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.     

The WC1(+) gammadelta T-cell population in cattle: a possible role in resistance to intracellular infection

Intracellular infections are important in veterinary medicine and detailed understanding of the associated immune responses is needed for optimal development of strategies based on diagnosis and vaccination. It is generally accepted that cell-mediated immune responses are of greatest importance in intracellular infections and recent studies from several bovine models of infection indicate that WC1(+) gammadelta T-cells have a number of possible levels of involvement, which remain incompletely defined. Investigations of experimental infection with Mycobacterium bovis in cattle have indicated that WC1(+) gammadelta T-cells are among the first cells to accumulate at initial sites of infection, an observation which has been linked with decreased numbers of these cells in the circulation within days of infection. These WC1(+) gammadelta T-cells have been shown to respond in vitro, both to protein antigens and to non-protein, phosphate containing antigens of M. bovis and to be capable of producing IFN-gamma. Studies of M. bovis infection in calves depleted of WC1(+) gammadelta T-cells by monoclonal antibody have suggested that the presence of these cells is associated with development of a Th1-biased acquired immune response. In combination, these observations allow speculation regarding a possible role for WC1(+) gammadelta T-cells as a link between the innate and acquired immune systems which is instrumental in establishing an appropriate response.     

Distribution and activation of T-lymphocyte subsets in tuberculous bovine lymph-node granulomas

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3(+), CD4(+), CD8(+), WC1(+), CD25(+)) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8(+) and CD25(+) cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8(+) cells in the initial attempt to restrain the granuloma growth. CD4(+) T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1(+) cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8(+) cells compared with CD4(+) cells. However, there was no sense of rimming effect for any of the investigated cell populations.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.     

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis

Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n=5) were vaccinated with M. bovis CFP formulated with either Emulsigen or Polygen adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-gamma (IFN-gamma) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-gamma responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-gamma responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen+CpG or CFP/Polygen+CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen+CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.     

Experimental production of infectious bovine keratoconjunctivitis: comparison of serological and immunological responses using pili fractions of Moraxella bovis.

The effect of vaccinating cattle and mice on the development of keratoconjunctivitis was studied. Cattle were vaccinated with whole cells, disrupted cells and pili fractions of three strains of Moraxella bovis. Mice were vaccinated with pili fractions of three strains. The resistance of all vaccinated animals was challenged with virulent cultures of M. bovis. In an attempt to correlate the response seen after vaccination and challenge with a pili fraction of M. bovis, vaccinated cattle and mice were grouped on the basis of signs of disease manifested and compared on the basis of serological responses. Serum samples were tested for antibodies by a gel diffusion precipitin test. A greater number of the sera of resistant cattle had antibodies to the homologous pili antigen than those of vaccinated nonresistant cattle. Cattle vaccinated with disrupted cells were not resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to challenge exposure by homologous than heterologous cultures. A greater number of the sera of resistant mice had antibodies to pili antigens than nonresistant mice.     

IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.     

Analysis of the antigen-specific IFN-gamma producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG.In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Safety and efficacy testing of a novel multivalent bovine bacterial respiratory vaccine composed of five bacterins and two immunogens

Bovine bacterial respiratory diseases have been one of the most serious problems due to their high mortality and economic loss in calves. The vaccinations of bovine bacterial respiratory vaccines have been complex because of no multivalent vaccine. In this study, novel multivalent bovine bacterial respiratory vaccine (BRV) was developed and tested for its safety and efficacy. BRV was composed of two immunogens and five bacterins. These were leukotoxoid and bacterin of Mannheimia haemolytica type A, outer membrane protein and bacterin of Pasteurella multocida type A, and bacterins of Haemophilus somnus, Mycoplasma bovis, and Arcanobacterium pyogenes. ELISA antibody titers to five bacterial antigens in vaccinated guinea pigs increased, compared with those in unvaccinated ones. BRV was safe for calves and pregnant cattle in this study. In calves challenged with M. haemolytica and P. multocida, the average daily weight gain and antibody titers of vaccinated calves increased, and respiratory symptoms (P<0.05) and treatment frequency (P<0.01) of vaccinated calves significantly decreased, compared with those of unvaccinated calves. Interestingly, the antibody titers of M. haemolytica leukotoxoid and Mycoplasma bovis were closely related with the reduction of respiratory symptoms. BRV would be an ecomonical measure for the protection against bovine bacterial respiratory diseases.     

Immunogenicity of a Moraxella bovis bacterin containing attachment and cornea-degrading enzyme antigens

An adjuvanted Moraxella bovis bacterin containing attachment antigens and cornea-degrading enzyme antigens protected cattle from infectious bovine keratoconjunctivitis (IBK) when experimentally challenged with homologous and heterologous challenge cultures of M. bovis. This bacterin also protected cattle against field exposure to M. bovis. Transmission electron microscopy and fluorescein labeled anti-M. bovis pili antiserum showed pili on the M. bovis bacterin strain. Scanning electron microscopy demonstrated a fibrillar glycocalyx. The bacterin strain of M. bovis, but not all strains of M. bovis, destroyed bovine corneal cell monolayers in vitro. Bovine corneal cells began to separate from each other within 5 min after M. bovis organisms were added and adhered to the cell monolayers. Moraxella bovis organisms remained attached to the disintegrating cells as the cell membrane separated and was digested. Vaccination stimulated bacterial agglutination antibodies. However, protection against experimental challenge was more closely related to the cornea-degrading enzyme content of the experimental bacterins. Twenty-two of 29 cattle (76%) vaccinated with bacterins containing a relative enzyme activity (REA) greater than 0.4 were protected in a rigorous challenge of immunity test. Only 1 of 21 non-vaccinated calves (5%) was free of IBK. Ninety-two percent (24/26) of calves vaccinated with a bacterin containing a REA greater than 0.29 remained free of IBK following field exposure, whereas 47% (8/17) non-vaccinated calves developed IBK. Only 8 of 12 calves (67%) vaccinated with a bacterin containing a REA of 0.09 remained free of IBK. In a larger field efficacy test consisting of 32 herds in six states, the incidence of IBK in individual herds ranged from 0% to 55%. The overall rate of infection was 11.2%. Vaccination of calves with an M. bovis bacterin that contained a REA of 0.63 reduced the incidence of IBK from 11.2% (217/1931) in the non-vaccinated controls to 4.3% (66/1520) in cattle vaccinated once and to 3.1% (48/1536) in cattle vaccinated twice.     

