E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.

Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.

Molecular detection of bovine alphaherpesvirus-1 in cases of reproductive disorders among cattle and buffaloes in Gujarat.

Bovine alphaherpesvirus-1 (BoAHV-1) is an important viral pathogen that causes significant economic losses to the dairy industry. The present study aimed to determine the prevalence of BoAHV-1 in cases of bovine reproductive disorder. Clinical samples were collected from various villages in Gujarat using specialized FTA® cards and were tested using real-time PCR assay targeting the gB gene of BoAHV-1. Out of 401 animals, 18.20% (95% CI: 14.74-22.28%) tested positive for BoAHV-1 DNA. The percentage positivity of BoAHV-1 was 20.37% in abortion cases and 19.55% in retention of fetal membrane cases, while only one out of nine metritis cases screened in the study was positive for BoAHV-1 DNA. A higher percentage positivity in buffaloes (22.14%) compared to cattle (16.30%) was recorded, but this difference was not statistically significant (p = 0.169). The frequency of BoAHV-1 detection was higher among crossbreeds (16.76%) and exotics (19.61%) than among indigenous cattle (8.82%), although this difference was not statistically significant (p = 0.400). There was also no significant difference in frequency distribution among animals of varying parity, ranging from 15.20 to 33.33% (p = 0.540). This study confirms the widespread circulation of BoAHV-1 and highlights the need for its control and prevention.

Seroprevalence to common infectious abortifacient and infertility causing agents in the dairy herds of India.

Background: Information on the prevalence of infectious agents in dairy farms forms the basis for formulating a suitable control strategy; especially in endemic situations. Aims: A cross-sectional study was undertaken to determine the prevalence of six economically important bovine diseases, causing reproductive disorders including bovine abortion in organized dairy herds in India. Methods: A total of 1,075 animals (cattle and buffaloes) from 09 dairy farms were screened by ELISA tests. Results: Bovine viral diarrhoea (BVD) was the most prevalent (56.5%) disease followed by infectious bovine rhinotracheitis (IBR) (45.4%). Prevalence of Q-fever (5.4%) and neosporosis (6.1%) were less on the farms. Although 16.3% of the samples turned positive for brucellosis, the contribution of calf-hood vaccination (B. abortus S19 vaccine) to the prevalence of antibodies cannot be ruled out. The overall prevalence of bovine anaplasmosis, known to cause sporadic abortions in dairy herds, was 34.1% in the 9 farms with a prevalence of less than 20% in 5 farms. Infection of multiple abortifacient (seroprevalence to more than two pathogens) was recorded in 56.8% of animals. A very strong association was observed between BVD and brucellosis (Odds ratio 14.2; P<0.001). Further, a positive association was also seen between seroprevalence of IBR and anaplasmosis, and neosporosis and Q fever (P<0.05). Conclusion: Viral diseases were found to be more common in the dairy herds than bacterial and protozoan diseases. Increased susceptibility of IBR seropositive cows to other bacterial and viral infections was observed.

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves.

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

A complex of DNA gyrase and RNA polymerase fosters transcription in Mycobacterium smegmatis.

We report here the existence of a complex between RNA polymerase (RNAP) and DNA gyrase in Mycobacterium smegmatis. The interaction between the two enzymes was detected during our attempts to purify DNA gyrase from M. smegmatis. RNAP subunits co-eluted along with DNA gyrase in two different affinity chromatography column procedures employed to purify the latter enzyme. A complex containing both the enzymes was isolated through gel filtration chromatography and sucrose density gradient centrifugation of the cell free extracts. The complex exhibited both DNA supercoiling and transcription activities. Reduction in the transcription activity of the complex in the presence of DNA gyrase inhibitor indicates a role for DNA gyrase in stimulating transcription.