Parenteral vaccination of domestic pigs with Brucella abortus strain RB51

William C. Stoffregen Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50010.

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Steven C. Olsen Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50010.

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Betsy J. Bricker Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50010.

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Abstract

Objective—To determine the immunogenicity and efficacy of Brucella abortus strain RB51 (SRB51) as a vaccine in domestic pigs.

Animals—Sixty-eight 6-week-old crossbred domestic pigs and twenty-four 4-month-old gilts.

Procedures—In experiment 1, pigs were vaccinated IM (n = 51) with 2 × 1010 CFUs of SRB51 or sham inoculated (17). Periodic blood samples were obtained to perform blood cultures, serologic evaluations, and cell-mediated immunity assays. Necropsies were performed at selected times between weeks 1 and 23 after vaccination to determine vaccine clearance. In experiment 2, gilts were similarly vaccinated (n = 18) or sham inoculated (8) and similar samples were obtained after vaccination. Gilts were bred and challenged conjunctivally with 5.0 × 107 CFUs of virulent Brucella suis strain 3B. Necropsies were performed on gilts and on fetuses or neonates after abortion or parturition, respectively. Bacterial cultures and serologic evaluations were performed on samples obtained at necropsy to determine vaccine efficacy.

Results—Humoral and cell-mediated immune responses did not differ between vaccinates and controls. After vaccination, SRB51 was not isolated from blood cultures of either group and was isolated from lymphoid tissues of 3 pigs at 2 weeks (n = 2) and 4 weeks (1) after vaccination. No differences were found in isolation of B suis or in seroconversion between vaccinated and control gilts and between their neonates or aborted fetuses.

Conclusions and Clinical Relevance—Parenteral vaccination with SRB51 does not induce humoral or cell-mediated immune responses. Vaccination with SRB51 did not protect gilts or their neonates and fetuses from virulent challenge with B suis.

Abstract

Objective—To determine the immunogenicity and efficacy of Brucella abortus strain RB51 (SRB51) as a vaccine in domestic pigs.

Animals—Sixty-eight 6-week-old crossbred domestic pigs and twenty-four 4-month-old gilts.

Procedures—In experiment 1, pigs were vaccinated IM (n = 51) with 2 × 1010 CFUs of SRB51 or sham inoculated (17). Periodic blood samples were obtained to perform blood cultures, serologic evaluations, and cell-mediated immunity assays. Necropsies were performed at selected times between weeks 1 and 23 after vaccination to determine vaccine clearance. In experiment 2, gilts were similarly vaccinated (n = 18) or sham inoculated (8) and similar samples were obtained after vaccination. Gilts were bred and challenged conjunctivally with 5.0 × 107 CFUs of virulent Brucella suis strain 3B. Necropsies were performed on gilts and on fetuses or neonates after abortion or parturition, respectively. Bacterial cultures and serologic evaluations were performed on samples obtained at necropsy to determine vaccine efficacy.

Results—Humoral and cell-mediated immune responses did not differ between vaccinates and controls. After vaccination, SRB51 was not isolated from blood cultures of either group and was isolated from lymphoid tissues of 3 pigs at 2 weeks (n = 2) and 4 weeks (1) after vaccination. No differences were found in isolation of B suis or in seroconversion between vaccinated and control gilts and between their neonates or aborted fetuses.

Conclusions and Clinical Relevance—Parenteral vaccination with SRB51 does not induce humoral or cell-mediated immune responses. Vaccination with SRB51 did not protect gilts or their neonates and fetuses from virulent challenge with B suis.

Swine brucellosis, which is caused by the bacterium Brucella suis, is found in domestic or feral pigs on all inhabited continents.1 Manifestations of swine brucellosis range from subclinical infection to abortions and infertility.1 In addition to its effects on female reproduction cycles and pregnancy, swine brucellosis may also manifest as infection and inflammation of primary and secondary reproductive organs of males as well as multicentric arthritis, diskospondylitis, and lymphadenitis in all aged pigs of both sexes.1 Of all the Brucella spp, B suis is most noteworthy for causing chronic and persistent infections.2,3Brucella suis is a zoonotic agent that has been known to infect slaughterhouse workers, farm workers, and hunters who have been exposed to tissues from B suis infected pigs.4,5

In the United States, the Cooperative State-Federal Brucellosis Eradication Program has led to the near elimination of swine brucellosis within domestic pigs. Currently, 49 states and Puerto Rico are classified as free of swine brucellosis (ie, stage III) while Texas is classified as stage II (> 1 B suis–infected herd identified in the past 2 years).6 However, periodic, isolated outbreaks of swine brucellosis still occur within the United States. Most of these outbreaks are attributed to contact with feral pigs. Feral pigs have also been responsible for infecting cattle with B suis.7

The reduction and near eradication of swine brucellosis from domestic herds within the United States have been achieved through a systematic program of herd testing, slaughter testing, traceback of reactors, and disposal of reactor herds. With the near complete eradication of swine brucellosis from domestic pigs in the United States, a reemergence of interest exists in new strategies, including candidate vaccines, to control swine brucellosis, particularly in light of the wide distribution of Brucella-infected feral pigs across the United States.

