While biosecurity, a central component of the One Health concept, is clearly defined, a harmonized definition of the term ´biosecurity measure´ (BSM) is missing. In turn, particularly at the farm and policy level, this leads to misunderstandings, low acceptance, poor implementation, and thus suboptimal biosecurity along the food animal production chain. Moreover, different views on BSMs affects making comparisons both at the policy level as well as in the scientific community. Therefore, as part of the One Health EJP BIOPIGEE project, a work group i) collected and discussed relevant inclusion and exclusion criteria for measures to be considered in the context of biosecurity and ii) conducted a systematic literature review for potentially existing definitions for the term BSM. This exercise confirmed the lack of a definition of BSM, underlining the importance of the topic. In the pool of articles considered relevant to defining the term BSM, specific research themes were identified. Based on these outcomes, we propose a definition of the term BSM:

Wildlife management, conservation interventions and wildlife research programs often involve capture, manipulation and transport of wild animals. Widespread empirical evidence across various vertebrate taxa shows that handling wildlife generally induces a severe stress response resulting in increased stress levels. The inability of individuals to appropriately respond to rapidly changing environmental conditions during and after manipulations may have deleterious and long-lasting implications on animal welfare. Therefore, mitigating stress responses in the frame of conservation interventions is a key animal welfare factor. However, we have a poor understanding of the metrics to adequately assess and monitor the dynamic physiological changes that animals undergo when subjected to stressful procedures in wild or captive conditions. A growing number of studies provide good evidence for reciprocal interactions between immune processes and stress. Here, we review the existing literature on a relatively new technique—Leukocyte Coping Capacity (LCC), a proxy for stress quantifying oxygen radical production by leukocytes. We discuss the strength and weaknesses of this immunological approach to evaluate stress, the individual capacity to cope with stress and the resulting potential implications for animal welfare. Additionally we present new data on LCC in captive roe deer (Capreolus capreolus) under long-time anesthesia and free-ranging Asiatic wild asses (Kulan; Equus hemionus kulan) were LCC was used to assess stress levels in animals captured for a reintroduction project.

To understand and reduce the concomitant effects of trapping and handling procedures in wildlife species, it is essential to measure their physiological impact. Here, we examined individual variation in stress levels in non-anesthetized European roe deer (Capreolus capreolus), which were captured in box traps and physically restrained for tagging, biometrics and bio-sampling. In winter 2013, we collected venous blood samples from 28 individuals during 28 capture events and evaluated standard measurements for stress (heart rate, body temperature, neutrophil to lymphocyte ratio, lactate and total cortisol). Additionally, we assessed stress using the immunological tool, Leukocyte Coping Capacity (LCC), a real-time proxy for stress measuring oxygen radical production by leukocytes. Finally, the behavioral response to handling was recorded using a scoring system. LCC and therefore stress levels were negatively influenced by the time animals spent in the box trap with human presence at the capture site prior to handling. In contrast, none of the classical stress measures, including total cortisol, nor the behavioral assessment, were correlated with the stressor tested (time of human presence prior to handling) and thus did not provide a clear depiction regarding the extent of the animals short-term stress response. Overall our study verifies the LCC as a strong method to quantify short-term stress reactions in wildlife. Moreover, our results clearly show that human presence at the trapping site prior to handling should be kept to an absolute minimum in order to reduce stress levels.

Hibernation is characterized by successive torpor bouts during which metabolic rate is down-regulated to 2–4% of euthermic levels along with core body temperatures (T_b) ranging between 0 and 10°C. One characteristic of the torpid state, which is periodically interrupted by a few hours of euthermic phases or arousals during hibernation, resides in an overall impairment of the immune system. The most striking change during torpor is the reduction of circulating white blood cells up to 90%, while their numbers rise to near summer euthermic level upon rewarming. However, potential changes in responsiveness and function of neutrophil granulocytes, accounting for the primary cellular innate immune defense, are unknown. Here we present the first data on shifts in oxidative burst capacity, i.e., the ability to produce reactive oxygen species (ROS), of neutrophils during hibernation. Using a chemiluminescence assay, we measured real-time ROS production in whole blood of hibernating garden dormice (Eliomys quercinus) in early or late torpor, and upon arousals. Accounting for changes in neutrophil numbers along the torpor-arousal cycle, we found significant differences, between torpid and euthermic states, in the neutrophil oxidative burst capacity (NOC), with shallow cell responses during torpor and a highly significant increase by up to 30-fold during arousals. Further, we observed a significant reduction of NOC from aroused animals with euthermic T_bof 36.95 ± 0.37°C, when tested at 6°C, whereas no change occurred in NOC from torpid individuals reaching constant T_bof 4.67 ± 0.42°C, when measured at 35°C. This dynamic indicates that the reduction in NOC during torpor may be temperature-compensated. These results linked to the understanding of immune function during the torpor-arousal cycle might have clinical relevance in the context of therapeutic hypothermia and reperfusion injury.

Despite the widely used application of standardized capture-handling protocols to collect blood and assess the physiological stress response, the actual sampling design (e.g., timing and the number of blood samples) often differs between studies, and the potential implications for the measured physiological endpoints remain understudied. We, therefore experimentally tested the effects of repeated handling and multiple blood sampling on the stress response in wintering free-living great tits (Parus major). We modified a well-established sampling protocol of avian studies by adding either an additional blood sample or a "sham-manipulation" (i.e., handling associated with the blood sampling procedure without venepuncture), to disentangle the effects of handling stress and blood loss. We combined three different stress metrics along the endocrine-immune interface to investigate the acute short-term stress response: total corticosterone concentrations (Cort), the heterophil/lymphocyte ratio (H:L), and the Leucocyte Coping Capacity (LCC). Our study provided three key results: i) no relationship between Cort levels, LCC and H:L, confirming that these three parameters represent different physiological endpoints within the stress response; ii) contrasting dynamics in response to stress by the measured parameters and iii) no difference in physiological stress levels 30 min after capture due to one additional blood sampling or handling event. By optimising the sampling design, our results provide implications for animal welfare and planning experimental procedures on stress physiology in passerine species.