Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.     

Climate Change Could Increase the Geographic Extent of Hendra Virus Spillover Risk

Disease risk mapping is important for predicting and mitigating impacts of bat-borne viruses, including Hendra virus (Paramyxoviridae:Henipavirus), that can spillover to domestic animals and thence to humans. We produced two models to estimate areas at potential risk of HeV spillover explained by the climatic suitability for its flying fox reservoir hosts, Pteropus alecto and P. conspicillatus. We included additional climatic variables that might affect spillover risk through other biological processes (such as bat or horse behaviour, plant phenology and bat foraging habitat). Models were fit with a Poisson point process model and a log-Gaussian Cox process. In response to climate change, risk expanded southwards due to an expansion of P. alecto suitable habitat, which increased the number of horses at risk by 175–260% (110,000–165,000). In the northern limits of the current distribution, spillover risk was highly uncertain because of model extrapolation to novel climatic conditions. The extent of areas at risk of spillover from P. conspicillatus was predicted shrink. Due to a likely expansion of P. alecto into these areas, it could replace P. conspicillatus as the main HeV reservoir. We recommend: (1) HeV monitoring in bats, (2) enhancing HeV prevention in horses in areas predicted to be at risk, (3) investigate and develop mitigation strategies for areas that could experience reservoir host replacements.     

Isolation and characterisation of polymorphic microsatellite loci in the vulnerable spectacled flying fox, Pteropus conspicillatus

The spectacled flying fox, Pteropus conspicillatus, is listed as vulnerable in Australia and is under threat from numerous impacts. Primers to amplify eight co-dominant microsatellite loci were designed for Pteropus conspicillatus, based on an enriched genomic library. Four loci were monomorphic in this species while the remaining four loci were highly polymorphic with 16–23 alleles. Two of the four monomorphic loci were found to be polymorphic in Pteropus alecto, a closely related congener. All but one of the six polymorphic loci were in Hardy Weinberg equilibrium. Additionally, six microsatellite loci isolated for Pteropus rodricensis were tested against individuals of P. conspicillatus with all loci amplifying reliably. These loci will be used to investigate population genetic structure in the vulnerable spectacled flying fox.     

Flying-Fox Roost Disturbance and Hendra Virus Spillover Risk

Bats of the genus Pteropus (flying-foxes) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The increased urban presence of flying-foxes often provokes negative community sentiments because of reduced social amenity and concerns of HeV exposure risk, and has resulted in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in HeV infection in flying-foxes, and an increased spillover risk. We sought to examine the impact of roost modification and dispersal on HeV infection dynamics and cortisol concentration dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood (REML). The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was small and non-significant (P = 0.440). Similarly, the difference in mean urine specific gravity-corrected urinary cortisol concentrations was small and non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find an underlying association between cortisol concentration and season, and cortisol concentration and region, suggesting that other (plausibly biological or environmental) variables play a role in cortisol concentration dynamics. The effect of roost disturbance on cortisol concentration approached statistical significance for region, suggesting that the relationship is not fixed, and plausibly reflecting the nature and timing of disturbance. We also found a small positive statistical association between HeV excretion status and urinary cortisol concentration. Finally, we found that the level of flying-fox distress associated with roost disturbance reflected the nature and timing of the activity, highlighting the need for a ‘best practice’ approach to dispersal or roost modification activities. The findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management.     

The cortisol stress response in male round goby (Neogobius melanostomus): effects of living in polluted environments?

Acute exposure to contaminants frequently induces stress, but prolonged exposures, such as those experienced by individuals in wild populations, can impair the capacity to mount a stress response. Using the round goby (Neogobius melanostomus), a small benthic fish and potential sentinel species for habitat contamination in the Great Lakes, we explored the impacts of living in highly polluted areas. Round goby were collected from highly contaminated and less contaminated areas of Hamilton Harbour (a well-known site of polycyclic aromatic hydrocarbon (PAH) and heavy metal contamination). The cortisol stress responses of fish from sites of high and low contamination were compared using an EIA assay on blood collected (n?=?112) either prior to or 0, 10, 30, 60, 240 min and 24 h following the application of a 4-min confined air exposure stressor. Plasma cortisol levels were elevated at 10 and 30 min post-stressor (100.3 and 87.5 ng/mL), and returned to levels similar to baseline (22.3 ng/mL) 1 h after the stressor. In contrast to predictions, round goby from areas of high and low contamination had similar cortisol levels at all timepoints. We also monitored stress responses immediately after a chasing stressor (n?=?19). During the chasing stressor, contaminated-site round goby exhausted faster than individuals from a less contaminated site, although they had similar levels of plasma cortisol (23.5 versus 20.9 ng/mL) and lactate (2.53 versus 1.98 mmol/L). Our results indicate that not all fishes may demonstrate impaired stress responses, even in highly contaminated habitats; however, such animals may still have increased vulnerability to predation.     

