LKTA and PlpE small fragments fusion protein protect against Mannheimia haemolytica challenge Carolina Guzmán-Brambila a , Saray Quintero-Fabián a , Celia González-Castillo a,b , Álvaro de Obeso-Fernández del Valle a , Beatriz Flores-Samaniego b , Germán de la Mora b , Argelia E. Rojas-Mayorquín c,d , Daniel Ortuño-Sahagún a,⇑ a Laboratorio de Desarrollo y Regeneración Neural, Instituto de Neurobiología, Departamento de Biología Celular y Molecular, CUCBA, Universidad de Guadalajara, camino Ing. R. Padilla Sánchez, 2100, Las Agujas, Zapopan 44600, Mexico b Laboratorio de Investigación y Desarrollo Bio-Zoo, S.A. de C.V, Mexico c Departamento de Ciencias Ambientales, Instituto de Neurociencias, CUCBA, Universidad de Guadalajara, Guadalajara, Jalisco 45100, Mexico d Departamento de Investigación Básica, Instituto Nacional de Geriatría – INGER. Periférico Sur No. 2767, Col. San Jerónimo Lídice, Deleg. Magdalena Contreras 10200, Mexico D.F., Mexico article info Article history: Received 11 January 2012 Accepted 2 July 2012 Keywords: Mannheimia haemolytica Respiratory bovine complex Leukotoxin PlpE Vaccine abstract Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle background- ing and feedlot industries. Mannheimia haemolytica is considered the most important pathogen associated with this disease. Vaccines against M. haemolytica have been prepared and used for many decades, but traditional bacterins have failed to demonstrate effective protection and their use has often exacerbated disease in vaccinated animals. Thus, the BRD complex continues to exert a strong adverse effect on the health and wellbeing of stocker and feeder cattle. Therefore, generation of recombinant proteins has been helpful in formulating enhanced vaccines against M. haemolytica, which could confer better protection against BRD. In the present study, we formulated a vaccine preparation enriched with recombinant small fragments of leukotoxin A (LKTA) and outer-membrane lipoprotein (PlpE) proteins, and demonstrated its ability to generate high antibody titers in rabbits and sheep, which protected against M. haemolytica bacterial challenge in mice. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle backgrounding and feedlot indus- tries (Gagea et al., 2006; Snowder et al., 2006). BRD research has provided significant understanding of the disease over the past 30 years (Fulton, 2009). Modern research tools have been used to generate advances in products including vaccines as well as tech- nological, biological, and pharmacological developments. The bac- terial component of the BRD complex continues to have a major adverse effect on health and wellbeing of stocker and feeder cattle (McVey, 2009; Griffin, 2010). BRD involves complex interactions among viral and bacterial pathogens that can lead to intense pulmonary inflammation (fibrinous pleuropneumonia) (Czuprynski, 2009). Among bacterial pathogens associated with the disease, two Gram negative bacteria are the more relevant: Histophilus somni, which causes respiratory disease, septicemia, thrombotic meningoencephalitis, myocarditis, arthritis, and abortion (Corbeil, 2007; O’Toole et al., 2009; Sandal and Inzana, 2010), and Mannheimia haemolytica, which is consid- ered the most important (Confer, 2009). Pathogens involved in the BRD complex have developed intricate mechanisms to thwart both the innate and adaptive immune responses of their hosts. These immune evasion strategies are likely to contribute to the failure of currently available vaccines to provide complete protec- tion to cattle against these pathogens (Srikumaran et al., 2007). M. haemolytica is an opportunist bacterium, gaining access to the lungs, when host defenses are compromised by stress or infec- tion with respiratory viruses or mycoplasma. Although, several ser- otypes act as commensals, A1 and A6 are the most common isolates from pneumonic lungs (Rice et al., 2007; Confer, 2009). Among potential virulence factors of M. haemolytica, leukotoxin A (LKTA), an exotoxin that is cytolytic for macrophages, neutrophils and all other leukocyte subsets (Berggren et al., 1981; Shewen and Wilkie, 1982), is critical in the induction of pneumonia (Jeyaseelan et al., 2002; Rice et al., 2007; Czuprynski, 2009) and antibodies 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.07.004 ⇑ Corresponding author. Address: Laboratorio de Desarrollo y Regeneración Neural, Departamento de Biología Celular y Molecular, P.O. Box 52-126, Guadalajara, Jalisco 45020, Mexico. Tel.: +52 33 37771191x33211; fax: +52 33 37771191. E-mail address: dortuno@cucba.udg.mx (D. Ortuño-Sahagún). Research in Veterinary Science 93 (2012) 1293–1300 Contents lists available at SciVerse ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc