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.

Kapsulotaenia tidswelli - an unusual cestode from the Australian goannas Varanus gouldii gouldii and V. giganteus.

Kapsulotaenia tidswelli is a proteocephalidean cestode that utilizes varanid lizards as definitive hosts. Fresh specimens of this cestode were observed with endogenous red pigmentation in the neck region that disappeared rapidly if specimens were not preserved in glutaraldehyde. The ultrastructural characteristics of the red pigment, which are described, suggest it is a carotenoid. Phylogenetic analysis confirmed a close relationship between K. tidswelli and other species of Kapsulotaenia for which sequence information is available. There is thus no reason to consider that the red pigmentation is because K. tidswelli is atypical, and it is proposed the carotenoids are likely to be associated with the diet of its varanid host.

Metabolic alkalosis, recovery and sprint performance.

Pre-exercise alkalosis and an active recovery improve the physiological state of recovery through slightly different mechanisms (e. g. directly increasing extracellular bicarbonate (HCO3 (-)) vs. increasing blood flow), and combining the two conditions may provide even greater influence on blood acid-base recovery from high-intensity exercise. Nine subjects completed four trials (Placebo Active ( PLAC A), sodium bicarbonate (NaHCO3) Active ( BICARB A), Placebo Passive ( PLAC P) and NaHCO3 Passive ( BICARB P)), each consisting of three, 30-s maximal efforts with a three min recovery between each effort. Pre-exercisealkalosis was evident in both NaHCO3 conditions, as pH and HCO3 (-) were significantly higher than both Placebo conditions (pH: 7.46 ± 0.04 vs. 7.39 ± 0.02; HCO3 (-): 28.8 ± 1.9 vs. 23.2 ± 1.4 mmol·L (-1); p<0.001). In terms of performance, significant interactions were observed for average speed (p<0.05), with higher speeds evident in the BICARB A condition (3.9 ± 0.3 vs. 3.7 ± 0.4 m·s (-1)). Total distance covered was different (p=0.05), with post hoc differences evident between the BICARB A and PLAC P conditions (368 ± 33 vs. 364 ± 35 m). These data suggest that successive 30-s high intensity performance may be improved when coupled with NaHCO3 supplementation.

Pre-exercise alkalosis and acid-base recovery.

The aim of this study was to observe the influence of pre-exercise sodium bicarbonate (NaHCO3) ingestion and varying recovery modes on acid-base recovery from a single bout of supramaximal exercise. Nine male subjects completed four separate, randomized cycle ergometer exercise trials to volitional fatigue at 120% maximum power output, under the following conditions: 0.3 g.kg(-1) BW NaHCO3 ingestion with passive recovery (BICARB P), 0.3 g.kg (-1) BW NaHCO3 ingestion with active recovery (BICARB A), placebo ingestion with passive recovery (PLAC P) and placebo ingestion with active recovery (PLAC A). Capillary blood samples were obtained every minute for 15 min during recovery. Significant main effects for pH were observed for time (F = 42.1, p < 0.001), intervention (BICARB and PLAC) (F = 1117.3, p < 0.001) and recovery condition (F = 150.0, p < 0.001), as the BICARB condition reduced acid-base perturbation. Significant interaction effects were observed between conditions (BICARB and PLAC) for active and passive recovery modes (F = 29.1, p < 0.001) as the active recovery facilitated H+ removal better than the passive condition. Pre-exercise alkalosis attenuates blood acid-base perturbations from supramaximal exercise to exhaustion, regardless of whether the recovery mode is active or passive. These findings suggest that individuals may benefit from introducing a pre-exercise alkalotic condition while including passive recovery during high-intensity training protocols.

Reproducibility of the blood lactate threshold, 4 mmol.l(-1) marker, heart rate and ratings of perceived exertion during incremental treadmill exercise in humans.

The aim of this study was to investigate the reproducibility of blood lactate measurements, heart rate (HR) and ratings of perceived exertion (RPE) during treadmill exercise at speeds corresponding to the lactate threshold ( v(Th,la)-) and a fixed blood lactate concentration of 4 mmol.l(-1)( v(la)-(,4)). Possible differences in reproducibility related to fitness levels were also investigated. A group of 20 men [mean (SD)] [age 20.5 (1.4) years] and 16 women [age 21.2 (0.9) years] took part in the study. The subjects performed two identical incremental exercise tests consisting of at least six 4 min stages. Blood lactate concentrations, HR and RPE were recorded at the end of each stage. Limits of agreement (LoA), correlation coefficients and 95% confidence intervals for the mean difference between tests were employed to investigate the level of agreement and reproducibility of blood lactate concentration, HR and RPE. For the group as a whole, the sample correlation coefficient for speed at v(Th,la)- was r=0.88, and was r=0.92 for the speed at v(la)-(,4). At v(Th,la) -, the correlation coefficients for the moderately fit and unfit were r=0.94 and r=0.36, respectively, and at v(la)-(,4) r=0.93 and r=0.68, respectively. The LoA for the moderately fit group indicated that a change of 1.62 km.h(-1) in v(Th,la)- would be necessary to be considered a change in training status. For HR and RPE, relationships between the tests were generally poor. The LoA suggested that changes in scores must be unacceptably large. These findings cast doubt on the sensitivity of testing for change of blood lactate concentration, HR and RPE in this population.