Serological response to Pasteurella multocida NanH sialidase in persistently colonized rabbits

Abstract

Pasteurella multocida is a mucosal pathogen that colonizes the upper respiratory system of rabbits. Respiratory infections can result, but the bacteria can also invade the circulatory system, producing abscesses or septicemia. P. multocida produces extracellular sialidase activity, which is believed to augment colonization of the respiratory tract and the production of lesions in an active infection. Previously, it was demonstrated that some isolates of P. multocida contain two unique sialidase genes, nanH and nanB, that encode enzymes with different substrate specificities (S. Mizan, A. D. Henk, A. Stallings, M. Meier, J. J. Maurer, and M. D. Lee, J. Bacteriol. 182:6874-6883, 2000). We developed a recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the NanH sialidase of P. multocida and demonstrated that rabbits that were experimentally colonized with P. multocida produce detectable anti-NanH immunoglobulin M (IgM) and IgG in serum, although they demonstrated no clinical signs of pasteurellosis. In addition, clinically ill pet rabbits infected with P. multocida possessed IgM and/or IgG antibody against NanH. The NanH ELISA may be useful for the diagnosis of P. multocida infections in sick rabbits as well as for screening for carriers in research rabbit colonies.

FIG. 1.

Derived amino acid sequence of P. multocida NanH sialidase acquired by PCR amplification of reference serotype strains (except those of serotypes 1 and 14, which were not amplified, and serotypes 2 and 6, which were not tested). The sequences exhibit 96.5% similarity and 78.8% identity among isolates; the sequence identity (dots) with the sequence of isolate 86-1913 is shown.

FIG. 2.

Phases of colonization of the respiratory system and infection of rabbits by P. multocida. After aerosol exposure, the replication of low-virulence strains may be limited to the mucus layer and the epithelial cell surface of the upper respiratory tract. Heavy colonization of the nasal turbinates and trachea can result without overt signs of illness. These animals may exhibit anti-NanH antibodies if they are persistently colonized. However, depression of mucosal defenses may enable bacterial invasion of the respiratory mucosa and consequent clinical symptoms of disease. Highly virulent strains of P. multocida may rapidly invade host cells and replicate in deep tissue, resulting in acute symptoms of localized or systemic disease. Clinically ill animals may possess anti-NanH antibodies if they are persistently colonized with P. multocida but probably exhibit antibodies against whole-cell lysates during chronic infection and during the convalescent period.

FIG. 3.

Serological response to NanH sialidase by two rabbits experimentally infected with a cocktail of three low-virulence P. multocida isolates. Colonization of the animals was confirmed by PCR detection of the bacteria in the trachea 14 weeks after exposure. Serum samples were tested for NanH (nonisotype-specific) IgG (at a 1:16 dilution) and IgM (at a 1:8 dilution) by ELISA. Serum samples (obtained 5 to 11 weeks postinfection) were tested by a whole-cell-lysate ELISA, as described by Kawamoto et al. (29). (A) Changes in the ODs of the samples, as determined by ELISA, over 11 weeks. Asterisks, positive readings; star, the sample positive by the Kawamoto ELISA. (B) The highest dilution (titer) that resulted in a positive OD.