Haemophilus parasuis es el agente etiológico de la enfermedad de Glässer, una enfermedad re-emergente porcina caracterizada por poliserositis fibrinosa, poliartritis y meningitis. Una de las características de H. parasuis es su alta variación antigénica. Se han descrito quince serotipos y existen muchas cepas que no pertenecen a ninguno de los serotipos descritos. También hay diferencias en la virulencia demostrada en la inoculación experimental de diferentes cepas, que van desde altamente virulentas a no patógenas. Esto podría estar relacionado con la existencia de un linaje no virulento de H. parasuis, comensal del tracto respiratorio superior de los cerdos. El control de la enfermedad de Glässer mediante la vacunación se ha intentado mediante el uso de bacterias inactivadas (bacterinas). Sin embargo, estas vacunas tienen un uso limitado por la falta de protección cruzada entre las cepas.

Investigadores del CReSA han utilizado un enfoque de vacunología inversa para determinar nuevos antígenos protectores. Esta metodología se basa en la secuenciación del genoma completo y el uso de análisis bioinformáticos para identificar nuevos candidatos vacunales, basándose en la predicción de que se encuentren expuestos en la membrana externa o que sean proteínas secretadas. Posteriormente, los candidatos son clonados y producidos como proteínas recombinantes. Las proteínas recombinantes se utilizan para probar su inmunogenicidad y capacidad protectora in vivo. Además, los genes de estos nuevos antígenos han sido utilizados para desarrollar diferentes herramientas moleculares para mejorar el diagnóstico de H. parasuis.

Cuatro publicaciones en revistas científicas resumen el trabajo realizado por los investigadores del CReSA para definir nuevos antígenos que podrían ayudar a mejorar el diagnóstico y las vacunas actuales frente a H. parasuis:

Pina S, Olvera A, Barceló A, Bensaid A. Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains. J Bacteriol. 2009 Jan;191(2):576-87.

This first work describes the identification of the first antigen candidates, the virulence-associated trimeric autotransporters (vtaA). In this study the whole genome of the highly virulent strain Nagasaki was sequenced. This allowed the in silico characterization of 13 trimeric autotransporters as assessed by the presence of a YadA C-terminal translocator domain. Their function was predicted to be related to adhesion of the bacteria to host cells, since the predicted structure of these proteins possess motifs commonly found in adhesins, hemagglutinins and invasins. In addition, they have various copies of collagen-like repeats in the middle of the proteins. The phylogenetic analysis of the translocator domains classified the vtaA into three groups. Then the genome comparison using microarray hybridization of the Nagasaki strain with different strains revealed that group 1 vtaA genes were divergent among virulent and non-virulent strains. This initial study allowed the development of different applications of the VtaA in diagnostics and vaccination.

Olvera A, Pina S, Pérez-Simó M, Oliveira S, Bensaid A. Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo. Vet Res. 2010 May-Jun;41(3):26.

Trimeric autotransporters are good vaccine candidates. For this reason the antigenic capacity of VtaA was evaluated. Fifteen recombinant VtaA passenger domains were produced as recombinant proteins and used to screen H. parasuis immune sera by immunoblotting. After infection of 4 animals with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 of the 13 VtaA of the Nagasaki strain was detected. IgA production against VtaA was detected in only one animal. Antibody cross-reaction with two of the corresponding proteins of strain HP1319 was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain. Taken together, these results indicate that the 6 VtaA were expressed during infection, inducing an antibody response. However, they are poorly expressed in the in vitro conditions used to produce the bacterin. Thus, VtaA are good immunogen candidates that could be used to improve the antigenicity of H. parasuis bacterines.

Olvera A, Pina S, Pérez-Simó M, Aragón V, Segalés J, Bensaid A. Immu
nogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA). Vaccine. 2011 Mar 24;29(15):2797-802.

After the description of the antigenicity of VtaA their immunogenic and protective capacity was evaluated. In this study 6 VtaA were produced as recombinant proteins and used to immunize snatch-farrowed, colostrum-deprived piglets. An in house developed ELISA demonstrated that immunized animals developed specific anti-VtaA systemic and mucosal antibodies. The protective capacity of the anti-VtaA antibodies was evaluated by the inoculation of 3×108 or 6×106 colony forming units (CFU) of the highly virulent strain Nagasaki. While non-vaccinated animals died in the first 2 days of the trial, vaccinated animals had a delayed course of disease and 33 or 57%, respectively, of the animals survived the lethal challenge. The protection achieved with the recombinant VtaA supports their potential as candidates to improve future vaccine formulations against H. parasuis.

Olvera A, Pina S, Macedo N, Oliveira S, Aragon V, Bensaid A. Identification of potentially virulent strains of Haemophilus parasuis using a multiplex PCR for virulence-associated autotransporters (vtaA). Vet J. 2011 Jan 17.

In this final study CReSA researchers evaluated by PCR the presence of the three groups of VtaA in 157 isolates. Group 3 vtaA genes were demonstrated in all H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains: 96% of the group 1 negative isolates were isolated from nasal passages of healthy animals, whereas no group 1 negative isolates were isolated from cases of Glässer’s disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.