- Email: [email protected]
Lipoarabinomannans from Mycobacterium avium subsp. avium affect bovine immune responses to different antigens
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
versions of gE produced in a procariotic system. Previous publications had identified regions within the gE which were toxic for bacteria and hindered expression of full length versions of the gene. Based on this, 2 fragments of 571 and 779 bp long were selected and expressed in E. coli. Soluble fractions of induced bacterial cultures were resolved by 12% SDS-PAGE and transferred to nitrocellulose strips. Preliminary results showed that gEc-specific mAbs recognized a single band for both constructs, while showing no reaction with seronegative samples or control antigen from non-transformed bacteria. All together these results showed the feasibility of developing effective and inexpensive diagnostic assays as essential complements for BHV-1 marker vaccines. doi:10.1016/j.vetimm.2008.10.227 Lipoarabinomannans from Mycobacterium avium subsp. avium affect bovine immune responses to different antigens Silvia Colavecchia ∗ , Ana Fernández, Silvia Mundo
Jolly, Ana
Stempler, Eloy
Area Inmunología, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarín 289, Buenos Aires, Argentina Keywords: Mycobacterium; Lipoarabinomannan; Immunomodulation; Bovine E-mail address: [email protected] (S. Colavecchia). Species: Ruminants Lipoarabinomannans (Lams) are the structurally more important outer cell wall component of all mycobacteria. Lams are ubiquitous of Mycobacteria spp. and appear as the most potent non-peptidic molecules to modulate the host immune response. Our objective was to determine the modulating effect of Lams on the immune response to soluble and particulates antigens in cattle. Lams were obtained from Mycobacterium avium subsp. avium (Maa), purified and characterized by ELISA and Western blot as described by Hamasur et al. (1999). Lams (1 mg) were injected three times to calves subcutaneously, in a mixture with the following antigens: 1 mg ovalbumin (OVA), 108 cells of canine erythrocytes (CE) or 2 mg of Maa whole bacteria. OVA, Maa and PBS (as control) were emulsified with Freund’s incomplete adjuvant (AFI) and CE was injected in PBS. Groups of calves were injected with the antigens without Lams as controls. Immune responses of calves were tested before and 21 days after the third immunization. Innate immune responses were evaluated by serum electrophoresis (EP) and respiratory burst (RB) on peripheral cells. Specific immune responses were evaluated by lymphoproliferation (LP) assay, serum ELISA and hemaglutination (HA) test in vitro and delayed-type hypersensitivity (DTH) in vivo. Differences between groups were analyzed by Kruskal Wallis or Student’s unpaired t-test. Our results show that Lams decrease significantly the RB of peripheral cells. This result agree with Leprae and Tuberculosis Lams effect described in human macrophages in vitro by Chan et al. (1991) who consider it as a survival mechanism to evade phagocytosis.
317
In addition, we detected increase in cellular immune response in Lam inoculated animals. On the other hand, specific antibodies titers were decreased by simultaneous inoculation with Lams. Our results confirm that simultaneous application of soluble and particulated antigens with Lams modifies the immune response against these antigens in cattle.
References Chan, J., et al., 1991. Infect. Immun. 59, 1755–1761. Hamasur, B., et al., 1999. FEMS Immunol. Med. Microbiol. 24, 11–17.
doi:10.1016/j.vetimm.2008.10.228 Safety of Brucella abortus RB51 vaccination of adult cows: Bacteriological evaluation of milk Karina Leite Miranda Universidade Federal de Minas Gerais, Belo Horizonte, MG. 30240-060, Brazil Keywords: Brucella abortus; Vaccine; Lipopolysaccharide Oantigen-deficient; S19 antigen E-mail address: [email protected]. Species: Ruminants Brucellosis caused by Brucella abortus is a chronic disease of cattle of worldwide public health and economic importance, resulting in abortion and infertility. B. abortus RB51 is a lipopolysaccharide O-antigen-deficient mutant of the virulent strain B. abortus 2308 used as an alternative vaccine to the S19, which induces antibodies that are detected by the routine serological tests. In Brazil, RB51 is approved for using in adult cows and there is only scanty data on the elimination of RB51 in milk. The aim of this study was to evaluate by culture the elimination of B. abortus in the milk of cows vaccinated with RB51. Ten brucellosis-free cows, vaccinated with S19 as calf, between 30 and 60 days after parturition were selected from the herd of Escola de Veterinária – UFMG, at Pedro Leopoldo – MG, Brazil. The cows were given subcutaneous injections of 1.0 mL of PBS containing 1.3 × 1010 CFU of B. abortus strain RB51, in the day 0 of experiment. Milk samples were collected from cows twice a day, in sterile polypropylene tubes on days 1, 2, 3, 4, 7, 14, 21, 28, 35, 42, 49, 56 and 63 after vaccination. The first milk streams of each teat were discarded, then 50 mL of milk samples were manually collected from all quarters and stored at −20 ◦ C. Samples were thawed and centrifuged at 2500 × g, for 15 min, the intermediated phase was discarded and the supernatant was mixed with the pellet. The mixtures were immediately inoculated in duplicate plates of tryptose agar with antibiotics (Farrel’s supplement). Moreover, 1.0 mL of each mixture was inoculated in 9.0 mL of enrichment media (tryptose broth with Farrel’s selective supplement) and incubated into 5% CO2 at 37 ◦ C for 5 days, and then inoculated in tryptose agar with antibiotics. All plates were incubated into 5% CO2 at 37 ◦ C for 9 days. No B. abortus was isolated from any sample tested. In order to confirm these results, all frozen