Brucella abortus strain RB51 is a laboratory-derived lipopolysaccharide O-side chain-deficient mutant of B abortus strain 2308.8Brucella abortus strain RB51 induces protective immunologic responses in cattle and bison against challenge exposure with virulent B abortus strains.9–11 Brucella abortus strain RB51 also does not induce antibody responses that react with conventional brucellosis serologic surveillance tests.12,13 Initial reports suggested that SRB51 induces an immune response and prevents transmission of B suis in pigs.14,15 The purposes of the study reported here were to determine the potential of parenteral administration of SRB51 to elicit an immune response as well as protection from challenge with virulent B suis in domestic pigs.

Materials and Methods

Bacterial cultures—A master seed stock of SRB51 was obtained.a After 1 passage on TSA, the seed stock was designated ARS/1. For experimental use in serologic and lymphocyte proliferation assays, SRB51 (ARS/1) bacteria were grown on TSAb for 48 hours at 37°C. Resulting cultures were suspended in PBS (0.15M) solution at a concentration of 1.3 × 1012 CFUs/mL and inactivated by γ-irradiation (1.4 × 106 rad). After irradiation, suspensions were washed in 0.15M NaCl solution and stored in 1.0-mL aliquots at −70°C.

For vaccination of pigs, SRB51 (ARS/1) was expanded on TSA for 48 hours at 37°C with 5% CO2. Harvested bacteria were suspended in PBS (0.15M NaCl) solution and then diluted to a concentration of 1.0 × 1010 CFUs/mL in PBS (0.15M NaCl) solution by use of an optical density method and spectrophotometer.c The final concentration was determined by standard plate counts on TSA after a 5-day incubation period at 37°C and 5% CO2.

The challenge culture B suis strain 3B (biovar 1) was originally obtained from an aborted fetus of a sow exposed to a polyvalent suspension of 3 strains of B suis. The 3 strains had previously been isolated from boars originating from 3 sources. Brucella suis strain 3B has been maintained as a lyophilized culture since 1942. Brucella suis strain 3B was grown on TSA for 48 hours at 37°C with 5% CO2. Bacteria were harvested and suspended in PBS (0.15M) solution and diluted to a concentration of 1 × 109 CFUs/mL by use of an optical density method and a spectrophotometer.c The final concentration was determined by standard plate counts on TSA after a 5-day incubation period at 37°C and 5% CO2.

Swine experiments and study design—The work reported herein was performed under the approval of the Institutional Animal Care and Use Committee of the National Animal Disease Center (Ames, Iowa). Vaccine clearance and induction of an immune response were initially assessed in weaned pigs (experiment 1). A total of sixty-eight 6-week-old crossbred domestic pigs were inoculated IM in the right cervical area with either 2.0 mL of PBS (0.15M) solution alone (n = 17) or 2.0 mL of PBS (0.15M) solution containing 2.0 × 1010 CFUs of SRB51 (51). All pigs were commingled in a single pen and fed ad libitum.

Induction of an immune response and protection from virulent challenge with B suis strain 3B were assessed in crossbred domestic gilts (experiment 2). A total of twenty-four 4-month-old gilts were inoculated IM in the right cervical area with 2.0 mL of PBS (0.15M) solution containing 2 × 1010 CFUs of SRB51 (n = 18) or sham inoculated with 2.0 mL of PBS (0.15M) solution (8). At approximately 10 months of age, gilts were bred by artificial insemination. Pregnancy was verified by use of ultrasonography. Fourteen vaccinates and 8 control gilts were successfully bred and challenged at approximately day 75 of gestation by bilateral conjunctival administration of 5.0 × 107 CFUs of B suis strain 3B (volume, 50 μL/eye).

Serologic evaluation—Blood was collected for serologic evaluation by cranial vena cava puncture in experiment 1 (weaned pig group) at weeks 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 after vaccination and in experiment 2 (gilt group) at weeks 0, 2, 4, 8, 12, 16, 20, and 24 after vaccination. Blood samples were allowed to clot, and serum was separated by centrifugation. Serum was divided into 1-mL aliquots and stored at −70°C until assays were performed. Serum antibody titers to SRB51 were determined by use of a previously described dot blot assay in which γ-irradiated SRB51 was used as the antigen and peroxidase-labeled rabbit anti-swine IgGd was used as the secondary antibody at a dilution of 1:500.16

Seroconversion as a result of challenge with the B suis strain was determined by use of fluorescence polarization,17 standard tube agglutination,18 and card agglutination assays18 with previously described methods that use B abortus antigen. Blood was collected on day 0 of challenge from all gilts and at necropsy from all gilts, neonates, and fetuses. Pigs that had a positive reaction on ≥ 1 of the 3 serologic assays were considered seroreactors.

