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.

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.

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.

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.

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.

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.

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.

Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity.

Two different tetrazolium compounds were compared for use in a colorimetric assay for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Brucella abortus. The tetrazolium compounds tested included 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sodium 3,3'-[1[(phenylamino)carbonyl]-3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT). The MTT and XTT colorimetric bactericidal assays were conducted by incubating antibody-opsonized bacteria with neutrophils in microtiter plates for 30 and 60 min at ratios of ten and 100 bacteria per neutrophil. Neutrophils were then lysed with saponin and samples were incubated 30 min with MTT or XTT plus coenzyme Q (CQ). Dead bacteria and lysed neutrophils did not react with MTT or XTT plus CQ. Live bacteria converted XTT to water soluble orange formazan in the presence of CQ and MTT to insoluble purple formazan. Absorption of formazan produced by bacteria from XTT was measured at 450 nm. Formazan produced by bacteria from MTT was solubilized by adding isopropanol and measured by absorption at 560 nm. Absorption of both types of formazan was directly related to viable bacteria cell number and used to determine the number of bacteria not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT or XTT plus CQ with known numbers of bacteria. The XTT and MTT colorimetric bactericidal assays produced comparable results when used to measure bovine neutrophil bactericidal activity against S. aureus, E. coli, L. monocytogenes, and B. abortus. However, the assay using XTT was quicker and easier to perform because bacteria converted XTT to a formazan that did not need to be solubilized before measuring absorption.

Effect of exercise on oxygen consumption, heart rate, and the electrocardiogram of pigs.

Pigs were exercised for 5 min at five different treadmill speeds (1.0-1.8 m X s-1) (3 degrees incline), while oxygen consumption (MO2), carbon dioxide production (MCO2), and the electrocardiogram (ECG) were recorded continuously. Data were taken at rest, during exercise, and at 2, 5, 15, and 30 min after exercise. Values for MO2, MCO2, and heart rate (HR) showed progressive increases with increasing treadmill speed. The respiratory exchange ratio (R) increased during exercise and approached 1.0, but peak values were seen shortly after exercise. Heart rate, MO2, MCO2, and R reached steady-state values after 2 min of exercise, which were maintained for the duration of exercise. In most cases, these variables had returned to control levels 15 min after exercise. A high correlation between HR and MO2 was found in these animals. Prominent increases in T-wave amplitude of the ECG were associated with exercise and early recovery. The metabolic and cardiac changes associated with exercise in these animals were all qualitatively similar to responses seen in exercising humans. Thus, this study further supports the belief that the pig is a good model for studying the cardiopulmonary responses to exercise in humans.

Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51.

Antibody and lymph node cell-mediated immune responses to recombinant Brucella abortus strain 19 Cu-Zn superoxide dismutase (rSOD) and to three synthetic strain 19 Cu-Zn SOD peptides were measured during 2 to 12 weeks following vaccination of cattle with B. abortus strain 19 or RB51. Cattle vaccinated with strain 19 or RB51 did not produce antibody to rSOD and to the SOD peptides. Lymph node cells from cattle vaccinated with strain 19, but not with strain RB51, proliferated when incubated with either rSOD or one of the three tested SOD peptides (GGDNYSDKPEPLGG). These results suggest that neither the strain 19 nor the strain RB51 vaccine induces antibody production to SOD and only the strain 19 vaccine induces lymph node cell-mediated immune responses to SOD.

Selective media for isolation of Brucella abortus strain RB51.

Brucella abortus strain RB51 (SRB51) is the standard vaccine used to protect cattle against brucellosis and is currently being used to vaccinate bison in the United States (US). Currently available media for culture of Brucella have not been evaluated for their ability to support growth of SRB51. In this study, five selective media for isolating brucellae, four commercially available media for gram-negative bacteria, and tryptose agar with 5% bovine serum (TSA) were compared to two SRB51 selective media developed in this study (rifampin brucellae medium (RBM), and malachite green brucellae medium (MGB)), for their ability to support growth and enhance recovery of SRB51. Four of the five media currently used for isolation of brucellae and two of the four media used for other Gram-negative bacteria did not support growth of SRB51. Modified Kuzdas and Morse (MKM), Brilliant Green, Skirrow's, RBM, and MGB supported growth of SRB51 in a manner similar to TSA. Recovery of SRB51 from tissues of SRB51-vaccinated bison was attempted on TSA, MKM, RBM, and MGB. From a total of 436 samples, SRB51 was isolated from 9.6, 4.3, 5.5, and 9.0% on TSA, MKM, RBM, or MGB media, respectively. Strain RB51 was recovered on only one medium (nine on TSA; three on RBM; and 9 on MGB) from 21 samples. Overgrowth of contaminating bacteria prevented potential detection of SRB51 from 9. 4, 5.5, 0.07, and 5.9% of samples on TSA, MKM, RBM, or MGB, respectively. These data suggest that the use of RBM and MGB, in combination with TSA, enhances the ability to recover SRB51 from tissue samples.

