human campylobacteriosis (Lin, 2009).
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Although, the above-mentioned control measures undoubtedly will help to decrease shedding of Campylobacter by chickens and may reduce the number of positive flocks, vaccination of poultry against Campylobacter will probably be most effective though this remains a major challenge. Outer membrane proteins (OMP) have been exploited for vaccine development because they are easily recognized by the immune system and have been demonstrated as promising candidates for vaccine against gram-negative bacteria (Stern et al., 1990; Lin et al., 2002a; Shoaf-Sweeney et al., 2008). Immunogenic OMP identified in Campylobacter include flagellum (Fla; Guerry, 1997), major outer membrane protein (major OMP; Zhang et al., 2000), cell-binding factor (Peb1; Pei and Blaser, 1993), multidrug efflux pump component (CmeC; Lin et al., 2002b, 2003; Lin et al., 2005), and ferric enterobactin receptor (CfrA; Zeng et al., 2008). In broiler chickens, Campylobacter infection leads to only a mild stimulation of immune system and colonization most likely involves immune tolerance (Hermans et al., 2012). Hence, we hypothesized that the use of C. jejuni crude OMP lysate that may strongly stimulate immune response in chicken could be an attractive vaccine candidate to prevent/ reduce shedding of C. jejuni from the intestinal tract of chickens. To effectively deliver OMP at the site of interest, we encapsulated OMP in the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP). The PLGA NP have been shown to protect encapsulated contents from early degradation and ensure controlled and effective sustained release of the active components. Therefore, the aim of this study was to investigate NP encapsulated OMP as a potential vaccine candidate against C. jejuni colonization of the chicken intestine.
MATERIALS AND METHODS Culture Growth The highly invasive strain C. jejuni 81–176 isolated from an outbreak associated with raw milk was used in the present study (Korlath et al., 1985). Campylobacter jejuni 81–176 strain was grown on Mueller-Hinton agar (MH; Becton Dickinson and Company, Sparks, MD) and incubated for 18 to 24 h at 42°C under microaerobic conditions (85% N2, 10% CO2, and 5% O2) in a DG250 Microaerophilic Workstation (Microbiology International, Frederick, MD).
Preparation of OMP of C. jejuni 81–176 The outer membrane proteins were extracted as described previously (Blaser et al., 1983) with a few modifications. Briefly, the cells grown on MH agar were harvested in MH broth and centrifuged at 6,000 × g for 15 min at 4°C. The pellet was resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride (Pefabloc SC, Boehringer, Mannheim, Germany) as protease in-
hibitor. The cells in suspension were sonicated 15 times with each sonication for 20 s duration and 30 s cooling between the sonications in a sonicator (Vibra-Cell, Sonics and Materials Inc., Newtown, CT) with setting of 3 pulser and 4 amplitude. The sonicated materials were centrifuged at 6,000 × g for 10 min at 4°C, and the supernatant was centrifuged again for at 10,000 × g for 2 h at 4°C and the pellet was freeze-dried and stored. The protein concentration was estimated using Bio-Protein Assay dye reagent kit (Bio-Rad, Hercules, CA) and 5 µg of OMP lysate was resolved on 12% SDS-PAGE and stained with Coomassie blue (Thermo Fisher Scientific, Rockford, IL) to check the quality of the extracted OMP.
