Insect physiology: the emerging story of ecdysis.

The discovery of a new insect peptide hormone that triggers ecdysis - shedding of an old cuticle - has revealed hidden layers of intricacy about an insect behavior previously thought to be mediated by a single neuropeptide.

Vaccines and animal welfare.

Vaccination promotes animal welfare by protecting animal health, but it also has other welfare benefits, e.g. recent investigations have looked at the potential of vaccines in immunoneutering such as immunocastration--a humane alternative to the painful traditional methods. Similarly, vaccination can be used during disease outbreaks as a viable alternative to stamping-out, thus avoiding the welfare problems that on-farm mass slaughter can cause. Protecting animal health through vaccination leads to improved animal welfare, and maintaining good welfare ensures that animals can respond successfully to vaccination (as poor welfare can lead to immunosuppression, which can affect the response to vaccination). It is clear that vaccination has tremendous advantages for animal welfare and although the possible side effects of vaccination can have a negative effect on the welfare of some individual animals, the harm caused by these unwanted effects must be weighed against the undoubted benefits for groups of animals.

Remodeling of the insect nervous system.

Our nervous systems and behavior are shaped by hormonally driven developmental changes that continue beyond the embryonic period. Key insights into this process have emerged from studies of the insect nervous system. During insect metamorphosis, the nervous system is remodeled through postembryonic neurogenesis, programmed cell death and the modification of persistent neurons. These changes are regulated to a large degree by gene cascades that are triggered by steroid hormones, the ecdysteroids. Current studies are attempting to reveal the molecular mechanisms involved in regulating these dramatic examples of developmental plasticity.

Assessing the welfare of genetically altered mice.

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.

Ethical challenges facing veterinary professionals in Ireland: results from Policy Delphi with vignette methodology.

Ethics is key to the integrity of the veterinary profession. Despite its importance, there is a lack of applied research on the range of ethical challenges faced by veterinarians. A three round Policy Delphi with vignette methodology was used to record the diversity of views on ethical challenges faced by veterinary professionals in Ireland. Forty experts, comprising veterinary practitioners, inspectors and nurses, accepted to participate. In round 1, twenty vignettes describing a variety of ethically challenging veterinary scenarios were ranked in terms of ethical acceptability, reputational risk and perceived standards of practice. Round 2 aimed at characterising challenges where future policy development or professional guidance was deemed to be needed. In round 3, possible solutions to key challenges were explored. Results suggest that current rules and regulations are insufficient to ensure best veterinary practices and that a collective approach is needed to harness workable solutions for the identified ethical challenges. Challenges pertaining mostly to the food chain seem to require enforcement measures whereas softer measures that promote professional discretion were preferred to address challenges dealing with veterinary clinical services. These findings can support veterinary representative bodies, advisory committees and regulatory authorities in their decision making, policy and regulation.

What do European veterinary codes of conduct actually say and mean? A case study approach.

Codes of Professional Conduct (CPCs) are pivotal instruments of self-regulation, providing the standards to which veterinarians should, and sometimes must, comply. Despite their importance to the training and guidance of veterinary professionals, research is lacking on the scope and emphasis of the requirements set out in veterinary CPCs. This paper provides the first systematic investigation of veterinary CPCs. It relies on a case study approach, combining content and thematic analyses of five purposively selected European CPCs: Federation of Veterinarians of Europe (FVE), Denmark, Ireland, Portugal and the UK. Eight overarching themes were identified, including 'definitions and framing concepts', 'duties to animals', 'duties to clients', 'duties to other professionals', 'duties to competent authorities', 'duties to society', 'professionalism' and 'practice-related issues'. Some differences were observed, which may be indicative of different approaches to the regulation of the veterinary profession in Europe (which is reflected in having a 'code of ethics' or a 'code of conduct'), cultural differences on the status of animals in society, and regulatory bodies' proactivity in adapting to professional needs and to societal changes regarding the status of animals. These findings will contribute to an improved understanding of the roles of CPCs in regulating the veterinary profession in Europe.

Identification of the cellular target for eclosion hormone in the abdominal transverse nerves of the tobacco hornworm, Manduca sexta.

The isolated abdominal central nervous system of Manduca sexta undergoes an increase in cyclic GMP (cGMP) when exposed to the insect peptide eclosion hormone (EH) before pupal ecdysis. Previously, cGMP immunocytochemistry revealed that the EH-stimulated increase in cGMP was contained in numerous filamentous processes within the transverse nerve associated with each abdominal ganglion. These processes seemed to be the axons of neurosecretory cells projecting to this neurohemal organ. In the present paper, we now show that the EH-stimulated cGMP is not present in neurosecretory terminals. There is no colocalization of the EH-stimulated cGMP with immunoreactivity of two peptides, known to be present in axons in the transverse nerves. Furthermore, there is no colocalization of EH-stimulated cGMP with the synaptic vesicle protein, synaptotagmin. The neurosecretory axons are localized to a narrow band at the anterior margin of the transverse nerve, whereas the cellular elements showing an EH-stimulated cGMP increase are primarily present in the posterior region. There are two cell types in this region: a granular and a nongranular type. The cGMP immunoreactivity seems to be contained within the nongranular type. During adult development, the cells of the posterior compartment spread in a thin layer between the transverse and dorsal nerves, become positive for myosin immunoreactivity between pupal stages 5 and 8, and seem to form the adult ventral diaphragm muscles. We conclude that the EH-sensitive filaments in the transverse nerves of Manduca are most likely to be intrinsic cells that subsequently develop into the ventral diaphragm muscles of the adult.

