Ricardo Brandão

Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

ABSTRACT Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

Assessment of the exposure to heavy metals in Griffon vultures (Gyps fulvus) from the Iberian Peninsula

Griffon vultures (Gyps fulvus), by virtue of their position at the top of the food chain and as obligate scavengers, are at risk of accumulating and concentrating heavy metals in their tissues and may be more predisposed to their toxic effects. The aim of this study is to investigate heavy metal concentrations in Griffon vultures in Portugal and Catalonia, Spain and to determine if heavy metal concentrations in the blood of weak and/or injured Griffon vultures admitted to wildlife rehabilitation centres (WRC) reflect contamination profiles in the local, free-living and outwardly healthy population. Whole-blood samples taken from 121 Griffon vultures caught in the wild or admitted to WRC in Portugal and Catalonia, Spain were examined for cadmium (Cd), mercury (Hg) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS). Cd and Hg were not detected in most samples (98.3% and 95%, respectively), while Pb was detected in all birds in concentrations ranging between 4.97 and 300.23 µg/dl. Birds admitted to WRC had significantly lower Pb concentrations (24.15 ± 15.07 and 25.98 ± 18.04 µg/dl in Portugal and Catalonia, Spain, respectively) than animals caught in the wild (29.67 ± 13.19 and 42.22 ± 50.08 µg/dl in Portugal and Catalonia, Spain, respectively) (p<0.05). This may be explained by the fact that malnutrition was the main cause of admission of Griffon vultures to WRC, as ingestion has been described as the most significant pathway for Pb exposure in raptors. Therefore Griffon vultures admitted to WRC do not seem to be representative of the local, free-flying populations, so it remains necessary to continue catching when one intends to monitor Pb exposure in this species. The population of vultures captured in Catalonia, Spain showed the highest mean blood Pb concentration, perhaps due to the municipal rubbish dump located near the feeding station, with rubbish providing a significant fraction of their trophic needs. The ingestion of game meat with bullet fragments in carcasses or with Pb shots embedded in their flesh could also be the cause of the high blood Pb concentrations found in some vultures. The potential risk of Pb exposure in Griffon vulture populations must be given consideration, since most individuals evaluated had Pb concentrations between 20 and 100µg/dl, which is considered to be subclinical exposure to Pb, and which is above the threshold level at which Pb can affect antioxidant system in this species (15 µg/dl).

Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from Iberian wolves (Canis lupus signatus) in Portugal

While much evidence supports the view that the total consumption of antimicrobials is the critical factor in selecting resistance, the possibility of resistant isolates and/or genes encoding resistance being transferred among different living communities has raised serious concerns. In the present study, Escherichia coli isolates recovered from faecal samples (n = 34) of Iberian wolves (Canis lupus signatus) were characterized for their antimicrobial drug susceptibility. Nearly two thirds of the isolates carried resistance to one or more antimicrobial drugs (in a panel of 19 antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. By screening a set of 20 multidrug-resistant E. coli for virulence genes, we found strains positive for cdt, chuA, cvaC, eaeA, paa and bfpA, which was the most common virulence trait. Phylogenetic analyses have shown that the majority of these E. coli strains fall into phylogenetic groups A and B1. In this study, the diversity of extended-spectrum β-lactamase-producing strains was expressed by both polymorphism of the pulsed-field gel electrophoresis patterns and the presence of various resistance and virulence genes profiles. Finding the specific implications of these multi-resistant bacteria (hosting several virulence factors) in wolf conservation is a challenging topic to be addressed in further investigations.

Hematology and serum biochemistry values of free-ranging Iberian wolves (Canis lupus) trapped by leg-hold snares

Hematology and serum biochemistry are important tools in assessing the health and physiological status of wildlife populations. Nevertheless, studies on free-ranging wolves (Canis lupus) are scarce, and no reference values are available neither for Iberian wolves nor for wolves captured with leg-hold snares. We report 37 hematology and serum biochemistry variables obtained from 26 free-ranging Iberian wolves captured with leg-hold snares between 2007 and 2014, including variables previously not reported in the literature. The values obtained are similar to the published reference intervals for Scandinavian wolves captured by darting from a helicopter, except for higher values for mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), leukocyte count, creatinine kinase (CK), γ-globulins, and total bilirubin (TBIL) and lower values for alkaline phosphatase (ALP). We propose that differences in leukocyte count, CK, and TBIL are related to the method of capture, while differences in RDW, MCHC, ALP, and γ-globulins could reflect physiological adaptations to environmental conditions, sampling, or pre-analytical artifacts. Lymphocyte count was lower and neutrophil/lymphocyte ratio was significantly higher in older, reproductive females, while ALP and phosphorus were higher in juvenile wolves. For the first time, we describe hematology and serum biochemistry values of free-ranging Iberian wolves captured with leg-hold snares. The data reported here is the first published reference for wolves captured with similar methods and for monitoring Iberian wolves populations’ physiological and health status.

Characterization and minimization of the stress response to trapping in free-ranging wolves (Canis lupus): insights from physiology and behavior

Abstract Objectives: Wildlife capture is an essential management tool that induces a reactive homeostasis response in the captured animals. The aim of this study was to characterize the reactive homeostatic response to trapping in free-ranging wolves and assess the mitigation achieved by reducing the duration of restraint. Materials and methods: Making use of wolves captured for ecological research as a model for wildlife acute stress, we characterize 25 reactive homeostasis mediators and we assess the effect on these mediators of reducing the duration of restraint in trap by using remote trap activation alarms. Results: Free-ranging wolves trapped by leg-hold snares (n = 15) showed higher stress leukogram, tissue injury and hematocrit; while lower glucose, ions and cardiac rate compared with captive wolves. They also showed higher leukocyte count and creatine kinase; but lower hematocrit, cardiac rate and rectal body temperature compared to wolves captured by darting from a helicopter. Daily distance travelled was significantly lower up to day 12 post-capture compared to the remainder of the telemetry follow-up and this effect was more noticeable on the nocturnal distance travelled. Reducing the duration of restraint on trap significantly lowered the stress leukogram and dehydration. Daily distance travelled during the night by wolves captured using trap-alarms was significantly lower only up to day 4 post-capture compared to up to day 28 for wolves captured without trap-alarms. Discussion: The capture method and duration of restraint influence the reactive homeostasis response of free-ranging wolves. Technological solutions that reduce the duration of restraint on trap significantly dampen this influence. Conclusions: Wildlife trapping actions should strive to minimize the delay from capture to manipulation.

MULTIDRUG RESISTANCE IN WILD BIRD POPULATIONS: IMPORTANCE OF THE FOOD CHAIN

Abstract:  The presence of multidrug-resistant (MDR) Escherichia coli has recently been reported in wild birds (gulls and birds of prey) that had no apparent exposure to antimicrobials. Little work has been done to assess the role of the food chain in the emergence and spread of MDR E. coli. In this study, we evaluated the presence of MDR E. coli in 29 fecal samples collected from wild birds living in a rehabilitation center (the center receives injured animals found in their natural habitat) and in eight feed samples. In total, 166 E. coli isolates were obtained: 129 from cloacal swabs and 37 from raw feed samples. The antimicrobial resistance profile of these isolates was determined, and we found that 75 isolates showed resistance to five or more drugs, resulting in a total of 38 different antimicrobial resistance patterns. Subsequently, the molecular characterization of 36 isolates, performed by pulsed-field gel electrophoresis, revealed a great similarity between isolates collected from various species of birds and also between these last ones and the ones found in their feed samples.

Assessment of DNA damage in Ardea cinerea and Ciconia ciconia: A 5-year study in Portuguese birds retrieved for rehabilitation.

Over the past decades, the presence of micronucleated blood cells has been used to detect genotoxic effects of xenobiotics in fish, amphibians and birds. This study assessed the frequency of micronuclei (MN) and other nuclear abnormalities in erythrocytes of individuals of Ardea cinerea and Ciconia ciconia retrieved for rehabilitation in order to evaluate the influence of age, temporal and spatial factors on the occurrence of DNA damage in Portuguese wild birds. Blood smears from 65 birds with different life-history backgrounds (e.g. geographic origin, age) were collected between 2007 and 2011 and the frequency of erythrocyte nuclear abnormalities (ENAs) was analysed. Differences in DNA damage between ages were observed to occur in C. ciconia, with chicks displaying significantly higher frequencies of ENAs (both when looking at total ENAs or only MN frequency) than juveniles and adults. Additionally, significant differences in ENAs frequencies were observed between different years and geographic origins, whereas MN frequency alone did not show significant alterations concerning spatial and temporal variations. These results suggest that the assessment of ENAs rather than MN frequency alone may be a useful and valuable tool to complement the evaluation of DNA damage in populations of birds, as prompted by individual life-history traits and environmental factors.

Treatment of a forelimb fracture and rehabilitation of a free-ranging Iberian Wolf (Canis lupus signatus)

The surgical treatment of an exposed compounded comminuted fracture of the right radius and ulna in a free-ranging adult female Iberian Wolf (Canis lupus signatus) with an osteosynthesis plate and screws and subsequent post-operative care are described. The evolution of the fracture healing was very similar to those expected in a dog of the same size. The prompt surgical intervention and a proper housing, feeding and wound management adapted to a free-ranging wolf, in view to reduce manipulation and post-operative complications, allowed the subsequent rehabilitation and release of the animal. After 10th post-operative weeks the wolf was fitted with a Global Positioning System (GPS) for wildlife tracking collar and released in the same area where it has been caught. GPS telemetry data showed that the animal covered increasingly large distances confirming a complete functionality of the right thoracic limb and its successfully return to the wild. This report could constitute the first detailed report of a long bone fracture treatment in a free-ranging wolf and its successfully rehabilitation, release and adaptation to the wild.