Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

Protection of rat atrial myocardium against electrical, mechanical and structural aspects of injury caused by exposure in vitro to conditions of simulated ischaemia.

1. Rat isolated and superfused atria were exposed for varying periods to a solution simulating the composition of extracellular fluid during myocardial ischaemia (SI). 2. Atria subjected to SI showed a loss of systolic contractile tension, a rise in diastolic tension, a shortening of electrical refractory periods, a slowing of action potential conduction velocity and disruption of the mitochondrial ultrastructure. All these features were reversible when the muscle was returned to normal superfusate. 3. Atria pretreated with a superfusate containing a calcium channel antagonist, a calmodulin inhibitor or an intracellular calcium antagonist showed fewer features of the response to SI than did controls. 4. Atria pretreated with a superfusate containing various non-steroidal anti-inflammatory agents did not show identical responses to SI. Sulphinpyrazone protected against all features of the response to SI but ibuprofen, flurbiprofen and GP25671 (a metabolite of sulphinpyrazone) had little effect. Flufenamate, phenylbutazone and salicylate enhanced the responses to SI.

The effects of some non-steroidal anti-inflammatory agents on membrane-associated calcium in rabbit peritoneal neutrophils.

Several non-steroidal anti-inflammatory agents (NSAIAs) (flufenamate, flurbiprofen, indomethacin, phenylbutazone, piroxicam and salicylate), one anti-inflammatory steroid (hydrocortisone) and three compounds known to affect cellular calcium metabolism [nifedipine, calcium ionophore A23187, 8-(diethylamino)octyl 3,4,5,-trimethoxy benzoate hydrochloride (TMB-8)] were tested for their effects on membrane-associated calcium in rabbit peritoneal neutrophils using the fluorescent probe chlortetracycline (CTC). The NSAIAs reduced the level of fluorescence attained by incubating neutrophils with CTC, and caused an immediate reduction in the fluorescence of CTC-loaded neutrophils, both effects being concentration-related. Nifedipine, A23187 and TMB-8 also reduced the fluorescence of CTC-loaded cells. However, these measured reductions in fluorescence were due in whole or in part to a drug-induced drop in the autofluorescence of the neutrophils. After applying a correction factor which took account of this effect there was still an immediate concentration-related reduction in CTC-dependent fluorescence after the addition of all the NSAIAs. A23187 also caused a reduction in CTC fluorescence, but nifedipine, TMB-8 and hydrocortisone were inactive. Flufenamate, indomethacin and piroxicam reduced the drop in fluorescence brought about by N-formyl-methionyl-leucyl-phenylalanine (FMLP). These findings suggest that NSAIAs and A23187 displace membrane-associated calcium. Electron microscopical findings confirm that indomethacin and salicylate both affect membrane-associated calcium. Using the fluorescent probe Quin 2, an attempt was made to determine whether or not the displacement of membrane-associated calcium by NSAIAs was accompanied by changes in cytoplasmic calcium ion concentration. This was unsuccessful, however, since the effects of the NSAIAs on the fluorescence of Quin 2-loaded neutrophils was accounted for almost entirely by their effects on the autofluorescence of the cells.

An in vitro method for assessing the effects of pro-inflammatory and anti-inflammatory compounds on microvascular permeability in the rat small intestine.

A method is described in which the blood vessels of the rat mesentery and small intestine were perfused for 15 min in vitro with a gelatin-containing physiological salt solution. Colloidal carbon (CC) was then added to the perfusate. In control preparations, very little CC was trapped in the microvessels of the small intestine, but if platelet-activating factor (PAF) was added for 5 min before the infusion of CC, many microvessels were "blackened." When the PAF antagonist BN52021 was included in the perfusate throughout, the "blackening" response to PAF was significantly reduced. Using micrographs of fixed specimens of gut, the amounts of "blackening" in the microvessels of the villi, the crypts of Lieberkuhn, and the muscularis were assessed using semiautomated image analysis. The technique provides a means of investigating the effects on microvascular permeability of pro-inflammatory and anti-inflammatory compounds. It is particularly useful for testing substances which, because of their highly toxic nature, cannot be administered systemically in vivo.

Increases in opacity and thickness induced by surfactants and other chemicals in the bovine isolated cornea.

