Development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera.

Serological tests, such as agglutination and indirect hemagglutination assay (IHA), have been used to identify antibodies against Pasteurella multocida in poultry sera, but none are highly sensitive. An enzyme-linked immunosorbent assays (ELISA) has been used with varying degrees of success in attempts to monitor seroconversion in vaccinated poultry, but are not suitable for diagnosis. Commercial ELISA kits are available for chickens and turkeys, but not for ducks. The present study reports development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between the two diagnostic tests. An in-house indirect ELISA was developed using a heat extract antigen of P. multocida strain X-73 as a coating antigen and horseradish peroxidase conjugated goat anti-duck IgG antibody (dIgG-HRP). The checkerboard titration method was done using sera from ducks immunized with P. multocida bacterin as positive sera and 1day old duckling sera as negative sera. The heat extract antigen at 1µg/ml, sample serum at a dilution of 1:100, and dIgG-HRP 1:2000 were optimal concentrations for the assay. The cut-off value was 0.200. Of the duck sera, 89.05% (244/274) were considered seropositive by ELISA. Estimates for sensitivity and specificity of ELISA were higher than prior values with medians of 94.7% [95% posterior probability interval (PPI)=89.6-98.2%] and 87.2% (PPI=68.2-98.3%). Estimates for sensitivity of IHA were lower than prior values (median=97.6, PPI=93.2-99.7%) while the specificity was close to the prior value (median=76.5, PPI=65.8-85.4%). This finding suggests that an in-house indirect ELISA can be used to detect duck antibody to fowl cholera.

Cross-protection conferred by immunization with an rOmpH-based intranasal fowl cholera vaccine.

A previous study demonstrated that a recombinant outer membrane protein H (rOmpH)-based intranasal fowl cholera vaccine elicited efficient homologous protection against the Pasteurella multocida strain X-73 (A:1) in chickens. The present study aimed to determine the cross-protectivity against heterologous P. multocida strains. The rOmpH was purified via electroelution and formulated with two kinds of adjuvants. The vaccine formulations in a total volume of 100 µl were 100 µg rOmpH with 3 µg of Escherichia coli enterotoxin B or 10 µg of CpG ODN2007. Chickens were assigned to three experimental groups depending on bacterial strain challenge exposure as well as three control groups. The chickens were immunized intranasally three times at three-week intervals. Challenge exposures were conducted by inoculation with homologous strain X-73 or heterologous strains P-1059 (A:3) or P-1662 (A:4) at four weeks after the final immunization. The specific antibody against rOmpH was produced in vaccinated birds. Sera IgY and secretory IgA antibody titres were significantly increased (P < 0.05) post-immunization. The stimulation index values of the vaccinated groups were significantly different from stimulation index values of the non-vaccinated groups (P < 0.05). Chicken survival rates after exposure to avian P. multocida strains ranged from 70% to 100%. There was no significant difference in protection between two kinds of adjuvants in vaccine formulations. Statistical analysis indicated no significant differences in protection among avian P. multocida strains challenge exposure. We conclude that an in-house rOmpH-based intranasal fowl cholera vaccine produced efficient cross-protectivity against heterologous strains of P. multocida.

Comparison of the Effect of Two Purification Methods on the Immunogenicity of Recombinant Outer Membrane Protein H of Pasteurella multocida Serovar A:1.

Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinity chromatography but this adversely affects its immunogenicity. The current study presents the results from an intervention study comparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography and native OmpH purified using electroelution and a nonimmunized control group. Chickens immunized with rOmpH purified using electroelution produced the highest ELISA antibody levels against P. multocida strains. Chickens in each of the 5 treatment groups were split into two subgroups for challenge with two different P. multocida strains. The average number of adhesions to CEF cells was statistically significantly lower in sera from chickens immunized with rOmpH or native OmpH purified using electroelution than in those of the three other treatment groups. The survival amongst chickens immunized with rOmpH or native OmpH purified using electroelution indicated high levels of protection. In contrast, survival probability was zero or low in the groups immunized with rOmpH purified using affinity chromatography and in the nonimmunized group. These findings show that the rOmpH purified using electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P. multocida infection.

Protectivity conferred by immunization with intranasal recombinant outer membrane protein H from Pasteurella multocida serovar A:1 in chickens.

Recombinant outer membrane protein H (rOmpH) is a potential fowl cholera vaccine candidate. The present study was aimed at developing rOmpH formulations for intranasal administration. The rOmpH was purified and formulated with either Escherichia coli enterotoxin B (LTB) or CpG oligodeoxynucleotides (ODN) as an adjuvant. Antibody responses in chickens intranasally immunized with rOmpH in combination with 2 different adjuvants were significantly increased (P<0.05) post immunization. Chicken survival rates showed that rOmpH formulated with ODN and LTB elicited 90% and 70% protection, respectively. Our findings indicated that rOmpH formulated with ODN elicited protection better than that formulated with LTB. Therefore, the vaccines formulations in the present study can be considered new intranasal vaccine formulations for fowl cholera in chickens.