A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage.

A 2-step microtiter plate assay was developed to simultaneously check wide values of MOIs of bacteriophages, ranging between MOI-0.0001 and MOI-10000 in the first step and optimize the most suitable MOI (lowest quantity of phage) for inhibiting the growth of the target bacteria in the second step. The results of the first step revealed that the effective MOI of coliphage-ɸ5 for controlling the growth of antimicrobial resistant (AMR) E. coli was between 4.36 and 43.6 for E.coli-EC-3; between 38.2 and 382 for E.coli-EC-7 and between 81.5 and 815 for E.coli-EC-11. The optimum MOI of coliphage-ɸ5 determined in the second step was 17.44, 191 and 326 for controlling the growth of E.coli-EC-3; E.coli-EC-7 and E.coli-EC-11, respectively. The effective MOI of vibriophage-ɸLV6 for controlling luminescent Vibrio harveyi in the first step was found to be between 18.3 and 183 and the optimum MOI as determined in the second step was 79. The sequential 2-step microtiter plate method yielded faster optimization of MOI and was economical compared to the conventional flask method. The measurement of OD values at 550 nm and 600 nm showed similar trend and replicate data from 5-wells and 3-wells yielded identical pattern indicating that the measuring absorbance data in 3-replicate wells at either OD550 or OD600 is sufficient to generate quantifiable phage lysis data. The 2-step microtiter plate assay finds application in phage therapy in human health care, agriculture and animal agriculture for determining the optimum MOIs for selected bacteriophages.

Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov.

Newcastle disease vaccines hitherto in vogue are produced from embryonated chicken eggs. Egg-adapted mesogenic vaccines possess several drawbacks such as paralysis and mortality in 2-week-old chicks and reduced egg production in the egg-laying flock. Owing to these possible drawbacks, we attempted to reduce the vaccine virulence for safe vaccination by adapting the virus in a chicken embryo fibroblast cell culture (CEFCC) system. Eighteen passages were carried out by CEFCC, and the pathogenicity was assessed on the basis of the mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index, at equal passage intervals. Although the reduction in virulence demonstrated with increasing passage levels in CEFCC was encouraging, 20% of the 2-week-old birds showed paralytic symptoms with the virus vaccine from the 18(th)(final) passage. Thus, a tissue-culture-adapted vaccine would demand a few more passages by CEFCC in order to achieve a complete reduction in virulence for use as a safe and effective vaccine, especially among younger chicks. Moreover, it can be safely administered even to unprimed 8-week-old birds.

Efficacy of mint (Mentha arvensis) leaf and citrus (Citrus aurantium) peel extracts as natural preservatives for shelf life extension of chill stored Indian mackerel.

Efficacy of mint (Mentha arvensis) leaf and citrus (Citrus aurantium) peel extracts in retarding the quality changes in Indian mackerel during chilled storage was investigated. Mint leaf extract showed higher quantity of phenolics and superior in-vitro antioxidant activities than citrus peel extract. Gutted mackerel were given a dip treatment in mint extract (0.5 %, w/v) and citrus extract (1 % w/v), packed in LDPE pouches and stored at 0-2 °C. The biochemical quality indices viz. total volatile base nitrogen (TVB-N), trimethylamine nitrogen (TMA-N), free fattyacids (FFA) were significantly (p < 0.05) lower in mint extract (ME) treated fishes compared to citrus extract (CE) treated and control fishes (C) without any treatment. Plant extract treatment significantly inhibited lipid oxidation in mackerel as indicated by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS). Aerobic plate count (APC) was markedly higher in C group followed by CE group throughout the storage period. As per sensory evaluation, shelf life of Indian mackerel was determined to be 11-13 days for C group, 13-15 days for CE group and 16-17 days for ME group, during storage at 0-2 °C.

Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov

Newcastle disease vaccines hitherto in vogue are produced from embryonated chicken eggs. Egg-adapted mesogenic vaccines possess several drawbacks such as paralysis and mortality in 2-week-old chicks and reduced egg production in the egg-laying flock. Owing to these possible drawbacks, we attempted to reduce the vaccine virulence for safe vaccination by adapting the virus in a chicken embryo fibroblast cell culture (CEFCC) system. Eighteen passages were carried out by CEFCC, and the pathogenicity was assessed on the basis of the mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index, at equal passage intervals. Although the reduction in virulence demonstrated with increasing passage levels in CEFCC was encouraging, 20% of the 2-week-old birds showed paralytic symptoms with the virus vaccine from the 18^th(final) passage. Thus, a tissue-culture-adapted vaccine would demand a few more passages by CEFCC in order to achieve a complete reduction in virulence for use as a safe and effective vaccine, especially among younger chicks. Moreover, it can be safely administered even to unprimed 8-week-old birds.