PBMC proliferation assays—Blood samples (45 mL) were collected into acid-citrate-dextrose solution from the cranial vena cava at weeks 8, 12, 14, 16, 18, and 20 after vaccination from pigs in experiment 1 and at weeks 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 after vaccination from the gilts in experiment 2. Peripheral blood mononuclear cells were enriched by density centrifugation with a Ficoll-sodium diatrizoate gradient.d

Fifty microliters of each cell suspension containing 5 × 105 PBMCs was added to each of 2 separate flat-bottom wells of 96-well microtiter plates that contained 100 μL of RPMI 1640 medium only or RPMI 1640 medium containing γ-irradiated SRB51 (105 to 109 bacteria/well). Cell cultures were incubated for 4 days at 37°C under 5% CO2. Plates were then pulsed with 1.0 mCi of [3H] thymidine/well for 18 hours. Cells were harvested onto glass filter mats and counted for radioactivity in a liquid scintillation counter.e

Necropsy examination—All pigs were euthanatized by administration of sodium pentobarbital into the cranial vena cava. Tissues collected for bacterial culture were collected with an aseptic technique, placed into individual containers, and immediately frozen at −70°C until processed. Tissues collected for histologic evaluation were immediately placed in neutral-buffered 10% formalin, processed by routine paraffin-embedding techniques, cut in 4-μm-thick sections, and stained with H&E.

In experiment 1, 4 vaccinates and 1 control pig were necropsied at weeks 1, 2, 3, 4, 5, and 6 after vaccination. Five vaccinates and 2 controls were necropsied at weeks 8, 12, 16, and 20 after vaccination, and the remaining 7 vaccinates and 3 controls were necropsied at week 23 after vaccination. Blood and urine were collected for bacterial culture at necropsy. The following tissues were collected for bacterial culture and histologic evaluation: liver, spleen, kidney, lung, pharyngeal tonsils, and lingual tonsil as well as prescapular, medial retropharyngeal, sternal, tracheobronchial, mediastinal, gastrohepatic, ileocecal, jejunal, renal, iliac, inguinal, prefemoral, popliteal, mandibular, and parotid lymph nodes.

In experiment 2, all neonates or fetuses were euthanatized and necropsied within a few hours of parturition or abortion, respectively. Samples of whole blood, CSF, and stomach contents and rectal swab specimens were obtained for bacterial culture, and lung, liver, spleen, and kidney tissue specimens were obtained for bacterial culture and histologic evaluation. Gilts were euthanatized and necropsied within 2 days of parturition (or abortion). Samples of whole blood, milk, and urine and vaginal swab specimens were obtained for bacterial culture, and lung, liver, spleen, kidney, mammary gland, uterus, and placenta tissue specimens as well as tracheobronchial, prescapular, medial retropharyngeal, mandibular, parotid, sternal, iliac, prefemoral, popliteal, gastrohepatic, and ileocecal lymph nodes were obtained for bacterial culture and histologic evaluation.

Bacterial culture—In experiment 1, whole blood was collected for bacterial culture at days 0, 7, 10, 14, 17, 21, 24, and 28 and weeks 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, and 23 after vaccination. In experiment 2, whole blood was collected for bacterial culture at day 0 and weeks 2, 4, 8, 12, 16, 20, and 24 after vaccination. Samples of whole blood and conjunctival, nasal, and vaginal swab specimens were collected after virulent challenge with B suis strain 3B on days 0, 3, 7, and 10 and weekly thereafter until parturition or abortion.

Whole blood for bacterial culture was immediately placed into tryptose broth containing acid citrate dextrose (1:1 [vol/vol]) and incubated for 7 days at 37°C and 5% CO2prior to plating on Brucella selective media. Urine, stomach contents, CSF, and milk were incubated in tryptose broth containing 5% bovine serum (1:3 [vol/vol]) for 7 days at 37°C and 5% CO2 prior to plating on Brucella selective media. After thawing, tissues were individually ground in approximately 10% (wt/vol) sterile PBS solution (pH, 7.2) with sterile glass grinders. Aliquots (100 μL) of each tissue homogenate or preincubated tryptose broth (blood, urine, stomach contents, CSF, and milk) were plated on 4 Brucella selective media. Media included tryptose agar containing 5% bovine serum; brilliant green agar (containing tryptose agar base with 5% bovine serum, brilliant green [0.001 μg/mL], bacitracin [25 U/mL], cycloheximide [100 μg/mL], nystatin [100 U/L], vancomycin [20 μg/mL], vancomycin [50 μg/mL], and EDTA [100 μg/mL]); Kuzdas-Morse selective medium (containing tryptose agar base with 5% bovine serum, bacitracin [25 U/mL], polymyxin B [6 U/mL], cycloheximide [100 μg/mL], and ethyl violet [1.4 μg/mL]); and RBM agar, a selective medium for SRB51.19 Inoculated plates were incubated at 37°C in 5% CO2 for 7 days.