Entrapment of OMP in PLGA NP The OMP entrapped PLGA NP were prepared by double-emulsion solvent evaporation method (waterin-oil-in-water, w/o/w) as described previously (Rajapaksa et al., 2010) with a few modifications. Briefly, 5% PLGA (85:15) polymer solution was prepared by dissolving 0.25 g of PLGA in 5 mL of methylene chloride/dichloromethane. Five milligrams of outer membrane protein suspended in 0.5-mL PBS was mixed with 0.25-mL of 2% polyvinyl alcohol solution prepared in 10 mM HEPES buffer (pH 7.5); then 0.05 mL of 20% sucrose and 0.05 mL of 20% magnesium hydroxide were added to the PLGA polymer solution and emulsified using probe sonication for 30 s (Branson-Sonifier 450, Ringwood, NJ) with a duty cycle of 30% and output control of 3. For the ease of sonication, the resulting emulsion (water-in-oil) was divided into 2 tubes and 11.5 mL of 2% polyvinyl alcohol and 1 mL of 12.5% Poloxamer 188 solution were added to each tube and emulsified by sonication for 30 s twice to obtain the final w/o/w emulsion. Contents of both the tubes were combined in a 100-mL beaker and stirred for 20 h at 4°C with a magnetic stirrer at 400 rpm to allow solvent evaporation. The NP were collected by centrifugation at 11,000 × g for 30 min at 4°C (FX6100 rotor, Beckman-Coulter, Brea, CA) for 30 min and resuspended in 45 mL of sterile distilled water; the washing step was repeated 3 times with sterile distilled water by repeated centrifugation and resuspension. The pellet of entrapped NP was finally suspended in 5 mL of 5% sucrose solution, freeze-dried for 16 to 18 h, and then stored at −20°C. Determination of OMP Entrapment Efficiency. The OMP entrapment in the PLGA NP was estimated using a bicinchoninic acid protein assay kit. Approximately 5 mg of freeze-dried NP was added to a 1-mL lysis buffer (5% SDS in 0.1 M NaOH) and incubated with gentle shaking for 1 h at 37°C. The solution was centrifuged at 11,000 × g for 5 min at 4°C, and the supernatant was collected and protein content was estimated using a bicinchoninic acid protein assay kit (Bio-Rad). The protein entrapment efficiency (wt/wt)
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was expressed as the amount of protein entrapped (in percentage) relative to the amount of protein used for entrapment. Scanning Electron Microscopy. The morphology of the protein-loaded NP was visualized using scanning electron microscopy. Briefly, the NP were placed on a double-sided adhesive tape attached to an aluminum stub and sputter-coated with gold/palladium beam for 2 min. The coated sample was imaged using Philips XL30-FEG scanning electron microscopy (Philips, Eindhoven, the Netherlands) at the Molecular and Cellular Imaging Center [Ohio Agricultural Research and Development Center (OARDC), Wooster, OH].
Vaccination and Challenge of Chickens One-day-old chickens were obtained from a local hatching facility at the Food Animal Health Research Program (OARDC, Wooster, OH). All chickens were confirmed to be Campylobacter negative by culturing cloacal swabs before vaccination. Animal experiments were performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. A total of 80 one-week-old chicks were divided randomly into 10 groups with 8 chicks in each group, and the chicks were vaccinated on d 7 followed by a booster vaccination at d 21. The vaccination schedule and the different treatment groups are listed in Table 1. Chickens were immunized either orally or subcutaneously (s/c) with OMP alone or with NP encapsulated OMP reconstituted in PBS. Because our main purpose was to optimize an oral vaccine for C. jejuni, only one dose (125 μg), representing a dose level between the high (250 μg) and low (25 μg) dose used for oral inoculation, was used for s/c administration. Because purified proteins may undergo degradation in the gastrointestinal tract and possibly fail to induce any antibody response when administered orally, the s/c injection also served as a quality control for our approach. The birds in all groups were orally challenged with 1 × 108 cfu/mL of C. jejuni 81–176 in 0.2-mL of PBS 14 d after booster vaccination and the control group received only 0.2 mL of PBS.
Collection and Processing of Chicken Samples The blood samples were collected and the sera were separated on the day of vaccine (d 7), 21, 35, and 42 d, and stored at −20°C until further use. Cloacal fecal samples were taken on d 0, 7, 14, 21, 28, 35, and 42, and in addition, cecal contents were taken on d 42 following euthanasia. The cloacal fecal samples from d 35 (day of challenge) were pooled for each treatment group. The cloacal fecal samples taken on other days were not pooled. For antibody response, cloacal fecal samples were suspended in 1 mL of PBS, suspension was centrifuged at 3,000 × g for 5 min at 4°C, and supernatants were stored at −20°C until further used for antibody detection. For cfu determination, the cecal and cloacal samples were weighed (weight range: ceca, 2 to 3 g; cloacal samples 1 to 2 g) and suspended in 3 and 1 mL of buffered peptone water, respectively, and homogenized. One hundred microliters of the homogenized suspension was serially (10-fold) diluted in buffered peptone water and plated on MH agar containing Campylobacter selective supplement (SR117E, Oxoid, Hampshire, UK) and incubated microaerobically at 42°C for 48 h. The Campylobacter colonies were counted and representative colonies were confirmed by PCR using Campylobacter genus specific 16s rRNA primers (Sanad et al., 2011). The cfu per gram of fecal sample was calculated considering respective weights of samples.
ELISA The ELISA was performed as described previously (Cawthraw et al., 1994), with few modifications. Briefly, to determine the level of C. jejuni specific IgY and IgA antibodies in serum and pooled fecal samples from different groups, ELISA plates (Nunc Maxisorp, Rochester, NY) were coated overnight at 4°C with 50 µL of pretitrated amounts of OMP (4 µg/mL for IgY; 6 µg/ mL for IgA) diluted in coating buffer (0.05 M carbonate buffer, pH 9.6). The plates were washed in PBS containing 0.1% Tween 20 (PBS-T), and 200 µL of
Table 1. Vaccination details for different treatment groups used in the study1 Treatment
n
Route of vaccination/volume (mL)
125 µg of OMP + NP 125 µg of OMP only NP only PBS only (control) 125 µg of OMP + NP 125 µg of OMP only 25 µg of OMP + NP 250 µg of OMP + NP 25 µg of OMP only 250 µg of OMP only
8 8 7 8 8 8 8 8 8 8
Oral/0.5 Oral/0.5 Oral/0.5 Oral/0.5 Subcutaneous/0.5 Subcutaneous/0.5 Oral/0.5 Oral/0.5 Oral/0.5 Oral/0.5
1OMP
Age at first vaccine (d)/booster (d)
= outer membrane protein of Campylobacter jejuni; NP = nanoparticle; n = sample size.
7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21 7/21
Age at challenge (d)/status (yes/no) 35/yes 35/yes 35/yes 35/no 35/yes 35/yes 35/yes 35/yes 35/yes 35/yes
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Figure 1. Scanning electron microscopy image of outer membrane protein (OMP) of Campylobacter jejuni entrapped in poly(lactide-coglycolide) (PLGA) nanoparticles (NP). Nanoparticles were placed on a double-sided adhesive tape attached to an aluminum stub and sputtercoated with gold/palladium beam for 2 min. The coated sample was imaged using a Philips XL30-FEG scanning electron microscope. a and b: Representative images at resolution levels 5,000× and 13,000×, respectively, of selected field of OMP entrapped in PLGA NP.
blocking buffer (5% skim milk suspension in PBS-T) was added to the wells and incubated at 37°C for 2 h. The plates were subsequently washed in PBS-T, and the test samples were diluted in blocking solution and then 50 µL was added to each well in duplicate and incubated at 37°C for 2 h. Serum samples were diluted 1:10 for IgA assay and in 4-fold dilutions starting from 1 in 64 for the IgY assay. Fecal supernatants were diluted 1:2 for both IgY and IgA assays. After 2 h of incubation, the plates were washed in PBS-T and incubated for 30 min at 37°C in 50 µL of peroxidase conjugated anti-IgA (1: 2,500) or anti-IgY (1:20,000) antibodies (Novus Biologicals, Littleton, CO) diluted in blocking solution. The plates were then washed in PBS-T and 50 µL of peroxidase substrate solution (TMB) was added to the wells (KPL, Gaithersburg, MD). The reaction was stopped with 1 N HCl solution after 10 min of color development. The plates were read at 450 nm using a Spectramax 340PC microplate reader (Molecular Devices, Sunnyvale, CA). Serum OMP-specific IgY antibody responses were measured by titrating serum samples by serial dilution, and antibody titers were determined using a cutoff of mean + 3 SD of the negative control group. Higher magnitude of serum IgY antibody responses allowed us to use the titration method. However, due to low magnitude of responses, the IgA titers were presented as OD values.
Statistical Analysis The ELISA results were analyzed with the JMP software (JMP, SAS Institute Inc., Cary, NC) using 1-way ANOVA followed by Tukey’s honestly significant difference test. For the chicken experiment, because sufficient replicates (n = 8) in each group were used to obtain statistically accurate data, the experiment was not repeated. Counts of Campylobacter cfu per gram were presented as means ± SE. The mean was determined by averaging the cfu counts from samples (n = 7 or 8);
for samples with below the level of detection of Campylobacter, detection limit was used as the minimum value. Data were log10 transformed and analyzed using 2-way ANOVA (GraphPad Prism v.5 Software Inc., La Jolla, CA). Correlation coefficient between antibody levels and cfu per gram were performed using GraphPad Prism v.5 software. A P-value of <0.05 was used to indicate statistically significant differences.
RESULTS Entrapment of OMP in PLGA NP Using the scanning electron microscopy, size of the OMP entrapped NP was determined and found to be ranging from 200 to 600 nm in diameter (Figure 1). The entrapment efficiency of OMP in PLGA NP was approximately 54%.
Campylobacter cfu Enumeration in Cecal and Cloacal Fecal Samples The vaccine trials were designed to check how effectively potential candidate vaccines under trial could reduce C. jejuni colonization (cfu/g) in intestinal contents when infected experimentally. Hence, we enumerated the cfu from cecal and cloacal contents from different vaccine trial groups. Our result showed that the groups were significantly different in the number of cfu per gram of tissues. The groups receiving the s/c vaccine had significantly lower cfu (below the detection limit) in cecal contents than all the groups receiving the oral vaccine (P < 0.05). The cfu per gram of cecal content was below the detection limit (<10 cfu) in the s/c groups that received either encapsulated OMP or OMP alone (Figure 2). The cecal cfu of the group given NP only orally was significantly higher than the other orally vaccinated groups except the group given 125 µg of OMP encapsulated in NP. The percentage of
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Figure 2. Campylobacter cfu per gram in cecal and cloacal fecal samples. The birds were orally challenged with 1 × 108 cfu of Campylobacter jejuni at 28 d post-first vaccination. The cecal contents and cloacal fecal samples were taken 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) indicate the groups differ significantly within sample from each organ (P < 0.05). OMP = outer membrane proteins; NP = nanoparticle; s/c = subcutaneous.
birds positive for Campylobacter in the cecal contents were also zero in groups vaccinated s/c with the encapsulated OMP or OMP alone (Table 2). The orally vaccinated groups showed presence of C. jejuni in the cecal contents: encapsulated OMP (88%), OMP alone (63%), and the group given NP alone (57%). The groups that received 25 or 250 µg of OMP alone were 67 and 50% positive for Campylobacter, respectively, in cecal contents. The groups that received 25 or 250 µg of encapsulated OMP were 63 and 38% positive for Campylobacter, respectively, in cecal contents. The group vaccinated with 125 µg of encapsulated OMP s/c had the lowest cfu in the cloacal fecal sample (below the detection limit) and was significantly dif-
ferent from all the other challenged groups. The group vaccinated with 125 µg OMP alone s/c was also different from the other orally vaccinated groups. The group given 250 µg OMP alone orally did not differ from the other orally vaccinated groups. The percentage of birds’ cloacal samples positive for C. jejuni was different in the oral groups and s/c groups. Sixty-three percent of chickens in groups vaccinated orally with 125 μg of OMP with or without encapsulation and 86% of chickens in group given NP alone were positive for C. jejuni. The cloacal samples from the group vaccinated s/c with encapsulated OMP (125 μg) had no detectable Campylobacter, whereas only 13% was positive in groups given OMP alone s/c. The groups that received
Table 2. Cecal and cloacal samples positive for Campylobacter expressed in percentage (n = 7 or 8 chicks/group)1 Cecum OMP + NP Amount of OMP (µg) 25 125 250
Cloaca OMP alone
s/c
Oral
s/c
Oral
NT BDL NT
63 88 38
NT BDL NT
67 63 50
OMP + NP
OMP alone
s/c
Oral
s/c
Oral
NT BDL NT
50 63 50
NT 13 NT
67 63 50
1OMP = outer membrane protein of Campylobacter jejuni; NP = nanoparticle; s/c = subcutaneous; BDL = below detection limit; NT = 25- and 250-µg doses were not tested by s/c route.
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25 or 250 µg of OMP alone were 67 and 50% positive for Campylobacter, respectively, in cloacal contents. The groups that received 25 or 250 µg of encapsulated OMP were both 50% positive for Campylobacter in cloacal contents (Table 2). The control group cecal and cloacal samples were negative for the Campylobacter. Because oral vaccination of birds with OMP (with and without encapsulation) using a maximum dose of 250 µg per bird did not induce any protection against C. jejuni colonization, only the serum and fecal samples from birds in groups vaccinated 125 μg (orally or s/c) were analyzed for antibody response.
