Type-specific seroprevalence of bluetongue in India during 2018 and 2019.

BACKGROUND AND AIM: Bluetongue (BT) is a major disease of sheep and goats and is endemic to India. It is known to cause significant economic losses to the sheep industry. The current study aimed to determine the type-specific seroprevalence of BT in sheep population of India during 2018-2019. MATERIALS AND METHODS: Blood samples (n=405) were collected from 6 months to 1 year old sheep from six districts (Nalgonda, Karimnagar, Khammam, Mahabubnagar, Warangal, and Ranga Reddy) of Telangana state, India. Group- and type-specific seroprevalence (against BT virus [BTV] serotypes BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24) was studied by competitive enzyme-linked immunosorbent assay and serum neutralization test, respectively. RESULTS: Results showed an overall seroprevalence of 14.81% (n=60) with the highest seroprevalence of 50% in Khammam district. Seroprevalence of BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24 was noted as 16.66%, 11.66%, 31.66%, 11.66%, 05%, 6.66%, 16.66%, 8.33%, 13.33%, 6.66%, and 16.66%, respectively. The majority of the sera neutralized more than 1 serotype, indicating superinfection or circulation of multiple serotypes in the sampled flocks. This mixed seroprevalence was observed in 43.33% of the sera with number of BTV serotype-specific antibodies ranging from two to eight in individual animals. CONCLUSION: Regular monitoring of circulating serotypes, especially in young herds, elucidates pattern of dominating serotypes in a particular area during a season. This knowledge can be applied to design appropriate vaccination strategies by including particular serotypes of virus as part of a multivalent vaccine for a particular period, in a particular area.

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.

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.

Prevalence and antimicrobial resistance pattern of Shiga toxigenic Escherichia coli in diarrheic buffalo calves.

AIM: Aim of the study was to investigate the prevalence, virulence gene profiles, and antimicrobial resistance pattern of Shiga toxigenic Escherichia coli (STEC) in diarrheic buffalo calves from Andhra Pradesh and Telangana States. MATERIALS AND METHODS: A total of 375 fecal samples from diarrheic buffalo calves of 1-7, 8-30, 31-60, and 61-90 days age were collected from which STEC were isolated, and virulence genes were detected using multiplex polymerase chain reaction. The antimicrobial resistance of isolates was tested by disk diffusion method. RESULTS: The prevalence of E. coli associated diarrhea in buffalo calves was 85.04%, of which 35.01% was STEC origin. In STEC, the combination of eaeA and, hlyA virulence genes was highest (42.45%) followed by stx1 (16.04%), stx1, stx2 and hlyA (13.21%), stx2 (12.64%), stx1, eae and hlyA (9.43%) and stx1 and hlyA (6.6%) genes were detected. Highest antimicrobial resistance was observed for tetracycline (63.21%) and ampicillin (48.11%), while chloramphenicol, gentamycin (96.33%) and imipenem (99.06%) antibiotics are susceptible. Multidrug resistance was detected in 69.81% of the STEC isolates from diarrheic buffalo calves. CONCLUSION: Higher prevalence of eaeA and hlyA genes carrying isolates of STEC may be a serious zoonotic threat and increased prevalence of multidrug resistance in E. coli may necessitate stringent selection of appropriate antimicrobial agent in treating buffalo calf diarrhea cases.

Type-specific seroprevalence of bluetongue in Andhra Pradesh, India, during 2005-2009.

Bluetongue (BT) is an infectious, arthropod-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), which is a double-stranded segmented RNA virus. Of the 26 confirmed BTV serotypes, 23 were reported in India based on the detection of antibodies or virus. In order to assess the prevalence of different serotypes in Andhra Pradesh, serum samples which were positive for BTV by group-specific antibody ELISA were subjected to type-specific neutralization of BTV serotypes 1, 2, 9, 10, 21 and 23. Of the 52 samples tested, 50.0, 44.23, 21.15, 26.92, 0, and 15.38 % neutralized BTV serotypes 1, 2, 9, 10, 21 and 23, respectively. However, 32.69 % of the ELISA positive sera could not neutralize any of these serotypes, indicating that there could be other serotype viruses (e.g., BTV-3 and -16) circulating in the State. This method can be used for surveillance of the circulating serotypes as well as for assessing the level of herd immunity, and assist in determining the vaccine strains to be used in multivalent vaccines.

Multi-component synthesis and in vitro and in vivo anticancer activity of novel arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides.

A three component one-pot protocol has been investigated for the synthesis of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1 from the commercially available materials. The title compounds 1 were also synthesized by a step-wise method and found to be identical with one-pot synthesis by spectral and analytical data. The newly synthesized compounds were evaluated for their in vitro anticancer activity against human cancer cell lines and in vivo anticancer activity on EAC-bearing mice. Compound 1a was found to be the most active both in in vitro and in vivo cytotoxic studies.

Evidence of bluetongue virus serotype 21 (BTV-21) divergence.

Bluetongue virus serotype 21 (BTV-21) was originally isolated from Australia, but has now been reported from India, Indonesia, China and Japan. We report the isolation, and sequencing of BTV-21 from India. The complete ORF sequence of VP2 gene of this isolate showed that it is closely related to recent BTV-21 isolates from Japan (93-94% identity), and distantly related to BTV-21 reference strain (86% identity). Our results, along with the available sequences of Japanese isolates, suggest that the currently circulating BTV-21 strains from India and Japan are divergent from the original strain(s) from Australia and shed light on designing molecular diagnostics for the detection of BTV.

Determination of the quaternary ammonium compound trospium in human plasma by LC-MS/MS: application to a pharmacokinetic study.

A highly sensitive, specific and evaporation free SPE extraction, LC-MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M(+)] cations, m/z 392-164 for trospium and m/z 400-172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05-10 ng/mL (r>0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.