Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis.

BACKGROUND: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect. METHODS AND RESULTS: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times. CONCLUSIONS: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column.

Short communication: preliminary observations on the serum levels of HSP70 and its correlation with serum cortisol, thyroid hormones, and acute-phase protein concentration in cattle naturally infected with foot-and-mouth disease virus.

The study aimed to explore the serum levels of HSP70 and identify its possible association with serum cortisol, thyroid hormones, and acute-phase protein concentrations in cattle naturally infected with foot-and-mouth disease (FMD) virus. After the FMD outbreak in an organized dairy cattle farm in India, blood samples were obtained from clinically infected (n = 40) and apparently healthy (n = 30) animals. Samples were processed and tested by an in-house DIVA assay for confirmation of FMD infection. Serum was analyzed for HSP70, cortisol, T4, T3, haptoglobin, and serum amyloid A by enzyme-linked immunosorbent assay (ELISA). HSP70 concentrations were significantly higher in the serum of clinically infected cattle (p < 0.01) than the healthy group. To the best of our knowledge, this is the first report describing the elevated serum levels of HSP70 under infectious diseases of bovines. Cortisol (p < 0.05), haptoglobin (p < 0.001), and serum amyloid A (p < 0.05) concentrations also markedly increased in the diseased animals; however, no differences (p > 0.05) were found in T4 and T3 levels between healthy and infected cattle. Elevated HSP70 concentration correlated positively with high cortisol (p < 0.05) and haptoglobin (p < 0.001) levels suggesting an essential link between these acute events during clinical infectious phase of FMD.

In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis.

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3ß1, HSD3ß6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 106 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant (P < 0.01) increase in testosterone production and up-regulation (P < 0.05) of CYP11A1, CYP17A1, HSD3ß1 and HSD3ß6 gene expression. In summary, the present study underscored the effects of UcOCN on testosterone biosynthesis, expression of crucial steroidogenic genes and interaction with GPRC6A receptors in buffalo Leydig cells, emphasizing its potential implications in andrology.

Production and characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and development of a sandwich ELISA for virus antigen detection.

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O.

Foot-and-mouth disease status in India during the second decade of the twenty-first century (2011-2020).

Foot-and-mouth disease (FMD) is a major disease of livestock in India and causes huge economic losses. The formal FMD control program started in 2003-04 in selected districts and was gradually expanded. The present study provides a descriptive review of the FMD outbreaks, prevalent serotypes, and genetic and antigenic features of the FMD virus (FMDV) that circulated in the country between 2011 and 2020. FMD outbreaks were regularly reported in cloven-hoofed domestic livestock and wildlife, with three serotypes including O, A, and Asia1. During the study period, a total of 2226 FMD outbreaks were documented and serotypes confirmed. FMDV serotype O dominated the outbreak scenario, accounting for about 92% of all outbreaks, followed by Asia1 (5% of all outbreaks) and A (3% of all outbreaks). Two major epidemics of FMD on an unprecedented scale during the years 2013 and 2018 by serotype O were recorded. The spatial distribution of FMD was characterized by a larger number of outbreaks in the southern region of the country. In an annual-scale analysis, 2020 was the year with the lowest outbreaks, and 2013 was the year with the highest. The month-scale analysis showed that outbreaks were reported throughout the year, with the highest numbers between October and March. The emergence of three major lineages (O/ME-SA/Ind2001d, O/ME-SA/Ind2001e, and O/ME-SA/Ind2018) of serotype O was observed during the period. In the cases of serotype A and Asia1, the appearance of at least one novel lineage/genetic group, including A/G-18/non-deletion/2019 and Asia1/Group-IX, was documented. While serotype A showed the advent of antigenic variants, serotypes O and Asia1 did not show any antigenic diversity. It was noticed during the course of an outbreak that animal movement contributes significantly to disease transmission. Except for 2018, when numerous FMD outbreaks were recorded, the number of annual outbreaks reported after 2016 has been lower than in the first half of the decade, probably due to mass vaccination and COVID-19 pandemic-linked movement restrictions. Even during outbreaks, disease symptoms in ruminant populations, including cattle, were found to be less severe. Regular six-monthly immunization certainly has a positive impact on the reduction of disease burden and should be followed without fail and delay, along with intensive disease surveillance.