ELISA Results Serum IgA. The serum optical density (OD) values due to secretory IgA response in the groups differed significantly at 14 d postvaccination (dpv; 21 d of age, P < 0.0001, Figure 3). The secretory IgA response of groups vaccinated with OMP by the s/c route with or without encapsulation was higher than the rest of the groups. However, the group vaccinated s/c with encapsulated OMP was different from the group vaccinated s/c with OMP alone. The rest of the groups’ serum OD values did not differ from one another. At 28 dpv (day of challenge, 35 d of age), the serum OD values of the group receiving OMP only by the s/c route had a higher OD value than the rest of the groups (P < 0.0001). The other groups did not differ from one another. At 7 d postchallenge (dpc, 42 d of age), the OD values of different groups did not differ from one another. Sig-
nificant negative correlation (r = −0.50, P < 0.0001) was observed between serum IgA antibody titers and cloacal cfu levels. Serum IgY. The serum IgY titers of the groups differed significantly at 14 dpv (21 d of age, P < 0.0001, Figure 4). The titers of groups vaccinated s/c with OMP or encapsulated OMP were higher than the rest of the groups. However, the groups s/c vaccinated with encapsulated OMP and OMP alone did not differ. The groups vaccinated with encapsulated OMP orally had higher titers than the control and lower titers than the s/c groups but did not differ from the groups given OMP or NP alone orally. At 28 dpv (day of challenge, 35 d of age), the serum titers of the different groups differed significantly (P < 0.0001). The differences between groups were similar to 14 dpv, and the s/c groups had higher titers than the rest of the groups. At 7 dpc (42 d of age), the serum titers of the groups were significantly different (P < 0.0001). The serum titers of s/c groups with encapsulated OMP or OMP alone were higher than the rest of the groups. However, the groups vaccinated s/c with encapsulated OMP or OMP alone did not differ. In addition, a significant negative correlation (r = −0.58, P < 0.001) was observed between serum IgY antibody titers and cloacal cfu levels. The oral groups given encapsulated OMP or OMP/NP alone did not differ from the control group. Fecal IgA. The OD values of the different groups at 7 dpv (14 d of age) differed significantly (<0.0001; Figure 5). The s/c group with encapsulated OMP had higher OD than the group administered encapsulated
Figure 3. Serum IgA in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The serum samples were taken at 14 and 28 d postvaccination and 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
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Figure 4. Serum IgY titers in birds vaccinated subcutaneously (s/c) and orally with outer membrane protein (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The serum samples were collected at 14 and 28 d postvaccination and 7 d postchallenge. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05).
OMP orally, and both groups had higher OD than the rest of the groups; the OD values of the rest of the groups did not differ. At 14 dpv (21 d of age), the s/c group given OMP alone had higher OD than the rest of the groups (P < 0.001), but the rest of the groups did not differ from each other. At 21 dpv (28 d of age), the groups differed from each other significantly (P < 0.001). The s/c group given encapsulated OMP had higher OD than the rest of the groups. The oral groups administered with encapsulated OMP or NP alone had significantly lower OD values than the rest of the groups. The groups given OMP s/c or orally did not differ significantly from the control group. At 7 dpc (42 d of age), the OD values of the groups differed significantly (P < 0.01). The s/c group with OMP alone had higher OD than the control group and the groups administered encapsulated OMP or OMP alone orally. However, the s/c group given OMP alone did not differ significantly from the group given NP alone orally. A significant negative correlation coefficient (r = −0.56, P = 0.0002) was observed between fecal IgA antibody titers and cloacal cfu levels. Fecal IgY. At 7 dpv (14 d of age), the s/c group with encapsulated OMP had a significantly higher OD value than the rest of the groups (P < 0.001; Figure 6). At 14 dpv (21 d of age), there was no significant differ-
ence between the groups. At 21 dpv (28 d of age), the group given OMP alone s/c differed from the rest of the groups (P < 0.01). At 7 dpc (42 d of age), the OD values of the groups differed significantly (P < 0.0001). The s/c group given OMP alone had higher OD value than the s/c group given encapsulated OMP, and both these groups had higher OD values than the rest of the groups.