Bacterial culture results—Suspect cultures of Brucella spp were identified on the basis of colony morphology, growth characteristics,18 and growth on selective media. Isolates were identified as Brucella spp by use of a PCR technique with Brucella specific primers to the omp2A region of the Brucella genome. Reactions consisted of 50 μL and contained 5 μL of the suspect culture of Brucella spp in tris-EDTA and 45 μL of reaction mixture consisting of 200μM each of dATP, dCTP, dGTP, and dTTPf; 1X PCR buffer IIf; 1.5mM MgCl2; 1.25 units of Taq polymeraseg; and 0.2mM of each upstream (5'-GCAACGGTGTTCTTCCACTC-3') and downstream (5'-GTATCAGGCTACGCAGAAGG-3') primer selected from the omp2A sequences of B abortus.20 Primers had 100% conservancy within the genomes of B suis and Brucella melitensis according to the results of a basic local alignment search tool (ie, BLAST) analysis. Tris-EDTA and a culture of Yersinia enterocolitica O:9 served as negative controls, and B suis strain 3B served as a positive control. Following a 10-minute activation at 95°C, reaction preparations were cycled in a ther mocyclerh for 40 cycles consisting of 30 sec onds at 95°C, 30 seconds at 44°C, and 60 seconds at 72°C. Products were analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide. Pigs were considered infected with B suis strain 3B if ≥ 1 sample yielded a positive Brucella culture result.

Cultures that had positive results for Brucella spp by the omp2A PCR assay were run in a second PCR assay to determine whether they were SRB51. This PCR assay used SRB51 specific primers targeted toward the insertion sequence 711.21,22 Each reaction mixture consisted of a volume of 25 μL containing 2.5 μL of the suspect culture of Brucella spp in Tris-EDTA, 1X PCR reaction buffer containing 50mM Tris; 1.5mM MgCl2; 10mM KCl; 50mM (NH4)2SO4; pH 8.3i; 200μM each of dATP, dCTP, dGTP, and dTTPf; 1X guanine-cytosine rich solutioni; 1.0 unit of DNA polymerasej; and 0.2μM of each upstream (5'-TGCCGATCACTTAAGGGCCTTCATTGCCAG-3') and downstream (5'-GCCAACCAACCCAAATGCTCACAA-3') primer. Thermocycling consisted of a single 5-minute incubation at 95°C followed by 40 cycles consisting of 15 seconds at 95°C, 30 seconds at 52°C, and 90 seconds at 72°C. Tris-EDTA and B suis strain 3B served as negative controls, and SRB51 served as the positive control. Products were analyzed by electrophoresis on 2.0% agarose gels stained with ethidium bromide.

Statistical analysis—For all analyses, a level of P < 0.05 was used to determine significant differences between vaccinates and controls. Serologic response data to SRB51 were converted to the logarithm of the titer for analysis. Resulting values were compared between vaccinates and controls by use of a repeated-measures, general linear-model procedure.k Proliferative responses to SRB51 were converted to the logarithm of the mean cpm and compared between vaccinates and controls by use of a general linear-model procedure and least square means.k A χ2 test was used to determine differences in positive bacterial culture results and seropositivity between neonates and fetuses from vaccinated versus control gilts.l The Fisher exact test was used to determine differences between vaccinates and controls on a per litter basis and to determine differences in positive bacterial culture results, seroconversion, and histologic lesions between vaccinated and control gilts or between neonates and fetuses from vaccinated or control gilts.l

Results

Serologic evaluation—The SRB51 dot blot titers were analyzed to determine serum antibody titers to SRB51 (Figures 1 and 2). No significant differences in serum antibody titers to SRB51 were found between vaccinates and controls in both experiments, with the exception of week 22 in experiment 1; however, at this time point, the mean titer of the control group was significantly higher than the vaccinated group.

Figure 1—
Figure 1—

Mean ± SEM serum antibody titer to SRB51 in vaccinated or control weaned pigs on the basis of γ-irradiated SRB51 dot-blot assay results. Pigs were vaccinated with 2.0 × 1010 CFUs of SRB51 (closed bars; n = 51) or sham inoculated with an equal volume of PBS solution (open bars; 17).

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1802

Figure 2—
Figure 2—

Mean ± SEM serum antibody titer to SRB51 in vaccinated or control gilts on the basis of γ-irradiated SRB51 dot-blot assay results. Gilts were vaccinated with 2.0 × 1010 CFUs of SRB51 (closed bars; n = 18) or sham inoculated with an equal volume of PBS solution (open bars; 8).

