Canine serum immunoreactivity to M. pachydermatis in vitro is influenced by the phase of yeast growth

In western immunoblotting studies of canine sera using Malassezia pachydermatis extracts, the reported patterns of immunoreactivity vary between different laboratories. Because the duration of culture influences the antigenic composition of lipid-dependent Malassezia spp. when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity using canine sera. Extracts of M. pachydermatis CBS 1879 grown in Sabouraud's liquid medium at 37 degrees C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and 98 and 68 kDa bands were recognized by five sera. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera used recognized more bands in exponential phase (d2) extracts when compared with decline phase (d8-d10) extracts, and two of these sera showed most bands in stationary phase (d4-d6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. There is variation in antigenic expression in different growth phases of M. pachydermatis, which might explain discrepancies between previous laboratory studies of canine immunity to this yeast.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

Effects of dietary cation-anion difference on blood cortisol and ACTH levels in reproducing ewes

Formulation of rations to induce a compensated metabolic acidosis in the post-partum cow has proved a useful strategy for prevention of milk fever. Such acidification improves the ability of the animal to maintain calcium homeostasis by promoting the absorption of calcium from the intestine and mobilization of calcium from the bone. In humans, an acidogenic diet results in mild metabolic acidosis in association with a state of cortisol excess and this increase in plasma cortisol may increase bone catabolism. The objective of our experiment was to induce acidification by anionic salt supplementation and to study the effects of anionic supplementation on plasma cortisol and adrenocorticotropic hormone (ACTH) levels in sheep. Twenty-seven twin-bearing sheep were assigned to two experimental groups and a control group, depending on dietary cation-anion difference (DCAD) (+272.6, -88.9 and +164.5 mEq/kg DM respectively). Sheep assigned to each dietary treatment received their respective rations beginning 6 weeks pre-partum and continuing until 12 days post-partum. Anionic diet induced a non-respiratory systemic acidosis in association with a mild increase in plasma cortisol concentration without changes in plasma ACTH levels. Our data suggest that the mild hypercortisolism observed in sheep fed the anionic diet may not be an effector for bone resorption induced by anionic salts. A mild hypercortisolism of this magnitude may lead to osteoporosis but this might require many years of adrenal hypersecretion while anionic salts are only used during the last weeks of pregnancy.     

Echocardiographic parameters in healthy young adult Sphynx cats

The objective of this retrospective study is to determine normal reference values for 2-Dimension (2D) and Motion-mode (M-mode) echocardiographic parameters in nonsedated healthy young adult Sphynx cats and to compare them to those of the domestic shorthair (DSH). 131 Sphynx cats underwent cardiac screening prior to breeding. The control group consisted of 30 healthy adult domestic cats. A complete cardiac ultrasound was performed on all cats using right parasternal long and short axis views. There were few echocardiographic parameters in the Sphynx that differed from those of the healthy DSH. Only the left atrial (LA) dimension in 2D and M-mode, the left atrial/aortic (LA/Ao) ratio and the internal dimension of the left ventricle in systole (LVIDs) measured with M-mode were different. In conclusion, although the heart of Sphynx cat can often have a particular 2-D echocardiographic appearance, the M-mode cardiac dimensions are similar to those of the DSH.     

Disseminated pheochromocytoma in a North American river otter (Lontra canadensis)

A 21-yr-old male North American river otter (Lontra canadensis) with a chronic history of degenerative osteoarthritis was evaluated for acute posterior paralysis. Because no definitive cause was identified and a poor prognosis was expected, the otter was euthanatized. A malignant neoplasm of adrenal gland origin with disseminated metastases to the central nervous system, lymph nodes, diaphragm, pancreas, spleen, and liver was diagnosed on postmortem examination. No clinical signs of disseminated neoplasia had been noted throughout the otter's history. The adrenal neoplasm was composed of nests of epithelial cells surrounded by a fine fibrovascular stroma. Neoplastic cells were immunohistochemically positive for chromogranin A, PGP9.5, metencephalin, and endorphin and negative for melan A and inhibin, confirming a diagnosis of a malignant pheochromocytoma. On the basis of the necropsy finding, metastasis of the pheochromocytoma might have contributed to the observed clinical signs.