Haematology and Plasma Biochemistry of Wild Black Flying-Foxes, (Pteropus alecto) in Queensland,Australia

This paper establishes reference ranges for hematologic and plasma biochemistry values in wild Black flying-foxes (Pteropus alecto) captured in South East Queensland, Australia. Values were found to be consistent with those of other Pteropus species. Four hundred and forty-seven animals were sampled over 12 months and significant differences were found between age, sex, reproductive and body condition cohorts in the sample population. Mean values for each cohort fell within the determined normal adult reference range, with the exception of elevated levels of alkaline phosphatase in juvenile animals. Hematologic and biochemistry parameters of injured animals showed little or no deviation from the normal reference values for minor injuries, while two animals with more severe injury or abscessation showed leucocytosis, anaemia, thrombocytosis, hyperglobulinemia and hypoalbuminemia.     

Bats Without Borders: Long-Distance Movements and Implications for Disease Risk Management

Fruit bats of the genus Pteropus (commonly known as flying-foxes) are the natural hosts of several recently emerged zoonotic viruses of animal and human health significance in Australia and Asia, including Hendra and Nipah viruses. Satellite telemetry was used on nine flying-foxes of three species (Pteropus alecto n = 5, P. vampyrus n = 2, and P. neohibernicus n = 2) to determine the scale and pattern of their long-distance movements and their potential to transfer these viruses between countries in the region. The animals were captured and released from six different locations in Australia, Papua New Guinea, Indonesia, and Timor-Leste. Their movements were recorded for a median of 120 (range, 47–342) days with a median total distance travelled of 393 (range, 76–3011) km per individual. Pteropus alecto individuals were observed to move between Australia and Papua New Guinea (Western Province) on four occasions, between Papua New Guinea (Western Province) and Indonesia (Papua) on ten occasions, and to traverse Torres Strait on two occasions. Pteropus vampyrus was observed to move between Timor-Leste and Indonesia (West Timor) on one occasion. These findings expand upon the current literature on the potential for transfer of zoonotic viruses by flying-foxes between countries and have implications for disease risk management and for the conservation management of flying-fox populations in Australia, New Guinea, and the Lesser Sunda Islands.     

Latitudinal range shifts in Australian flying‐foxes: A re‐evaluation

Recent range shifts towards higher latitudes have been reported for many animals and plants in the northern hemisphere, and are commonly attributed to changes in climate. Relatively little is known about such changes in the southern hemisphere, although it has been suggested that latitudinal distributions of the fruit‐bats Pteropus alecto and Pteropus poliocephalus changed during the 20th century in response to climate change in eastern Australia. However, historical changes in these species distributions have not been examined systematically. In this study we obtained historical locality records from a wide range of sources (including banding and museum records, government wildlife databases and unpublished records), and filtered them for reliability and spatial accuracy. The latitudinal distribution of each species was compared between eight time‐periods (1843–1920, 1921–1950, five 10‐year intervals between 1950 and 2000, and 2001–2007), using analyses of both the filtered point data (P. alecto 870 records, P. poliocephalus 2506) and presence/absence data within 50 × 50 km grid cells. The results do not support the hypothesis that either species range is shifting in a manner driven by climate change. First, neither the northern or southern range limits of P. poliocephalus (Mackay, Queensland and Melbourne, Victoria respectively) changed over time. Second, P. alecto's range limit extended southward by 1168 km (approximately 10.5 degrees latitude) during the twentieth century (from approximately Rockhampton, Queensland to Sydney, New South Wales). Within this zone of southward expansion (25–29°S), the percentage of total records that were P. alecto increased from 8% prior to 1950 to 49% in the early 2000s, and local count data showed that its abundance increased from several hundred to more than 10 000 individuals at specific roost sites, as range expansion progressed. Pteropus alecto expanded southward at about 100 km/decade, compared with the 10–26 km/decade rate of isotherm change, and analyses of historical weather data show that the species consequently moved into recently‐colder regions than it had previously occupied. Neither climate change nor habitat change could provide simple explanations to explain P. alecto's observed rapid range shift. More generally, climate change should not be uncritically inferred as a primary driver of species range shifts without careful quantitative analyses.     

