Desarrollo de un inmunógeno prototípico para el control de mastitis bovinas causadas por Staphylococcus aureus

Tesis para la obtención del grado académico de Doctora en Ciencias Biológicas, de la Universidad Nacional del Litoral, en 2012Staphylococcus aureus es el patógeno más frecuentemente aislado de casos de mastitis bovina en Argentina y el mundo. Las particulares características de las infecciones intramamarias por S. aureus, han llevado a la búsqueda de alternativas para coadyuvar al control de esta enfermedad, siendo una de ellas la inmunización. El objetivo general de este trabajo fue desarrollar una vacuna prototípica contra mastitis bovinas causadas por S. aureus. Se compararon las respuestas inmunes generadas en bovinos por bacterinas de S. aureus productoras de polisacárido capsular 5 (CP5), formuladas con Al(OH3), o Iscom Matrix. La formulación con Iscom Matrix potenció la producción de anticuerpos específicos en sangre y leche, con elevada capacidad opsónica. Posteriormente, se compararon las respuestas inmunes generadas en bovinos por una bacterina y un lisado de S. aureus CP5 formulados con Iscom Matrix. La formulación con lisado bacteriano mostró un mejor desempeño en cuanto a la producción de anticuerpos específicos y funcionales, y la expresión de citoquinas con potencial importancia en el control de infecciones por S. aureus. Finalmente, se obtuvo un inmunógeno prototípico compuesto por lisado de S. aureus CP5, suplementado con los antígenos recombinantes proteína de unión a fibronectina A (FnBP-A), clumpling factor A (ClfA) y β-toxina, formulado con Iscom Matrix. La inmunización de vaquillonas con el inmunógeno multicomponente potenció la respuesta de anticuerpos en sangre y leche dirigidos hacia los antígenos recombinantes. Los mismos resultaron eficientes en la opsonización del microorganismo y en la neutralización de la actividad de la β-toxina nativa de S. aureus in vitro.Staphylococcus aureus is the most frequently isolated pathogen from bovine mastitis in Argentina and worldwide. The peculiar pathogenic characteristics of S. aureus lead to the development of alternatives measures, like immunization, to control this disease. The aim of this study was to develop a prototype vaccine against S. aureus bovine mastitis. Immune responses induced in heifers by S. aureus bacterins from a capsular polisacharide 5 (CP5) producing strain, formulated either with Al(OH)3 or Iscom Matrix™, were compared. Formulation of experimental immunogen with Iscom MatrixTM strengthened humoral in blood and milk, with an augmented opsonic capacity. In addition, immune responses generated in heifers by either a S. aureus CP5 bacterin or lysate, formulated with Iscom MatrixTM were evaluated. The bacterial lysate formulation showed a better performance regarding production of specific and functional antibodies and induction of cytokines involved in early immune responses to the immunogen. Finally, a prototype immunogen composed by S. aureus CP5 lysate, supplemented with recombinant antigens fibronectin binding protein A (FnBP-A), clumping factor A (ClfA) and β-toxin, adjuvanted with Iscom MatrixTM, was obtained. Heifers’ immunization with the multicomponent immunogen strengthened antibody responses against recombinant antigens in blood and milk. These antibodies were efficient in opsonizing S. aureus and blocking native β-toxin hemolytic activity in vitro.EEA RafaelaFil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentin

Risk factors of S. aureus intramammary infection in pre partum dairy heifers under grazing conditions and molecular characterization of isolates from heifers and cows