DISCUSSION The commensal nature of C. jejuni in avian species is still a debated topic, but it is beyond doubt that the experimental C. jejuni infection triggers both systemic and mucosal immune responses in chickens (de Zoete et al., 2007). Moreover, the elevated levels of Campylobacter-specific serum antibodies correlated with the reduced colonization level of Campylobacter, suggesting a protective role of antibodies in Campylobacter infection in chickens. The high levels of Campylobacter maternal antibodies in younger chickens may partly contribute to the lack of Campylobacter infection in young chickens in natural environments during the first 2 wk of life, which was also supported by experimental challenge studies (Sahin et al., 2001, 2003b). Our findings on the protective nature of Campylobacter-specific antibodies further
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Figure 5. Fecal IgA in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The fecal samples were taken at 7-d intervals postvaccination and 7 d postchallenge. The cloacal fecal samples within each treatment group were pooled for 28 d postvaccination (35 d of age) collection and have no error bars. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
Figure 6. Fecal IgY in birds vaccinated subcutaneously (s/c) and orally with outer membrane proteins (OMP) of Campylobacter jejuni either encapsulated or not encapsulated in nanoparticles (NP). The fecal samples were taken at 7-d intervals postvaccination and 7 d postchallenge. The cloacal fecal samples within each treatment group were pooled for 28 d postvaccination (35 d of age) collection and have no error bars. Bars (mean of 7 or 8 birds ± SE) without common letters (a–c) differ significantly within each sampling day (P < 0.05). OD = optical density.
CAMPYLOBACTER JEJUNI OUTER MEMBRANE PROTEIN VACCINE
support the practicability of developing immunizationbased approaches to curtail Campylobacter infection in chickens. To date, there is no vaccine available to control Campylobacter colonization in poultry. In previous research studies, attempts were made to develop an effective vaccine against C. jejuni using immunogenic flagellar proteins administered intranasally (Lee et al., 1999), formalin inactivated C. jejuni administered orally (Rice et al., 1997), and inner membrane antigen of C. jejuni with Salmonella carrier administered orally (Wyszyńska et al., 2004, 2008). Widders et al. (1996) demonstrated that the intraperitoneal route of vaccination with killed C. jejuni was more effective than the oral or s/c route in reducing Campylobacter intestinal colonization in chickens. In addition, Noor et al. (1995) reported that an in ovo vaccine was capable of eliciting early antibody response in chicken. However, none of these vaccine trials were successful in completely preventing the Campylobacter infection in chickens. Here we designed a vaccine trial using potent immunogenic OMP of C. jejuni entrapped in NP and administered through oral and s/c routes (Islam et al., 2010, Zeng et al., 2010). To increase the mucosal immune response, encapsulation of the OMP in NP was attempted in this study. Previous studies have demonstrated that vaccine particles encapsulated in PLGA NP are efficient in eliciting potent immune response (Sarti et al., 2011; Dwivedi et al., 2012). The NP are used in mucosal vaccines because mucosal candidate vaccines on their own are not very effective due to degradation in the gastrointestinal tract. The NP-encased vaccines efficiently elicit immune response if administered by the s/c, intranasal, or oral route (Dwivedi et al., 2012). The PLGA NP are readily taken up by the Peyer’s patches in the ileum and can stimulate antigen presenting cells and subsequently T cells (Igartua et al., 1998; Chong et al., 2005; Waeckerle-Men and Groettrup, 2005). The NP-encased vaccines administered orally in mice induced gut-specific IgA response as well as IgG response (Sarti et al., 2011). Intranasal administration of Chitosan-DNA NP carrying flagellar protein genes of C. jejuni in chickens increased the immune response, but was not very effective in reducing C. jejuni infection (Huang et al., 2010). The present study highlighted the importance of administration of the OMP in controlling intestinal colonization of C. jejuni, and it correlated with the induction of Campylobacter-specific immunoglobulins when delivered s/c but not orally. Oral vaccines that contain only protein may not be effective due to protein degradation in the gut (Garinot et al., 2007). Therefore, attempts have been made to encapsulate the proteins in NP so that they can be released slowly and taken up by the intestinal microfold cells, resulting in induction of effector and memory immune responses (Brayden et al., 2005; des Rieux et al., 2006). Therefore, we encapsulated the OMP in PLGA NP, but this approach failed to elicit the desired immunity when the vaccine