Are Flying-Foxes Coming to Town? Urbanisation of the Spectacled Flying-Fox (Pteropus conspicillatus) in Australia

Urbanisation of wildlife populations is a process with significant conservation and management implications. While urban areas can provide habitat for wildlife, some urbanised species eventually come into conflict with humans. Understanding the process and drivers of wildlife urbanisation is fundamental to developing effective management responses to this phenomenon. In Australia, flying-foxes (Pteropodidae) are a common feature of urban environments, sometimes roosting in groups of tens of thousands of individuals. Flying-foxes appear to be becoming increasingly urbanised and are coming into increased contact and conflict with humans. Flying-fox management is now a highly contentious issue. In this study we used monitoring data collected over a 15 year period (1998–2012) to examine the spatial and temporal patterns of association of spectacled flying-fox (Pteropus conspicillatus) roost sites (camps) with urban areas. We asked whether spectacled flying-foxes are becoming more urbanised and test the hypothesis that such changes are associated with anthropogenic changes to landscape structure. Our results indicate that spectacled flying-foxes were more likely to roost near humans than might be expected by chance, that over the period of the study the proportion of the flying-foxes in urban-associated camps increased, as did the number of urban camps. Increased urbanisation of spectacled flying-foxes was not related to changes in landscape structure or to the encroachment of urban areas on camps. Overall, camps tended to be found in areas that were more fragmented, closer to human habitation and with more urban land cover than the surrounding landscape. This suggests that urbanisation is a behavioural response rather than driven by habitat loss.     

Blood hormone profiles,physiological variables,and behavioral criteria in Corriedale ewes fed different TMR moisture levels during thermal–humidity exposure

In this study, blood hormone profiles, physiological variables, and behavioral criteria in Corriedale ewes fed total mixed ration (TMR) at different moisture levels during thermal–humidity exposure were evaluated. Nine non-pregnant Corriedale ewes (ave. BW = 41 ± 3.5 kg) were individually fed diets based on maintenance requirements in metabolic crates. Ewes were assigned to three treatment groups according to a 3 × 3 Latin Square design for 3 periods of 21 days duration each (9 ewes per treatment). Treatments were TMR (CP (crude protein) = 16.1, TDN (total digestible nutrients) = 69.1%) moisture levels at 40, 50, and 60%. No differences were found in blood hormone profiles including cortisol (μg/dL), immunoglobulin G (mg/dL), triiodothyronine (ng/mL), thyroxin (μg/dL), growth hormone (ng/mL), prolactin (ng/mL), insulin (μU/mL), insulin-like growth factor 1 (ng/mL), aldostrone (ng/dL), antidiuretic hormone (pg/mL), and creatinine (mg/dL) among all treatment groups (p > 0.05). Measurements of physiological variables indicated that heart rate (number of beats/min) in the afternoon was higher in 50 and 60% TMR group than in the 40% group (p < 0.05). No differences were observed in respiratory rate (number/min), panting score, and fecal score among the groups (p > 0.05). The behavior criteria including urine excretion frequency (number/d), fecal excretion frequency (number/d), standing frequency (number/d), resting frequency (number/d), standing duration (min/d), and resting duration (min/d) showed no differences among the treatment groups (p > 0.05). Conclusions drawn indicate the minor impacts of TMR moisture levels up to 60% on behavioral criterions of Corriedale ewes during thermal–humidity exposure, but help smooth down the intensified heat stress conditions over physiological variables and endocrine system.     

Blood plasma biomarkers of citrinin and ochratoxin A exposure in young adults in Bangladesh

Citrinin (CIT) and Ochratoxin A (OTA) are nephrotoxic mycotoxins which can co-occur in food commodities, resulting in internal exposure. Studies in many countries reported on the presence of OTA in human blood; however, such biomonitoring data for CIT is still scarce. This study was conducted to characterize both CIT and OTA biomarker levels in plasma of volunteers since food analysis data are insufficient to assess human exposure in Bangladesh. In total 104 blood samples were collected from university students in 2013 (sampling 1: n?=?64, midsummer) and 2014 (sampling 2: n?=?40, end winter) for analysis of CIT and OTA and their metabolites HO-CIT and OTα by LC-MS/MS and HPLC-FD techniques, respectively. CIT and HO-CIT were detected in 90% (max 2.70 ng/mL) and 85% (max 1.44 ng/mL) of all samples. Mean levels in sampling 2 (CIT 0.47 ng/mL; HO-CIT 0.40 ng/mL) were higher than in sampling 1 (0.25 ng/mL; 0.37 ng/mL) indicative of variable CIT exposure. OTA was present in all (max 6.63 ng/mL) and OTα in 98% (max 0.99 ng/mL) of the samples. In sampling 1, mean OTA (0.85 ng/mL) was higher than in sampling 2 (0.51 ng/mL); the reverse situation was found for OTα mean levels. The calculated dietary OTA intake among the students (mean 9.9; max 91.7 ng/kg bw/week) was lower than the tolerable weekly intake for this mycotoxin (120 ng/kg bw/week) set by EFSA. But frequent co-exposure to CIT should be considered, and the results of this study indicate the necessity to identify major sources of CIT and OTA intake in the Bangladeshi population.     

