Meningococcal omp85 in detergent-extracted outer membrane vesicle vaccines induces high levels of non-functional antibodies in mice.

The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6-12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same opsonic titres. Thus, the genetically modified vaccine with high Omp85 antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen.

Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Interleukin-22 protects against non-typeable Haemophilus influenzae infection: alteration during chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is a major health problem becoming a leading cause of morbidity and mortality worldwide. A large part of these disorders is associated with acute exacerbations resulting from infection by bacteria, such as non-typeable Haemophilus influenzae (NTHi). Our understanding of the pathogenesis of these exacerbations is still elusive. We demonstrate herein that NTHi infection of mice chronically exposed to cigarette smoke (CS), an experimental model of chronic obstructive pulmonary disease (COPD), not only causes acute pulmonary inflammation but also impairs the production of interleukin (IL)-22, a cytokine with potential anti-bacterial activities. We also report that mice lacking IL-22, as well as mice exposed to CS, have a delayed clearance of NTHi bacteria and display enhanced alveolar wall thickening and airway remodeling compared with controls. Supplementation with IL-22 not only boosted bacterial clearance and the production of anti-microbial peptides but also limited lung damages induced by infection both in IL-22-/- and CS-exposed mice. In vitro exposure to CS extract altered the NTHi-induced IL-22 production by spleen cells. This study shows for the first time that a defect in IL-22 is involved in the acute exacerbation induced by NTHi infection during experimental COPD and opens the way to innovative therapeutic strategies.

Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella.

The lipopolysaccharide (LPS) is up to now the only identified major virulence determinant of Brucella. This bacterium is responsible for brucellosis in animals and for Malta fever in humans. Several monoclonal antibodies (mAbs) directed against various LPS epitopes have been characterized. Two mAbs, named A15-6B3 and B66-2C8, directed against distinct LPS epitopes have been used to select peptides from 11 phage display libraries. The sequences of the selected peptides contain an overrepresentation of either proline or tryptophan residues when selected with either A15-6B3 or B66-2C8 mAbs, respectively. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed. These data suggest that a subset of the selected peptides are mimotopes of the LPS epitopes.

Stable expression of biologically active recombinant bovine interleukin-4 in Trypanosoma brucei.

We have explored the potential of Trypanosoma brucei as a eukaryotic expression system. Procyclic forms, which correspond to an insect-adapted stage, can easily be cultured in vitro. The cells grow to densities approximately 10-fold greater than higher eukaryotic cells and are not infectious for mammals. An expression vector which can stably integrate into the genome was used to express high levels of recombinant bovine interleukin-4 (IL-4). Trypanosome-derived IL-4 is released into the medium and is biologically active. The recombinant protein down-regulates CD14 expression in human macrophages and inhibits NO production by stimulated bovine macrophages.

Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain.

The 40 N-terminal amino acids of the 20 kDa antigen A2 from Brucella melitensis were sequenced and showed important similarities with 4 bacterioferrins. A monoclonal antibody raised against this antigen cross-reacted with Escherichia coli bacterioferritin. Hybridization of two sets of degenerate primers with B. melitensis HindIII-digested genomic DNA identified a 3.8 kb fragment. This fragment was shown to contain a bacterioferritin gene (bfr) encoding a 161-amino acid protein. The sequence of the Brucella bacterioferritin is 69% similar to that of E. coli, and many of the ferroxidase centre and haem-ligation residues are conserved.

Identification of the major T-cell antigens present in the Brucella melitensis B115 protein preparation, Brucellergene OCB.

Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-gamma (IFN-gamma) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two-spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17)-showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella.

Infection of cattle with Yersinia enterocolitica O:9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests.

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of brucellosis was reported in these animals and no Brucella abortus strains were isolated. After two years, no brucellosis outbreak was registered in all of the herds concerned. On this basis, all the serological reactions observed were classified as false positive. An ELISA using Yersinia Outer membrane Proteins (YOPs) as antigens was developed in order to discriminate between a Yersinia enterocolitica O:9 infection and a Brucella abortus infection. Antibodies against YOPs were detected in sera from Y. enterocolitica O:9 experimentally infected cattle (n = 4) but not in sera from B. abortus experimentally infected cattle (n = 4). In a field study, 66.7% of the 174 serum samples from cattle presenting false positive serological reactions showed anti-YOPs antibodies whereas only 10% of 454 sera, classified negative by the brucellosis screening tests, showed anti-YOPs antibodies. Our results suggest that infections with Y. enterocolitica O:9 may cause false positive reactions in brucellosis testing.