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1802

Seroconversion rates for control and vaccinated gilts that were challenged with B suis strain 3B during gestation were 7 of 8 and 11 of 14 gilts, respectively (P = 0.40). Seropositivity rate was 27.8% (25/90) for neonates and fetuses from control gilts and 35.1% (60/171) for neonates and fetuses from vaccinated gilts (P = 0.23).

PBMC proliferation assays—Analysis of data from PBMC proliferation assays revealed that at all time points in both experiments, no significant difference was found in mean cpm between the vaccinated and control groups (Figures 3 and 4). At each time point, the pokeweed mitogen positive control wells yielded a mean cpm of > 100,000 for the control and vaccinated groups.

Figure 3—
Figure 3—

Mean cpm ± SEM in PBMC blastogenesis assays of weaned pigs vaccinated with 2.0 × 1010 CFUs of SRB51 (closed bars, stimulated; gray bars not stimulated; n = 9) or sham inoculated with an equal volume of PBS solution (open bars, stimulated; hatched bars, not stimulated; 6).

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1802

Figure 4—
Figure 4—

Mean cpm ± SEM in PBMC blastogenesis assays of gilts vaccinated with 2.0 × 1010 CFUs of SRB51 (closed bars, stimulated; gray bars, not stimulated; n = 18) or sham inoculated with an equal volume of PBS solution (open bars, stimulated; hatched bars, not stimulated; 8).

Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1802

Bacterial culture results—In experiment 1, SRB51 was isolated from 3 of 51 vaccinated pigs at the following time points: the lingual tonsil and sternal lymph node in 1 pig at 2 weeks after vaccination; the tracheobronchial lymph node from 1 pig at 2 weeks after vaccination; and the prescapular lymph node from 1 pig at 4 weeks after vaccination. Brucella abortus strain RB51 was not isolated from blood cultures from any pigs at any time point in experiments 1 and 2, and SRB51 was also not isolated from samples obtained at necropsy from sham-inoculated control pigs in experiment 1.

Recovery of B suis after inoculation and from tissues collected at necropsy from control and vaccinated gilts and their neonates and fetuses was recorded (Table 1). Polymerase chain reaction analysis of all isolates obtained after inoculation and also at necropsy confirmed that none were SRB51. Pigs were considered infected with B suis strain 3B if ≥ 1 sample (ie, tissues, blood, swab specimens, or fluids) yielded a positive Brucella culture result. No significant differences in positive bacterial culture result rates were found between control and vaccinated gilts for samples from the after inoculation-antemortem period (P = 0.14) and from samples obtained at necropsy (P = 0.39). Likewise, no significant differences were found in positive bacterial culture result rates between neonates and fetuses from control gilts or vaccinated gilts when examined on a per neonate-fetus basis (P = 0.46) or a per litter basis (P = 0.47).

Table 1—

Recovery of Brucella suis from individual neonates and farrowed litters at necropsy and gilts after inoculation and at necropsy.*

VariablesCulture results
PositiveNegative
Neonates and fetuses
No. from vaccinates96 (56.1%)75 (43.9%)
No. from control gilts55 (61.1%)35 (38.9%)
Litters
No. from vaccinates12 (85.7%)2 (14.3%)
No. from control gilts7 (87.5%)1 (12.5%)
Gilts after inoculation, antemortem
No. of vaccinates8 (57.1%)6 (42.9%)
No. of control gilts7 (87.5%)1 (12.5%)
Gilts at necropsy
No. of vaccinates12 (85.7%)2 (14.3%)
No. of control gilts8 (100%)0 (0%)

Gilts were challenged with 5.0 × 107 CFUs of B suis strain 3B on day 75 of gestation.

Lesions—No gross lesions were observed in pigs of experiment 1, and with the exception of purulent endometrial exudate in 1 vaccinated gilt, no gross lesions were observed in gilts of experiment 2. No noteworthy histologic lesions were found in pigs from experiment 1. Histologic lesions in the gilts from experiment 2 and their neonates and fetuses were recorded. Purulent lymphadenitis characterized by mild to moderate numbers of neutrophils present within sinusoids of interfollicular areas of multiple lymph nodes was present in 5 gilts (5 vaccinates). Lymph nodes affected included medial retropharyngeal (2), mandibular (1), gastrohepatic (1), sternal (1), popliteal (1), iliac (1), and tracheobronchial (1). Portal hepatitis characterized by a low to moderate number of neutrophils, lymphocytes, and plasma cells surrounding portal vessels and bile ducts was present in 5 gilts (2 controls and 3 vaccinates). Five gilts (2 controls and 3 vaccinates) had purulent endometritis characterized by low to moderate numbers of neutrophils within the mucosa and lamina propria of the uterus. In 1 control gilt, moderate numbers of lymphocytes and plasma cells were present in addition to neutrophils. Five gilts (1 control and 4 vaccinates) had interstitial nephritis characterized by multifocal areas of low to moderate number of neutrophils, lymphocytes, and plasma cells within cortical interstitial areas. No significant (P = 0.34) difference was found between the number of control and vaccinated gilts that had histologic lesions consistent with Brucella infection.