Changes in the opacity and thickness of the bovine isolated cornea in response to incubation with surfactants and other chemicals have been monitored over a period of 4.5 h. Although there was some correlation between opacity and thickness for the anionic and non-ionic surfactants and for some of the chemicals, there was no correlation for cationic surfactants. It is suggested that the measurement of opacity in the isolated cornea gives a more reliable indication of likely in vivo eye irritancy than does the measurement of corneal thickness.

Stimulation of protein kinase C activity may increase microvascular permeability to colloidal carbon via alpha-isoenzyme.

The vasculature of the isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution in vitro. Various phorbol-related compounds that are known to have different affinities for the protein kinase C (PKC) isoenzymes, and bradykinin (BK), were tested for their ability to cause the microvascular endothelium to become permeable to injected colloidal carbon (CC). Phorbol 12,13-dibutyrate (PDB), 12-deoxyphorbol 13-phenylacetate (DOPPA), thymeleatoxin (TMX), and resiniferatoxin (RFX), each at a concentration of 1 microM, were found to increase permeability. Pretreatment with the PKC inhibitor Ro 31-8220 (1 microM) significantly reduced the response to all of these compounds. Indomethacin (1 microM), on the other hand, reduced only the effect of RFX. 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA) (1 microM) and BK (10 microM) did not increase CC leakage. These results suggest that the Ca(2+)-dependent PKC alpha-isoenzyme was involved in the increase in endothelial permeability. BK does not appear to stimulate PKC activity in this experimental situation.

Possible involvement of microtubules in platelet-activating factor-induced increases in microvascular permeability in vitro.

The blood vessels of the rat small intestine were perfused in vitro with a gelatin-containing physiological salt solution (GPSS). The addition of platelet-activating factor (PAF, 5 microM), podophyllotoxin (50 microM), colcemid (50 microM), or nocodazole (50 microM) to the GPSS for 5 min caused an increase in vascular permeability. This was manifested as an increased trapping of circulating colloidal carbon (CC) within the walls and was assessed using semiautomated image analysis. Pretreatment for 10 min with taxol (5 microM) in the perfusate significantly reduced the permeability-enhancing effects of all four agonists. Since podophyllotoxin, colcemid, and nocodazole are all microtubule-disrupting agents, and since taxol is a microtubule-stabilizing agent, these results suggest that microtubules are involved in the response of the microvessels to PAF. An explanation based on "tensegrity" or "force-counterbalance" is put forward to account for these findings.

In vitro effects of PAF on venous endothelial cell actin disposition.

Venous endothelial cells have been stained in vitro for the contractile protein actin, using an immunofluorescence technique. Semi-automated image analysis showed that treatment with platelet-activating factor (PAF 5 x 10(-7) M) caused elongation of endothelial cells and alteration in their actin-staining characteristics. These changes are prevented by the PAF antagonist BN52021 (1 x 10(-5) M and 1 x 10(-6) M) and the absence of extracellular Ca++. It is suggested that it is the resultant decrease in intracellular Ca++ levels which is responsible for preventing the effects of PAF.

Effects of PAF and PAF antagonists on the shape of venous endothelial cells in vitro.

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cells in vitro. BN52021 had a significant protective action at concentrations of 1 microM and 0.1 microM, but at 100 microM had a damaging effect of its own. CV3988 (0.1 microM and 1 microM) and L652,731 (20 microM) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 microM and 100 microM, L652,731 100 microM) both compounds alone caused significant changes of shape. BN52021 (0.1 microM) was also effective against leukotriene (LT) C4, at 1 microM against bradykinin and LTE4, and at 10 microM against LTD4 and the calcium ionophore A23187. BN52021 (10 microM) was ineffective against shape changes induced by histamine, prostaglandin (PG) E2 and lysophosphatidylcholine (LPC). Neither indomethacin (100 microM) nor verapamil (20 microM) altered the response to PAF. Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.

The effects of TMB-8 on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents.

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 microM) in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 microM), calcium ionophore A23187 (10 microM), histamine (300 microM), bradykinin (2 microM), prostaglandin E2 (PGE2) (30 microM), or leukotrienes (LT) C4, D4 or E4 (1 microM). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.

Inhibition by calcium antagonists of shape changes induced in vitro by pro-inflammatory mediators in venous endothelial cells.