Comparative effects of bovine cytokines on cattle and bison peripheral blood mononuclear cell proliferation.

Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines. Bovine interleukin 1 beta (BoIL-1 beta), interleukin 2 (BoIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens. These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherichia coli 055:B5 lipopolysaccharide (LPS) at 10-0.1 micrograms/ml. BoIL-2 alone, but not BoIL-1 beta and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens. In addition, BoIL-1 beta and BoIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens. These results indicate that BoIL-1 beta and BoIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation.

Role of immune responses to a GroEL heat shock protein in preventing brucellosis in mice vaccinated with Brucella abortus strain RB51.

Resistance to infection with virulent Brucella abortus strain 2308 and antibody and lymphocyte proliferative responses to a recombinant 60 kDa B. abortus GroEL heat shock protein were measured in mice vaccinated with attenuated B. abortus strain RB51. Mice at 12-20 weeks after vaccination with 5 x 10(8) colony forming units (CFU) of strain RB51 had increased resistance to infection with strain 2308 and increased antibody and lymphocyte proliferative responses to GroEL following challenge infection with 2308. However, these mice at 12-20 weeks after vaccination did not have greater resistance to infection than mice vaccinated with 5 x 10(6) CFU of strain RB51, which had no increased antibody or lymphocyte proliferative response to GroEL. These results indicate that mice vaccinated with strain RB51 can have antibody and cell-mediated immune responses to GroEL during infection with virulent strain 2308, although neither response appeared to have an essential role in vaccine-induced immunity to brucellosis.

In vitro effects of live and killed Brucella abortus on bovine cytokine and prostaglandin E2 production.

Live and gamma-irradiated-killed Brucella abortus strain 2308 increased interleukin 1 (IL-1), but not interleukin 2 (IL-2), interferon-gamma (IFN-gamma), or prostaglandin E2 (PGE2) production when incubated with normal bovine peripheral blood mononuclear cells (PBMC). Live B. abortus was more effective than killed B. abortus in stimulating IL-1 production by normal PBMC. Both live and killed B. abortus were equally effective in suppressing IL-2 and IFN-gamma production by Concanavalin A-stimulated PBMC. Incubation of PBMC with the cyclo-oxygenase inhibitor, indomethacin, blocked PGE2 synthesis, but did not further enhance IL-1 production or prevent suppressed IL-2 and IFN-gamma production that was induced by live and killed B. abortus. These results suggest that B. abortus-induced suppression of IL-2 and IFN-gamma production did not appear to be mediated by the suppressive prostaglandin, PGE2, or other cyclo-oxygenase metabolites.

Comparative analysis of immune responses in cattle vaccinated with Brucella abortus strain 19 or strain RB51.

Immune responses were measured for 12 weeks following vaccination of cattle with either Brucella abortus strain (S) 19 or SRB51. Cattle vaccinated with S19, but not with SRB51, produced antibodies that agglutinated B. abortus S1119 in the standard tube agglutination test. Cattle vaccinated with S19 or SRB51 produced antibodies to the surface antigens of SRB51 when measured by a dot enzyme-linked immunosorbent assay. Superficial cervical lymph node (LN) cells obtained by biopsy at 10 and 12 weeks from cattle given the S19 or SRB51 vaccine exhibited similar proliferative responses when incubated in vitro with gamma-irradiated B. abortus S2308. At 10 and 12 weeks after vaccination, LN cells obtained from cattle given S19 or SRB51 proliferated to 22 protein fractions (106-18 kDa proteins) of B. abortus S2308 that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Twelve of the same 22 fractions, which contained 49-27 kDa proteins, produced a stimulation index of greater than 10 when incubated with LN cells taken from S19-vaccinated or SRB51-vaccinated cattle. Two factions, which contained 27 kDa proteins of S2308, induced the highest proliferative response (stimulation index 25 or greater) by LN cells in cattle given either S19 or SRB51. These results suggest that cattle vaccinated with S19 or SRB51 have similar LN immune responses to S2308, but unlike S19, SRB51 does not induce positive results in the standard tube agglutination test used to diagnose brucellosis in cattle.

Cloning bovine cytokine cDNA fragments and measuring bovine cytokine mRNA using the reverse transcription-polymerase chain reaction.

Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-gamma activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression.

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative.

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.