Histologic lesions consistent with Brucella infection were present in 21.2% (19/90) of neonates and fetuses from control gilts and 23.3% (40/171) of neonates and fetuses from vaccinated gilts. Diffuse, mild to moderate, purulent interstitial pneumonia characterized by low to moderate numbers of neutrophils infiltrating alveolar septa was present in 18.9% (17/90) of neonates and fetuses from control gilts and 19.9% (34/171) of neonates and fetuses from vaccinated gilts. Multifocal, purulent, portal hepatitis characterized by mild to moderate numbers of neutrophils surrounding vessels and bile ducts within portal areas of the liver was present in 2 neonates and fetuses from control gilts and 6 neonates and fetuses from vaccinates gilts. No significant (P = 0.83) difference was found between the number of neonates and fetuses from control gilts and vaccinated gilts that had histologic lesions consistent with Brucella infection.

Discussion

Results of our study indicate that single-dose parenteral vaccination with SRB51 is not sufficiently immunogenic to protect against virulent challenge with B suis strain 3B in domestic pigs. In experiments 1 and 2, vaccinated pigs failed to develop antibody titers against γ-irradiated SRB51 that were significantly higher than those of the nonvaccinated controls. Serum antibody titers examined in cattle in which SRB51 has been found to be immunogenic and efficacious were significantly higher in vaccinated pigs than in nonvaccinated controls at weeks 4 to 20 after vaccination23,24 Also, no significant difference was found in seroconversion between control and vaccinated gilts and between neonates and fetuses from control and vaccinated gilts after challenge with virulent B suis.

No evidence exists from our study indicating that SRB51 elicits a strong cell-mediated immune response after parenteral vaccination in pigs. Results of PBMC proliferation assays after stimulation with γ-irradiated SRB51 did not significantly differ between controls and vaccinated pigs in experiments 1 and 2. Brucella abortus strain RB51 has been shown to elicit robust proliferative responses in other species. Bison parenterally vaccinated with 1.2 to 6.1 × 1010 CFUs of SRB51 have significantly higher PBMC proliferation responses than nonvaccinated controls at weeks 12 and 18 after vaccination,25 and the same dosage of SRB51 in a subsequent study11 was demonstrated to be protective in bison. Likewise, cattle parenterally vaccinated with 1 × 1010 CFUs of SRB51 had PBMC proliferative responses that were significantly higher than those of controls at weeks 10, 12, 14, and 16 after vaccination, and cattle parenterally vaccinated with 3 × 109 CFUs of SRB51 had PBMC proliferative responses that were significantly higher than those of controls at weeks 14 and 18 after vaccination.23,24 Both doses were associated with protection from virulent B abortus challenge.23,24 It has been shown that pigs that are infected with B suis are capable of eliciting a significantly higher PBMC proliferative response over noninfected controls.26

Data from the weaned pigs of experiment 1 suggest that SRB51 is cleared quickly in pigs. Isolation of SRB51 was achieved in only 3 pigs (2 at week 2 after vaccination and 1 at week 4 after vaccination). The elicitation of a protective immune response has been associated with longer persistence of SRB51 within regional lymph nodes. In bison, bacterial titers of SRB51 were found to peak at week 2 after vaccination and steadily decline through week 18 after vaccination, with total vaccine clearance achieved by week 24 after vaccination.25,27,28 In cattle, SRB51 has been shown to persist until week 14 after vaccination within the regional lymph nodes of some cattle that were vaccinated with protective doses.29

The hallmark of Brucella vaccine efficacy has long been the reduction in recovery of Brucella organisms in maternal and fetal tissues after virulent challenge.11,24,29 Our study failed to show a significant reduction in the recovery of B suis organisms on a per gilt, per neonatefetus, or per litter basis.

Results of our study are contrary to those reported by Lord et al.15 In their study, SRB51 was used to parenterally vaccinate domestic gilts IM and also PO at doses of 106 to 109 CFUs. In that study, the lipopolysaccharide O-side chain from B abortus or B suis was also used to vaccinate pigs IM or PO. It was concluded that all vaccine preparations provided 100% protection on the basis of the lack of seroconversion, lack of abortions, and increased litter size over those of the control groups. A major difference between the study by Lord et al15 and our study is that in their study, the challenge dose was not defined; vaccinated gilts were bred to boars previously determined to be shedding B suis in their semen; however, the culture status of the semen from the boars used for sire was not determined on the days of insemination. Our study used a defined challenge dose of a single strain of virulent B suis for conjunctival administration. In the study by Lord et al,15 the Brucella-culture status of all pigs was not examined.15 Cultures were performed only on vaginal swab specimens from sows that aborted and on tissues from aborted fetuses. Samples from pigs that had full-term pregnancies were not cultured. In our study, we determined the culture status on the basis of a full complement of tissues from vaccinated and control gilts as well as from their neonates and fetuses to assess the efficacy of vaccination with SRB51.

