Evidence of orthohantavirus and leptospira infections in small mammals in an endemic area of Gampaha district in Sri Lanka.

BACKGROUND: Orthohantaviruses and leptospira are emerging zoonotic pathogens of high public health significance. The epidemiology of orthohantavirus infections and leptospirosis is similar and presents related clinical pictures in humans. However, a paucity of data on actual reservoir hosts for orthohantaviruses and leptospira exists. Therefore, this study aimed at determining the occurrence of orthohantaviruses and leptospira in small mammals captured in an endemic region of Sri Lanka. METHODS: Rodents and shrews were morphologically and/or genetically identified using morphological keys and DNA barcoding techniques targeting the cytochrome oxidase b subunit gene (Cytb). Lung tissues and sera were subsequently analyzed for the presence of orthohantavirus RNA using qRT-PCR. Sera of rats were tested for IgG antibodies against orthohantaviruses and leptospira. RESULTS: Forty-three (43) small mammals representing: Rattus (R.) rattus (black rat) or R. tanezumi (Asian rat), Suncus murinus (Asian house shrew), R. norvegicus (brown rat) and Mus musculus (house mouse) were investigated. No orthohantavirus RNA was detected from the lung tissue or serum samples of these animals. Elevated levels of IgG antibodies against Puumala orthohantavirus (PUUV) and/or Seoul orthohantavirus (SEOV) antigens were detected in sera of 28 (72%) out of the 39 rats analysed. Interestingly, 36 (92%) of the 39 rats also showed presence of anti leptospira-IgG antibodies in their serum, representing dual infection or dual exposure in 26/39 (66.7%) of examined rats. CONCLUSIONS: This project targets important public health questions concerning the occupational risk of orthohantavirus infections and/or leptospirosis in an endemic region of Sri Lanka. Most rats (72%) in our study displayed antibodies reacting to orthohantavirus NP antigens, related to PUUV and/or SEOV. No correlation between the orthohantavirus and leptospira IgG antibody levels were noticed. Finally, a combination of both morphological and DNA barcoding approaches revealed that several species of rats may play a role in the maintenance and transmission of orthohantavirus and leptospira in Sri Lanka.

Intranasal oxygen reverses hypoxaemia in immobilised free-ranging capybaras (Hydrochoerus hydrochaeris).

Capybara (Hydrochoerus hydrochaeris) is the main host of tick-borne pathogens causing Brazilian spotted fever; therefore, controlling its population is essential, and this may require chemical restraint. We assessed the impact of chemical restraint protocols on the partial pressure of arterial oxygen (PaO2) and other blood variables in 36 capybaras and the effect of different flows of nasal oxygen (O2) supplementation. The capybaras were hand-injected with dexmedetomidine (5 µg/kg) and midazolam (0.1 mg/kg) and butorphanol (0.2 mg/kg) (DMB, n = 18) or methadone (0.1 mg/kg) (DMM, n = 18). One-third of the animals were maintained in ambient air throughout the procedure, and one-third were administered intranasal 2 L/min O2 after 30 min whereas the other third were administered 5 L/min O2. Arterial blood gases, acid-base status, and electrolytes were assessed 30 and 60 min after drug injection. The DMB and DMM groups did not vary based on any of the evaluated variables. All animals developed hypoxaemia (PaO2 44 [30; 73] mmHg, SaO2 81 [62; 93] %) 30 min before O2 supplementation. Intranasal O2 at 2 L/min improved PaO2 (63 [49; 97] mmHg and SaO2 [92 [85; 98] %), but 9 of 12 capybaras remained hypoxaemic. A higher O2 flow of 5 L/min was efficient in treating hypoxaemia (PaO2 188 [146; 414] mmHg, SaO2 100 [99; 100] %) in all the 12 animals that received it. Both drug protocols induced hypoxaemia, which could be treated with intranasal oxygen supplementation.

Measurement of catestatin and vasostatin in wild boar Sus scrofa captured in a corral trap.

OBJECTIVE: Our aim was to analyse the chromogranin A-derived peptides vasostatin and catestatin in serum from wild boar (Sus scrofa) captured in a corral trap. Acute capture-related stress quickly leads to a release of adrenalin and noradrenalin, but these hormones have a short half-life in blood and are difficult to measure. Chromogranin A (CgA), a glycoprotein which is co-released with noradrenalin and adrenalin, is relatively stable in circulation and the CgA-derived peptides catestatin and vasostatin have been measured in domestic species, but not yet in wildlife. RESULTS: Vasostatin and catestatin could be measured and the median (range) serum concentrations were 0.91 (0.54-2.86) and 0.65 (0.35-2.62) nmol/L, respectively. We conclude that the CgA-derived peptides vasostatin and catestatin can be measured in wild boar serum and may thus be useful as biomarkers of psychophysical stress.

STANDING SEDATION WITH XYLAZINE AND REVERSAL WITH YOHIMBINE IN JUVENILE ASIAN ELEPHANTS (ELEPHAS MAXIMUS).

Evaluation and improvement of immobilization methods are important for wildlife welfare and biodiversity conservation. The sedative and physiological effects of xylazine (50-110 mg per elephant; 0.09-0.15 mg/kg IM) were evaluated in 15 juvenile Asian elephants (Elephas maximus) in Sri Lanka. The time from xylazine injection until first sign of sedation, handling, and reversal with yohimbine (0.009-0.03 mg/kg IV) were recorded. Behavioral signs, level of sedation (no effect, light, moderate, or deep) and response to handling were assessed. Rectal temperature, pulse, and respiratory rates were recorded and arterial blood samples were analyzed 30 and 45 min after xylazine injection. The first sign of sedation occurred within 5-18 min. Standing sedation was induced in all elephants, but the level of sedation varied differently over time for each elephant. Twelve elephants remained standing throughout the sedation period, while 3 elephants became laterally recumbent. Sedative effects included lowered head and trunk, droopy ears, snoring, and penis protrusion. Pulse rate, respiratory rate, and rectal temperature ranged between 30-45 beats/min, 4-12 breaths/min, and 35.6-37.2°C, respectively, at 30 min after xylazine injection, and there were no changes over time. Pulmonary function and acid-base balance were adequate (range partial pressures of arterial oxygen 73-123 mmHg and carbon dioxide 33-52 mmHg, arterial hemoglobin oxygen saturation 96-99%, pH 7.34-7.54, lactate 0.9-2.5 mmol/L). Yohimbine was administered 46-110 min after the injection of xylazine, and the first sign of recovery occurred within 1-4 min. Resedation after reversal with yohimbine was observed in two elephants. In conclusion, xylazine at the doses used induced light to deep sedation with stable physiology and most elephants remained standing.

Wild boar behaviour during live-trap capture in a corral-style trap: implications for animal welfare.

BACKGROUND: Wildlife traps are used in many countries without evaluation of their effect on animal welfare. Trap-capture of wild animals should minimise negative effects on animal welfare, irrespective of whether the animals are trapped for hunting, research, or management purposes. Live-trap capture of wild boar (Sus scrofa) followed by killing inside the trap by gunshot is a recently introduced but disputed hunting method in Sweden. Approval of trap constructions is based on gross necropsy findings of 20 trapped and shot wild boars. For improved animal welfare evaluation, our aim was to study wild boar behaviour during live-trapping in a 16 m2 square corral-style trap. Behavioural assessments were conducted after filming 12 capture events of in total 38 wild boars (five adults, 20 subadults, 13 piglets). Selected behavioural traits were compared with pathological changes (trap-related lesions) found at necropsy of the 20 subadults, to determine if these variables were useful proxies of capture-induced stress in wild boar. RESULTS: The wild boars spent less time resting in the evening than in the night and morning. Using Friedman's ANOVA, there was an overall difference in the time spent foraging. However, we only found a difference between the evening and morning in the Wilcoxon matched pairs test after the Sequential Bonferroni correction, where the wild boars spent more time foraging in the evening than in the morning. Single captured individuals showed more escape behaviours and reacted more strongly to external stimuli than individuals captured in a group. It was more common for animals to charge against the mesh walls of the trap upon human approach compared to upon initial capture when the trap door closed. Trap-related pathological findings due to trauma were documented in 13 of the 20 subadults that were necropsied. Behavioural alterations indicative of capture-induced stress (e.g. charging into the trap walls) were documented in trapped wild boars with no or minor physical injuries (e.g. skin abrasions, subcutaneous haemorrhage). CONCLUSIONS: Behavioural assessment provided valuable information for determination of capture-induced stress in wild boar when evaluating live-trapping in a corral-style trap, whereas pathological evaluation through necropsy did not fully reflect the animal welfare aspects of live-trapping. We emphasize the inclusion of species-specific behavioural data assessment for evaluation of capture-related stress during live-trapping and for testing of new trap constructions before approval.

