Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens

Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.     

Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens

Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.     

Brucella abortus strain RB51 vaccination in elk. II. Failure of high dosage to prevent abortion

Brucella abortus strain RB51 is used as a vaccine because it induces antibodies that do not react on standard serologic tests for brucellosis allowing differentiation between vaccination and infection. Strain RB51 was evaluated in captive elk (Cervus elaphus) to determine if vaccination protected against abortion following experimental challenge. Thirty elk were vaccinated intramuscularly with 1.0 x 10(10) colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 x 10(10) CFU exactly 1 yr later. All vaccinated elk seroconverted via a modified dot blot assay to strain RB51 with the booster group having higher titers (P < or = 0.001). Seventeen other elk served as unvaccinated controls. All elk were bred and determined pregnant using pregnancy-specific protein B analysis. Elk were challenged in March 2000 with 1.1 x 10(7) CFU of B. abortus strain 2308 administered intraconjunctivally and all elk seroconverted to strain 2308. Fifteen of 17 control elk aborted; 16 of 16 elk given a single vaccination aborted (P = 0.44); and 13 of 14 elk given a booster aborted (P = 0.86). There were two viable calves in the control group and one in the booster group. Strain 2308 was recovered from fetuses and nonviable calves in all groups. Based on the results of this and other studies, the use of strain RB51 to prevent abortion in elk cannot be recommended.     

Combined S99/RB51 antigen for complement fixation test for serological diagnosis of brucellosis in cattle and sheep

AIMS: To assess the efficiency of a single antigen for the complement fixation (CF) test, prepared by combining Brucella abortus smooth strain 99 (S99) with Brucella abortus rough strain RB51(RB51), in detecting cattle and sheep infected or vaccinated with Brucella spp. METHODS AND RESULTS: Serum samples from B. abortus-infected and RB51-vaccinated cattle were tested by the CF test using S99, RB51 and the combined S99/RB51 as antigens. Likewise, serum samples from Brucella melitensis-infected, RB51-vaccinated and Brucella ovis-infected sheep were tested by the CF test using S99, RB51, hot saline (HS) and combined S99/RB51 as antigens. Comparative analysis of the CF results showed that no reduction of sensitivity or specificity occurs when S99/RB51 antigen is used instead of specific antigens used separately. CONCLUSIONS: The results of this study indicated that combined S99/RB51 antigen used in the CF test, because of its specificity and sensitivity, could be used in animal brucellosis surveillance systems to improve the efficiency of the preliminary screening of herds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes an improved antigen for the CF test for the epidemiological survey of animal brucellosis. It could represent advantages over standard protocols because of its ability to detect antibody responses following infection or vaccination withBrucella strains of rough and smooth phenotype.     

Immune responses of elk to vaccination with Brucella abortus strain RB51

In a study conducted from January to August 2000, elk (Cervus elaphus) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6) or injected with 0.15 M NaCl solution (n = 3) at approximately 6 mo of age. Beginning at 2 wk and continuing to 25 wk after vaccination, SRB51-vaccinated elk had greater antibody responses (P < 0.05) to SRB51 when compared to nonvaccinated elk. Peripheral blood mononuclear cells (PBMC) from SRB51-vaccinated elk had greater (P < 0.05) proliferative responses to SRB51 at 18 wk after vaccination when compared to responses of nonvaccinated elk. Strain RB51 was recovered from blood samples of all vaccinates at 2 wk, and three of six vaccinates at 4 wk after vaccination. The SRB51 vaccine strain was recovered from the superficial cervical lymph node of all vaccinates sampled at 6 wk after vaccination. but not from lymph node samples obtained from vaccinates at 12 or 18 wk after vaccination. At 34 wk after vaccination, SRB51 was recovered from the bronchial lymph node of one of five vaccinates but not from other tissues. Strain RB51 was not recovered at any time from samples obtained from nonvaccinated elk. This study suggests that following vaccination with SRB51, elk remain bacteremic for a prolonged period of time, rapidly develop high antibody titers, and are slower to develop detectable proliferative responses in PBMC when compared to responses of cattle or bison (Bison bison).     