Assessment of the cell-mediated immunity in cattle infection after bovine herpesvirus 4 infection, using an in vitro antigen-specific interferon-gamma assay.

The cell-mediated immunity (CMI) following bovine herpesvirus 4 (BHV4) infection has been poorly investigated in cattle. The in vivo response measured by a delayed type of hypersensitivity (DTH) assay has been reported to be positive in only few animals showing serological evidences of BHV4 infection. We have investigated the CMI following BHV4 infection by an in vitro antigen-specific interferon gamma (IFN-gamma) release assay, as an indicator of an actively acquired immunity to BHV4. Our preliminary results using a partially purified antigen suggest that there was a measurable CMI in 75 out of 168 animals (44.4%) originating from a farm with a clinical history and serological evidences (76.3% seropositivity) of BHV4 infection. If the results of serological tests and BHV4 IFN-gamma test are interpreted in parallel, 81.5% of the animals are classified positive, demonstrating the complementarity of these tests. The specificity of the BHV4 IFN-gamma test was supported by the absence of a measurable CMI in 41 animals originating from a farm with no clinical history or serological evidence of BHV4 infection. In an allied study, we developed a bovine herpesvirus 1 (BHV1) IFN-gamma test. This allowed us to measure the antigen specific IFN-gamma release after stimulation with a mixture of BHV1 and BHV4 antigens. Animals that were classified negative by the BHV4 IFN-gamma test and by the BHV1 IFN-gamma test, were classified negative after stimulation with a mixture of both antigens. Animals that were classified positive by the BHV4 IFN-gamma test or the BHV1 IFN-gamma test, were classified positive after stimulation with a mixture of both antigens. Taken together these results suggest that the in vitro assessment of the CMI after BHV4 infection should be further investigated as a specific and valuable alternative to the DTH assay.

Quantitative assessment by flow cytometry of T-lymphocytes producing antigen-specific gamma-interferon in Brucella immune cattle.

Peripheral blood mononuclear cells (PBMC) isolated from cattle infected with Brucella secreted gamma-interferon (IFN-gamma) after antigen-specific stimulation with Brucellergene, which is a mixture of cytoplasmic proteins of rough Brucella melitensis B115. Following the depletion of the monocyte-macrophages from the PBMC, the enriched lymphocyte populations stimulated with Brucellergene did not produce IFN-gamma. Two-colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules identified the cells producing IFN-gamma among the PBMC stimulated with Brucellergene. Moreover, this method could be used to estimate the number of T-cells specifically producing IFN-gamma. For a given animal, there is a significant correlation (p < 0.05) between the production of IFN measured by an ELISA of the supernatant of whole blood stimulated with Brucellergene and the number of T-cells producing IFN-gamma after in vitro stimulation with Brucellergene. The development of the immunofluorescence staining technique provides a new tool for analysing and for measuring the T-cell immune response in cattle.

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.

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4.

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Detection of Yersinia enterocolitica serogroup O:3 by a PCR method.

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.

Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes.

A thus far unknown gene encoding a Brucella abortus protein has been isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep. Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa (calculated molecular mass). The recombinant B. abortus protein, expressed in Escherichia coli, and the corresponding Brucella melitensis protein migrated at the same apparent molecular masses as shown by Western blotting (immunoblotting). Among a series of serum samples from B. melitensis- or B. abortus-infected sheep and cows, 51 and 39%, respectively, showed a signal at 17 kDa on Western blot analysis of total protein extract from Brucella bacteria. These figures amount to 70 and 61% for sheep and cattle, respectively, in a competitive enzyme-linked immunosorbent assay with a specific monoclonal antibody. These data indicate that the 17-kDa antigen may be useful for serological diagnosis of Brucella infection.

Specific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production.

