Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization.

Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage 'ϕLd'. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 µg protein and 120 µg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 µg protein and 60 µg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

Isolation of bacteriophages infecting Xanthomonas oryzae pv. oryzae and genomic characterization of novel phage vB_XooS_NR08 for biocontrol of bacterial leaf blight of rice.

Bacterial leaf blight (BLB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases worldwide in rice-growing regions. The Ineffectiveness of chemicals in disease management has increased the interest in phage therapy. In this study, we isolated 19 bacteriophages, infecting Xoo, from a rice field, which belonged to phage families Siphoviridae, Myoviridae, and Podoviridae on the basis of electron microscopy. Among 19 phages, Phage vB_XooS_NR08, a member of the Siphoviridae family, expressed antibacterial activity against all Xoo strains tested and did not lyse X. campestris and other unrelated bacterial hosts. Phage NR08 showed more than 80% viability at a temperature range of 4°C-40°C, pH range of 5-9, and direct exposure to sunlight for 2 h, whereas UV light and chemical agents were highly detrimental. In a one-step growth curve, NR08 has a 40-min latent period, followed by a 30-min burst period with a burst size of 250 particle/bacterium. The genome of NR08 is double-stranded DNA, linear having a size of 98,812 bp with a G + C content of 52.9%. Annotation of the whole-genome sequence indicated that NR08 encodes 142 putative open reading frames (ORFs), including one ORF for tRNA, namely, trna1-GlnTTG. Comparative genome analysis of NR08 showed that it shares maximum similarity with Pseudomonas phage PaMx42 (40% query coverage, 95.39% identity, and acc. Length 43,225) and Xanthomonas phage Samson (40% query coverage, 96.68% identity, and acc. Length 43,314). The average alignment percentage (AP) of NR08 with other Xoophages was only 0.32 to 1.25 since the genome of NR08 (98.8 kb) is almost double of most of the previously reported Xoophages (43-47 kb), thus indicating NR08 a novel Xoophage. In in vitro bacterial challenge assay, NR08 showed bacteriostasis up to 24 h and a 99.95% reduction in bacterial growth in 48 h. In rice pot efficacy trials, single-dose treatment of NR08 showed a significant reduction in disease up to 90.23% and 79.27% on 7 and 21 dpi, respectively. However, treatment using 2% skim milk-supplemented phage preparation was significantly less effective as compared to the neat phage preparation. In summary, this study characterized a novel Xoophage having the potential as a biocontrol agent in the mitigation of BLB in rice.

Apparent prevalence and risk factors associated with occurrence of Coxiella burnetii infection in goats and humans in Chhattisgarh and Odisha, India.

Coxiella burnetii is one of the most contagious pathogen associated with Q fever in humans, while, ruminants act as important source of infection for humans. In the present cross sectional study, a total of 464 samples were collected from 218 goats comprising of 218 sera, 218 blood and 28 milk from various parts of Chhattisgarh and Odisha region, India. Besides, environmental (33; soil- 4, faecal- 10, feed-6, drainage water- 6, drinking water- 7) and rodent (38) samples were also collected from the premises of the animals. Human sera samples (93) were collected from same sampling area comprised of workers at an organized dairy farm (43), and farmers (50). The samples were subjected to PCR targeting the trans and com1 genes and detection of antibodies using commercial ELISA kits. An overall 14.22% (95% CI: 10.2-19.47%) of the goat samples were positive using either PCR or ELISA. While, by using PCR and ELISA, 11.93% (26/218) and 9.63% (21/218) of the samples were positive for C. burnetii. A higher seropositivity (46.24%; 95% CI: 36.46-56.32%) was observed for antibodies against C. burnetii in samples collected from humans. None of the human, environmental and rodent samples were positive for C. burnetii using PCR. This seems to be the first cross sectional study to focus the hidden threat of coxiellosis among goat population and associated risk factors in India.

Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken.

Resiquimod (R-848), an imidazoquinoline compound, is a potent synthetic Toll-like receptor (TLR) 7 agonist. Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 µg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes.