Facial action units, activity and time spent with dam are effective measures of pain in response to mulesing of Merino lambs.

Repeatable measures of pain in ruminants following husbandry procedures are required to validate responses to pain relief. This study tested the hypotheses that facial action units, activity and time spent with dam can be used to assess the efficacy of pain relief in lambs following mulesing. Merino lambs (n = 120) were allocated to one of six treatments implemented at mulesing: (1) lambs that were not mulesed or lambs that were mulesed and administered (2) no pain relief, (3) meloxicam 15 min before mulesing, (4) Tri-Solfen®, (5) a combination of meloxicam 15 min before mulesing and Tri-Solfen after mulesing and (6) meloxicam at mulesing. Facial action units detected a difference in pain between mulesed and non-mulesed lambs at 1 and 5 h post-mulesing (P = 0.005 and <0.001) but not at 26 h post-mulesing. Lambs that were not mulesed were more active and spent more time with their dams than mulesed lambs (P < 0.001). No differences were observed between lambs that were mulesed with or without pain relief. Therefore, facial action units, activity of the lamb and time spent with dam can detect pain in response to mulesing in Merino lambs but cannot detect any changes associated with pain relief.

Behavioural measures reflect pain-mitigating effects of meloxicam in combination with Tri-Solfen® in mulesed Merino lambs.

Flystrike costs the Australian industry $173 to 280 M per annum and 70% to 80% of Merino lambs are currently mulesed to reduce the risk of flystrike. To alleviate welfare concerns there has been widespread adoption of analgesics to mitigate the pain associated with mulesing. The objective of this experiment was to determine the effectiveness of Tri-Solfen® and meloxicam (Metacam® 20) at reducing pain-related behavioural responses to mulesing in Merino lambs. One hundred and forty Merino lambs were allocated to one of seven treatment groups: (1) non-mulesed (Control); (2) mulesed with no pain relief; (3) subcutaneous (s.c.) meloxicam administered 15 min before mulesing; (4) Tri-Solfen® administered at time of mulesing; (5) Tri-Solfen® and saline injection (s.c.) 15 min before mulesing; (6) Tri-Solfen® and meloxicam (s.c.) 15 min before mulesing; and (7) meloxicam (s.c.) at time the of mulesing. Behavioural responses such as standing, walking and lying were measured every 15 min for 6 h on the day of marking and for up to 2 h for 4 days thereafter. Standing (hunched v. normal) and walking (stiff v. normal) behaviours were then categorised into pain- and normal-related behaviours while lying remained in its own category. Mulesed lambs with no pain relief displayed significantly more pain-related behaviours than Control lambs during the 6 h post-mulesing (1.22 v. 0.22 out of a total score of 3; RSD=1.15). Lambs that received a combination of pain relief displayed significantly less pain-related behaviour than mulesed lambs with no pain relief on the day of mulesing (0.85 v. 1.22 out of a total score of 3; RSD=1.15). Administration of meloxicam or Tri-Solfen® on their own had minimal if any significant effect on pain-related behaviours on the day of mulesing. The results of this experiment support the use of pain-related behaviours to measure the efficacy of analgesics and the use of multimodal analgesia during mulesing of lambs.

An evaluation of semiautomatic approaches to contour segmentation applied to fungal hyphae.

Semiautomatic image analysis techniques are particularly useful in biological applications, which commonly generate very complex images, and offer considerable flexibility. However, systematic study of such methods is lacking; most research develops fully automatic algorithms. This paper describes a study to evaluate several different semiautomatic or computer-assisted approaches to contour segmentation within the context of segmenting degraded images of fungal hyphae. Four different types of contour segmentation method, with varying degrees and types of user input, are outlined and applied to hyphal images. The methods are evaluated both quantitatively and qualitatively by comparing results obtained by several test subjects segmenting simulated images qualitatively similar to the hyphal images of interest. An active contour model approach, using control points, emerges as the method to be preferred to three more traditional approaches. Feedback from the image provider indicates that any of the methods described have something useful to offer for segmentation of hyphae.

Development of a sandwich ELISA for ovine granulocyte/macrophage colony-stimulating factor.

Recombinant ovine granulocyte/macrophage colony-stimulating factor (rOv GM-CSF) has been expressed in Chinese hamster ovary cells. A stable, cloned line of these cells has been established which secretes high levels (40 mu g ml(-1)) of rOv GM-CSF. Three murine monoclonal antibodies (mAbs) were produced which reacted with rOv GM-CSF on Western blots. These mAbs also neutralised the activity of both recombinant and native Ov GM-CSF in a bone marrow haemopoietic progenitor cell assay. Two of the mAbs, which recognise mutually exclusive epitopes, were selected for the development of a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GM-CSF in biological samples of ovine origin.

