A new multiplex RT-PCR assay for serotyping of bluetongue virus

219-225Among all viral diseases affecting small ruminants, bluetongue is the one that affects adversely to an alarming extent. The current available diagnosis/serotyping of bluetongue virus (BTV) is time consuming, costly, and requires to screen individually for each of the 29 distinct serotypes. The present study was conducted with the objective of developing a multiplex reverse transcription PCR (mRT-PCR) assay for serotyping of BTV, especially for serotypes BTV-1, 2, 9, 12, 16, 21 and 23 predominantly circulating in India. The type specific primers for the selected BTV serotypes were designed targeting the serotype specific segment-2 region of BTV based on the reference serotype sequences of Indian isolates available in GenBank. The mRT-PCR was conducted in two groups - group A for BTV-1, 9, 12, 21 and group B for BTV-2, 16 and 23. A panel of 25 BT suspected clinical samples were typed by mRT-PCR. The results were further validated by the gold standard serum neutralization test (SNT). A seroprevalence of 60% for BTV- 2, 10% for BTV- 9, 15% for BTV- 1, 10% for BTV- 16 and 5% for BTV- 23 were observed. Further, we noticed that there was a mixed serotype infection in 10% of BTV positive samples. In conclusion, we report the development of a novel mRT-PCR assay for a rapid and cost-effective nucleic acid based serotyping of BTV having the specificity same as SNT

Antagonistic effect of ursolic acid on Staphylococcal biofilms

Aim: The present study was carried out to study the effect of ursolic acid (UA) as a potential anti-biofilm agent in dispersing the biofilm generated by Staphylococcus aureus isolated from milk samples of crossbred dairy cows on the day of drying. Further, in the S. aureus isolates, the presence of intracellular adherence gene locus involved in biofilm production (icaD) was investigated. Materials and Methods: A total of 50 S. aureus strains were isolated over a period of 3 months from 200 milk samples collected from crossbred dairy cows on the day of drying. These isolates were subjected for biofilm detection by Congo red agar (CRA), microtiter plate assay (MTP), and polymerase chain reaction specific for icaD gene. The antagonistic effect of biofilm formation by UA was studied using different concentrations (30 μg/ml and 60 μg/ml) of UA and compared with the control group. Results: Among the 50 S. aureus subjected for biofilm detection, 34 and 40 isolates were detected as biofilm agents by CRA and MTP methods, respectively. The in vitro studies on the effect of UA in inhibiting biofilm formation by S. aureus using MTP assay showed 71.5% and 48.6% inhibition at UA concentrations of 60 μg/ml and 30 μg/ml, respectively, with a significant difference (p<0.05) between the treated and untreated isolates, which was further evident by scanning electron microscopy. Interestingly, the isolates that were tested to be resistant through Antibiotic Sensitivity Test to commonly used antibiotics were found to be sensitive to all the tested antibiotics following UA treatment at both the tested concentrations. Furthermore, molecular detection of icaD gene for biofilm detection revealed that all the isolates that were positive by MTP had icaD gene. Conclusion: Increased incidence of biofilm agents in dairy infections must be considered as an alarming situation. UA treatment significantly enhanced the sensitivity of the microbial pathogens to commonly used antibiotics. Hence, attention must be paid toward implementation of new strategies such as therapeutic regimes with a combination of antibiotic and anti-biofilm agents for effective treatment of infections in dairy farms

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

Aim: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. Materials and Methods: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription -PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. Results: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269Ex103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. Conclusion: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample

Infection kinetics and antibody responses in Deccani sheep during experimental infection and superinfection with bluetongue virus serotypes 4 and 16

Aim: The current study was designed to understand the infection kinetics and antibody responses of major circulating serotypes of bluetongue virus (BTV) in India, i.e., BTV-4 and BTV-16 through experimental infection and superinfection of Deccani sheep, a popular breed of sheep found in the southern states of India. Materials and Methods: Experimental infection with 106 TCID50/ml BTV-4 was followed by superinfection with BTV-16 and vice versa. Along with observing for clinical signs and immunological responses in the experimentally infected sheep, the effect of infection of one specific serotype on the outcome of superinfection with a different serotype was also studied. Results: Certain interesting findings have been made in the course of experimental infection, such as prominent signs of infection in BTV-4 infection, mild or no clinical signs in BTV-16-infected and superinfected animals, and non-seroconversion of one of the BTV-16-superinfected animals. In addition, BTV was isolated from infected sheep in all the experimental conditions except BTV-16 superinfection. Furthermore, it was observed that immune response in the form of type-specific antibodies was slower with BTV-16 superinfection. Conclusion: Superinfection of a sheep with more than one serotype of BTV is a common phenomenon in BT endemic countries like India. Such situation was replicated in an experimental infection in the current study, and the findings to our knowledge are first of a kind and are likely to aid in unfolding the newer aspects of BTV pathogenesis and virulence

Abstracts of National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental Biotechnology

This book contains the abstracts of the papers presented at the National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental Biotechnology (NCB4EBT-2021) Organized by the Department of Biotechnology, National Institute of Technology Warangal, India held on 29–30 January 2021. This conference is the first of its kind organized by NIT-W which covered an array of interesting topics in biotechnology. This makes it a bit special as it brings together researchers from different disciplines of biotechnology, which in turn will also open new research and cooperation fields for them. Conference Title: National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental BiotechnologyConference Acronym: NCB4EBT-2021Conference Date: 29–30 January 2021Conference Location: Online (Virtual Mode)Conference Organizer: Department of Biotechnology, National Institute of Technology Warangal, Indi