Narayanan Krishnaswamy

Synergy of lipopolysaccharide and resiquimod on type I interferon, pro-inflammatory cytokine, Th1 and Th2 response in chicken peripheral blood mononuclear cells

Toll-like receptors (TLRs) recognize conserved molecular structures of invading pathogens and initiate an immune response to curtail the infection prior to the development of more powerful and specific adaptive immunity. Understanding the interactions between different TLRs in terms of immune response genes is a pre-requisite for using various TLR agonists alone or in combination as adjuvants or as stand-alone agents against various diseases. Lipopolysaccharide (LPS) and resiquimod (R-848) are TLR agonists that are recognized by TLR4 and TLR7, respectively. In this study, the effect of LPS and/or R-848 on chicken peripheral blood mononuclear cells (PBMCs) was investigated. LPS and R-848 synergistically up-regulated the transcripts of interferon-β (IFN-β), IFN-γ, IL-4 and IL-1β as compared to the individual response (P<0.05). The results indicate that these agonists synergistically interact and enhance type-I IFN, pro-inflammatory cytokine as well as Th1 and Th2 responses in chicken PBMCs, suggesting their potential as an adjuvant candidate to be used in combination with various poultry vaccines.

Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken

Resiquimod (R-848), an imidazoquinoline compound, is a potent synthetic Toll-like receptor (TLR) 7 agonist. Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes.

Prophylactic potential of resiquimod against very virulent infectious bursal disease virus (vvIBDV) challenge in the chicken

The study evaluated the prophylactic potential of resiquimod (R-848), a synthetic TLR7 agonist, against very virulent infectious bursal disease virus (vvIBDV) infection in chicken. Specific pathogen free White Leghorn chicks of three week age were treated with R-848 (50μg/bird, intramuscular) or PBS (n=26/group). Twenty four hour later, half of the birds from each group were challenged with 10(5) ELD50 of vvIBDV and observed for 10days. To understand the effect of R-848, immune response genes such as interferon (IFN)-β, IFN-γ, IL-1β, IL-4, iNOS and TLR7 were analyzed at 24 and 48h post-challenge in PBMCs ex vivo by real-time PCR (n=6/group). On day 4 post-challenge, representative birds (n=3/group) were sacrificed to study the bursal damage and IBDV antigen clearance. Immunosuppression was assessed by antibody response against live Newcastle disease virus (NDV) vaccine, which was administered on day 10 post-challenge. R-848 pre-treatment significantly upregulated the transcripts of each immune response gene studied (P<0.05). There was 50% mortality on vvIBDV challenge in control birds, while it was only 20% with R-848 group. R-848 pre-treatment reduced the bursal damage as indicated by lower bursal lesion score in histopathology, reduced IBDV antigen signal in immunohistochemistry and improved antigen clearance in agar gel immunodiffusion test. Further, it protected significantly against vvIBDV induced immunosuppression as indicated by HI antibody titre. It is concluded that pre-treatment of R-848 conferred partial protection from mortality and bursal damage while complete protection against immunosuppression in chicken when challenged with vvIBDV, which could be due to the upregulation of immune response genes.

Administration of TLR7 agonist, resiquimod, in different types of chicken induces a mixed Th1 and Th2 response in the peripheral blood mononuclear cells

This study evaluated the variation in immune response in peripheral blood mononuclear cells (PBMCs) of broiler, White Leghorn (WL) and Kadaknath breeds of chicken following administration of TLR7 agonist, resiquimod (R-848). Expression of different immune related genes viz., interferon-β (IFN-β), IFN-γ, IL-1β, IL-4, TLR7 and iNOS was assessed by quantitative real time PCR over a period of 24 h. The results indicated that there was a significant up-regulation in the relative expression of immune response genes post R-848 administration (P < 0.01). In conclusion, the transcriptional expression of IFN-β, IFN-γ, IL-1β, IL-4, iNOS and TLR7 genes in the PBMCs was significantly up-regulated over 24 h in broiler, WL and Kadaknath breeds of birds after the administration of R-848. Overall, R-848 induced a mixed Th1 and Th2 response in PBMCs of chicken origin ex vivo.

Spatial molecular epidemiology of carbapenem‐resistant and New Delhi metallo beta‐lactamase (blaNDM)‐producing Escherichia coli in the piglets of organized farms in India

A cross‐sectional study was conducted in 10 government‐organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli.

Endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra

Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (n = 18). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/group) were categorized into hyperplastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was inconclusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (n = 6) served as control. The mean fold change (2-ΔΔCt) for the target genes was determined using β-actin as endogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of COX-2 and PGFS. Further, correlation matrix with Spearmans rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (P < 0.05). It is concluded that histological grading of pyometra into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic pathway in the canine endometrium that has diagnostic potential under clinical settings.

