Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Use of oral fluids to detect anti Lawsonia intracellularis antibodies in experimentally infected pigs / Utilização de fluidos orais para detecção de anticorpos anti Lawsonia intracellularis em suínos experimentalmente infectados

Several pathogens and antibodies derived from serum or produced in tissues associated with the oral cavity are present in the oral fluid (OF). Considering the applicability of this alternative sample, recent studies in veterinary medicine have tested OF as a replacement for serum in diagnostic assays. The aim of this study was to standardize the immunoperoxidase monolayer assay (IPMA) to detect anti-Lawsonia intracellularis immunoglobulin A (IgA) and immunoglobulin G (IgG) in OF samples from experimentally infected pigs. Sixty-two pigs were divided into two groups: control (T1, n=30) and inoculated with L. intracellularis (T2, n=32). Blood, OF and fecal samples were collected at 0, 7, 14, 21, 28 and 42 days post-inoculation (dpi). Some adaptations of the standard technique for serum were made to IPMA for the detection of IgA and IgG in OF. The IPMA showed high specificity and sensitivity for serum samples and high specificity and moderate sensitivity for the detection of IgA and IgG in OF. There was high agreement between the results of serum IgG and OF IgA and IgG. Based on our results, oral fluid samples may be used for the evaluation and determination of anti-L. intracellularis antibodies in pigs, but not for individual diagnosis of swine proliferative enteropathy.(AU)

Vários patógenos e anticorpos derivados do soro ou produzidos em tecidos associados a cavidade oral estão presentes no fluido oral (FO). Considerando a aplicabilidade dessa amostra alternativa, estudos recentes em medicina veterinária têm testado o FO como substituto do soro para testes diagnósticos. O objetivo desse estudo foi padronizar a imunoperoxidase em monocamada de célula (IPMC) para a detecção de imunoglobulina A e imunoglobulina G anti-Lawsonia intracellularis em amostras de FO de suínos experimentalmente infectados. Um total de 62 suínos foram divididos em dois grupos: controle (T1, n=30) e inoculados com L. intracellularis (T2, n=32). Sangue, FO e amostras de fezes foram coletados aos 0, 7,14, 21, 28 e 42 dias após a inoculação (dpi). Algumas adaptações da técnica foram realizadas na técnica padrão da IPMC para a detecção de IgA e IgG. A IPMC demostrou alta especificidade e sensibilidade para amostras de soro e alta especificidade de moderada sensibilidade para a detecção de IgA e IgG em FO. Houve alta concordância entre resultados de detecção de IgG em soro com a IgA e IgG em amostras de FO. Baseado em nossos resultados, amostras de fluido oral podem ser usadas em avaliações e detecção de anticorpos anti-L. intracellularis em suínos, porém não de forma individual.(AU)

Experimental reproduction of proliferative enteropathy and the role of IFN-gamma in protective immunity against Lawsonia intracellularis in mice

Proliferative enteropathy was reproduced in IFN-gamma receptor knockout (IFN-gamma R-) mice by experimental infection with Lawsonia intracellularis (L. intracellularis). The cecum and the colon of the infected mice were evidently enlarged 2 weeks post infection. The presence of L. intracellularis was identified in the stool and the cecum of the mice after infection. However, high levels of IFN-gamma were detected in the sera of the infected mice 2 weeks PI. These data indicated that the IFN-gamma produced in the infected mice should have been utilized by it's receptor to elicit protective immune responses against L. intracellularis infections.