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1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, Texas 77843-4467, USA
2 Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, 440 Reynolds Medical Building, College Station, Texas 77843-1114, USA
3 Department of Veterinary Science, Louisiana State University Agricultural Center, Louisiana State University, Baton Rouge, Louisiana 70803, USA
4 Corresponding author (email: aarenas{at}cvm.tamu.edu)
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Brucellosis eradication programs in North America have been successful in controlling the pathogen in domestic livestock but not in wildlife populations (Ragan, 2002). Currently, elk (Cervus elaphus nelsoni) and bison (Bison bison) are the wildlife reservoirs of B. abortus in the Greater Yellowstone area, and transmission from wildlife to cattle has occurred (Thorne, 1980).
Effective vaccines to control brucellosis in wildlife are not currently available. Commercially available vaccine strains used for brucellosis eradication in cattle have been tested in wildlife species (Davis and Elzer, 2002), but results from elk vaccination trials have shown that efficacy is reduced in comparison to cattle. Additional vaccination-related problems include interference with diagnosis (Schurig et al., 2002), resistance to antibiotics, and potential virulence for animals and humans (Berkelman, 2003; Ashford et al., 2004). The B. abortus strain 19 (S19) appeared to be safe in adult elk but has been shown to reduce abortion rates only by 30% (Thorne et al., 1981). The S19 vaccine also does not cause morbidity or mortality in pronghorn antelope (Antilocapra americana; Elzer et al., 2002), bison (Davis et al., 1991) or coyotes (Canis latrans). Another Brucella vaccine, SRB51, has been shown to be safe in a wider range of nontarget species, including ravens (Corvus corax), Richardson ground squirrels (Spermophilus richardsonii), and deer mice (Peromyscus maniculatus; Januszewski et al., 2001); bighorn sheep (Ovis canadensis), pronghorn antelope, mule deer (Odocoileus hemionus), and moose (Alces alces shirasi; Kreeger et al., 2002b); and black bears (Ursus americanus; Olsen et al., 2004). However, if administered parenterally, SRB51 did not protect against abortion in elk (Cook et al., 2002; Kreeger et al., 2002a).
The distribution of the disease appears to be correlated with high animal densities associated with winter feeding (Etter and Drew, 2006). Infected and susceptible elk commingling on feed grounds ensure exposure of animals to B. abortus, enhancing the probability of transmission. Control of brucellosis should be focused on these sites to prevent or reduce exposure of the pathogen to naïve animals, thus breaking the chain of transmission. Difficulties with integrating Brucella vaccination strategies into control efforts have been associated not only with the low efficacy of S19 and RB51 in elk but also with the delivery method used to immunize the animals. Currently, elk vaccination uses a S19 biobullet ballistic approach, and problems arising from this methodology include excessive time and labor, logistics, and high cost.
During April 2004 to November 2006, we evaluated the potential for delivering a live RB51 vaccine to elk via a controlled microencapsulated release vehicle. The capsule was made of alginate, a naturally occurring biopolymer that offers the advantages of biocompatibility, low toxicity, and encapsulation conditions that are compatible with live organisms (Wee and Gombotz, 1998). In an attempt to enhance the efficacy of the capsule, we also incorporated a novel protein from the eggshell precursor of the parasite Fasciola hepatica, Vitelline protein B (VpB). This recombinant 31-kDa protein possesses an unusual resistance to proteolytic breakdown (Rice-Ficht et al., 1992), which may reduce erosion time and release of the capsule content. To further explore the alternatives of using this method, PO delivery of the microencapsulated vaccine was also investigated, principally because this is the most cost-effective way to deliver a vaccine in wildlife populations.
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MATERIALS AND METHODS |
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Fifty-four 1–2-yr-old red deer (Cervus elaphus elaphus) females from a privately owned tuberculosis-free and brucellosis-free commercial herd were used as an animal model for Rocky Mountain elk (Cervus elaphus nelsoni) because of their close genetic relationship. Upon arrival, animals were retested for specific anti-Brucella immunoglobulin G (IgG) levels (total IgG) by enzyme-linked immunosorbent assay (ELISA) and were dewormed (moxidectin, Cydectin; Wyeth, Madison, New Jersey, USA). Deer were acclimated for 3 mo before vaccination. All animal care and experimental procedures were performed in compliance with the institutional animal-care protocol.
Bacterial strains
Bacterial strains used in these experiments included the vaccine strains SRB51 and S19. Bacteria were grown on tryptic soy agar (TSA; Difco, BD, Sparks, Maryland, USA) at 37 C with 5% CO2. Three days postincubation, SRB51 plates were harvested and bacteria were pelleted and standardized for subcutaneous or PO vaccination at a dose of 1.5x1010, whether encapsulated or nonencapsulated. For animal challenge, a dose of 1x109 of vaccine S19 was standardized using a klett meter and plating onto TSA plates retrospectively to confirm the dose.
