Characterization of the duration of immunity of Brucella abortus strain RB51 vaccination in cattle after experimental challenge.

Fifty-two, Hereford heifers were obtained from brucellosis-free herds and randomly assigned to Brucella abortus strain RB51 (RB51) vaccination (n = 32) or control (n = 20) treatments. Vaccinates received 1010 colony-forming units (CFU) of a commercial lyophilized RB51 vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody, interferon-γ responses, and proliferative responses to RB51 antigens in cattle vaccinated with RB51 as compared to controls. A subgroup of control and vaccinated cattle were experimentally challenged at approximately 4, 5, and 6 years after inoculation with 107 CFU of B. abortus strain 2308 at 170-180 days gestation. After experimental challenge, 6 of 14 (43 %) control animals aborted at a higher rate (P < 0.05) when compared to RB51 vaccinates in years 4 and 5, but not year 6 (0 %, 10 %, and 50 %, respectively). When comparing recovery of Brucella from all tissues except head lymph nodes draining the site of challenge, RB51 vaccinates had reduced infection rates (P < 0.05) after experimental challenge at 4 years (14 %), but not at 5 or 6 years (78 % and 67 %, respectively) when compared to non-vaccinated cattle (93 %). Our data suggests that calfhood vaccination with RB51 does not induce lifelong immunity and suggests implementation of booster vaccination by 4-5 years of age should be utilized in endemic areas to maintain high levels of protection.

Immune responses and clinical effects of experimental challenge of elk with Brucella abortus strain 2308.

To assess the effects of challenge dose and stage of gestation on infection and abortion, 35 elk were conjunctivally challenged with virulent Brucella abortus strain 2308 (S2308) during pregnancy. Seventeen elk were experimentally challenged early in the second trimester of gestation (December) with high (approximately 108 CFU) or low challenge (approximately 107 CFU) treatments having 8 and 9 pregnant elk, respectively. Other pregnant elk were experimentally challenged at a later challenge time (approximately early third trimester, February), with high and low challenge treatments having 8 and 10 elk, respectively. Conjunctival swabs from all animals were culture positive for the S2308 strain at 7 days after experimental challenge. All animals seroconverted on a B. abortus ELISA but optical density readings were not influenced (P > 0.05) by time of challenge or by challenge dosage. In the early challenge group, abortions occurred in 2 of 9 (22%) in the low challenge treatment and 3 of 8 (37%) in the high challenge treatment, whereas in the later challenge group, 1 of 8 (12.5%) in the low challenge treatment and 2 of 10 (20%) in the high challenge treatment aborted. The ability to recover B. abortus from samples obtained at necropsy did not differ (P > 0.05) between early and late challenges or between high and low challenge treatments. Despite the lack of abortions observed after experimental challenge, recovery from maternal tissues ranged from 50% (low dose, late challenge) to 77% (low dose, early challenge). Our data suggests that naïve elk do not abort as frequently after experimental infection with B. abortus strain 2308 as compared to similar data in cattle and bison.

Swine brucellosis: current perspectives.

Brucella suis is a significant zoonotic species that is present in domestic livestock and wildlife in many countries worldwide. Transmission from animal reservoirs is the source of human infection as human-to-human transmission is very rare. Although swine brucellosis causes economic losses in domestic livestock, preventing human infection is the primary reason for its emphasis in disease control programs. Although disease prevalence varies worldwide, in areas outside of Europe, swine brucellosis is predominantly caused by B. suis biovars 1 and 3. In Europe, swine are predominantly infected with biovar 2 which is much less pathogenic in humans. In many areas worldwide, feral or wild populations of swine are important reservoir hosts. Like other Brucella spp. in their natural host, B. suis has developed mechanisms to survive in an intracellular environment and evade immune detection. Limitations in sensitivity and specificity of current diagnostics require use at a herd level, rather for individual animals. There is currently no commercial vaccine approved for preventing brucellosis in swine. Although not feasible in all situations, whole-herd depopulation is the most effective regulatory mechanism to control swine brucellosis.

Immune responses of bison and efficacy after booster vaccination with Brucella abortus strain RB51.

Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 10(10) CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1ß (IL-1ß) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 10(7) CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against brucellosis in bison.

Advancement of knowledge of Brucella over the past 50 years.

Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. At that time, the genus was considered to contain only 3 species: Brucella abortus, Brucella melitensis, and Brucella suis. Since the early 1960s, at least 7 new species have been identified as belonging to the Brucella genus (Brucella canis, Brucella ceti, Brucella inopinata, Brucella microti, Brucella neotomae, Brucella ovis, and Brucella pinnipedialis) with several additional new species under consideration for inclusion. Although molecular studies have found such high homology that some authors have proposed that all Brucella are actually 1 species, the epidemiologic and diagnostic benefits for separating the genus based on phenotypic characteristics are more compelling. Although pathogenic Brucella spp have preferred reservoir hosts, their ability to infect numerous mammalian hosts has been increasingly documented. The maintenance of infection in new reservoir hosts, such as wildlife, has become an issue for both public health and animal health regulatory personnel. Since the 1960s, new information on how Brucella enters host cells and modifies their intracellular environment has been gained. Although the pathogenesis and histologic lesions of B. abortus, B. melitensis, and B. suis in their preferred hosts have not changed, additional knowledge on the pathology of these brucellae in new hosts, or of new species of Brucella in their preferred hosts, has been obtained. To this day, brucellosis remains a significant human zoonosis that is emerging or reemerging in many parts of the world.

Recent developments in livestock and wildlife brucellosis vaccination.

Live attenuated brucellosis vaccines have been available for protecting domestic livestock against Brucella melitensis and B. abortus for more than 60 years. Current vaccines are effective in preventing abortion and transmission of brucellosis, but poor at preventing infection or seroconversion. In addition, they can induce abortions in pregnant animals and are infectious to humans. It can be argued that current vaccines were developed empirically in that the immunological mechanism(s) of action were not determined. Current knowledge suggests that both the innate and adaptive immune responses contribute to immunity against intracellular pathogens and that binding of pathogen structures onto pattern recognition receptors (PRRs) is crucial to the development of adaptive immunity. The phagosome appears to be vital for the presentation of antigens to T-cell subtypes that provide protective immunity to intracellular pathogens. The observation that killed bacteria or subunit vaccines do not appear to fully stimulate PRRs, or mimic Brucella trafficking through phagosomes, may explain their inability to induce immunity that equals the protection provided by live attenuated vaccines. Brucella appears to have multiple mechanisms that subvert innate and adaptive immunity and prevent or minimise immunological responses. New technologies, such as DNA vaccines and nanoparticles, may be capable of delivering Brucella antigens in a waythat induces protective immunity in domestic livestock or wildlife reservoirs of brucellosis. Because of the re-emergence of brucellosis worldwide, with an increasing incidence of human infection, there is a great need for improved brucellosis vaccines. The greatest need is for new or improved vaccines against B. melitensis and B. suis.

Brucellosis: 'One Health' challenges and opportunities.

Brucellosis is an ancient disease with host-specific evolutionary mechanisms that allow itto hide from or manipulate cellular immunity and achieve intracellular persistence. The disease yields low fatality rates but can cause substantial disabilities. Zoonotic brucellosis remains widespread and neglected in many areas despite notable advances in science, technology, and management in the 19th and 20th Centuries. The burden appears to remain greatest, and yet most under-prioritised globally, amongst pastoral peoples and small-scale livestock farmers. Capacity building for zoonotic brucellosis diagnosis, surveillance, management, and treatment in developing countries faces numerous challenges. Adaptive risk management can provide a framework to build stakeholder support for addressing the complexities and uncertainties, and learning from management actions. The challenges and opportunities for brucellosis management must be recognised as fundamentally multivariate, multifaceted, and integrative; it is thus crucial for veterinary, public health, and wildlife/conservation professions to collaboratively develop, adopt and promulgate a brucellosis One Health paradigm.

Biosafety considerations for in vivo work with risk group 3 pathogens in large animals and wildlife in North America.

Regulations in the United States require animal biosafety level 3 (ABSL-3) or biosafety level 3 agriculture (BSL-3-Ag) containment for many endemic zoonotic pathogens and etiologic agents of foreign animal diseases. In an effort to protect public health, billions of dollars were invested in regulatory programs over many years to reduce the prevalence of zoonotic pathogens such as Brucella and Mycobacterium bovis in domestic livestock. In addition to research needs in domestic livestock hosts, the establishment of brucellosis and tuberculosis in wildlife in the United States has created a need for research studies addressing these zoonotic diseases. As guidelines in the Biosafety in Microbiological and Biomedical Laboratories (BMBL, 2009) for BSL-3 and BSL-3-Ag facilities are primarily directed toward laboratory or vivarium facilities, additional issues should be considered in designing large animal containment facilities for domestic livestock and/or wildlife. Flight distance, herd orientation, social needs, aggressiveness, and predictability are all factors we considered on a species by species basis for designing our containment facilities and for work practices with large ruminants. Although safety risk cannot be completely eliminated when working with large animals, studies in natural hosts are critical for advancing vaccine and diagnostic development, and providing basic knowledge of disease pathogenesis in natural hosts. Data gathered in these types of studies are vital for state and national regulatory personnel in their efforts to design strategies to control or eradicate diseases such as brucellosis and tuberculosis in their natural hosts, whether it is domestic livestock or wildlife. It is likely that failure to address the prevalence of disease in wildlife reservoirs will lead to re-emergence in domestic livestock. The overall benefit of these studies is to protect public health, provide economic benefits to producers, and protect the economic investment made in regulatory programs.

