Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings.

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and -25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 µg of protein. Group I ducks received 3762 µg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

RIG-I expression pattern and cytokine profile in indigenous ducks infected with duck plague virus.

The present study was undertaken to elucidate mRNA expression pattern of RIG-I and serum cytokines profile alterations in indigenous ducks of Assam, India viz. Pati, Nageswari and Cinahanh in response to natural infections of duck plague virus. Field outbreaks of duck plague virus were attended during the study period for collection of tissue and blood samples. The ducks under study were divided into three distinct groups as per health status i.e. healthy, duck plague infected and recovered. Results from the study revealed that RIG-I gene expression was significantly upregulated in liver, intestine, spleen, brain and PBMC of both infected and recovered ducks. However, fold changes in RIG- I gene expression was lower in recovered ducks as compared to infected ones which indicated continued stimulation of RIG-I gene by the latent viruses. Both serum pro and anti-inflammatory cytokines were elevated in infected ducks as compared to healthy and recovered ducks, indicating activation of inflammatory reactions in the ducks due to virus invasion. The results from the study indicated that innate immune components of the infected ducks were stimulated in order to make an attempt to resist the virus from the infected ducks.