Edmonds et al14 also examined SRB51 vaccination of domestic pigs with 109 to 1012 CFUs/dose.14 Results of that study indicate that some pigs can develop a humoral immune response against SRB51 after SC or oral vaccination. Unlike our study, the study by Edmonds et al14 did not have any nonvaccinated control pigs to which the results of the SRB51 vaccinated pigs could be compared.

Results of our study are similar to those of other studies30,31 in which B abortus strain 19 was examined as a vaccine candidate for control of swine brucellosis. Brucella abortus strain 19 was found to not confer demonstrable immunity and protection against virulent challenge with B suis.30,31 Cedro et al,32,33 however, reported efficacy of a live B abortus vaccine strain when coadministered with heat-killed B suis and lipopolysaccharide from B suis.32,33 This compound vaccine was found to decrease abortion rates as well as culture recovery of B suis.

Although Brucella vaccines traditionally have not been 100% efficacious under experimental conditions in preventing maternal infection, fetal infection, and lesions, the SRB51 and B abortus strain 19 vaccines greatly reduce the occurrence of all of these after virulent challenge in cattle. In our study, none of these conditions were met; therefore, SRB51 does not appear to be a suitable vaccine candidate for the control of B suis infection in pigs.

ABBREVIATIONS

SRB51

Brucella abortus strain RB51

TSA

Tryptose serum agar

PBMC

Peripheral blood mononuclear cell

cpm

Counts per minute

a.

Dr. Gerhardt Schurig, Virginia Tech, Blacksburg, Va

b.

Difco Laboratories, Detroit, Mich

c.

Beckman, Palo Alto, Calif

d.

Sigma Chemical Co, St Louis, Mo.

e.

1450 Microbeta scintillation counter, Wallac Inc, Gaithersburg, Md

f.

Boehringer Mannheim, Indianapolis, Ind

g.

AmpliTaq Gold polymerase, Perkin Elmer, Branchburg, NJ

h.

MJ Research Inc, Watertown, Mass

i.

FastStart DNA polymerase, Roche Molecular Biochemicals, Indianapolis, Ind

j.

Roche Diagnostics, Basel, Switzerland

k.

PROC GLM LSMEANS, SAS Statistical Software, SAS Institute Inc, Cary, NC

l.

PROC FREQ CHISQ, SAS Statistical Software, SAS Institute Inc, Cary, NC.

References

  • 1.

    MacMillan AP, Schleicher H & Korslund J, et al. Brucellosis. In: Straw BE, Zimmerman JJ, D'Allaire S, et al, eds. Diseases of swine. 9th ed. Ames, Iowa: Blackwell Publishing, 2006;603612.

    • Search Google Scholar
    • Export Citation
  • 2.

    Deyoe BL. Immunology and public health significance of swine brucellosis. J Am Vet Med Assoc 1972;160:640643.

  • 3.

    Deyoe BL, Manthei CA. Sites of localization of Brucella suis in swine. Proc Annu Meet U S Anim Health Assoc 1967;71:102108.

  • 4.

    Starnes CT, Talwani R & Horvath JA, et al. Brucellosis in two hunt club members in South Carolina. J S C Med Assoc 2004;100:113115.

  • 5.

    Trout D, Gomet TM & Bernard BP, et al. Outbreak of brucellosis at a United States pork packing plant. J Occup Environ Med 1995;37:697703.

  • 6.

    Veterinary Services. National Center for Animal Health Programs. Status of current eradication programs. Available at: www.aphis.usda.gov/vs/nahps/domestic-animal.html. Accessed Jan 24, 2006.

    • Search Google Scholar
    • Export Citation
  • 7.

    Ewalt DR, Payeur JB & Rhyan JC, et al. Brucella suis biovar 1 in naturally infected cattle: a bacteriological, serological, and histological study. J Vet Diagn Invest 1997;9:417420.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Schurig GG, Roop RM & Bagchi T, et al. Biological properties of RB51; a stable rough strain of Brucella abortus. Vet Microbiol 1991;28:171188.

  • 9.

    Cheville NF, Stevens MG & Jensen AE, et al. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus. Am J Vet Res 1993;54:15911597.

    • Search Google Scholar
    • Export Citation
  • 10.