Safety and efficacy of Brucella abortus strain RB51 vaccine in captive pregnant elk

Brucella abortus strain RB51 is a laboratory-derived rough mutant of virulent B. abortus strain 2308 used as a vaccine because it induces antibodies that do not react on standard brucellosis serologic tests. Strain RB51 vaccine was evaluated in pregnant captive elk (Cervus elaphus) to determine (1) if it induced abortion and (2) if it protected against abortion following subsequent challenge. The time period of this study (February-June, 1998) was similar to field conditions where elk are vaccinated and possibly exposed to B. abortus. Fourteen elk were randomly and equally divided into vaccinated and control groups. The vaccinated group was vaccinated intramuscularly with 1.03 x 10(10) colony-forming units (CFU) of strain RB51 and seroconverted postvaccination. Antibodies to strain RB51 were detected by a modification of an existing dot-blot assay. Both groups were challenged 40 days postvaccination with 9.8 x 10(6) CFU of B. abortus strain 2308 administered intraconjunctivally. The first abortion occurred 38 days postchallenge. Abortion occurred in all control elk and in five of seven vaccinated elk 5 to 12 wk postchallenge (P = 0.23). Mixed strain RB51 and 2308 infections were present in fetuses and vaginas from the vaccinated group whereas only strain 2308 was cultured from control group fetuses and vaginal swabs. Further evaluation of strain RB51 will be necessary to determine if it will be safe and efficacious in free-ranging pregnant elk.     

Immune responses of bison to ballistic or hand vaccination with Brucella abortus strain RB51

From January through July of 2000, a study was conducted to evaluate clearance, immunologic responses, and potential shedding of Brucella abortus strain RB51 (SRB51) following ballistic or subcutaneous (SQ) vaccination of 7 mo old bison (Bison bison) calves. Ten bison calves were vaccinated SQ with 1.4 x 10(10) colony-forming units (CFU) of SRB51 and five calves were inoculated SQ with sterile 0.15 M sodium chloride. An additional 10 bison calves were ballistically inoculated in the rear leg musculature with 1 x 10(10) CFU of SRB51 and five calves were ballistically inoculated with an empty Biobullet. Serologic responses were monitored at 0, 2, 4, 6, 8, 12, 18, and 24 wk using the standard tube agglutination test and a dot-blot assay. Swabs from rectal, vaginal, nasal, and ocular mucosal surfaces, and blood were obtained for culture from all bison at 2, 4, 6, and 8 weeks post-inoculation to evaluate potential shedding by vaccinated bison or persistent septicemia. The superficial cervical lymph node was biopsied in eight ballistic and eight hand vaccinated bison at 6 or 12 wk to evaluate clearance of the vaccine strain from lymphatic tissues. Lymphocyte proliferative responses to irradiated SRB51 bacteria were evaluated in peripheral blood mononuclear cells (PBMC) at 4, 6, 8, 12, 18, and 24 wk after inoculation. Serum obtained from hand or ballistically vaccinated bison demonstrated antibody responses on the dot-blot assay that were greater than control bison (saline or empty Biobullet) at 2, 4, 6, and 8 wk after vaccination. Antibody titers of ballistically vaccinated bison did not differ (P > 0.05) from hand vaccinated bison at any sampling time. Blood samples obtained from all bison at 2, 4, 6 and 8 wk after vaccination were negative for SRB51. One colony of SRB51 was recovered from the vaginal swab of one ballistically vaccinated bison at 2 wk after vaccination. All other ocular, vaginal, nasal, and rectal swabs were culture negative for SRB51. Strain RB51 was recovered from superficial cervical lymph nodes of hand and ballistic vaccinated bison at 6 (two of four and two of four bison, respectively) and 12 wk (three of four and one of four bison, respectively). Serologic tests and bacterial culture techniques failed to demonstrate infection of nonvaccinated bison. Peripheral blood mononuclear cells obtained from hand vaccinated bison had greater (P < 0.05) proliferative responses to strain RB51 bacteria when compared to PBMC from nonvaccinated and ballistically vaccinated bison. Proliferative responses of PBMC from ballistically vaccinated bison did not differ (P > 0.05) at any sampling time from proliferative responses of PBMC from control bison. Serum alpha 1-acid glycoprotein concentrations, plasma fibrinogen, and total protein concentrations were not influenced by treatments. Ballistic delivery of SRB51 did not induce adverse effects or influence clearance of the vaccine strain. There were no proliferative responses of PBMC to SRB51 in bison ballistically vaccinated with SRB51; whereas bison inoculated with SRB51 by hand injection had greater proliferative responses than control or ballistically vaccinated bison. Our study suggests that ballistic delivery may require a greater dose of SRB51 to induce cell-mediated immune responses in bison that are comparable to those induced by hand injection, and that ballistic or hand delivery of 1 x 10(10) CFU of SRB51 is safe in bison calves.     

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.     

Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.     

Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.     

Caspase-2-Dependent Dendritic Cell Death, Maturation, and Priming of T Cells in Response to Brucella abortus Infection

Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming.     

Maternal antibody blocks humoral but not T cell responses to BVDV.

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.     

Control of Tissue Reactions in Monkeys Vaccinated with Viable Coccidioides immitis by Prevaccination with Killed Coccidioides immitis.

Converse, J. L. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.), G. A. Deauville, E. M. Snyder, J. G. Ray, and M. E. Seaquist. Control of tissue reactions in monkeys vaccinated with viable Coccidioides immitis by prevaccination with killed Coccidioides immities. J. Bacteriol. 90:783-788. 1965.-Control of undesirable tissue reactions resulting from the subcutaneous injection of 150 viable arthrospores of Coccidioides immitis (strain D-76) was obtained by four injections of formalin-killed arthrospores 14, 12, 8, and 4 weeks (total dose, 36 mg) before injection of the viable arthrospores. Only 6 and 12% of these vaccinated animals exhibited ulceration and lymphadenopathy, respectively, as compared with 100 and 83% of the animals receiving only the viable vaccine. Agar-gel immunodiffusion precipitin titers of approximately 1:64 were evident 3 months after vaccination in animals receiving both vaccines, as compared with 1:128 in those injected with the viable vaccine alone. The above data indicated that somatic reactions to injection of a viable vaccine could be eliminated by preinjection of a killed vaccine. However, 6 months after vaccination, respiratory challenge (7,500 strain Cash arthrospores) indicated that this treatment also impaired the protective effect of the viable vaccine. All animals receiving both vaccines developed mild pulmonary coccidioidomycosis, whereas only 50% of the animals receiving only the viable vaccine were infected. In addition, the group receiving both vaccines demonstrated a more rapid and higher postchallenge precipitin titer. All vaccinated animals (those receiving the killed, the viable, or a combination of the two vaccines) survived for 4 months after challenge, as compared with 88% mortality (50% within 14 days) in the nonvaccinated controls.     

Development of an antibody ELISA for potency testing of furunculosis (Aeromonas salmonicida subsp salmonicida) vaccines in Atlantic salmon (Salmo salar L)

The study was conducted in Atlantic salmon to establish the initial and basic scientific documentation for an alternative batch potency test for salmon furuculosis vaccines. We assessed the antibody response development for Aeromonas salmonicida vaccines at different immunisation temperatures (3, 12 and 18 °C), by an enzyme-linked-immunosorbent assay (ELISA) 3, 6, 9 and 12 weeks post vaccination, and the correlation between antibody response and protection in cohabitation challenge experiments performed 6 and 12 weeks post vaccination. Fish immunised with a vaccine containing full antigen dose had a significant increase in antibody response after 252 day degrees and the measured values correlated well with protection after 500 day degrees. Fish vaccinated with a reduced antigen dose showed a significant lower antibody response than fish vaccinated with the full dose vaccine at all samplings, and showed a similar low relative percent survival (RPS) in the challenges. The results from this study indicate that an antibody ELISA can discriminate between vaccines of different antigen content and the method may replace challenge tests in batch potency testing of furunculosis vaccines in Atlantic salmon. An immunisation temperature of 12 °C and sampling after 6-9 weeks, seemed to be the most appropriate time for using antibody responses to confirm batch potency.     

Safety,Immunogenicity and Duration of Immunity Elicited by an Inactivated Bovine Ephemeral Fever Vaccine

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.     