Neurons involved in nitric oxide-mediated cGMP signaling in the tobacco hornworm, Manduca sexta.

Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244-7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3;,5;-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment-specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS.

DNA fingerprinting of spontaneously hypertensive and Wistar-Kyoto rats: implications for hypertension research.

Probes to hypervariable minisatellite regions of DNA identify multiple loci scattered over the autosomal chromosomes and produce a complex Southern blot pattern of fragments termed a DNA 'fingerprint'. As concern has been raised that different stocks of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) may not be biologically identical, we have compared the DNA of SHR and WKY from several sources using two such probes which identify different sets of minisatellite sequences. While the DNA fingerprints of SHR from the various sources were identical, variability was observed in those of WKY, indicating genetic heterogeneity between different WKY stocks. In animals from one of the commercial suppliers even inter-rat variability in DNA fingerprints was seen, suggesting genetic heterogeneity within that single colony. These observations indicate that experimental results obtained using WKY from different sources may not be directly comparable and could provide an explanation for some of the conflicting data that exist on the comparative characteristics of SHR and WKY. In separate studies, direct comparisons both of the DNA fingerprints of SHR and WKY and of SHR and stroke-prone spontaneously hypertensive rats (SHRSP) showed multiple differences between the strains. The polymorphisms seen could provide useful linkage markers in locating the chromosomal sites of the genetic loci responsible for raised blood pressure in the SHR and the propensity to strokes in the SHRSP.

Subtractive hybridization strategy using paramagnetic oligo(dT) beads and PCR.

Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%-0.5% of the poly(A)+ RNA pool.

Reactions of hemocytes of immune and non-immune Galleria mellonella larvae to Proteus mirabilis.

Immune larval Galleria mellonella removed live Proteus mirabilis from the hemolymph less effectively than did non-immune larvae. This was attributed to a decline in total hemocyte counts, levels of plasmatocytes and granulocytes and hemocyte adhesion capacity. Immune serum possessed factors which reduced bacterial adhesion to the hemocytes. This was not due to altering bacterial surfaces but rather to irreversible suppression of hemocyte activity.

Identification of a developmentally regulated gene, esr16, in the tracheal epithelium of Manduca sexta, with homology to a protein from human epididymis.

Events related to both ecdysis and metamorphosis require the expression of a new set of genes, the majority of which are regulated by the changes in ecdysteroid levels. We have initiated studies to identify genes whose expression is up-regulated between 24 and 4 h before pupal ecdysis in Manduca sexta. In this paper we report the partial characterization of one such gene, esr16. The transcript of esr16 is detected by Northern blot analysis in nervous tissue, muscle and trachea isolated from animals 4 h before, but not 24 h before pupal ecdysis. In situ hybridization showed that the transcript was expressed in epithelial cells of the large tracheae surrounding the nervous system and muscle. Sequence analysis suggested that the gene encoded a secreted protein with 35% identity to HE1, a human epididymal-specific gene.

The effect on fertility of immunizing female sheep with ram sperm acrosin and hyaluronidase.

Female sheep were injected with highly purified and partially purified preparations of ram sperm acrosin and hyaluronidase. The fertility and immune response of the sheep were monitored. Fertility was not significantly reduced in any single group, though a positive correlation was observed between high antibody titres against acrosin and reduced fertility. Studies on the direct action of sera from the ewes on ejaculated ram spermatozoa did not show any evidence of sperm agglutination or immobilization. Similar studies with denuded spermatozoa (detergent induced 'acrosome reaction') sometimes resulted in agglutination and enzyme inhibition was also seen; there was no correlation between any of these parameters and pregnancy.

Experimental epikeratophakia with biological adhesive.

Successful experimental epikeratophakia grafting was done with the use of a biological adhesive. The use of an adhesive eliminated the need for any suturing of the corneal lenticule to the host cornea, and this method reduced the length of the operative procedure to only one third of the time that is taken when sutures are used. Tisseel (Immuno AG, Vienna), a commercially available two-component adhesive system based on human fibrinogen, which is activated by thrombin, was used. The glue was used in combination with an antifibrinolytic agent. With alteration of the operative technique and lenticule design, 70% of glued epikeratophakia grafts in a rabbit model were retained, compared with a 50% success rate with grafts applied without the use of an adhesive.