    Cheville NF, Olsen SC & Jensen AE, et al. Effects of age at vaccination on efficacy of Brucella abortus strain RB51 to protect cattle against brucellosis. Am J Vet Res 1996;57:11531156.

    • Search Google Scholar
    • Export Citation
  • 11.

    Olsen SC, Jensen AE & Stoffregen WC, et al. Efficacy of calfhood vaccination with Brucella abortus strain RB51 in protecting bison against brucellosis. Res Vet Sci 2003;74:1722.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Stevens MG, Hennager SG & Olsen SC, et al. Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51. J Clin Microbiol 1994;32:10651066.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Olsen SC, Evens D & Hennager SG, et al. Serologic responses of calfhood-vaccinated cattle to Brucella abortus strain RB51. J Vet Diagn Invest 1996;8:451454.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Edmonds MD, Samartino LE & Hoyt PG, et al. Oral vaccination of sexually mature pigs with Brucella abortus vaccine strain RB51. Am J Vet Res 2001;62:13281331.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Lord VR, Cherwonogrodzky JW & Schurig GG, et al. Venezuelan field trials of vaccines against brucellosis in swine. Am J Vet Res 1998;59:546551.

  • 16.

    Olsen SC, Stevens MG & Cheville NF, et al. Experimental use of a dot blot assay to measure serologic responses of cattle vaccinated with Brucella abortus strain RB51. J Vet Diagn Invest 1997;9:363367.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Paulo PS, Vigliocco AM & Ramondina RF, et al. Evaluation of primary binding assays for presumptive serodiagnosis of swine brucellosis in Argentina. Clin Diagn Lab Immunol 2000;7:828831.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Alton GG, Jones LM & Angus RD, et al. Techniques for the brucellosis laboratory. Paris: Institut National de la Recherche Agronomique, 1988;17136.

    • Search Google Scholar
    • Export Citation
  • 19.

    Hornsby RL, Jensen AE & Olsen SC, et al. Selective media for isolation of Brucella abortus strain RB51. Vet Microbiol 2000;73:5160.

  • 20.

    Ficht TA, Bearden SW & Sowa BA, et al. DNA sequence and expression of the 36 kilodalton outer membrane protein gene of Brucella bortus. Infect Immun 1989;57:32813291.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Vemulpalli R, McQuiston JR & Schurig GG, et al. Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains. Clin Diagn Lab Immunol 1999;6:760764.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Bricker BJ, Halling SM. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol 1995;33:16401642.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Olsen SC. Responses of adult cattle to vaccination with a reduced dose of Brucella abortus strain RB51. Res Vet Sci 2000;69:135140.

  • 24.

    Olsen SC. Immune responses and efficacy after administration of a commercial Brucella abortus strain RB51 to cattle. Vet Ther 2000;1:183191.

    • Search Google Scholar
    • Export Citation
  • 25.

    Olsen SC, Jensen AE & Palmer MV, et al. Evaluation of serologic responses, lymphocyte proliferative responses, and clearance from lymphatic organs after vaccination of bison with Brucella abortus strain RB51. Am J Vet Res 1998;59:410415.

    • Search Google Scholar
    • Export Citation
  • 26.

    Kaneene JM, Anderson RK & Johnson DW, et al. Cell-mediated immune responses in swine from a herd infected with Brucella suis. Am J Vet Res 1978;39:16071611.

    • Search Google Scholar
    • Export Citation
  • 27.

    Olsen SC, Cheville NF & Kunkle RA, et al. Bacterial survival, lymph node pathology, and serological responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19. J Wildl Dis 1997;33:146151.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Olsen SC, Kreeger TJ, Schultz W. Immune responses of bison to ballistic or hand vaccination with Brucella abortus strain RB51. J Wildl Dis 2002;38:738745.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Olsen SC, Bricker B & Palmer MV, et al. Responses of cattle to two dosages of Brucella abortus strain RB51: serology, clearance and efficacy. Res Vet Sci 1999;66:101106.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Manthei CA. Research on swine brucellosis by the bureau of animal industry (1941–1947). Am J Vet Res 1948;9:4045.

  • 31.

    Juerado FR, Cedro VCF, Morán BL. Vacunacion de porcinos con Brucella abortus cepa 19. Ministerio de Agricultura y Ganaderia Publicacion Miscelanea 1950;327:127.

    • Search Google Scholar
    • Export Citation
  • 32.

    Cedro VCF, Cisale HO & Cacchione RA, et al. Vacunacion antibrucelica de porcinos a campo. Revista de Investigaciones Ganaderas 1959;7:297314.

  • 33.

    Cedro VCF, Cisale HO, Barrantes R. Vacunacion contra la brucellosis porcina 2° ensayo a campo. Revista de Investigaciones Ganaderas 1960;10:337345.

    • Search Google Scholar
    • Export Citation
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