The changes in shape of endothelial cells (ECs) lining rat inferior venae cavae in response to exposure in vitro to platelet activating factor (PAF, 5 x 10(-7) M), or 5-hydroxytryptamine creatinine sulphate (5HT, 3 x 10(-5) M), or calcium ionophore A23187 (1 x 10(-5) M), or cytochalasin B (CB, 1 x 10(-5) M) were assessed using an immunofluorescence technique and semi-automated image analysis. Most of these shape changes were prevented by lowering intracellular Ca2+ levels in several ways. Thus, pre-treatment with ryanodine (1 x 10(-6) M), verapamil HCl (2 x 10(-5) M), indomethacin (1 x 10(-4) M), isoprenaline sulphate (2 x 10(-6) M) or dibutyryladenosine 3'5' cyclic monophosphate (DbcAMP, 1 x 10(-5) M) significantly reduced the effects of PAF, 5HT and A23187. Pre-treatment of ECs with 8(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (TMB-8, 1 x 10(-5) M) or the nominal absence of Ca2+ in the extracellular medium prevented the effects of PAF and 5HT, but not those of A23187. The responses to 5HT were also reduced by cyproheptadine HCl (3 x 10(-6) M). However, only the absence of extracellular Ca2+ reduced the effects of CB. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide HCl (W7, 1 x 10(-5) M) and the protein kinase C inhibitor 1-(5-isoquinolinyl sulphanyl)-2-methylpiperazine 2HCl (H7, 1 x 10(-5) M) were without effect against all the above agonists, but H7 and verapamil prevented EC shape changes induced by phorbol myristate acetate (PMA, 1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents.

Histamine (300 microM), bradykinin (2 microM), prostaglandin E2 (PGE2) (30 microM), or the leukotrienes (LT) C4 and E4 (1 microM) but not D4 (1 microM) applied in vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 microM) or verapamil (20 microM) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.

A morphological study of the effects of 5-hydroxytryptamine and indomethacin on rat mesenteric venules.

A method is described for preparing venules of the rat mesentery for electron microscopy after the application of 5-hydroxytryptamine (5-HT) and pretreatment with indomethacin. Local application of 5-HT caused the leakage of colloidal carbon and the emigration of leucocytes into the venule wall. 5-HT also caused endothelial cells to bulge and their nuclei to contort. It increased the number of protrusions on both the luminal and abluminal surfaces of the endothelium and increased the width of the subendothelial space, and the degree of vesiculation in the endothelial cells. Systemic treatment with indomethacin significantly decreased the amount of carbon passing through the endothelium after the local application of 5-HT, but enhanced some of the other effects of 5-HT. Thus it increased the bulging of endothelial cells and contortion of their nuclei, and further increased the number of surface protrusions and the subendothelial space. It had no effect on the emigration of leucocytes resulting from the application of 5-HT.

Action of histamine on endothelial cells of guinea-pig isolated hepatic portal vein and its modification by indomethacin or removal of calcium.

Methods are described for preparing guinea-pig hepatic portal vein endothelium for light microscopy and electron microscopy. Histamine (100 mug/ml) caused damage to the endothelium which was visible with both the light and electron microscopes. The damage was reduced in the absence of calcium. The reduction was more apparent with the electron microscope than with the light microscope. Indomethacin (100 mug/ml) protected the endothelial cells against the damaging effects of histamine. Possible modes of action of histamine and indomethacin are discussed.

A morphological study of the effects of indomethacin, flufenamate, salicylate and calcium ions on rabbit peritoneal neutrophil polymorphs.

Rabbit peritoneal neutrophil polymorphs have been examined by transmission electron microscopy to determine the effects of removing and replacing extracellular calcium. Degranulation, disruption of the cell membrane and vesiculation are all more marked in the presence of calcium than in its absence. Cooling the cells to 4 degrees has a protective effect. The addition of indomethacin, flufenamate or salicylate to a calcium-free incubating medium decreases degranulation, protects the membrane and reduces vesiculation, particularly at 4 degrees. When extracellular calcium is replaced at 37 degrees indomethacin and salicylate slightly reduce the amount of degranulation; flufenamate and salicylate significantly reduce the signs of damage. Higher concentrations of indomethacin and flufenamate cause considerable degranulation and damage. When extracellular calcium is present in the incubating medium throughout, the addition of indomethacin, flufenamate or salicylate shows varying effects. High concentrations of all these drugs, however, cause extensive degranulation and damage.