The effect on fertility of vaccinating female sheep with biochemically defined fractions of ram spermatozoa.

Sixty sheep were vaccinated with six biochemically defined fractions of ram spermatozoa obtained by differential extraction procedures with 0.1% Triton X-100 and 2 M MgCl2, followed by ion-exchange chromatography. No one group showed a significant reduction in fertility as compared with the controls, but there was some evidence that MgCl2 extracts were not more potent infertility-inducing agents. Some of the mechanisms by which the immune response could influence fertility are also discussed.

Experimental epikeratophakia using tissue lathed at room temperature.

This report presents for the first time the results of carrying out epikeratophakia with tissue lathed at room temperature. Using an experimental model of epikeratophakia in the rabbit, we evaluated tissue handling techniques for the preparation of donor lenticules. Details of the technique are described and the in-vivo and histopathological findings reported.

Radiofrequency thermal balloon angioplasty in an experimental model of peripheral arterial stenosis.

RATIONALE: To assess the effect of thermal balloon angioplasty on surgically created peripheral arterial stenoses. METHODS: Unilateral femoral arterial stenoses were created in 17 neonatal lambs (Dorset X Suffolk/Mule), using absorbable sutures. Six to 8 weeks later, the stenoses were dilated using either a standard (N=5), or thermal balloon angioplasty (N=9). Immediate angiography, flow and pressure gradient measurements were made to assess the acute result following either procedure. The survivors were followed up for a further period of 4 to 6 weeks, and a terminal angiographic study performed. The thermally treated segments of vessels were removed for qualitative histologic analysis. RESULTS: Thermal angioplasty appeared to be acutely successful in eight of nine animals, compared with one of five successful procedures following standard angioplasty. Higher therapeutic temperatures (> or = 80 degrees C) were associated with vascular complications. At the terminal study, stenoses had recurred in four of six survivors successfully treated with thermal angioplasty. Histologic studies demonstrated non-uniform effects of thermal dilation on the vessel wall, with variable changes ranging from partial or full-thickness tears, fibroblastic or myocyte proliferation, and disorganization of the vessel wall layers. CONCLUSION: Thermal angioplasty appears to be acutely more beneficial compared with standard angioplasty. However there is a significant recurrence of stenoses, and non-uniform changes in the vessel wall.

Some failings of pulsatility index and damping factor.

Pulsatility index (PI) is a commonly used method of objective assessment of the Doppler waveform. PI falls with increasing proximal stenosis and is raised by increasing peripheral resistance. Damping factor (DF) for an arterial segment is calculated by dividing the proximal by the distal PI. DF rises with increasing severity of disease of the arterial segment. DF is not, however, sufficiently accurate to be used alone but is usually combined with transit time measurements to provide information of diagnostic use. Both PI and DF have been examined in a canine model of combined segment disease. With increasing stenosis, distal PI falls as expected but so also does proximal PI. Such a stenosis is, in effect, a flow-throttling resistance so that although the characteristics of blood flow are altered by its presence, similar changes are observed both above and below the stenosis. The reduction of PI by a stenosis distal to the insonation site may result in the false interpretation of a low PI as indicating disease proximal to the insonation site. The observed similarity between PI proximal and distal to a stenosis reduces the usefulness of pulsatility index damping factor, particularly in the assessment of the femoro-popliteal segment in combined segment disease.

Quantitative assessment of human proteinases as agents for chemonucleolysis.

A rabbit model system is described. It allows accurate measurement of the dose-dependent loss of glycosaminoglycan from the nucleus pulposus of lumbar intervertebral discs after injection of a proteinase. At the dose equivalent to that of chymopapain used in human chemonucleolysis, two human serine proteinases, cathepsin G and chymotrypsin, were as effective as chymopapain in removing up to 80% of the glycosaminoglycan from the disc. A cysteine proteinase, cathepsin B released no more than 45% of glycosaminoglycan. X-ray films clearly showed narrowing of the disc space when 30-40% of glycosaminoglycan was removed. The degradation of the nucleus pulposus was seen histologically as loss of toluidine blue metachromasia.

Effects of acute and chronic restraint on the adrenal gland weight and serum corticosterone concentration of mice and their faecal output of oocysts after infection with Eimeria apionodes.

Experiments were designed to investigate the potential stress of procedures commonly used for restraining mice and to discover whether habituation of the adrenocortical response occurred when chronic restraint was used. The study also examined the effect of chronic restraint on the faecal output of oocysts by mice trickle infected with Eimeria apionodes. The results showed that restraining mice for one hour was stressful and that restraining them repeatedly for one hour daily for seven or 21 days did not lead to habituation of the adrenocortical response. Restraint for one hour daily for seven days did not cause the recurrence of a clinical infection in mice which were chronically infected with E apionodes. Restraining mice for one hour daily for seven days before and for 14 days during the infection also had no effect on the prepatency, patency or intensity of an E apionodes infection.