80 jorge lopez vaccine 2014

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Vaccine 32 (2014) 6805–6811

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Vaccine journal homepage: www.elsevier.com/locate/vaccine

Oral immunization against porcine pleuropneumonia using the cubic phase of monoolein and purified toxins of Actinobacillus pleuropneumoniae Jorge Lopez-Bermudez a , David Quintanar-Guerrero b , Horacio Lara Puente e , d ´ ´ ¨ ´ Carrasco a , Jorge Tortora Perez c , Francisco Suarez Guemez , Abel Ciprian Susana Mendoza Elvira a,∗ a

Laboratorio de Virología-FES-Cuautitlán, UNAM, Mexico Laboratorio en Tecnología Farmacéutica FES-Cuautitlán, UNAM, Mexico c Laboratorio de Patología-FES-Cuautitlán, UNAM, Mexico d Facultad de Medicina Veterinaria y Zootecnia, UNAM, Mexico e Laboratorios Avi-Mex, S.A. de C.V., Mexico b

a r t i c l e

i n f o

Article history: Received 4 June 2014 Received in revised form 11 September 2014 Accepted 24 September 2014 Available online 19 October 2014 Keywords: Porcine pleuropneumonia Apx I Apx II Apx III Immunity Oral vaccine

a b s t r a c t The main goal of this work was to obtain an orally administered immunogen that would protect against infections by Actinobacillus pleuropneumoniae. The Apx I, II and III toxins were obtained from the supernatants of cultures of serotypes 1 and 3 of A. pleuropneumoniae. The capacity of monoolein gel to trap and protect the Apx toxins, and the effect of their incorporation on the stability of the cubic phase were evaluated. The gel was capable of trapping a 400-␮g/ml concentration of the antigen with no effects on its structure. Approximately 60% of the protein molecules were released from the gel within 4 h. Four experimental groups were formed, each one with four pigs. All challenges were conducted in a nebulization chamber. Group A: Control (−) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: vaccinated twice intramuscularly with ToxCom (a commercial toxoid) at an interval of 15 days and then challenged; and Group D: vaccinated orally twice a week for 4 weeks with ToxOral (an oral toxoid) and challenged on day 28 of the experiment with a same dose of 2.0 × 104 UFC of A. pleuropneumoniae serotypes 1 and 3. The lesions found in group B covered 27.7–43.1% of the lungs; the pigs in group C had lesions over 12.3–28%; and those in group D over 15.4–32.3%. No lesions were found in the Group A pigs. A. pleuropneumoniae induced macroscopic lesions characteristic of infection by and lesions microscopic detected by histopathology. The etiologic agent was recovered from the infected lungs, tonsils and spleen. The serotypes identified were 1 and 3. An indirect ELISA test identified the antibodies against the Apx toxins in the serum of the animals immunized orally. © 2014 Published by Elsevier Ltd.

1. Introduction Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiologic agent of contagious porcine pleuropneumonia (CPP), a highly transmissible respiratory illness responsible for large economic losses on pig farms [1]. To date, 15 serotypes of App have been identified, based on capsular antigens, all with different degrees of virulence [2,3]. CPP is characterized by

∗ Corresponding author at: Facultad de Estudios Superiores – Cuautitlán, UNAM, Laboratorio de Virología, Av. 1о , De Mayo S/N Campo I, Santa María las Torres, CP 54700 Cuautitlán Izcalli, Estado de México, Mexico. Tel.: +55 5623 2058; fax: +52 55 56232058. E-mail address: seme@unam.mx (S. Mendoza Elvira). http://dx.doi.org/10.1016/j.vaccine.2014.09.056 0264-410X/© 2014 Published by Elsevier Ltd.

manifestations of variable clinical signs in the acute or chronic phases [4,5]. The bacteria present a series of actions coordinated with diverse virulence factors, such as capsular antigens [6,7], external membrane protein antigens [8,9], external membrane lipopolysaccharides [10,11], the Ca2+ -dependent Apx I, II, III and IV toxins, with hemolytic and/or cytotoxic activity. These toxins have been identified in all 15 serotypes of A. pleuropneumoniae [3,4,12–15]. The virulence of A. pleuropneumoniae is multifactorial, several studies have shown that virulence is strongly associated with production of the Apx toxins [7,11,16]. The various types of vaccines, commercially available combine bacterins, bacterins/toxoids and toxoids, but they continue to be applied by parenteral route. Thus, developing oral immunization methods with carrier and protection systems could be of great importance in controlling CPP [17–19].


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