The cytokine response of afferent lymph following orf virus reinfection of sheep.

Abstract The in vivo dynamics of differentiated cells and interleukin (IL)-lß, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ titres in afferent lymph were compared following orf virus reinfection and inactivated virus injection of previously infected sheep. The biphasic lymphoblast and cytokine response in the lymph to virus reinfection is consistent with a response initially to orf virus as recall antigen followed by a response to viral replication. CD4 T cells increased in output over other cell types in the lymph in both groups. A rapid immune/inflammatory response was detected in lymph plasma as an increase in cytokine titres within 24 h of virus reinfection or injection. Lymph cells producing IL-1ß and IL-8 appeared prior to those producing GM-CSF in both groups. In spite of variations in the concentration of individual cytokines in lymph following reinfection, both the size of the orf lesion and the time to resolve were similar in all cases. Résumé- Les dynamiques in vivo des cellules différenciées et des taux d'IL-1ß, de GM-CSF et d'interferon γ dans la lymphe afférente furent comparés chez des moutons antéricurement infectés après une réinfection par le virus de l'ecthyma contagieux et après injection d'un virus inactivé. La réponse biphasique lymphoblastique et des cytokines à la réinfection virale est compatibles avec une réponse primaire au virus de l'ecthyma contagieux comme antigène mémoire suivie par une réponse secondaire à la réplication virale. Dans les 2 groupes, le nombre de TCD4 est plus élevé que les autres populations cellulaires mises en évidence dans la lymphe. Une réponse de type immune/inflammatoire est révélée dans le plasma par une élévation des taux de cytokines dans les 24 heures qui suivent la réinfection virale ou l'injection de virus inactivé. Les lymphocytes producteurs dTL-1ß et d'IL-8 apparaissent avant les lymphocytes producteurs de GM-CSF dans les deux groupes. En dépit des variations de concentration des cytokines individuelles dans le lymphe après reinfection, à la fois la taille des lesions d'ecthyma et les délais de guérison sont identiques dans tous les cas. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Effet sur les cytokines de la lymphe afferente d'une reinfection par les virus de l'ecthyma contagieux chez le mouton.) Veterinary Dermatology 1996; 7: 11-20.] Resumen Se comparó la actividad de células diferenciadas y los titulos de interleuquina (IL)-1ß, IL-8, factor de estimulación de colonias de granulocitos y macrófagos (GM-CSF) e interferon (IFN)-y entre reinfecciones por el virus del ectima contagioso e inyección de virus inactivado de ovinos previamente infectados. La respuesta a la reinfección en forma bifásica linfoblástica y de citoquinas en la linfa está de acuerdo con una respuesta inicial al virus del ectima por estimulación antigénica, seguida por una respuesta a la replicación viral. Las células T CD4 aumentaron respecto a otros tipos celulares en la linfa de ambos grupos. Se detectó una respuesta inmune/inflamatoria rápida en el linfa-plasma en forma de aumento de los titulos de citoquinas dentro de las 24 h de reinfección o inyección del virus. Las células linfáticas productoras de IL-1ß e IL-8 aparecicron antes que las productoras de GM-CSF en ambos grupos. A pesar de las variaciones en la concentración de citoquinas individuates en la linfa después de la reinfección, tanto el tamaño de la lesión por el virus del ectima contagioso como el tiempo de resolución fueron similares en todos los casos. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Produccion de citoquinas en el ganglio linfatico afferente tras la reinfección por el virus del ectima contagioso.) Veterinary Dermatology 1996; 7: 11-20.] Zusammenfassung- Es wurde die in-vivo-Dynamik der Titer differenzierter Zellen und Interleukin (1L)-1ß, IL-8, Granulozytenmakrophagenkolonien-stimulierender Faktor (GM-CSF) und Interferon (IFN)-γ in afferenter Lymphe nach einer Reinfektion mit ORF-Virus und einer Injektion inaktivierten Virusmaterials von früher infizierten Schafen verglichen. Die biphasische Lymphoblasten- und Zytokin-Reaktion in der Lymphe auf die Virusreinfektion stimmte mit einer initialen Reaktion gegenüber ORF-Virus überein, da der Wiederabruf von Antigen von einer Reaktion auf die Virusreplikation gefolgt wird. CD4-T-Zellen vermehrten sich im Output stärker als andere Zelltypen in der Lymphe beider Gruppen. Es wurde eine rasche immunologische/entzündliche Reaktion im Lymphplasma in Form eines Anstieges von Zytokin-Titern innerhalb von 24 Stunden nach Virusreinfektion oder -injektion festgestellt. Lymphatische Zellen, die IL-1ß und IL-8 produzieren, erschienen vor dem Auftreten von solchen, die GM-CSF produzieren, in beiden Gruppen. Trotz des Schwankens der Konzentration der individuellen Zytokine in der Lymphe nach Reinfektion, waren sowohl die Größe der ORF-Veränderungen und die Zeit der Heilung ähnlich in alien Fällen. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Die ZytokinReaktion afferenter Lymphe nach einer Reinfektion mit orf-virus beim schaf.) Veterinary Dermatology 1996; 7: 11-20.].

