Risk factors for bovine rotavirus infection and genotyping of bovine rotavirus in diarrheic calves in Bangladesh.

Bovine rotavirus (BRV) is considered the leading cause of calf diarrhea worldwide, including Bangladesh. In this study we aimed to identify risk factors for BRV infection and determine the G and P genotypes of BRV strains in diarrheic calves. Fecal samples were collected from 200 diarrheic calves in three districts between January 2014 and October 2015. These samples were screened to detect the presence of BRV using rapid test-strips BIO K 152 (RTSBK). The RTSBK positive samples were further tested by polyacrylamide gel electrophoresis and the silver staining technique to detect rotavirus dsRNA. Risk factors were identified by multivariable logistic regression analysis. The G and P genotypes of BRV were determined by RT-PCR and sequencing. A phylogenetic tree was constructed based on the neighbor-joining method using CLC sequence viewer 8.0. About 23% of the diarrheic calves were BRV positive. The odds of BRV infection were 3.8- (95% confidence interval [95% CI]: 1.0-14.7) and 3.9-times (95% CI:1.1-14.2) higher in Barisal and Madaripur districts, respectively, than Sirrajganj. The risk of BRV infection was 3.1-times (95% CI: 1.5-6.5) higher in calves aged ≤ 5 weeks than those aged >5 weeks. Moreover, the risk of BRV infection was 2.6-times (95% CI:1.1-5.8) higher in crossbred (Holstein Friesian, Shahiwal) than indigenous calves. G6P[11] was the predominant genotype (94.4%), followed by G10P[11] (5.6%). The BRV G6 strains were found to be closest (98.9-99.9%) to Indian strains, and BRV G10 strains showed 99.9% identities with Indian strain. The VP4 gene of all P[11] strains showed >90% identities to each other and also with Indian strains. The most frequently identified BRV genotype was G6P[11]. About 23% of calf diarrhea cases were associated with BRV. To control disease, high-risk areas and younger crossbred calves should be targeted for surveillance and management. The predominant genotype could be utilized as the future vaccine candidate or vaccines with the dominant genotype should be used to control BRV diarrhea in Bangladesh.

Application of extrinsic fluorescence spectroscopy for the high throughput formulation screening of aluminum-adjuvanted vaccines.

High throughput screening (HTS) of excipients for proteins in solution can be achieved by several analytical techniques. The screening of stabilizers for proteins adsorbed onto adjuvants, however, may be difficult due to the limited amount of techniques that can measure stability of adsorbed protein in high throughput mode. Here, we demonstrate that extrinsic fluorescence spectroscopy can be successfully applied to study the physical stability of adsorbed antigens at low concentrations in 96-well plates, using a real-time polymerase chain reaction (RT-PCR) instrument. HTS was performed on three adjuvanted pneumococcal proteins as model antigens in the presence of a standard library of stabilizers. Aluminum hydroxide appeared to decrease the stability of all three proteins at relatively high and low pH values, showing a bell-shaped curve as the pH was increased from 5 to 9 with a maximum stability at near neutral pH. Nonspecific stabilizers such as mono- and disaccharides could increase the conformational stability of the antigens. In addition, those excipients that increased the melting temperature of adsorbed antigens could improve antigenicity and chemical stability. To the best of our knowledge, this is the first report describing an HTS technology amenable for low concentration of antigens adsorbed onto aluminum-containing adjuvants.