Efficacy of dart or booster vaccination with strain RB51 in protecting bison against experimental Brucella abortus challenge.

This study characterized the efficacy of the Brucella abortus strain RB51 vaccine in bison when delivered by single intramuscular vaccination (hand RB51), by single pneumatic dart delivery (dart RB51), or as two vaccinations approximately 13 months apart (booster RB51) in comparison to control bison. All bison were challenged intraconjunctivally in midgestation with 10(7) CFU of B. abortus strain 2308 (S2308). Bison were necropsied and sampled within 72 h of abortion or delivery of a live calf. Compared to nonvaccinated bison, bison in the booster RB51 treatment had a reduced (P < 0.05) incidence of abortion, uterine infection, or infection in maternal tissues other than the mammary gland at necropsy. Bison in single-vaccination treatment groups (hand RB51 and dart RB51) did not differ (P > 0.05) from the control group in the incidence of abortion or recovery of S2308 from uterine, mammary, fetal, or maternal tissues at necropsy. Compared to nonvaccinated animals, all RB51 vaccination groups had reduced (P < 0.05) mean colonization or incidence of infection in at least 2 of 4 target tissues, with the booster RB51 group having reduced (P < 0.05) colonization and incidence of infection in all target tissues. Our data suggest that booster vaccination of bison with RB51 enhances protective immunity against Brucella challenge compared to single vaccination with RB51 by hand or by pneumatic dart. Our study also suggests that an initial vaccination of calves followed by booster vaccination as yearlings should be an effective strategy for brucellosis control in bison.

Immune responses and safety after dart or booster vaccination of bison with Brucella abortus strain RB51.

One alternative for management of brucellosis in Yellowstone National Park bison (Bison bison) is vaccination of calves and yearlings. Although Brucella abortus strain RB51 vaccination protects bison against experimental challenge, the effect of booster vaccinations was unknown. This study characterized immunologic responses after dart or booster vaccination of bison with Brucella abortus strain RB51. In two studies, 8- to 10-month-old female bison were inoculated with saline (n = 14), hand vaccinated with 1.1 × 10(10) to 2.0 × 10(10) CFU of RB51 (n = 21), or dart vaccinated with 1.8 × 10(10) CFU of RB51 (n = 7). A subgroup of hand vaccinates in study 1 was randomly selected for booster vaccination 15 months later with 2.2 × 10(10) CFU of RB51. Compared to single vaccinates, booster-vaccinated bison had greater serologic responses to RB51. However, there was a trend for antigen-specific proliferative responses of peripheral blood mononuclear cells (PBMC) from booster vaccinates to be reduced compared to responses of PBMC from single vaccinates. PBMC from booster vaccinates tended to have greater gamma interferon (IFN-γ) production than those from single vaccinates. In general, dart vaccination with RB51 induced immunologic responses similar to those of hand vaccination. All vaccinates (single hand, dart, or booster) demonstrated greater (P < 0.05) immunologic responses at various times after vaccination than nonvaccinated bison. Booster vaccination with RB51 in early gestation did not induce abortion or fetal infection. Our data suggest that booster vaccination does not induce strong anamnestic responses. However, phenotypic data on resistance to experimental challenge are required to fully assess the effect of booster vaccination on protective immunity.

Comparison of abortion and infection after experimental challenge of pregnant bison and cattle with Brucella abortus strain 2308.

A comparative study was conducted using data from naive bison (n = 45) and cattle (n = 46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered during midgestation. The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (P < 0.05) in bison than in cattle. In animals that did abort, the time between experimental challenge and abortion was shorter (P < 0.05) for bison than for cattle. Brucella colonization of four target tissues and serologic responses on the standard tube agglutination test at the time of abortion did not differ (P > 0.05) between cattle and bison. The results of our study suggest that naive bison and cattle have similarities and differences after experimental exposure to a virulent B. abortus strain. Although our data suggest that bison may be more susceptible to infection with Brucella, some pathogenic characteristics of brucellosis were similar between bison and cattle.