Detection, Identification, and Antimicrobial Susceptibility of Campylobacter spp. and Salmonella spp. from Free-ranging Nonhuman Primates in Sri Lanka.

Infections with Campylobacter spp. and Salmonella spp. are the most frequently reported causes of human bacterial enteritis. Warm-blooded animals, including livestock, pets, and wildlife, can be carriers of the bacteria and may contaminate the environment and food products. The present study investigated the occurrence of Campylobacter spp. and Salmonella spp. in fecal pat samples from free-ranging toque macaques (Macaca sinica) and tufted gray langurs (Semnopithecus priam) collected in March-May 2015 in Sri Lanka. In 58 samples from toque macaques, Campylobacter jejuni was isolated in 10 (17%), Campylobacter coli in four (7%), and Salmonella enterica subsp. enterica serovar Virchow in two (3%). None of the bacteria were isolated in the 40 samples from tufted gray langurs. Pulse-field gel electrophoresis and multilocus sequence typing identified six profiles and four clonal complexes of C. jejuni. The isolated Campylobacter spp. showed varying susceptibility to antimicrobial substances. All Campylobacter spp. isolates were susceptible to chloramphenicol, erythromycin, florfenicol, gentamicin, and streptomycin. Four of the C. jejuni were resistant to at least one of the following: ampicillin, ciprofloxacin, nalidixic acid, and tetracycline, and one of the isolates was multidrug resistant. All four C. coli were resistant to ampicillin, whereas the two Salmonella Virchow strains were susceptible to all antibiotics tested. The presence of Campylobacter spp. and Salmonella spp. in toque macaques may have an impact on the conservation of endangered primates and public health in Sri Lanka.

COMPARISON OF THE EFFICACY AND SAFETY OF MEDETOMIDINE-KETAMINE VERSUS MEDETOMIDINE-AZAPERONE-ALFAXALONE COMBINATION IN FREE-RANGING ROCKY MOUNTAIN BIGHORN SHEEP ( OVIS CANADENSIS).

Chemical immobilization is a key aspect of wildlife management. To minimize dose-dependent adverse effects, immobilization protocols often include two or more synergistic agents, which allows for reductions in individual drug dosages. Free-ranging bighorn sheep ( Ovis canadensis) in Canada ( n = 74) were remotely injected with a combination of medetomidine (0.16 ± 0.04 mg/kg) and ketamine (4.0 ± 1.4 mg/kg) (MK), or combination of medetomidine (0.14 ± 0.06 mg/kg), azaperone (0.21 ± 0.11 mg/kg), and alfaxalone (0.45 ± 0.21 mg/kg) (MAA). Once recumbency was achieved, arterial blood samples were collected and immediately analyzed for blood gas and acid-base status. Rectal temperature, heart rate, and respiratory rate were recorded upon recumbency and throughout anesthesia at 5-15 min intervals. At conclusion of the procedures, medetomidine was reversed by intramuscular atipamezole at five times the medetomidine dose. Induction times (mean ± standard deviation) of animals that became immobilized with one dart (8.7 ± 3.2 min, 7.3 ± 3.9 min) and recovery times of all animals (3.4 ± 1.5 min, 3.9 ± 1.6 min) were not significantly different between MK and MAA groups, respectively. Both MK and MAA groups experienced severe hypoxemia (PaO2 42 ± 9 mmHg, 40 ± 10 mmHg, respectively). PaCO2 was significantly higher ( P = 0.0248) in the MK group (median 54 mmHg) than the MAA group (median 48 mmHg) with a trend towards lower pH (7.40 vs 7.42, respectively, P = 0.07). Initially, MK animals had higher heart rates than MAA animals (median 49 vs 40 beats/min), which decreased over time. In bighorn sheep, both MK and MAA produced reliable, reversible immobilization with smooth inductions and recoveries. However, less respiratory depression was seen with MAA than MK.