The aims of the research reported here were to identify potential risk factors associated with the presence of Staphylococcus aureus intramammary infection (IMI) in pre partum dairy heifers on 17 dairy farms from three provinces of Argentina and to characterize, at molecular level, isolates from those heifers and lactating cows from two selected herds. A total of 1474 heifers and 4878 lactating cows were studied. The prevalence of Staphylococcus aureus IMI in the heifers, heifers at quarter level and lactating cow mammary quarters was 14.41, 4.82, and 14.65%, respectively. Univariate analysis showed the key variables associated with S. aureus IMI presence in the heifers were: S. aureus IMI prevalence in cows of the lactating herd, the time calves stayed with their dam after birth, the calf rearing system, the place of rearing (own farm or other dairy farm) and fly control on the farm. None of the variables included in the multivariable analysis was associated with the presence of S. aureus IMI in the pre partum heifers, probably due to low variability among management practices used by the farms for rearing the heifer calves. At the molecular level, S. aureus isolates were grouped into three main PFGE clusters and several genotypes within the clusters. Isolates from mammary secretion of pre partum heifers and milk of lactating cows comprised different PFGE clusters in both herds, although two exceptions occurred. The absence of gene fnbpB, which codifies for a virulence factor protein involved in cell invasion by S. aureus, was significantly more frequent in pre partum heifer secretion isolates than in isolates from lactating cow milk. These results suggest that, under these management conditions, isolates from mammary secretions of pre partum heifers do not originate from the milk of lactating cows, but rather other sources to which the heifer is exposed.EEA RafaelaFil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Molineri, Ana Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Neder, Veronica Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina.Fil: Vitulich, Carlos Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina.Fil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina

Humoral immune response of pregnant goats to two Corynebacterium pseudotuberculosis bacterin formulations

Caseous lymphadenitis is a chronic suppurative bacterial disease caused by Corynebacterium pseudotuberculosis, and it has a high prevalence among small ruminants. Different vaccine preparations have been employed to control this disease. The adjuvant used in the formulation is a critical factor for the improvement of vaccine efficacy. No commercial vaccines are available in Argentina. The aims of the present work were to compare the specific antibody response against a C. pseudotuberculosis whole cell vaccine formulated with a classical and an immunoestimulant adjuvant in pregnant goats, and to assess the transfer of vaccine-specific antibodies from the goat to the offspring through colostrum. Twenty three pregnant goats were vaccinated. The animals were randomly allocated to three groups: control, aluminum hydroxide Al(OH)3 adjuvant and a new cage-like particle adjuvant (ISPA). Antibodies (IgG) against this antigen were measured by ELISA. A robust humoral immune response was detected in vaccinated animals, with production of specific IgG. No significant differences were found between the vaccines formulated with different adjuvants. High levels of specific antibodies were detected in colostrum samples from both immunised groups, which were passively transferred from goats to offprings. No adverse local reactions in the vaccines injection sites were detected

Humoral immune response of pregnant goats to two Corynebacterium pseudotuberculosis bacterin formulations

Caseous lymphadenitis is a chronic suppurative bacterial disease caused by Corynebacterium pseudotuberculosis with a high prevalence in small ruminants. Different vaccine preparations have been employed to control this disease. The adjuvant used in the formulation is a critical factor for the improvement of vaccine efficacy. No commercial vaccines are available in Argentina. The aims of the present work were to compare the specific antibody response against a C. pseudotuberculosis whole cell vaccine formulated with a classical and an immunoestimulant adjuvant in pregnant goats, and to assess the transfer of vaccine- specific antibodies from the goat to the offspring through colostrum. Twenty three pregnant goats were vaccinated. The animals were randomly allocated to three groups: control, aluminum hydroxide Al(OH)3 adjuvant and a new cage-like particle adjuvant (ISPA). Antibodies (IgG) against this antigen were measured by ELISA. A robust humoral immune response was detected in vaccinated animals, with production of specific IgG, without finding significant differences between the vaccines formulated with different adjuvants. High levels of specific antibodies were detected in colostrum samples from both immunized groups, which were passively transfered from goats to offprings. No adverse local reactions in the vaccines injection sites were detected.Fil: Auad, Jordana. Universidad Católica de Córdoba; ArgentinaFil: Cerutti, Julieta. Universidad Católica de Córdoba; ArgentinaFil: Cooper, Laura Gabriela. Universidad Católica de Córdoba; ArgentinaFil: Camussone, Cecilia María. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Lozano, Natalia A.. Universidad Católica de Córdoba; ArgentinaFil: Crespo, Francisco M.. Establecimiento Los Talas. Potrero de Garay, Córdoba; ArgentinaFil: Lozano, Alejandro. Universidad Católica de Córdoba; Argentin

Functional role of antibodies generated in heifers through immunization with Staphylococcus aureus vaccines in invasion and phagocytosis assays