Histological observations of bovine tuberculosis in lung and lymph node tissues from British deer

Deer are recognized as hosts of Mycobacterium bovis and assessing the role of wild cervids in perpetuating tuberculosis among cattle has motivated extensive research on several continents. In this paper, the histopathology of lymph node and lung tuberculous granulomas in M. bovis positive British deer is presented. The overall aim was to seek further insights into the potential for onward transmission from infected deer to other species, including cattle. Samples were obtained from an extensive survey of wild mammals in South-West England and from statutory tuberculosis surveillance. M. bovis culture-positive samples were characterised microscopically as to their stage of lesion advancement, number of acid-fast bacilli and granuloma encapsulation. Seventy percent of the deer developed granulomas containing far greater numbers of M. bovis bacilli than typically reported in cattle. Red and fallow deer had the largest number of poorly encapsulated granulomas often containing many hundreds of bacilli. The results are consistent with infected wild British deer being a potential source of environmental contamination and onward transmission to other species. However, further work on levels of bacillary shedding is required before this can be confirmed.     

Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control infection.     

Bovine γδ T cells: cells with multiple functions and important roles in immunity

The γδ T-cell receptor (TCR)-positive lymphocytes are a major circulating lymphocyte population in cattle, especially in young calves. In contrast, human and mice have low levels of circulating γδ TCR(+) T cells (γδ T cells). The majority of the circulating γδ T cells in ruminants express the workshop cluster 1 (WC1) molecule and are of the phenotype WC1(+) CD2(-) CD4(-) CD8(-). WC1 is a 220000 molecular weight glycoprotein with homology to the scavenger receptor cysteine-rich (SRCR) family, closely related to CD163. The existence of 13 members in the bovine WC1 gene family has recently been demonstrated and although murine and human orthologues to WC1 genes exist, functional gene products have not been identified in species other than ruminants and pigs. Highly diverse TCRδ usage has been reported, with expanded variable genes in cattle compared to humans and mice. Differential γ chain usage is evident between populations of bovine γδ T cells, this may have implications for functionality. There is a growing body of evidence that WC1(+) γδ T cells are important in immune responses to mycobacteria and may have important roles in T cell regulation and antigen presentation. In this review, we will summarize recent observations in γδ T cell biology and the importance of γδ T cells in immune responses to mycobacterial infections in cattle.     

Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

The presence of CD25 on bovine WC1+ gammadelta T cells is positively correlated with their production of IL-10 and TGF-beta, but not IFN-gamma

WC1+ cells in cattle exhibit both regulatory and effector activities. However, it has not been elucidated whether they are so plastic that both activities co-exist in one cell or there are separate subpopulations of effector and regulatory cells. Since the production of IFN-gamma and IL-10 seems to be related to WC1+ cells' effector and regulatory function, respectively, the main aim of this study was to determine whether those cytokines are produced by separate subpopulations of WC1+, or are co-produced by the same cells. Due to increasingly frequent emphasised role of consumption of IL-2 in the mechanism of suppressor action of mouse CD25+CD4+ T regulatory cells, expression of the receptor's alpha chain for interleukin 2 (CD25) on WC1+ lymphocytes has been evaluated. An average of 5.21% of WC1+ cells obtained from PBMCs of 12-month-old heifers show constitutive expression of the CD25 molecule, with CD25(high)WC1+ and CD25(low)WC1+ cells accounting for 1.05% and 4.10% of WC1+ lymphocytes, respectively. For detection of intracellular cytokine production, PBMCs were stimulated with concanavalin A. Both IFN-gamma(-) and IL-10-producing cells within the CD25(-)WC1+ and CD25+WC1+ subpopulations were mainly separate subpopulations. The average percentage of IFN-gamma(+)IL-10(-), IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ cells among CD25(-)WC1+ lymphocytes was 4.03%, 2.67% and 0.51%, respectively. A positive correlation was observed between the presence of the CD25 molecule on WC1+ lymphocytes and production of IL-10 and TGF-beta, because the average percentage of IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ among CD25+WC1+ lymphocytes was 3 and 4.5 times higher as compared to the corresponding cells in the CD25(-)WC1+ subpopulation, whereas the percentage of IFN-gamma(+)IL-10(-) cells in both the subpopulations was not significantly different. The percentage of TGF-beta+ cells within the CD25+WC1+ subpopulation was 2.72 times as high as that of CD25(-)WC1+ lymphocytes. Therefore, with respect to the production of IFN-gamma, IL-10 and TGF-beta, CD25+WC1+ lymphocytes turn out to have a more suppressor profile than CD25(-)WC1+.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis. METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 x 105 colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50-51 days after challenge were euthanised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis. RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05). CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.