Responsiveness of prepubertal crossbred bull calves to exogenous GnRH and its impact on reproductive hormones under tropical conditions.

This study investigated the age related variations in luteinizing hormone (LH), androstenedione, testosterone, and total estrogens response to exogenous gonadotropin-releasing hormone (GnRH) in Holstein-Friesian (HF) × Tharparkar bull calves. Fifteen bull calves were selected and, based on their age, were divided into Group I (14-16 months, n = 5), Group II (9-12 months, n = 5), and Group III (6-8 months, n = 5). All bull calves were administered with 10 µg of GnRH intramuscularly. Blood samples were collected at an interval of 30 min commencing 1 h prior to GnRH treatment until 4 h post-GnRH treatment and thereafter, at an interval of 1 h for the next 3 h. Endocrine response in terms of pretreatment values, peak values, area under curve, and time taken to attain peak values for LH, androstenedione, testosterone, and total estrogens was evaluated in all the bull calves. Significant differences were observed in pretreatment values, peak concentrations, and area under curve for androstenedione and testosterone between the groups; with response being higher in Group I bull calves. The results indicated that the HF × Tharparkar bull calves of 14 months age and above respond to exogenous GnRH by secreting significant amounts of testosterone.

Development and validation of a simple, sensitive enzyme immunoassay for quantification of androstenedione in bull plasma.

As an alternative to radioimmunoassay a simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for androstenedione quantification in plasma of Karan Fries bulls using second antibody coating technique. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was performed to analyze androstenedione directly in 40 µl of bull plasma. The androstenedione standards ranged from 0.20 to 200 pg/40 µl /well and the sensitivity of the assay was 5 pg/ml plasma. Serially diluted bull plasma containing high endogenous androstenedione showed good parallelism with bovine androstenedione standard curve. Intra- and inter-assay coefficients of variation (CV) were found to be 8 and 9%, respectively. Peripheral plasma androstenedione concentrations determined in young and adult bull samples ranged between 104-990 pg/ml and 184-2040 pg/ml, respectively.

Effects of supplementation of Tinospora cordifolia to crossbred cows peripartum.

Guduchi (Tinospora cordifolia), a medicinal plant used in ayurveda, is well documented for its immunomodulatory properties. Since the crossbred periparturient cow is highly susceptible to various diseases that effectively reduces its reproductive performance postpartum we explored the possibility of enhancing the reproductive performance of crossbred cows by guduchi supplementation peripartum. A total of 15 pregnant Karan Fries cows were selected and divided into two groups: treatment group of 8 cows which were supplemented with guduchi at 60 g/day for 45 days prepartum and 120 g/day for 45 days postpartum and unsupplemented control group of 7 cows. Jugular blood samples were collected from all cows during the periparturient period for analysis of endocrine (progesterone, total estrogens and PGFM), immunological and hematological parameters. Incidence of retention of fetal membranes, endometritis, pyometra and calf mortality were higher in control group of cows in comparison to those recorded in treated group. The guduchi supplemented cows exhibited faster uterine involution (28 days vs. 42 days) and early commencement of cyclicity (37 days vs. 58 days; based on plasma progesterone profiles) in comparison to untreated control group of cows. Mean birth weight of calves from treatment group of cows was significantly higher than those from control group however no significant difference was observed in average daily body weight gain of calves in both the groups. A higher total leukocyte, lymphocyte, neutrophil count along with increased neutrophil lymphocyte ratio was recorded in guduchi supplemented cows in comparison to untreated cows although plasma total antioxidant activity was similar between the two groups. Prepartum plasma progesterone concentration was significantly lowered in the treated group however there was no significant change in peripartum plasma total estrogens and PGFM levels due to guduchi supplementation.