A successful Staphylococcus aureus vaccine should elicit a long-term antibody response that prevents establishment of the infection. The aim of the present study was to evaluate the functional role of antibodies raised against different S. aureus CP5 vaccines in invasion to bovine mammary epithelial cells (MAC-T) and phagocytosis by bovine milk macrophages in vitro. Sera and whey from cows immunized with a whole-cell S. aureus CP5 vaccine adjuvanted with Al(OH)3 or with ISCOM Matrix, significantly reduced internalization of S. aureus in MAC-T cells without significant differences between both groups. The effect of antibodies generated by a S. aureus whole-cell and a lysate vaccine formulated with ISCOM Matrix was also evaluated. Sera and whey from both immunized groups significantly reduced S. aureus internalization in MAC-T cells without significant differences between both groups. Whey antibodies against whole-cell and lysate vaccines were also able to inhibit internalization in MAC-T cells of a heterologous S. aureus strain. In addition, sera from animals vaccinated with S. aureus lysate or bacterin promoted milk macrophage phagocytosis. These results provide an insight into the potential mechanisms by which these vaccines can afford protection to the mammary gland against S. aureus intramammary infection.EEA RafaelaFil: Renna, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Pereyra, Elizabet Amanda Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Veterinarias del Litoral. Laboratorio de Biología Celular y Molecular Aplicada; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Baravalle, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Veterinarias del Litoral. Laboratorio de Biología Celular y Molecular Aplicada; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dallard, Bibiana Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Veterinarias del Litoral. Laboratorio de Biología Celular y Molecular Aplicada; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Marcipar, Iván Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentin

Comparison of phenotypic tests for detecting penicillin G resistance with presence of blaZ gene in Staphylococcus aureus isolated from bovine intramammary infections

Few studies have described the relationship between genotypic and phenotypic methods for detecting penicillin resistance in Staphylococcus aureus isolated from bovine intramammary infection (IMI). Six phenotypic methods for penicillinase detection were compared with a genotypic method testing the presence of the β-lactamase gene blaZ in Staph. aureus (n = 150) isolated from bovine IMI. Highest sensitivities and specificities were observed for disk diffusion (DD) (93 and 97·4%), minimum inhibitory concentration (MIC) (90·3 and 97·4%), Cefinase™ (85·9 and 97·4%) and Diatabs™ (85·7 and 98·7%). The estimated cut-off points estimated in the present study can be considered close to the ones indicated by CLSI (2013). The molecular detection of blaZ gene is the only method that may indicate the real or potential capacity of producing β-lactamase in Staph. aureus. Considering that from a clinical standpoint a false negative result from a phenotypic test is the most unfavourable situation, a combination of standard DD with Diatabs™ or Cefinase™ should be performed by routine mastitis laboratories to minimise false negative results.EEA RafaelaFil: Russi, Norma B.. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Maito, Julia. Lactodiagnóstico Sur; ArgentinaFil: Dieser, Silvana Andrea. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales; ArgentinaFil: Renna, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neder, Veronica Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Pol, Martín. Lactodiagnóstico Sur; ArgentinaFil: Tirante, Liliana. Lactodiagnóstico Sur; ArgentinaFil: Odierno, Liliana Mónica. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentin

Presencia de <i>Moraxella bovoculi</i> y <i>Moraxella bovis</i> en rodeos bovinos lecheros con antecedentes de brote de queratoconjuntivitis infecciosa bovina

La queratoconjuntivitis infecciosa bovina (QIB) es una enfermedad altamente contagiosa, de distribución mundial, caracterizada por afectar el ojo de los bovinos. En Argentina existe escasa información actualizada sobre la epidemiología de la enfermedad. Si bien, M. bovis es el principal agente aislado en casos de QIB, poco se ha investigado sobre la presencia de otras especies de Moraxella en su patogenia. El objetivo de este trabajo fue contribuir al conocimiento de la epidemiología de la QIB evaluando la prevalencia de especies de Moraxella (M. bovis, M. bovoculi, M. ovis) presentes en rodeos lecheros de la cuenca santafesina. Se utilizaron animales de dos rodeos lecheros con antecedentes de brotes de QIB. Se tomaron muestras de 47 animales (crianza artificial y recría), en diferentes estadios de la enfermedad, mediante hisopado de la mucosa y conjuntiva ocular.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

A safe and effective vaccine against bovine leukemia virus

peer reviewedPrevious attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1