Aasif Ahmad Sheikh

Effect of in vitro zinc supplementation on HSPs expression and Interleukin 10 production in heat treated peripheral blood mononuclear cells of transition Sahiwal and Karan Fries cows

The changing climatic scenario with apprehended rise in global temperature is likely to affect the livestock adversely vis-à-vis production and reproduction. This has prompted more focus in addressing the unfavorable effects of thermal stress in livestock system. Presuming that the trace element zinc is indispensible for cellular antioxidant system and immune function, the present study was designed to investigate the effect of zinc treatment on heat stress alleviation and immune modulation in peripheral blood mononuclear cells (PBMC) of indigenous and crossbred transition cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were selected for the experiment. The blood samples were collected at -21, 0 and +21 days in relation to expected date of calving. The experiment was carried out in vitro after isolating PBMC from whole blood. The 48h cultured PBMC were subjected to assorted levels of exposures viz. 37°C, 42°C to impose heat stress and 42°C+zinc to alleviate heat stress and modulate immunity. The PBMC viability was 86%, 69% and 78%, respectively. The mRNA expression of heat shock proteins (HSP 40, 70 and 90α) and Interleukin-10 (IL-10) production varied between the two breeds vis-à-vis days and levels of exposure. The mRNA expression of HSP40 and HSP70 was significantly (P<0.05) higher in Karan Fries than the Sahiwal cows. Both the breeds showed maximum expression of HSP on the day of parturition, more so in KF than Sahiwal. There was a significant (P<0.05) difference in the HSP mRNA expression at different levels of exposure. Zinc treatment to heat stressed PBMC caused a significant (P<0.05) down regulation of HSP. For immune status, anti-inflammatory cytokine, IL-10 in the culture supernatant was accessed. The IL-10 was significantly (P<0.05) higher in Karan Fries (168.18±14.09pg/ml) than the Sahiwal cows (147.24±11.82pg/ml). The IL-10 concentration was highest on the day of calving. Zinc treatment reduced the IL-10 concentration. From the study, it could be concluded that the zinc supplementation in heat stressed PBMC can ameliorate thermal stress and modulate immune response which can act as a model for reducing heat stress during the periparturient period in tropical livestock.

Alteration in some pro and anti-inflammatory cytokines associated with complete and incomplete gestation cycle of cows

Abstract The maternal immune response during pregnancy is regulated by a complex array of pro and anti-inflammatory cytokines which provide optimum conditions for the embryo implantation and survival. The possible role of pro and anti-inflammatory cytokines during the complete gestation cycle of ruminants and the difference in their circadian rhythmicity between successful and unsuccessful pregnancies is still unknown. To study this, blood samples were collected from three groups of cows, pregnant (P), non-pregnant (NP) and aborted (ABORT) cows starting from the day of Artificial Insemination (AI) till calving in P cows and till stages of non pregnancy in other cows. Various pro and anti-inflammatory cytokines were estimated by bovine-specific ELISA test and compared. Successful pregnancies had lower levels of pro-inflammatory cytokines (IL-2, IL-6 and IL-8) but higher anti-inflammatory cytokine (IL-10) mainly during implantation. Significant (p < 0.05) increase in pro-inflammatory cytokines and low IL-10 levels was noticed at abortion and at calving. This study indicates that temporal and spatial aspects of reducing the release of various pro-inflammatory cytokines and maintaining high anti-inflammatory cytokines during pregnancy are essentially required for maintenance of pregnancy. Any disturbance in the cytokine equilibrium may lead to persistent inflammation and pregnancy failure.

Effect of tropical thermal stress on peri-implantation immune responses in cows

Pregnancy losses during the peri-implantation period cause a negative impact on the reproductive and economic performance of dairy herds. In this study, we investigated the possible immunological factors which may contribute to pregnancy loss during the peri-implantation period under different seasons of tropical conditions. Eighteen Karan Fries cows, six cows in each season (W: winter; HH: hot-humid; HD: hot-dry) were selected. These cows exhibited heat and were brought for artificial insemination (AI; day 0). Blood was collected on days 0, 10, 14, 16, 18, 21 and 28 post-AI. Pregnancy was confirmed by non-return to heat, progesterone assay and ultrasonography. Blood neutrophils were isolated and tested for their number, myeloperoxidase (MPO) concentrations and expression of cell adhesion molecules (CD11b, CD14, CD25, CD47), interferon tau stimulated genes (ISG15, MX1, OAS1) and chemokine receptors (CXCR1, CCL2). Plasma cortisol, progesterone, IL-2 and IL-10 were also estimated. Neutrophil number, MPO levels, the relative expression of various neutrophil receptors and plasma IL-2 were low between days 14-21 post-AI in all seasons. However, plasma cortisol and IL-10 were higher during the same period. The inflammatory activity of neutrophils, plasma IL-2 and cortisol were highest in HH, intermediate in HD and lowest in W season. However, plasma progesterone and IL-10 were highest in W season and lowest in HH season. Our results show that blood neutrophils sense the implanting embryo and downregulate their activity to ensure successful implantation; however, under harsh environmental conditions, it is a great challenge for the immune system to maintain such balance and thus it may negatively affect the outcome of pregnancy.