Discovery of herpesviruses in Canadian wildlife.

Herpesviruses (HVs) have a wide range of hosts in the animal kingdom. The result of infection with HVs can vary from asymptomatic to fatal diseases depending on subtype, strain, and host. To date, little is known about HVs naturally circulating in wildlife species and the impact of these viruses on other species. In our study, we used genetic and comparative approaches to increase our understanding of circulating HVs in Canadian wildlife. Using nested polymerase chain reaction targeting a conserved region of the HV DNA polymerase gene, we analyzed material derived from wildlife of western and northern Canada collected between February 2009 and Sept 2014. For classification of new virus sequences, we compared our viral sequences with published sequences in GenBank to identify conserved residues and motifs that are unique to each subfamily, alongside phylogenetic analysis. All alphaherpesviruses shared a conserved tryptophan (W856) and tyrosine (Y880), betaherpesviruses all shared a serine (S836), and gammaherpesviruses had a conserved glutamic acid (E835). Most of our wildlife HV sequences grouped together with HVs from taxonomically related host species. From Martes americana, we detected previously uncharacterized alpha- and beta-herpesviruses.

ANIMAL WELFARE FROM MOUSE TO MOOSE--IMPLEMENTING THE PRINCIPLES OF THE 3RS IN WILDLIFE RESEARCH.

The concept of the 3Rs (replacement, reduction, and refinement) was originally developed for improving laboratory animal welfare and is well known in biomedical and toxicologic research. The 3Rs have so far gained little attention in wildlife research, and there could be several reasons for this. First, researchers may prioritize the welfare of populations and ecosystems over the welfare of individual animals. The effects of research on individual animals can, however, impact welfare and research quality at group and population levels. Second, researchers may find it difficult to apply the 3Rs to studies of free-living wildlife because of the differences between laboratory and wild animals, species, research environment, and purpose and design of the studies. There are, however, several areas where it is possible to transfer the 3R principles to wildlife research, including replacement with noninvasive research techniques, reduction with optimized experimental design, and refinement with better methods of capture, anesthesia, and handling. Third, researchers may not have been trained in applying the 3Rs in wildlife research. This training is needed since ethics committees, employers, journal publishers, and funding agencies increasingly require researchers to consider the welfare implications of their research. In this paper, we compare the principles of the 3Rs in various research areas to better understand the possibilities and challenges of the 3Rs in wildlife research. We emphasize the importance of applying the 3Rs systematically throughout the research process. Based on experiences from laboratory research, we suggest three key factors to enhance implementation of the 3Rs in wildlife research: 1) organizational structure and management, 2) 3R awareness, and 3) research innovation, validation, and implementation. Finally, we encourage an interdisciplinary approach to incorporate the 3R principles in wildlife research. For improved animal welfare and increased research quality, researchers have moral obligations to include the 3Rs into all research areas, including wildlife research.

Pulmonary gas exchange and acid-base status during immobilisation of black rhinoceroses (Diceros bicornis) in Zimbabwe.

When immobilising wildlife, adverse side effects can include hypoxaemia, acidosis and hypertension. Pulmonary gas exchange and acid-base status were evaluated during immobilisation of 25 free-ranging and one boma-held black rhinoceros (Diceros bicornis) in Zimbabwe. The effect of different body positions on arterial oxygenation was evaluated. A combination of the following drugs was used: an opioid (etorphine or thiafentanil), azaperone and an a2 -adrenoceptor agonist (detomidine or xylazine). Respiratory and heart rates, rectal temperature and pulse oximetry-derived haemoglobin oxygen saturation were recorded. Serial arterial blood samples were analysed immediately in the field. Marked hypoxaemia and hypercapnia were recorded in immobilised free-ranging black rhinoceroses. Arterial oxygenation was higher during sternal compared to lateral recumbency. Most rhinoceroses developed acidaemia of respiratory and metabolic origin. Initially high lactate concentrations in free-ranging rhinoceroses decreased during immobilisation. Pulse oximetry was unreliable in the detection of hypoxaemia. Positioning in sternal recumbency and routine use of oxygen supplementation are recommended in the management of immobilised rhinoceroses as measures to improve arterial oxygenation.