Effect of intravenous infusion of recombinant ovine leptin on feed intake and serum concentrations of GH, LH, insulin, IGF-1, cortisol, and thyroxine in growing prepubertal ewe lambs

In sheep, serum concentrations of leptin change congruently with increases or decreases in nutritional status, while intracerebroventricular infusions of leptin dramatically suppress feed intake in well-fed lambs, and may also increase growth hormone (GH), and/or luteinizing hormone (LH) in undernourished lambs. The objective of the present study was to determine the effects of peripherally delivered ovine leptin, via intravenous infusions, on feed intake and serum concentrations of GH, LH, insulin, IGF-1, cortisol, and thyroxine. Twelve ewe lambs weighing 29.4 +/- 0.7 kg were infused intravenously with a linearly increasing dose of leptin or saline (n = 6 per group) for 10 days, reaching a maximum dose delivered of 0.5mg/h on day 10. Feed intake was assessed twice daily, and blood samples were collected every 10 min for 6 h on days 0, 2, 5, 8, and 10. Serum concentrations of leptin increased in leptin-treated lambs by day 2 (P = 0.05), and continued to increase to concentrations 9-fold greater than saline-infused lambs by day 10 (P < 0.001). Despite the substantial increase in serum leptin, feed intake did not differ between leptin and saline-infused lambs except on day 3.5 (P = 0.01). Furthermore, intravenous infusions of leptin did not significantly influence serum concentrations of insulin, cortisol, IGF-1, thyroxine, LH, or GH. Collectively, these observations contrast with the potent hypophagic effects of leptin when delivered intracerebroventricularly into well-fed lambs. The reasons for the disparate response of lambs treated intravenously with leptin, versus that reported for lambs treated intracerebroventricularly with leptin are not known, but may provide insight into the mechanism(s) of leptin resistance.     

Pheasant ataxia: a new condition in pheasant poults

Between 1995 and 1997 a neurological condition in pheasant poults from 24 sites in England and Scotland was investigated. Affected birds showed varying degrees of ataxia and incoordinated movements and, in severe cases, recumbency, but generally remained alert with their heads held upright. The condition characteristically affected poults from seven weeks of age and the incidence on any one site was low. No significant bacteria were isolated consistently from brain tissue. The condition was characterised histologically by a non-suppurative meningoencephalitis, in which lesions were found predominantly in the cerebellum in 61 of 81 samples examined (75.3 per cent). A non-suppurative myelitis was recorded in 16 of 20 spinal cords examined. No lesions were recorded in peripheral neural tissue and lesions were rare in other tissues. The condition appeared not to have been recorded previously in pheasants. A viral aetiology was suspected but Newcastle disease virus was not involved.     

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.     

Ultrasonography of the equine neonatal kidney

Ultrasonography is important in the clinical examination of the foal. The ultrasonographic appearance and size of the neonatal kidneys were defined and an imaging protocol established in 6 normal Thoroughbred foals (mean age +/- s.d. 5.0 +/- 3.2 days). Characteristically, in both the heart-shaped right kidney and bean-shaped left kidney, the renal cortex was more echogenic than the medulla. The terminal recesses, renal crest and pelvis were identified, as was the ureter, which contained anechoic urine in its lumen. The renal, interlobar and arcuate vessels were seen. For the right kidney, the ultrasonographic probe was placed at the 14-17th intercostal spaces and paralumbar fossa. For the left kidney, the probe was at the 16th or 17th intercostal spaces and paralumbar fossa. Perirenal structures, including the caudate lobe of the liver, the dorsal extremity of the spleen, the adrenals, the aorta and caudal vena cava were also identified. An understanding of the ultrasonographic appearance of the normal neonatal kidney, accompanied by a routine imaging protocol to ensure that all regions of each kidney are examined, permit a more informed interpretation of renal images in the first few days postpartum.     

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.