Preparation of B. abortus SRB51 antigen-loaded microspheres
Alginate beads, loaded with 1.5x1010 colony-forming units (cfu)/ml of the vaccine SRB51, were prepared as previously described (Abraham et al., 1996) with some modifications. Briefly, enumerated, live SRB51 vaccine strain (total 1.5x1011 for 10 doses) was resuspended in a total of 100 µl of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10mM MOPS, 0.85% NaCl, pH 7.4) and mixed with 10 ml of alginate solution (1.5% sodium alginate, 10mM MOPS, 0.85% NaCl, pH 7.3). Spheres (300 µm) were obtained by extruding the suspension through a 200-µm nozzle into a 100 mM calcium chloride solution that was stirred for 15 min using an Inotech encapsulator I-50 (Inotech Biosystems International, Rockville, Maryland, USA). For a permanent cross-linking of the capsule, microspheres were washed three times with 200 ml of MOPS buffer for 10 min and further stirred in a solution containing 0.05% poly-L-lysine (molecular weight = 22,000) for 15 min. Following two successive washes, the beads were stirred in a solution of 0.03% alginate for 5 min to apply a final outer coating. All capsules were stored at 4 C in MOPS buffer until use. To determine the number of bacteria per 1 ml of capsules, spheres were removed from the encapsulator before the permanent cross-linking and were washed three times with 50 ml of MOPS buffer, and particles were dissolved using 10 ml of depolymerization solution (50mM sodium citrate, 0.45% NaCl 10mM MOPS, pH 7.2). Enumeration of bacteria was determined by plating onto TSA plates.
The addition of VpB as a component of the alginate core was achieved by the addition of 1 mg of VpB to the bacteria-alginate suspension described above. Extrusion and capsule formation used the same preparation conditions.
Immunization of red deer
Fifty-four 1–2-yr-old, female red deer were randomly distributed into six different treatments (n=9/group). Three groups were inoculated subcutaneously with a total dose of 1.5x1010 cfu of either nonencapsulated SRB51, encapsulated SRB51 with alginate, or encapsulated RB51 with alginate and VpB. Two groups were vaccinated by the PO route by squirting the vaccine into their mouth; one group received 1.5x1010 cfu of encapsulated SRB51 with alginate, and the second group received with encapsulated RB51 with alginate and VpB. The control group received a subcutaneous injection of 1 ml of empty capsules (no bacteria entrapped). A single vaccination dose was given to all animals.
Detection of Brucella-specific antibody levels.
To determine anti-Brucella–specific antibody in serum, blood samples were collected by jugular venipuncture immediately before vaccination and 6, 12, 17, and 28 wk postvaccination. Serum samples were analyzed for anti-Brucella IgG (total IgG) determination by ELISA. Heat-killed SRB51 cell antigen was used to coat 96-well plates (Nunc-Immuno plates, high binding protein; Thermo Fisher Scientific, Rochester, New York, USA) at a concentration of 25 µg/well. After overnight incubation at 4 C, plates were washed using phosphate-buffered saline (PBS) containing 0.05% Tween-20, blocked (0.25% w/v bovine serum albumin), and incubated with the diluted deer-serum samples (1:100 in blocking buffer) for 2 hr. Following three more washes, goat anti-deer IgG horseradish peroxidase (KPL Systems, Silver Spring, Maryland, USA) conjugate was added at a dilution of 1:1,000 and incubated at room temperature for 1 hr. After incubation, plates were washed, and o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich, St. Louis, Missouri, USA) was added following manufacturer’s instructions for 20 min. The reaction was stopped by the addition of 50 µl of 0.5M NaOH. The absorbance was measured at 450 nm (A450). All assays were performed in triplicate and repeated at least two times.
Lymphocyte proliferation assay from peripheral blood mononuclear cells
At 12 wk postvaccination, mononuclear cells were isolated from peripheral-blood buffy coats as previously described (Waters et al. 2002) with some modifications. Briefly, 2x105 cells/well were seeded in 96-well plates (Falcon, Becton Dickinson, San Jose, California, USA) in RPMI medium containing 10% (v/v) fetal bovine serum, 1mM L-glutamine, and 1mM nonessential amino acids. Cells were stimulated, with B. abortus wild-type S2308 lysate at a concentration of 12.5 µg/ml, concanavalin A (5 µg/ml), or medium alone, and were incubated for 6 days at 37 C with 5% CO2. After this incubation period, 1 µCi of methyl-[3H] thymine was added to each well. Following 18 hr of incubation, cells were harvested onto fiber filters using a 96-well plate cell harvester, and the incorporated radioactivity was measured by liquid-scintillation counting. Lymphocyte proliferation data is represented as mean counts per minute (cpm) ± standard deviation.