Effect of whey protein isolate on rehydration after exercise

Studies have examined adding protein to carbohydrate–electrolyte rehydration drinks, but the effects of protein in isolation remain unknown. Ten subjects completed two trials in which they were dehydrated (~2 % of pre-exercise body mass) by intermittent cycling in the heat. Subjects then rehydrated (150 % total mass loss) over 1 h with mineral water (W) or mineral water plus 20 g L^?1 whey protein isolate (WP) and remained in the laboratory for a further 4 h. Blood and urine samples were provided pre-exercise, post-exercise, post-rehydration and every hour thereafter. From blood samples, serum osmolality, change in plasma volume and plasma albumin content was determined, whilst the volume and osmolality of urine samples were determined. There was no difference between trials for total urine volume [W: 1,234 (358) mL; WP: 1,306 (268) mL; P = 0.409], drink retention [W: 40 (14) %; WP: 37 (14) %; P = 0.322] or net fluid balance [W: ?605 (318) mL; WP: ?660 (274) mL; P = 0.792] 4-h post-rehydration. Plasma volume was greater 3 and 4 h post-drinking during WP, and plasma albumin content relative to pre-exercise was increased 1–4 h post-drinking in WP only. These results suggest that addition of 20 g L^?1 whey protein isolate neither enhances nor inhibits post-exercise rehydration, when a volume equivalent to 150 % of sweat losses is ingested in 1 h. As post-exercise nutritional requirements are multifactorial (rehydration, glycogen resynthesis, myofibrillar/mitochondrial protein synthesis), these data demonstrate that when post-exercise protein intake might benefit recovery or adaptation, this can be achieved without compromising rehydration.     

Hormonal regulation of leucine catabolism in mammary epithelial cells

Branched-chain amino acids (BCAA) are actively taken up and catabolized by the mammary gland during lactation for syntheses of glutamate, glutamine and aspartate. Available evidence shows that the onset of lactation is associated with increases in circulating levels of cortisol, prolactin and glucagon, but decreases in insulin and growth hormone. This study determined the effects of physiological concentrations of these hormones on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 3 mM d-glucose, 0.5 mM l-leucine, l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine, and 0–50 μU/mL insulin, 0–20 ng/mL growth hormone 0–200 ng/mL prolactin, 0–150 nM cortisol or 0–300 pg/mL glucagon. Increasing extracellular concentrations of insulin did not affect leucine transamination or oxidative decarboxylation, but decreased the rate of oxidation of leucine carbons 2–6. Elevated levels of growth hormone dose dependently inhibited leucine catabolism, α-ketoisocaproate (KIC) production and the syntheses of glutamate plus glutamine. In contrast, cortisol and glucagon increased leucine transamination, leucine oxidative decarboxylation, KIC production, the oxidation of leucine 2–6 carbons and the syntheses of glutamate plus glutamine. Prolactin did not affect leucine catabolism in the cells. The changes in leucine degradation were consistent with alterations in abundances of BCAA transaminase and phosphorylated levels of branched-chain α-ketoacid dehydrogenase. Reductions in insulin and growth hormone but increases in cortisol and glucagon with lactation act in concert to stimulate BCAA catabolism for glutamate and glutamine syntheses. These coordinated changes in hormones may facilitate milk production in lactating mammals.     