Fluctuation in the number, type and activity of blood neutrophils in cows exhibiting successful and unsuccessful completion of gestation cycle

Abstract Neutrophils (first line of cellular defense) are capable of detecting presence of foreign genome in the mother’s womb. Role of neutrophils during full gestation cycle of ruminants and the difference in their number, type, and activity in successful and unsuccessful pregnancies is not known. To evaluate this, blood samples were collected at artificial insemination (0 day) and on days 10, 14, 16, 18, and 21 in non-pregnant (NP) cows. However in pregnant (P) cows, samples were collected as indicated above and every 30 days for the complete gestation. In aborted cows, samples were collected as above till abortion. Higher total leukocyte counts were observed in NP and aborted cows at abortion. Neutrophil: lymphocyte ratio increased significantly (p < 0.05) in NP and aborted cows. Phagocytic activity (PA) and myeloperoxidase concentrations were significantly higher (p < 0.05) on day 18 post insemination in NP cows. PA and myeloperoxidase also increased significantly (p < 0.05) at abortion in aborted cows. Neutrophils exhibited limited decrease in their number and activity in successful pregnancies during implantation. After that their number and activity were constantly maintained throughout the gestation cycle. Any increase in the number and inflammatory activity of neutrophils may lead to non-pregnancy or loss in pregnancy.

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4‐hr duration. The IFNτ pathway was determined through real‐time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon‐tau stimulated gene expression in blood neutrophils.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ± 0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.

Interferon tau stimulated gene expression and proinflammatory cytokine profile relative to insemination in dairy cows

Abstract A profitable and scientific dairy farming can be achieved by identifying the non-pregnant animals at an early date post-insemination. The present study was executed to identify the genes for early pregnancy diagnosis in bovine. The blood samples were collected from the Karan Fries cows on days 0, 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 relative to the date of insemination. The experimental animals were grouped into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. Each group comprised of 6 cows. The plasma TNF-α concentration was higher in pregnant than EEM and LEM cows. The IL-8 concentration was higher in EEM than pregnant and LEM cows. The mRNA expression of CXCL17 and IFIT2 was significantly (p < 0.05) higher from day 4–35 than day 0 in pregnant and LEM cows. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The experiment reveals that the time-dependent changes in the IFNτ stimulated gene expression in blood neutrophils coupled with proinflammatory cytokine profile could be useful biomarkers for bovine gestation.

Development of enzyme-linked immunosorbent assay for early pregnancy diagnosis in cattle

The prerequisite for enhancing reproductive efficacy and successful calving is an early and accurate diagnosis of pregnancy. A highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing the second antibody coating technique and the horseradish peroxidase conjugate as a label for the determination of Interferon-stimulated protein, 15 kDa (ISG15) in dairy cows was developed. To validate a neutrophil lysate based ELISA for early pregnancy diagnosis, blood samples from healthy multiparous Karan Fries (KF) cows were collected on day 0 (day of AI), 10, 14, 16 and 21 post artificial insemination. The pregnancy and non-pregnancy in cows were confirmed by plasma progesterone assay, ultrasonography and per rectal palpation. The detection range of the assay was 0.312 to 25 ng/ml with a sensitivity of 0.13 ng/ml. The inter-assay and intra-assay coefficient of variation was 12.1% and 10.1%, respectively. The parallelism for measured and expected concentrations had an r2 value of 0.90 and 0.93, respectively. It was observed that ISG15 concentration was greater (P < 0.01) in pregnant as compared to non-pregnant cows, the greatest being 9.36 ± 0.50 ng/ml on day 16 in pregnant cows. With the use of immunocytochemistry, there was a positive staining for ISG15 with greater staining in the neutrophils of day 16 pregnant cows. The study reveals that ISG15 protein in a neutrophil lysate can be utilized as a biomarker for early pregnancy diagnosis in cows.

PCR–RFLP based genotyping of Indian isolates of Toxoplasma gondii

Toxoplasma gondii is an apicomplexan parasite capable of infecting a wide variety of warm-blooded animals, including birds and humans and is zoonotically important too. Felidae serve its definitive hosts and most infections are inoccous while in various intermediate hosts (e.g. sheep), it is responsible for abortion, still births. Humans which are immune compromised are also susceptible to toxoplasmosis. Most of the epidemiological studies have revealed it to be belonging to three clonal types with exceptions in South Africa having atypical isolates. Current genotyping was carried out at 11 genetic loci (SAG1, 5′-SAG2, 3′-SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358 and PK1) using multiplex-nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR–RFLP). SAG1, alt SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358 and PK1 could differentiate our strain/isolates as type I (T. gondii RH) and type III (T. gondii isolates from Chennai and Izatnagar). 5′SAG2 and 3′SAG2 in combination confirmed these as above mentioned genotypes. Further, the T. gondii RH was assigned Toxo DB#10 and local isolates of T. gondii were assigned Toxo DB#2. The present study is the first report on existence of Type III T. gondii lineage from animal population of Indian subcontinent based on PCR–RFLP.