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of cytoplasmic proteins from Brucella melitensis B115 was used as a specific antigenic stimulus in bovine whole blood culture. Supernatants harvested at 18 to 24 h after the in vitro antigenic stimulus were assayed for their gamma interferon (IFN-gamma) content by using a commercial sandwich enzyme-linked immunosorbent assay kit. The IFN-gamma assay was evaluated with 10 heifers during the course (80 days) of an experimental infection and with 14 cows from an ongoing brucellosis outbreak. All of these animals were slaughtered, and pertinent organs were subjected to classical bacteriological analyses. In addition, we analyzed 23 field cases in which false-positive serological reactions occurred. The IFN-gamma results were compared with those of the standard DTH and a battery of serological assays, and they were correlated with bacteriological data. Both for the experimental infection and for the field brucellosis outbreak, the IFN-gamma assay detected infection in more animals than any combination of the serological tests, and it detected infection earlier than these tests. Finally, none of the samples from cows showing false-positive serological reactions was classified as positive by the IFN-gamma assay, attesting to its specificity and to its usefulness in interpreting ambiguous serological results. A rapid and convenient alternative to the DTH, the IFN-gamma assay appears to be an ideal method that is complementary to the serological diagnosis protocols.

O-Polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry.

Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody.

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.

Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay.

Brucellosis research is currently focused on the identification of nonlipopolysaccharide (LPS) antigens which could potentially be useful for the specific serologic diagnosis of brucellosis as well as for vaccinal prophylaxis. On the basis of previous reports, we selected eight Brucella proteins (OMP36, OMP25, OMP19, OMP16, OMP10, p17, p15, and p39) as candidate antigens to be further evaluated. The genes encoding these proteins were cloned, sequenced, and overexpressed in Escherichia coli. The recombinant proteins were purified with a polyhistidine tag and metal chelate affinity chromatography and evaluated in an indirect enzyme-linked immunosorbent assay (iELISA). The specificity of the iELISA was determined with sera from healthy cattle, sheep, and goats and ranged from 95 to 99%, depending on the recombinant antigen and the species tested. Sera from experimentally infected, and from naturally infected, animals were used to evaluate the sensitivity of the iELISA. The antiprotein antibody response was often delayed when compared to the anti-smooth LPS (S-LPS) response and was limited to animals which developed an active brucellosis infection (experimentally infected pregnant animals and sheep and goats from areas where brucellosis is still endemic). Among the recombinant antigens, the three cytoplasmic proteins (p17, p15, and p39) gave the most useful results. More than 80% of the animals positive in S-LPS serology were also positive with one of these cytoplasmic proteins alone or a combination of two of them. None of the recombinant antigens detected experimentally infected nonpregnant cows and sheep or naturally infected cattle. This study is a first step towards the development of a multiprotein diagnostic reagent for brucellosis.

Effects of bovine herpesvirus type 1 infection in calves with maternal antibodies on immune response and virus latency.

The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of bovine herpesvirus type 1 (BHV-1) was performed to determine whether they could become seronegative after the disappearance of maternal antibodies while latently infected with BHV-1. Four uninfected calves were controls. All calves were monitored serologically for 13 to 18 months. In addition, the development of a cell-mediated immune response was assessed by an in vitro antigen-specific gamma interferon (IFN-gamma) production assay. All calves had positive IFN-gamma responses as early as 7 days until at least 10 weeks after infection. However, no antibody rise was observed after infection in the three calves with the highest titers of maternal antibodies. One of the three became seronegative by virus neutralization test at 7 months of age like the control animals. This calf presented negative IFN-gamma results at the same time and was classified seronegative by enzyme-linked immunosorbent assay at around 10 months of age. This calf was latently infected, as proven by virus reexcretion after dexamethasone treatment at the end of the experiment. In conclusion, this study demonstrated that BHV-1-seronegative latent carriers can be obtained experimentally. In addition, the IFN-gamma assay was able to discriminate calves possessing only passively acquired antibodies from those latently infected by BHV-1, but it could not detect seronegative latent carriers. The failure to easily detect such animals presents an epidemiological threat for the control of BHV-1 infection.

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus.

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.