A combined vaccine against <Emphasis Type="Italic">Brucella abortus</Emphasis> and infectious bovine rhinotracheitis

The present study was undertaken to study the immune response in calves vaccinated with Brucella abortus strain 19, infectious bovine rhinotracheitis (IBR) vaccines in monovalent form and combined vaccine containing both antigen. The seroconversion of monovalent and combined vaccines was tested in seronegative cattle calves. IBR vaccine alone and combination with live Brucella abortus S19 vaccine elicited an anamnestic response on day 60 post booster but started declining from day 90 onwards against IBR. B. abortus S19 alone and in combination with IBR vaccine gave more than 2 log protection in mice two weeks post challenge. Fluorescence polarization assay analysis with sera samples of calves vaccinated with B. abortus S19 monovalent vaccine alone and in combination with IBR vaccine revealed the presence of B. abortus antibodies. The components of the combined vaccine did not show any evidence of interference in the development of immunity. This combined vaccine may provide economical and affordable biological for the control of brucellosis and IBR.     

Safety of Brucella abortus strain RB51 in black bears

In two studies conducted from October 1999 to March 2000 and December 2000 to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4-3.1 x 10(10) colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n=12) or 2 ml of 0.15 M NaCl solution (saline, n=11). We did not detect a difference (P>0.05) in antibody titers to SRB51 in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study 1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51 did not appear to influence (P>0.05) reproductive performance.     

Safety of revaccination of pregnant bison with Brucella abortus strain RB51

From December 1998 through February 1999, a study was conducted in a Brucella-infected bison herd to evaluate the safety of booster vaccination of adult bison (Bison bison) with 6 x 10(9) colony forming units (CFU) of Brucella abortus strain RB51 (SRB51) that had previously been vaccinated as yearlings with 1 x 10(10) CFU of SRB51. Abortions or other adverse effects were not observed after SRB51 booster vaccination. At 10 wk after adult vaccination, pregnant and nonpregnant bison (n = 65) were randomly selected for bacteriologic sampling of targeted maternal tissues during abattoir processing. Fetal tissues were also sampled in pregnant bison. The SR351 recovered from tissue samples of eight of 48 pregnant bison and none of 17 nonpregnant bison. In three of the eight culture-positive bison, SRB51 was recovered from fetal tissues. In three additional bison, one pregnant and two nonpregnant, B. abortus biovar 1 field strain was recovered from internal iliac or supramammary lymphatic tissues. Results of this study suggest the possibility that the SRB51 vaccine can be safely used to booster vaccinate pregnant bison in a Brucella-infected bison herd. Our data also reaffirms the potential for B. abortus field strains to persist in bison until attainment of reproductive age, despite extensive use of vaccination and serologic testing.     

Immune Response of Calves Vaccinated with Brucella abortus S19 or RB51 and Revaccinated with RB51

Brucella abortus S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with B. abortus S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6–1.2 x 10¹¹ CFU) or RB51 (1.3 x 10¹⁰ CFU) on day 0, and revaccinated with RB51 (1.3 x 10¹⁰ CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4⁺ and CD8⁺ T-cells; IFN-ɣ and IL-17A production by CD4⁺ T-cells; cytotoxic CD8⁺ T-cells; IL-6 secretion; CD4⁺ and CD8⁺ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4⁺ memory cells, induction of CD21⁺ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4⁺ and CD8⁺ T-cells, cytotoxic CD8⁺ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4⁺- or CD8⁺-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.     

Immunogenicity of a new lot of Francisella tularensis live vaccine strain in human volunteers

Abstract A new lot of Francisella tularensis live vaccine strain (LVS) was tested for immunogenicity in 19 human volunteers. Scarification vaccination induced specific cell-mediated and humoral immune responses. We noted a significant rise in antibodies against irradiation-killed LVS, formalin-killed virulent strain SCHU4, and an ether extracted antigen preparation (EEx) beginning 14 days after vaccination. A main target of the humoral immune response was lipopolysaccharide. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx by day 14 and 100% of vaccinees responded positively by day 21. Background IgA titers were lower than corresponding IgG or IgM titers. No early IgM rise was noted with any antigen. By day 14 after vaccination, in vitro lymphocyte responses to LVS, the rough variant of LVS, and EEx were significantly increased compared to controls. Seventy percent of volunteers had a positive in vitro lymphocyte response to EEx within 14 days of vaccination. We predict that EEx will be a usefull antigen for diagnosing tularemia and for evaluating the immunogenicity of vaccines against tularemia. We are testing this antigen using sera from human cases of tularemia and control sera.