Host response to subsequent B. abortus S19 exposure
At 7 mo postvaccination, three to four animals from each vaccination group (except RB51/alginate SC; n=2) were exposed conjunctivally, as previously described (Kreeger et al., 2000), using a challenge dose of 1x109 cfu/deer of B. abortus S19. Dose exposure was confirmed by serial dilutions and plating onto TSA plates. At 2 wk post-challenge, animals were euthanized, and spleens were harvested, weighed and homogenized. For homogenization, 1 ml of peptone saline was added to 1 g of tissue. Each sample was treated for 5–10 min using a stomacher. From each sample, 100 µl was plated onto Farrell’s media (Oxoid LTD, Basingstoke, Hampshire, England) in duplicate. At 3–5 days postincubation, bacteria were enumerated. Results are represented as the mean cfu/g of tissue±standard error of the mean (SEM).
Statistical procedures
Anti-Brucella IgG levels elicited from vaccination were expressed as the mean absorbance at 450±SD for each group. For lymphocyte proliferation, the cpm from each group were expressed as the mean cpm±SD. Bacterial load from S19 challenge was expressed as mean log cfu±SEM for each group. The significance of differences between groups was determined by analysis of variance (ANOVA); a P value <0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two different capsular formulations were prepared using the same alginate base. Variation of the formulation included the addition of VpB within the capsule to modify the degradation kinetics and release of the organism. When capsules were analyzed using light microscopy, all of the capsule formulations appeared spherical and uniform with a mean diameter of 310 µm (Fig. 1
). Furthermore, bacterial viability following encapsulation exceeded 95%, as demonstrated by recovery of the organism following dissolution of the capsules (data not shown).
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At 12 wk postvaccination, animals that received the encapsulated vaccine with VpB in the formulation (regardless of the immunization route) were the only individuals that had a statistically significant proliferative response compared with the controls (P<0.0005 PO vaccinates, P<0.005 SC group; Fig. 2). Interestingly, the cpm counts in animals that received encapsulated RB51 with VpB PO were also higher than in deer that received the same formulation via SC (P<0.3). None of the animals that received nonencapsulated vaccine had a significant cellular response compared with naïve nonvaccinated animals.
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Immunization with RB51 elicited an anti-Brucella IgG response that was clearly detectable by 6 wk postvaccination (Fig. 3). During the initial 17 wk, anti-Brucella IgG levels were higher in animals that received the injected vaccine compared with the groups that were immunized PO (P<0.05). Between 17 to 28 wk, anti-Brucella IgG levels in animals that were PO-vaccinated had an increase in anti-Brucella IgG compared with deer SC-vaccinated.
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The host protective response was determined by subtracting the mean cfu of S19 recovered per gram of spleen from deer vaccinated with the nonencapsulated or encapsulated vaccine from the mean cfu per gram recovered from naïve non-vaccinated but infected deer. At 2 wk postchallenge, only animals that received encapsulated SRB51 with VpB had a significant decrease in bacterial load in the spleen (Fig. 4). Red deer that received the vaccine PO were the only group that was statistically significant compared with the nonencapsulated, injected SRB51 (P<0.04). Animals that were PO-immunized with the VpB capsules had a 1.27 log reduction in spleen counts compared with animals vaccinated with nonencapsulated SRB51 and a 1.68 log reduction compared with naïve, nonvaccinated, but S19 exposed, animals. Brucella abortus strain 19 spleen counts in deer that received the VpB capsules via SC were also diminished by 1.21 log compared with the nonencapsulated RB51 and by 1.62 log compared with non-SRB51 vaccinated controls (P<0.2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Recent data indicate that the manner in which antigen reaches the lymph organs and how it is delivered to the antigen-presenting cells are fundamental in the induction of an optimal immune response. There is experimental evidence to support the observation that microencapsulation serves to modify the uptake and processing of antigen (Eyles et al., 2001; Sun et al., 2003). Also, it has been suggested that prolonged persistence of the vaccine strain in the host is needed for the development of a suitable anti-Brucella immunity (Kahl-McDonagh and Ficht, 2006). In an effort to develop a more efficient way to present the current vaccine SRB51 to the lymphoid tissue and to increase the exposure time of the organism to the host cells, we developed a controlled-release strategy in which SRB51 was encapsulated into alginate-VpB composite microspheres. We were successfully able to entrap SRB51 and develop uniform spherical batches of capsules, even when VpB was added to the formulation. By adding this component to the capsules, we modified the efficiency of the capsule as demonstrated by the difference in cellular and humoral responses observed in animals that received this formulation.