Increased blubber cortisol in ice-entrapped beluga whales (<Emphasis Type="Italic">Delphinapterus</Emphasis><Emphasis Type="Italic">leucas</Emphasis>)

Entrapments of whales in sea ice occur occasionally in the Arctic and often last several weeks or months, resulting in emaciation or death of whales. These events provide a unique opportunity for investigating the physiological response to a prolonged or chronic stress in an otherwise healthy population of marine mammals. By measuring cortisol in blubber, a peripheral tissue, we expect to see a reflection of long-term or chronic stress rather than short-term or acute stress. Adipose tissue should be less subject to rapid changes compared to blood cortisol, reflecting stressors experienced over a longer period of time, and should not be affected by potential stress associated with sampling. We measured blubber cortisol of 29 beluga whales (Delphinapterus leucas) entrapped in November 2006 in Husky Lakes basin and 26 whales from the same population (Eastern Beaufort Sea) during regular seasonal harvests in July of 2006 and 2007. Mean cortisol concentrations (±SEM) were seven times higher in blubber from entrapped whales (1.76 ± 0.32 ng/g wet weight) compared to whales from regular seasonal harvests (0.26 ± 0.042 ng/g wet weight) and appeared to increase with whale age. Our results provide a measure of blubber cortisol from a prolonged stress and demonstrate blubber cortisol as a useful indicator of longer-term exposure to stress in beluga whales.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Human exposure to mycotoxins in Egypt

This investigation examined the exposure of Egyptian infants to Aflatoxin M₁ (AfM₁) and of lactating mothers to Aflatoxin B₁, using AfM₁ in human milk as a biomarker for exposure to AfB₁. The presence of ochratoxin A (OA) in human milk was also investigated to determine the levels of infants exposure to OA from human milk. The results indicated that AfM₁ was found in 66 (55 %) of 120 human milk samples with a mean of 0.3 ± 0.53 ng/mL (range 0.02 to 2.09 ng/mL). OA was found in 43 (35.8 %) of 120 human milk samples with a mean of 21.1 ± 13.7 ng/mL (range 5.07 to 45.01 ng/mL), which will cause a daily intake of OA from human milk exceeding the suggested tolerable dose of 5 ng/kg_-1 of OA body weight. On the other side AfM₁ was found in 25 % of blood samples (5 out of 20 samples), at a mean of 1.18 ng/mL, but it was detected only in one urine sample (1 out of 20 samples). OA was detected only in 2 out of 13 blood samples (15.4 %) with an average 3.67 ng/mL. Whereas OA was not detected in all analyzed urine samples.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.     

Serum histones as biomarkers of the severity of heatstroke in dogs

Heatstroke is associated with systemic inflammatory response syndrome, leading to multiple organ dysfunction and death. Currently, there is no specific treatment decreasing hyperthermia-induced inflammatory/hemostatic derangements. Emerging studies indicate that histones leaking from damaged cells into the extracellular space are toxic, pro-inflammatory, and pro-thrombotic. We therefore hypothesize that serum histones (sHs) are elevated during heatstroke and are associated with the severity of the disease. Sixteen dogs with heatstroke and seven healthy controls were included in the study. Median serum histones (sHs) upon admission in dogs with heatstroke were significantly higher (P = 0.043) compared to that in seven controls (13.2 vs. 7.3 ng/mL, respectively). sHs level was significantly higher among non-survivors and among dogs with severe hemostatic derangement (P = 0.049, median 21.4 ng/mL vs. median 8.16 ng/mL and P = 0.038, 19.0 vs. 7.0 ng/mL, respectively). There were significant positive correlation between sHs and urea (r = 0.8, P = 0.02); total CO2 (r = 0.661, P = 0.05); CK (r = 0.678, P = 0.04); and prothrombin time (PT) 12 h post presentation (r = 0.888, P = 0.04). The significant positive correlation between sHs and other heatstroke severity biomarkers, and significant increase among severely affected dogs, implies its role in inflammation/oxidation/coagulation during heatstroke. sHs, unlike other prognostic and severity biomarkers in heatstroke, can be pharmacologically manipulated, offering a potential therapeutic target.     

Hapten Synthesis and Monoclonal Antibody-Based Immunoassay Development for the Analysis of Thidiazuron

Thidiazuron (TDZ) is a synthetic plant growth regulator, which is widely used in agriculture and tissue culture. A sensitive and specific monoclonal antibody (mAb3D6F5B2) was obtained. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb3D6F5B2. The established icELISA possessed a 50 % inhibition concentration (IC₅₀) of 0.61 ng/mL and a quantitative range of 0.15–2.49 ng/mL. The recoveries of TDZ fortified in water and soil samples were 76–126 %. The residual level of TDZ in soil samples detected by icELISA was confirmed with high performance liquid chromatography-mass spectrometry (LC/MS). The correlation coefficient (R ²) between the two assays was 0.9898, demonstrating this icELISA is suitable to detect TDZ residues in environmental and agricultural samples.