The 175 antigen expressed on myeloid and erythroid cells during differentiation is associated with serine protease activity.

Monoclonal antibody 175 recognizes a cell-surface antigen on more than 80% of nucleated ovine bone marrow cells (BMC). The distribution is unusual, as the majority of differentiated myeloid and erythroid cells express the antigen (175 antigen), whereas mast cells, basophils, and the majority of lymphocytes do not. The level of 175 antigen expression has been shown to increase as BMC differentiate during hematopoiesis. Previous attempts to identify the 175 antigen have been unsuccessful. In this study, the 175 antigen was affinity-purified and shown to contain serine protease activity. Immunoblot analysis following sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of bone marrow cell lysates run under reducing or nonreducing conditions showed two closely adjacent protein bands (a doublet) of 28 to 30 kD molecular weight. N-linked deglycosylation showed that the 30-kD band was a glycosylated form of the 28-kD protein. Both protein bands shared the same N-terminal amino acid sequence over 20 residues, with high homology with serine proteases. Affinity-purified 175 antigen was proteolytic in substrate gels, the activity being inhibited by the 175 monoclonal antibody (Mab) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), but not by metallo, thiol, or acid protease-specific inhibitors. The 175 antigen is therefore part of a growing family of cell-surface proteases associated with hematopoietic cell differentiation.

Ultrastructural Studies of Orf Virus Infection and Replication in Fetal Lamb Fibrocytes.

Résumé- Le cycle de réplication du virus de l'ecthyma contagieux a été identifié dans des études in vitro et un modèle hypothétique a été développé. Pendant la premiére phase, qui dure à peu près 5 heures, le virus pénètre la cellule par le biais d'un processus de phagocytose, et perd ses enveloppes. La phase d'éclipse, pendant laquelle le virus est apparemment intégréà l'ADN de l'hôte, dure environ 8 à 10 heures. Pendant la phase finale, les virions se développent dans des zones biens définies du viroplasme à partir desquelles les viriojns matures vont migrer jusqu'aux bords de la cellule. Là, ils sortent soit par exocytose, soit à l'intérieur de projections microvilleuses qui sont pincées à leur base, soit encore par désintégration de la cellule hôte. [Onwuka, S.K., Jenkinson, D. Mc, Inglis, L., Pow, I., GRAY, E.W., Reid, H.W. Ultrastructural studies of orf virus infection and replication in fetal lamb fibrocytes (Etudes ultrasturcturales de l'infection par le virus de l'ecthyma contagieux et de sa réplication dans les fibrocytes de foetus d'agneau). Resumen- Se identificó el ciclo de replicación del virus del ectima contagioso en estudios temporales in vitro y se desarroló un posible modelo experimental. Durante la primera fase, que dura unas 5 h, el virus penetra en la células por fagocitosis y se libera de la cubierta. La fase de "eclipse", con el virus presentándose como hebras de DNA, dura aproximadamente de 8 a 10 h. En la fase final los viriones se desarrollan dentro de zonas bien definidas en el viroplasma desde las cuales los viriones maduros migran hasta los limites celulares. A partir de alii parecen salir por exocitosis o en proyecciones de microvellosidades "pinzadas" hacia el exterior; también pueden ser liberados como consecuencia de la desintegración de la célula huésped. [Onwuka, S.K., Jenkinson, D. Mc, Inglis, L., Pow, I., GRAY, E.W., Reid, H.W. Ultrastructural studies of orf virus infection and replication in fetal lamb fibrocytes (Estudios ultraestructurales de la infección por el virus del ectima contagioso y replicación en fibrocitos fetales de carnero). Abstract- The cycle of replication of orf virus was identified in temporal in vitro studies and a putative model was developed. During the first phase, which lasts about 5 h, the virus enters the cells by a phagocytic process and uncoats. The "eclipse" phase, with the virus apparently present as strands of DNA, lasts for approximately the next 8-10 h. In the final phase virions develop within well-defined zones of viroplasm from which mature virions migrate to the margins of the cell. There they apparently exit either by exocytosis or within microvillous projections which are "pinched off"; they can also be released by disintegration of the host cell.

Hepatic recruitment of mast cells occurs in rats but not mice infected with Schistosoma mansoni.