Resiquimod enhances mucosal and systemic immunity against avian infectious bronchitis virus vaccine in the chicken

Abstract Adjuvant enhancing mucosal immune response is preferred in controlling many pathogens at the portal of entry. Earlier, we reported that a toll-like-receptor 7 (TLR7) agonist, resiquimod (R-848), stimulated the systemic immunity when adjuvanted with the inactivated Newcastle disease virus vaccine in the chicken. Here, we report the effect of R-848 when adjuvanted with live or inactivated avian infectious bronchitis virus (IBV) vaccines with special emphasis on mucosal immunity. Specific pathogen free (SPF) chicks (n = 60) were equally divided into six groups at two weeks of age and immunized with either inactivated or live IBV vaccine adjuvanted with or without R-848. Groups that received either PBS or R-848 served as control. A booster was given on 14 days post-immunization (dpi). R-848 enhanced the antigen specific humoral and cellular immune responses when co-administered with the vaccines as evidenced by an increase in the antibody titre in ELISA and stimulation index in lymphocyte transformation test (LTT) till 35 dpi and increased proportion of CD4+ and CD8+ T cells on 21 dpi in the flow cytometry. Interestingly, it potentiated the IgA responses in the tear and intestinal secretions when used with both live and inactivated IBV vaccines. The combination of IBV vaccine with R-848 significantly up-regulated the transforming growth factor beta 4 (TGFβ4) transcripts in the peripheral blood mononuclear cells (PBMCs) than that of the respective vaccine per se. An enhanced secretory IgA response is likely due to the up-regulation of TGFβ4, which is responsible for class switching to IgA. In conclusion, co-administration of R-848 with inactivated or live IBV vaccine enhanced the systemic as well as mucosal immune responses in the chicken.

Molecular characterisation of bla OXA48 carbapenemase, extended spectrum beta −lactamase (ESBL) and Shiga toxin producing Escherichia coli isolated from farm piglets of India

OBJECTIVES The aim of this study was to characterise carbapenemase-, extended-spectrum β-lactamase (ESBL)- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India. METHODS Faecal samples (n=741) from 10 organised pig farms, including non-diarrhoeic (n=546) and diarrhoeic (n=195) piglets, were processed for isolation of carbapenem-resistant and ESBL-producing E. coli. RESULTS A total of 27 and 243 isolates were phenotypically confirmed as carbapenem-resistant and ESBL-producers, respectively. The meropenem minimum inhibitory concentration (MIC) of carbapenem-resistant isolates ranged from 8-128μg/mL. On genotypic screening of the 27 carbapenem-resistant isolates, 3 isolates were positive for the blaOXA-48 carbapenemase gene; no other carbapenemase genes were detected. The 243 ESBL-producing isolates were positive for blaCTX-M-1 (n=135), qnrA (n=92), qnrB (n=112), qnrS (n=49), tetA (n=42), tetB (n=45) and sul1 (n=43). The Shiga toxin virulence markers stx1 and stx2 were detected in 41 and 38 of the 243 phenotypic ESBL-producing isolates, respectively. Multilocus sequence typing (MLST) of blaOXA-48-positive E. coli isolates showed ST10- and ST5053-like sequence types. CONCLUSION This is the first report on the presence of blaOXA-48-carrying E. coli in piglets in India, which pose a potential risk to public health.

Oestrus synchronization in goats using impregnated intravaginal progesterone sponge and buck effect

A study was conducted with the objective to compare the efficacy of the intravaginal progesterone sponge and buck effect on oestrus synchronization, conception rate and oestrous behaviour during non-breeding season in non-descript goats. The study was carried out from November, 2015 to February, 2016 and does (n=18) were divided into three equal groups. T1 group was treated with intravaginal progesterone (P4) sponge kept in situ for 15 d (350mg P4/sponge) and T2 group was teased with a sexually active aproned buck for 6 h/dy for 15 d. The does in control group were kept without any treatment. The does in T1 group exhibited 100% oestrus as against 50% in T2 group within 72 h of treatment. The interval between removal of sponge and onset of oestrus was significantly (P<0.05) shorter in T1 (36.00±5.58 h) than introduction of buck in T2 (174.5±73.52 h) and control (245.30±79.90 h) groups. The conception rate (CR) between control (50%) and T1 (100%) group differed significantly (P<0.05). The study revealed that the intravaginal progesterone sponge synchronized the oestrus and fertility.

Endometritis Increases Pro-inflammatory Cytokines in Follicular Fluid and Cervico-vaginal Mucus in the Buffalo Cow

ABSTRACT Emerging evidence shows that some of the pro-inflammatory cytokines are elevated not only in the endometrium but also in the follicular fluid of cows with endometritis. Developing a cervico-vaginal mucus (CVM) based test has the potential for becoming a pen-side test because of the ease of sample collection. The present study describes the results of two different experiments. The first experiment was conducted to investigate the influence of endometritis on the proinflammatory cytokines of follicular fluid based on the reproductive tracts of buffalo collected at a slaughter house Buffalo genitalia were categorized into purulent endometritis (PE), cytological endometritis (CE), and non-endometritis (NE) based on the white-side test and endometrial cytology, respectively (n = 14/group). Each group was subdivided into follicular and mid-luteal stage (n = 7/stage) and the follicular fluid was collected from the largest follicle. Second experiment was done to study the difference in the levels of proinflammatory cytokines in the CVM of repeat breeders with subclinical endometritis presented to the clinic. CVM was collected from the repeaters (n = 10) and non-repeaters (n = 10) through aseptic trans-vaginal aspiration. The pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα were quantitated through bovine specific ELISA kits. Significantly higher concentrations of pro-inflammatory cytokines (IL-1β, IL-8, IL-6, and TNFα) along with low intra-follicular estradiol in buffaloes of PE and CE groups suggest that endometritis impedes the follicular steroidogenesis. Significantly higher concentration of IL-1β and TNF-α in the CVM of repeaters indicate their potential as a pen-side diagnostic test for CE.