A study of the effects of indomethacin, flufenamate and salicylate on the localization of intracellular calcium in rabbit neutrophil polymorphs using an antimonate staining method.

An antimonate staining technique was used to localize intracellular calcium in rabbit peritoneal neutrophil polymorphs. When calcium was added to a previously calcium-free extracellular medium neutrophils showed a slight loss of intracellular stainable calcium after 1 h incubation. There was a significant increase in the number of damaged cells and a slight increase in degranulation. Indomethacin (5 X 10(-5)M and 8 X 10(-4)M), both in the absence and the presence of extracellular calcium, enhanced the loss of stainable calcium, and at 37 degrees, increased the amount of damage, but increased degranulation only at the higher concentration and in the presence of calcium. Indomethacin may act as a membrane labilizer. Salicylate (2.5 X 10(-3)M and 1 x 10(-2)M at 37 degrees) slightly decreased the loss of intracellular calcium in the presence of extracellular calcium and, in the absence and presence of calcium and at the higher concentration, decreased degranulation and decreased the amount of damage. Salicylate may act as a membrane stabilizer. Flufenamate (3 x 10(-5)M and 2.4 x 10(-4)M) showed variable effects.

Modification by some antagonists of the shape changes of venous endothelial cells in response to inflammatory agents in vitro.

Endothelial cells lining guinea pig inferior venae cavae change shape when exposed to histamine, bradykinin, A23187 or platelet-activating factor (PAF) in vitro. Pre-treatment of the endothelium with isoprenaline or quin 2 significantly reduced the shape changes produced in response to histamine, bradykinin and A23187, but not those to PAF. Since both isoprenaline and quin 2 may reduce the concentration of cytoplasmic Ca++, the former by raising cyclic AMP (cAMP) levels and the latter by acting as a Ca++ buffer, the results provide further evidence for the involvement of Ca++ in the responses of large vein endothelial cells to inflammatory agents in vitro. The effects of pre-treating the endothelium with the histamine receptor-blockers mepyramine (H1) or cimetidine (H2), or the bradykinin receptor-blockers des-arg9[leu8] bradykinin (B1) or des-arg[Hyp3, Thi5,8,D-Phe7] bradykinin (B2) suggest that the response to histamine is both H1 and H2 receptor-mediated, while the response to bradykinin is only B2 receptor-mediated.

The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium.

The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent the in vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.

Vasoconstriction in rat isolated mesentery and small intestine in response to various activators of protein kinase C.

The vasculature of rat isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution (GPSS). When 5-hydroxytryptamine (5HT, 1 x 10(-4) M), or the calcium ionophore A23187 (1 x 10(-4) M), or 12-deoxyphorbol 13-phenylacetate (DOPPA, 1 x 10(-6) M), or 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA, 1 x 10(-6) M) or thymeleatoxin (TMX, 1 x 10(-6) M) was added to the GPSS for 5 min there was a gradual rise in perfusion pressure, whereas resiniferatoxin (RFX, 1 x 10(-6) M) was without effect. Pre-treatment of the tissue with the protein kinase C (PKC) inhibitor Ro 31-8220 (1 x 10(-6) M) significantly reduced the rise in perfusion pressure in response to 5HT, DOPPA, DOPPAA and TMX, but not that to A23187. Platelet-activating factor (PAF, 5 x 10(-6) M) caused an almost immediate but transient rise in perfusion pressure, followed by a more gradual rise, neither response being blocked by Ro 31-8220. When blood vessels of the mesentery alone were perfused with gelatin-free PSS, PAF caused a transient rise in perfusion pressure, but with no subsequent gradual rise over 5 min. After Ca(2+)-depletion this transient response was also absent. In contrast, when blood vessels were perfused with gelatin-free PSS, DOPPA and TMX still caused gradual rises in perfusion pressure, which were totally abolished by Ro 31-8220. TMX had no effect at all when the tissue was depleted of Ca2+, whereas the response to DOPPA was only partially reduced.(ABSTRACT TRUNCATED AT 250 WORDS)