The pathogenesis of infection with Schistosoma mansoni in rats is distinct from that in mice. Rats are non-permissive hosts and infection is terminated in the liver before egg laying commences whereas the parasites completes its life cycle in mice. Comparison of the mast cell responses in the two species reveals that a pronounced hepatic mastocytosis occurs in the rat and this is concomitant with the demise of the parasite. The majority of recruited hepatic mast cells contain the highly soluble granule chymase, rat mast cell protease-II, which is released systemically into blood during the period of parasite elimination. In contrast, very few mast cells are found in livers of parasitized mice and none contain the soluble granule chymase mouse mast cell protease-1. However, during egg deposition in the gut, an intraepithelial mastocytosis occurs in parasitized mice. These intraepithelial cells are typical mucosal mast cells as determined by their content of mouse mast cell protease-1. Recruitment of mucosal mast cells occurs in the intestinal lamina propria of infected rats soon after the parasites migrate to the liver. These findings suggest that mast cells of the mucosal phenotype are involved in the pathogenesis of the hepatic response to infection in the rat but that, in the mouse, mucosal mastocytosis is associated with intestinal sensitization by egg antigens.

Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells.

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase-I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.

Quantitative and cytochemical studies of mast cell proteases in rat ovaries and uteri in various reproductive states.

A role for mast cell proteases (RMCP I and II) in the cyclical remodelling of ovarian and uterine tissues of rats was investigated in the oestrous and pregnancy cycles using immunocytochemistry and enzyme-linked immunosorbent assays. The concentrations of RMCP I exceeded that of RMCP II by 100-fold in both tissues, but were always much higher in uteri than ovaries. Most of the protease activity in the uterus was located in the myometrium, whereas it was more focally distributed in the hilus and medulla of the ovary. Protease activity was confined to mast cells identified by metachromatic staining and no single cell contained both proteases. The concentrations of RMCP I and II in the two organs did not fluctuate throughout the 4-day oestrous cycle. Neither were RMCP I concentrations in the uterus altered by administration of diethylstilboestrol to ovariectomized animals, although total amounts per uterus were substantially greater than in the controls. Concentrations of RMCP I were substantially reduced in the uterus after day 6 of pregnancy and rose during the puerperium. The reduction was greater in pregnant than in pseudopregnant horns and tended to be lower in the vicinity of conceptuses than between them. The physiological significance of the lower mast cell protease concentrations is unclear, although their absence may contribute to the decreased protein catabolism during pregnancy.

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.

Characterisation of ovine mast cells derived from in vitro culture of haemopoietic tissue.

Ovine mast cells generated in vitro from bone marrow (BMMC) were compared with mucosal mast cells (MMC) isolated from parasitised abomasum. Ultrastructurally, the granules of BMMC were partially developed and immature. Both cells types contained beta-hexosaminidase, arylsulfatase, histamine, dopamine and sheep mast cell proteinase (SMCP). Greater amounts of beta-hexosaminidase, but less SMCP, histamine and arylsulfatase were present in BMMC. Stimulation with calcium ionophore A23187 caused the secretion of granule constituents and generation of leukotriene C4 by BMMC in a dose-dependent manner. An additional [3H]diisopropylfluorophosphate-binding 31,500 mol. wt. serine esterase, antigenically related to SMCP (27,000 mol. wt.) was present in cultures of BMMC but was not detected in isolated MMC. Both enzymes were detected in BMMC by Day 7 of culture and were secreted concomitantly following stimulation of BMMC with ionophore.

Actions of bovine skin washings and sera on the motile zoospores of Dermatophilus congolensis.

Concentrated skin washings, even from vaccinated animals, failed to inhibit the motility of the infective zoospores of Dermatophilus congolensis, or to prevent them from germinating or infecting cattle; their constituent immunoglobulins did not attach to the flagella although IgA and IgG2 did bind to the cell bodies. It is concluded that the specific antibodies at the skin surface of ruminants are unlikely to have a role in zoospore immobilisation. Post vaccination sera rapidly immobilised and clumped the zoospores by means of a coat around the flagella, in which immunoglobulins, particularly IgM, were detected. IgM and IgG1 also attached to the cell bodies of the zoospores.

Familial trends in low birth weight.

The reproductive performance of sisters and sisters-in-law of 185 women who had delivered "light-for-dates" and "premature expulsion" low birth weight infants was studied. Percentile birth weights were compared taking into account length of gestation, fetal sex, and the height, weight, parity, and smoking habits of the mother. Sisters of women who had delivered light-for-dates babies had lighter babies than the general population, their sisters-in-law, or the sisters of women in the premature expulsion group. These other groups, however, had the expected distribution of percentile birth weights. Data on familial trends in smoking habits and unknown gestation are also presented. The results are consistent with the theory that the mother's own intrauterine experience affects her reproductive performance but could also be explained by shared family learning experience of as yet unidentified microsocial factors related to pregnancy performance.