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was delivered by the oral route. Previous studies with mucosal vaccines against Campylobacter have shown mixed results in eliciting mucosal antibody production (Huang et al., 2010; Zeng et al., 2010). However, NP encapsulated OMP vaccine delivered by the s/c route resulted in higher IgY and IgA titers in cloacal feces. The group given encapsulated OMP s/c had higher and earlier fecal IgA response (7 and 14 dpv) and postchallenge. The fecal IgY was higher in both the s/c groups (encapsulated and nonencapsulated OMP). The serum IgA and IgY responses also appeared earlier and at higher levels in the s/c groups. The fecal and cecal C. jejuni cultures were negative (below the detection limit) for the group injected s/c with encapsulated OMP, whereas other vaccinated groups were positive for C. jejuni (Figure 2, Table 2). Therefore, incorporation of Campylobacter immunogenic antigens in a biodegradable NP administered parenterally may have promise in providing better protection from C. jejuni colonization in chickens. Control of Campylobacter infection would reduce the likelihood of contamination of chicken carcasses with Campylobacter during processing and transportation thus will have a significant impact on zoonosis. Our data suggest that the s/c route of vaccination with encapsulated OMP or OMP alone is highly effective in inducing protective antibody responses and preventing C. jejuni colonization in the chicken. The s/c vaccination with OMP provided significant protection against Campylobacter infection by the virtue of higher IgY and IgA responses in both serum and feces. These results point toward superiority of the s/c vaccine over oral vaccine in the control of intestinal infection with Campylobacter. Although the s/c administered vaccines may not be practical for large-scale application in broilers, the vaccine can still be of use in layer and breeder hens. Campylobacter can persist in laying hens for a prolonged period; therefore, if layers/breeders are free of the organism, the possibility of zoonotic threats from the organism transmitted through contaminated eggs or environment or management practices can be minimized. Although, vertical egg transmission of the organism is extremely rare (Sahin et al., 2003a), C. jejuni has been isolated from reproductive organs of chickens (Camarda et al., 2000) and chickens can be infected by ingestion of the bacteria from contaminated shells (Cox et al., 2012). Reducing the Campylobacter colonization in the layer and breeder hen population, therefore, will reduce the environmental contamination and possibility of cross contamination of eggs, thereby minimizing the risk of ingestion of the organism by chicks and subsequent colonization. In summary, our findings suggest the usefulness of NP-based delivery of C. jejuni antigens to confer protection against C. jejuni colonization in chickens; however, further research is needed to optimize the oral delivery of modified NP-based vaccines for cost-effective and practical use in the current broiler production system.
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ACKNOWLEDGMENTS We thank Juliette Hanson and Kingsly Berlin (both from Food Animal Health Research Program, Wooster OH) for their technical assistance with chicken experiment. G. Rajashekara’s laboratory is supported by the OARDC, The Ohio State University, and the Agriculture and Food Research Initiative grant #2012-6800319679, USDA.
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Widders, P. R., R. Perry, W. I. Muir, A. J. Husband, and K. A. Long. 1996. Immunisation of chickens to reduce intestinal colonisation with Campylobacter jejuni. Br. Poult. Sci. 37:765–778. Wyszyńska, A., A. Raczko, M. Lis, and E. K. Jagusztyn-Krynicka. 2004. Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 22:1379–1389. Wyszyńska, A., J. Zycka, R. Godlewska, and E. K. JagusztynKrynicka. 2008. The Campylobacter jejuni/coli cjaA (cj0982c) gene encodes an N-glycosylated lipoprotein localized in the inner membrane. Curr. Microbiol. 57:181–188. Zeng, X., F. Xu, and J. Lin. 2008. Ferric enterobactin receptor CfrA is an attractive vaccine candidate against Campylobacter jejuni. Abstract E-014, 108th General Meeting of American Society for Microbiology, Boston, MA. Zeng, X., F. Xu, and J. Lin. 2010. Development and evaluation of CmeC subunit vaccine against Campylobacter jejuni. J Vaccines Vaccin. 1:112. Zhang, Q., J. C. Meitzler, S. Huang, and T. Morishita. 2000. Sequence polymorphism, predicted secondary structures, and surface-exposed conformational epitopes of Campylobacter major outer membrane protein. Infect. Immun. 68:5679–5689.