Live vaccines are more efficacious than vaccination with heat-killed organisms or cellular extracts and provide a significant level of immunity for protection against brucellosis (Zhan et al., 1995; Schurig et al., 2002). During microsphere formulation, SRB51 was exposed to relatively mild conditions that preserved bacterial viability (95%). This is in contrast to many standard encapsulation procedures, which employ harsh conditions to affect polymerization, including direct exposure of the bacteria to organic solvents, shear stress, and ultrasound homogenization (Lima and Rodrigues, 1999).
Oral bait administration of vaccines is the most practical and cost-effective method to vaccinate wildlife, and successful techniques and strategies for PO immunization of foxes (Vulpes vulpes) against rabies have been extensively demonstrated (Schneider, 1995; Pastoret and Brochier, 1996). Many pathogens, including Brucella spp, access the body via the mucosal surfaces; neutralization of the microorganism at the mucosal site would be an ideal situation to prevent brucellae from infecting the host. Our data indicate that increased efficacy with current Brucella vaccine strains can be achieved if the antigens are presented PO in a controlled-release format. The composite microspheres may serve to enhance the viability of bacteria in the ruminant digestive tract while providing immunization in a controlled-release format. The capsule might also provide a vaccine package that could be combined with baits for easy delivery.
Humoral immunity was assessed within all the SRB51 vaccine formulations and routes of vaccination. Immunization with SRB51 induced elevated anti-Brucella IgG levels (total IgG); however, levels were lower in deer vaccinated with the nonencapsulated vaccine than those induced by the microcapsules. In PO vaccinates, increasing levels of IgG between 17 to 28 wk were observed, which may reflect the protective benefits of the capsule for the live vaccine during exposure to conditions in the digestive tract. It is important to mention that all groups except controls, had increased levels of anti-Brucella IgG. Only the PO RB51 vaccinated groups had statistically significant increase compared with deer vaccinated via SC (P<0.05). The overall increases observed in all groups may have been associated with the seasonal hormonal cycles in deer.
Induction of specific cell-mediated immune responses following immunization is a hallmark for the establishment of a protective immune response. In elk, both cellular and humoral responses might be needed to generate a strong immunity toward Brucella infections (Kreeger et al., 2002a). Our results with the encapsulated SRB51 indicated that by 12 wk alginate/ VpB capsules administered SC or PO stimulated a statistically significant higher cellular response compared with nonencapsulated SRB51. These data suggest that by incorporating the SRB51 vaccine into a delivery vehicle, the necessary conditions needed to trigger a protective cellular response are created. Moreover, a low cellular response elicited by nonencapsulated RB51 was observed, corroborating the results previously reported by other researchers (Cook et al., 2002; Kreeger et al., 2002a).
After the initial cellular and humoral responses were assessed, three or four animals from each group were challenged with S19 to determine the degree of protection conferred by the encapsulated vaccine to subsequent Brucella exposure. This strain was used because it has been previously shown that S19, by itself, is able to cause prolonged infection in deer and can be cultured from the spleen by 2 wk postinoculation. Using a challenge dose of 1x109 cfu, a significantly (P<0.04) lower infection rate was observed in animals that were immunized with the encapsulated vaccine. This was especially true for deer that received VpB in the formulations. In the case of PO vaccination, results not only corroborate the observed cellular and humoral responses but also support our idea that the capsule serves as a vehicle necessary for proper immunogenicity. The fact that reduction of infection was afforded to such a degree via PO delivery is highly promising and relevant to the current needs for a practical vaccination strategy. Further investigations with a higher number of animals, and actual challenge with wild-type organisms, are still needed.
In summary, our findings indicate that alginate-VpB encapsulation of live Brucella might be used not only to enhance vaccine efficacy in elk but also to provide a practical means of vaccination. Enhanced immune responses were observed in red deer that were vaccinated with encapsulated formulations, especially containing the VpB additive. Oral vaccination with VpB encapsulated formulations was able to invoke both humoral- and cell-mediated responses in red deer and to offer protection from a challenge of live S19, as evidenced by organism recovery from the spleen. These data support the hypothesis that an enhanced and prolonged host response because of a mucosal immune stimulation can be achieved via PO vaccination. Finally, the results observed using nonencapsulated RB51 were similar to those obtained in previous studies (Olsen et al., 2002) in which RB51, by itself, is not sufficient to induce a good cellular response or reduce infection in elk (Kreeger et al., 2002a) to a significant degree.
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ACKNOWLEDGMENTS |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Received for publication 22 March 2007.
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