versions of gE produced in a procariotic system. Previous publications had identified regions within the gE which were toxic for bacteria and hindered expression of full length versions of the gene. Based on this, 2 fragments of 571 and 779 bp long were selected and expressed in E. coli. Soluble fractions of induced bacterial cultures were resolved by 12% SDS-PAGE and transferred to nitrocellulose strips. Preliminary results showed that gEc-specific mAbs recognized a single band for both constructs, while showing no reaction with seronegative samples or control antigen from non-transformed bacteria. All together these results showed the feasibility of developing effective and inexpensive diagnostic assays as essential complements for BHV-1 marker vaccines. doi:10.1016/j.vetimm.2008.10.227 Lipoarabinomannans from Mycobacterium avium subsp. avium affect bovine immune responses to different antigens Silvia Colavecchia ∗ , Ana Fernández, Silvia Mundo
Jolly, Ana
Stempler, Eloy
Area Inmunología, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarín 289, Buenos Aires, Argentina Keywords: Mycobacterium; Lipoarabinomannan; Immunomodulation; Bovine E-mail address: [email protected] (S. Colavecchia). Species: Ruminants Lipoarabinomannans (Lams) are the structurally more important outer cell wall component of all mycobacteria. Lams are ubiquitous of Mycobacteria spp. and appear as the most potent non-peptidic molecules to modulate the host immune response. Our objective was to determine the modulating effect of Lams on the immune response to soluble and particulates antigens in cattle. Lams were obtained from Mycobacterium avium subsp. avium (Maa), purified and characterized by ELISA and Western blot as described by Hamasur et al. (1999). Lams (1 mg) were injected three times to calves subcutaneously, in a mixture with the following antigens: 1 mg ovalbumin (OVA), 108 cells of canine erythrocytes (CE) or 2 mg of Maa whole bacteria. OVA, Maa and PBS (as control) were emulsified with Freund’s incomplete adjuvant (AFI) and CE was injected in PBS. Groups of calves were injected with the antigens without Lams as controls. Immune responses of calves were tested before and 21 days after the third immunization. Innate immune responses were evaluated by serum electrophoresis (EP) and respiratory burst (RB) on peripheral cells. Specific immune responses were evaluated by lymphoproliferation (LP) assay, serum ELISA and hemaglutination (HA) test in vitro and delayed-type hypersensitivity (DTH) in vivo. Differences between groups were analyzed by Kruskal Wallis or Student’s unpaired t-test. Our results show that Lams decrease significantly the RB of peripheral cells. This result agree with Leprae and Tuberculosis Lams effect described in human macrophages in vitro by Chan et al. (1991) who consider it as a survival mechanism to evade phagocytosis.
317
In addition, we detected increase in cellular immune response in Lam inoculated animals. On the other hand, specific antibodies titers were decreased by simultaneous inoculation with Lams. Our results confirm that simultaneous application of soluble and particulated antigens with Lams modifies the immune response against these antigens in cattle.
References Chan, J., et al., 1991. Infect. Immun. 59, 1755–1761. Hamasur, B., et al., 1999. FEMS Immunol. Med. Microbiol. 24, 11–17.
doi:10.1016/j.vetimm.2008.10.228 Safety of Brucella abortus RB51 vaccination of adult cows: Bacteriological evaluation of milk Karina Leite Miranda Universidade Federal de Minas Gerais, Belo Horizonte, MG. 30240-060, Brazil Keywords: Brucella abortus; Vaccine; Lipopolysaccharide Oantigen-deficient; S19 antigen E-mail address: [email protected]. Species: Ruminants Brucellosis caused by Brucella abortus is a chronic disease of cattle of worldwide public health and economic importance, resulting in abortion and infertility. B. abortus RB51 is a lipopolysaccharide O-antigen-deficient mutant of the virulent strain B. abortus 2308 used as an alternative vaccine to the S19, which induces antibodies that are detected by the routine serological tests. In Brazil, RB51 is approved for using in adult cows and there is only scanty data on the elimination of RB51 in milk. The aim of this study was to evaluate by culture the elimination of B. abortus in the milk of cows vaccinated with RB51. Ten brucellosis-free cows, vaccinated with S19 as calf, between 30 and 60 days after parturition were selected from the herd of Escola de Veterinária – UFMG, at Pedro Leopoldo – MG, Brazil. The cows were given subcutaneous injections of 1.0 mL of PBS containing 1.3 × 1010 CFU of B. abortus strain RB51, in the day 0 of experiment. Milk samples were collected from cows twice a day, in sterile polypropylene tubes on days 1, 2, 3, 4, 7, 14, 21, 28, 35, 42, 49, 56 and 63 after vaccination. The first milk streams of each teat were discarded, then 50 mL of milk samples were manually collected from all quarters and stored at −20 ◦ C. Samples were thawed and centrifuged at 2500 × g, for 15 min, the intermediated phase was discarded and the supernatant was mixed with the pellet. The mixtures were immediately inoculated in duplicate plates of tryptose agar with antibiotics (Farrel’s supplement). Moreover, 1.0 mL of each mixture was inoculated in 9.0 mL of enrichment media (tryptose broth with Farrel’s selective supplement) and incubated into 5% CO2 at 37 ◦ C for 5 days, and then inoculated in tryptose agar with antibiotics. All plates were incubated into 5% CO2 at 37 ◦ C for 9 days. No B. abortus was isolated from any sample tested. In order to confirm these results, all frozen