Immune responses and protection against experimental Brucella suis biovar 1 challenge in nonvaccinated or B. abortus strain RB51-vaccinated cattle.

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 10(10) CFU of the Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody and proliferative responses to RB51 antigens in cattle vaccinated with RB51 than in the controls. Pregnant cattle received a conjunctival challenge at approximately 6 months of gestation with 10(7) CFU of B. suis bv. 1 strains isolated from naturally infected cattle. The fluorescence polarization assay and the buffered acid plate agglutination test had the highest sensitivities in detecting B. suis-infected cattle between 2 and 12 weeks after experimental infection. Serologic responses and lymphocyte proliferative responses to B. suis antigens did not differ between control and RB51 vaccinees after experimental infection. No abortions occurred in cattle in either treatment group after challenge, although there appeared to be an increased incidence of retained placenta after parturition in both the control and the RB51 vaccination treatment groups. Our data suggest that the mammary gland is a preferred site for B. suis localization in cattle. Vaccination with RB51 did not reduce B. suis infection rates in maternal or fetal tissues. In conclusion, although B. suis is unlikely to cause abortions and fetal losses in cattle, our data suggest that RB51 vaccination will not protect cattle against B. suis infection after exposure.

Brucellosis in the United States: role and significance of wildlife reservoirs.

Regulatory programs for brucellosis in domestic livestock have been active in the U.S. for almost 80 years. Wildlife reservoirs of brucellosis include bison (Bison bison) and elk (Cervus elaphus nelsonii) for Brucella abortus whereas Brucella suis is the predominant species infecting feral swine. The persistence of brucellosis in wildlife reservoirs poses a risk for reintroduction of Brucella into domestic livestock. Reducing the prevalence of brucellosis in wildlife reservoirs is anticipated to be complicated and costly, and the problem is unlikely to be quickly resolved. Although some tools are currently available for use in the wildlife reservoirs, development of new vaccines, diagnostics, and management procedures will most likely be needed for effective control of brucellosis.

Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 or RB51 overexpressing superoxide dismutase and glycosyltransferase genes.

Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 (RB51) or a recombinant RB51 strain overexpressing superoxide dismutase (sodC) and glycosyltransferase (wboA) genes (RB51+sodC,wboA). Bison were vaccinated with saline only or with 4.6 x 10(10) CFU of RB51 or 7.4 x 10(10) CFU of RB51+sodC,wboA (n = eight animals/treatment). Bison vaccinated with RB51 or RB51+sodC,wboA had greater (P < 0.05) antibody responses, proliferative responses, and production of gamma interferon to RB51 after vaccination than did nonvaccinates. However, bison vaccinated with RB51+sodC,wboA cleared the vaccine strain from draining lymph nodes faster than bison vaccinated with the parental RB51 strain. Immunologic responses of bison vaccinated with RB51+sodC,wboA were similar to responses of bison vaccinated with RB51. Pregnant bison were intraconjunctivally challenged in midgestation with 10(7) CFU of B. abortus strain 2308. Bison vaccinated with RB51, but not RB51+sodC,wboA vaccinates, had greater protection from abortion, fetal/uterine, mammary, or maternal infection than nonvaccinates. Our data suggest that the RB51+sodC,wboA strain is less efficacious as a calfhood vaccine for bison than the parental RB51 strain. Our data also suggest that the RB51 vaccine is a currently available management tool that could be utilized to help reduce brucellosis in free-ranging bison.

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay.

Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk.

An aerosolized Brucella spp. challenge model for laboratory animals.

To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M) in a laboratory animal model of brucellosis, dosages of 10(3)-10(10) colony forming units (CFU) were nebulized to mice. Although tissue weights were minimally influenced, total CFU per tissues increased beginning at 10(6)-10(7) CFU dosages, with 10(9) CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery. At 12 weeks after vaccination with 10(7) CFU of B. abortus strain RB51 (SRB51) or saline (control), mice were challenged intraperitoneally (i.p.) (6.4 x 10(4) CFU) or via aerosol (1.76 x 10(9) CFU) with S2308. Mice vaccinated with SRB51 had reduced (P < 0.05) splenic, liver and lung colonization (total CFU and CFU/g) after i.p. challenge with S2308 as compared with control mice after i.p. S2308 challenge. Control and SRB51-vaccinated mice did not differ (P > 0.05) in splenic, liver or lung colonization after aerosol S2308 challenge. Failure to demonstrate vaccine protection was not because of a high aerosol challenge dosage as colonization of spleen and liver tissues was lower (P < 0.05) after aerosol challenge when compared with control mice after i.p. S2308 challenge.

Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle.

The Bovigam assay is approved for use within the United States as a complementary tuberculosis test. Prior to whole blood culture and the ensuing ELISA to detect interferon-(IFN)-gamma, samples are subjected to various holding time/temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. To evaluate these effects, 5-month-old Holstein calves (n = 7) received 10(3) cfu Mycobacterium bovis by aerosol. Heparinized blood was collected 2 months after challenge and held at 4 or 22 degrees C for 0, 8 or 24 h prior to culture with mycobacterial antigens or pokeweed mitogen (PWM). Responses of samples held for 8 or 24 h were comparable and lower than responses of cultures prepared immediately after collection, regardless of holding temperature. Differences in responses of samples held at 4 degrees C versus 22 degrees C were also minimal. A subset of samples was held for 2 h at 37 degrees C at the beginning of the holding period. This subset of samples had diminished responses to all stimulants and increased holding times (i.e., 24 h versus 8 h) negatively impacted the response. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, reduces IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this assay of regulatory importance.

Immune responses of elk to initial and booster vaccinations with Brucella abortus strain RB51 or 19.

Previous studies have suggested that currently available brucellosis vaccines induce poor or no protection in elk (Cervus elaphus nelsoni). In this study, we characterized the immunologic responses of elk after initial or booster vaccination with Brucella abortus strains RB51 (SRB51) and 19 (S19). Elk were vaccinated with saline or 10(10) CFU of SRB51 or S19 (n=seven animals/treatment) and booster vaccinated with a similar dosage of the autologous vaccine at 65 weeks. Compared to nonvaccinates, elk vaccinated with SRB51 or S19 had greater (P<0.05) antibody responses to SRB51 or S19 after initial vaccination and after booster vaccination. Compared to nonvaccinated elk, greater (P<0.05) proliferative responses to autologous antigen after initial vaccination occurred at only a few sample times in SRB51 (6, 14, and 22 weeks) and S19 (22 weeks) treatment groups. In general, proliferative responses of vaccinates to nonautologous antigens did not differ (P>0.05) from the responses of nonvaccinated elk. Gamma interferon production in response to autologous or nonautologous Brucella antigens did not differ (P>0.05) between controls and vaccinates after booster vaccination. Flow cytometric techniques suggested that proliferation occurred more frequently in immunoglobulin M-positive cells, with differences between vaccination and control treatments in CD4+ and CD8+ subset proliferation detected only at 22 weeks after initial vaccination. After booster vaccination, one technique ([3H]thymidine incorporation) suggested that proliferative responses to SRB51 antigen, but not S19 antigen, were greater (P<0.05) in vaccinates compared to the responses of nonvaccinates. However, in general, flow cytometric and other techniques failed to detect significant anamnestic responses to autologous or nonautologous Brucella antigens in S19 or SRB51 vaccinates after booster vaccination. Although some cellular immune responses were detected after initial or booster vaccination of elk with SRB51 or S19, our data suggest that responses tend to be transient and much less robust than previously reported in SRB51-vaccinated cattle (Bos taurus) or bison (Bison bison). These data may explain why the vaccination of elk with S19 and SRB51 induces poor protection against brucellosis.

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus).

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.

Photopolymerized hydrogel carriers for live vaccine ballistic delivery.

Photopolymerized poly(ethylene glycol) (PEG)-crosslinked hydrogels were assessed for their ability to serve as a payload vehicle to deliver a viable bacterial vaccine (Brucella abortus strain RB51 (RB51) to bison in Yellowstone National Park) ballistically using thermoplastic degradable Biobullets. PEG modified with degradable glycolide or lactide oligomers capped with photopolymerizable methacrylate groups served to crosslink the hydrogel vaccine carrier inside commercial hydroxypropylcellulose Biobullets. Release of 1 microm diameter model fluorescent particles from hydrogels followed known degradation trends for glycolide- and lactide-modified PEG hydrogels. All particles were released from PEG-co-glycolide hydrogels after approximately 10 days and PEG-co-lactide hydrogels after approximately 45 days following gel degradation. Minimal particle release was observed from pure PEG dimethacrylate hydrogels over 40 days. P. aeruginosa (strain PAO1) and RB51 live vaccines exhibit excellent viability following exposure to photopolymerization encapsulation within these gel matrices. Hydrogels photopolymerized into the payload chamber of Biobullets exhibit similar ballistic properties to commercially available Biobullets and penetrate and remain intact when fired intramuscularly into live elk for release of their gel payload in the host.