Interferon Alpha Characterization and Its Comparative Expression in PBM Cells of Capra hircus and Antelope cervicapra Cultured in the Presence of TLR9 Agonist

TLR9 plays pivotal role in innate immune responses through upregulation of costimulatory molecules and induction of proinflammatory cytokines like type I interferons including interferon alpha (IFNA). The present study characterized IFNA cDNA and predicted protein sequences in goat and black buck. Response of the PBM cells to TLR9 agonist CpG ODN C and Phorbol Myristate Acetate (PMA) was evaluated by realtime PCR. IFNA coding sequences were amplified from leukocyte cDNA and cloned in pGEMT-easy vector for nucleotide sequencing. Sequence analysis revealed 570 bp, IFNA ORF encoding 189 amino acids in goat and black buck. Black buck and goat IFNA has 92.1% to 94.7% and 93% to 95.6% similarity at nucleotide level, 86.3% to 89.5% and 70.9% to 91.6% identity at amino acid level with other ruminants, respectively. Nonsynonymous substitutions exceeding synonymous substitutions indicated IFNA evolved through positive selection among ruminants. In spite of lower total leukocyte count, the innate immune cells like monocytes and neutrophils were more in black buck compared to goat. In addition, CpG ODN C-stimulated PBM cells revealed raised IFNA transcript in black buck than goat. These findings indicate sturdy genetically governed immune system in wild antelope black buck compared to domestic ruminant goat

Effectiveness of Action in India to Reduce Exposure of Gyps Vultures to the Toxic Veterinary Drug Diclofenac

Contamination of their carrion food supply with the non-steroidal anti-inflammatory drug diclofenac has caused rapid population declines across the Indian subcontinent of three species of Gyps vultures endemic to South Asia. The governments of India, Pakistan and Nepal took action in 2006 to prevent the veterinary use of diclofenac on domesticated livestock, the route by which contamination occurs. We analyse data from three surveys of the prevalence and concentration of diclofenac residues in carcasses of domesticated ungulates in India, carried out before and after the implementation of a ban on veterinary use. There was little change in the prevalence and concentration of diclofenac between a survey before the ban and one conducted soon after its implementation, with the percentage of carcasses containing diclofenac in these surveys estimated at 10.8 and 10.7%, respectively. However, both the prevalence and concentration of diclofenac had fallen markedly 7–31 months after the implementation of the ban, with the true prevalence in this third survey estimated at 6.5%. Modelling of the impact of this reduction in diclofenac on the expected rate of decline of the oriental white-backed vulture (Gyps bengalensis) in India indicates that the decline rate has decreased to 40% of the rate before the ban, but is still likely to be rapid (about 18% year−1). Hence, further efforts to remove diclofenac from vulture food are still needed if the future recovery or successful reintroduction of vultures is to be feasible

Estimates of changes between three surveys of ungulate carcasses (T1, T2, T3) in the true prevalence <i>f</i> of diclofenac, the arithmetic mean concentration of diclofenac in livers of animals in which it was present (ppm wet weight), the estimated mean percentage of vultures killed by a meal of mixed tissues, and the annual percentage rate of decline of the vulture population.

<p>The interval between meals <i>F</i> was assumed to be two or three days and annual adult survival in the absence of diclofenac <i>S₀</i> was assumed to be either 0.90 or 0.97. Ratios of parameter estimates and their bootstrap 95% confidence limits are shown for each pairwise comparison of surveys.</p

Comparisons between the residual deviance and Akaike Information Criterion (AIC) of various logistic regression models of the variation among site clusters (S) and survey time periods (T) in the apparent prevalence of diclofenac (the proportion of liver samples with detectable levels of the drug).

<p>A null model in which the proportion was assumed to be constant (C) across site clusters and time periods was compared with models in which the odds of a sample having detectable diclofenac varied either among site clusters or time periods or was given by the product of a site-cluster effect and a time-period effect (denoted S+T). A full model with proportions specific to each site-time combination is denoted S.T.</p

Estimates of the parameters of a model which describes the true prevalence <i>f</i> of diclofenac in liver samples taken during three surveys of ungulate carcasses (T1, T2, T3) and the scale <i>a</i> and shape <i>b</i> parameters of the Weibull distribution of diclofenac concentrations (ppm wet weight).

<p>The value <i>b</i> is assumed to be common to all three surveys. Also shown is the arithmetic mean concentration of diclofenac (ppm wet weight) for those samples which contained the compound, calculated from <i>a</i> and <i>b</i>. Parameter estimates and their bootstrap 95% confidence limits are shown for each of three surveys.</p

Estimates of the mean percentage of vultures killed by a meal of mixed tissues assuming that the interval between meals <i>F</i> was two or three days, and the annual percentage rate of decline of the vulture population, assuming the two values of <i>F</i> and annual adult survival in the absence of diclofenac <i>S₀</i> of either 0.90 or 0.97.

<p>Parameter estimates and their bootstrap 95% confidence limits are shown for each of three surveys of ungulate carcasses (T1, T2, T3).</p

Comparison of probability density functions of diclofenac concentrations in ungulate liver before and after the ban on the veterinary use of diclofenac.

<p>Fitted probability density functions are shown of diclofenac concentration (ppm wet weight) in ungulate liver samples from three surveys: red = T1, pre-ban, dark green = T2, soon after the ban, dark blue = T3, 7–31 months after the ban. The curves are derived from a Weibull model in which both the true prevalence of diclofenac <i>f</i> (including those with concentrations < LOQ) and the scale parameter <i>a</i> of the Weibull distribution of concentrations of diclofenac in those samples are determined by a site-cluster effect and a survey period effect. The shape parameter <i>b</i> of the Weibull distribution is assumed not to vary with site-cluster or survey period. Values of <i>f</i> and <i>a</i> in all three surveys were adjusted so that the results simulate those expected if the 21 site-clusters covered by the T1 (pre-ban) survey had been covered at the same sampling intensity in the second T2 and third T3 surveys.</p

Locations of sampling site clusters in India.

<p>The map shows centroids of 21 site clusters at which liver samples were obtained from carcasses of domesticated ungulates. Numbers next to the symbols identify site clusters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019069#pone-0019069-t001" target="_blank">Table 1</a>. Triangles show clusters sampled in all three surveys (T1, T2, T3), squares show clusters sampled in T1 and T2, diamonds, T1 and T3, and circles T1 only.</p

Comparison of the distributions of diclofenac concentrations before and after the ban on the veterinary use of diclofenac.

<p>Cumulative distributions of diclofenac concentration (ppm wet weight) in ungulate liver samples from three surveys: red = T1, pre-ban, green = T2, soon after the ban, blue = T3, 7–31 months after the ban are shown by the stepped lines. The curves show cumulative Weibull distributions fitted separately to the data for each survey. Fitted values of prevalence <i>f,</i> the scale <i>a</i> and shape <i>b</i> parameters respectively were T1, 0.110, 1.336 and 0.592; T2, 0.122, 1.458 and 0.597; T3, 0.061, 1.844 and 0.673.</p