Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions.

Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs.

Giardia duodenalis Colonization Slightly Affects Gut Microbiota and Hematological Parameters in Clinically Healthy Dogs.

Giardia duodenalis (Giardia) is a worldwide cause of acute diarrheal disease both in humans and animals. The primary aim of this study was to investigate possible variations in gut microbiota in a population of asymptomatic dogs (n = 31), naturally infected or not by Giardia. Gut microbiota and the hematological, biochemical, and fecal parameters related to intestinal function were investigated. Giardia infection was associated with a significant shift of beta diversity, showing a relevant reduction of Gammaproteobacteria and an increase of Fusobacteria in male-positive dogs if compared with negatives. A significant imbalance of different bacterial taxa, with particular reference to the Erysipelotrichales, Lactobacillales, Clostridiales, and Burkholderiales orders, was observed, with the first two being higher in Giardia-positive dogs. Giardia-positive males displayed significantly higher values of cCRP than negative males as well as positive females, supporting the presence of a pro-inflammatory state. Taken together, these results indicate that the presence of Giardia does not substantially modify the microbial ecology of the intestine nor the hematological markers of disease. Thus treatments against Giardia should be considered with caution in asymptomatic subjects.

How Do Avalanche Dogs (and Their Handlers) Cope with Physical Exercise? Heart Rate Changes during Endurance in a Snowy Environment.

This study aimed to assess the heart rate (HR) responses of avalanche SAR dogs and handlers under working field conditions. Thirteen SAR units (dogs and handlers) performed an exercise (Endurance) consisting of approximately 5.5 km of rough tracks through deep snow, at an altitude of 1991-2250 m.a.s.l. The exercise was repeated twice for each of the two different tracks. Both handlers and dogs were equipped with a global positioning satellite/heart rate (GPS/HR) system (Polar®). Multivariable models were used to evaluate the effects of environmental (i.e., gradient, altitude, track, and time) and intrinsic (i.e., speed, repetition, and breed) factors on changes from baseline HR (Δ%HR). The dog's Δ%HR was greater in the flat and uphill compared with downhill, and increased progressively as the speed increased (p < 0.001). Moreover, it rose at altitudes above 2100 m.a.s.l. and peaked after 30 min of the Endurance activity (p < 0.01). These findings indicated that HR monitors could be a valuable tool to contribute to the evaluation of avalanche dogs' fitness in their real working environment. In contrast, the lack of correlation between the dogs' and handlers' HR changes suggests that handlers might not perceive the physical conditions of their dog in real-time. Thus, implementing protocols to monitor avalanche SAR dogs' fitness using a GPS/HR monitoring system could help handlers to tailor the training and workload and to detect the risk factors for physical distress of working dogs.

Extracellular circulating miRNAs as stress-related signature to search and rescue dogs.

Our research explores serum extracellular circulating miRNAs (ecmiRNAs) involved in dog stress response immediately after the search and rescue (SAR) of missing people. The experimental plan considers four arduous SAR simulations. The SAR dogs are trained by the Alpine School of the Military Force of Guardia di Finanza (Passo Rolle, Italy). The First SAR Trial analyzed dog serum samples at rest time (T0), and immediately after SAR performance (T1) using the miRNome-wide screening next-generation sequencing (NGS). T1 versus T0 NGS results revealed a different expression level of let-7a and let-7f. Subsequently, in a large sample size including: 1st (n = 6), 2nd (n = 6), 3rd (n = 6), and 4th (n = 4) trials, let-7a and let-7f were validated by qPCR. Bioinformatics analysis with TarBase (v.8) and the Diana-mirPath (v.3) revealed a functional role of let-7a and let-7f in the p53 pathway to restore cellular homeostasis. Let-7a and let-7f, highly expressed at T1, could stop MDMs-p53 inhibition inducing the p53 increase in level. In addition, let-7a and let-7f, via p53 post-transcriptional regulation, buffers p53 transcription spikes. During SAR stress, the possibility of p53 preconditioning could explain the phenomenon of "stress hardening" where the tolerance of particular stress increases after preconditioning.

A simulated avalanche search and rescue mission induces temporary physiological and behavioural changes in military dogs.

Saving human lives is of paramount importance in avalanche rescue missions. Avalanche military dogs represent an invaluable resource in these operations. However, their performance can be influenced by several environmental, social and transport challenges. If too severe, these are likely to activate a range of responses to stress, which might put at risk the dogs' welfare. The aim of this study was to assess the physiological and behavioural responses of a group of military dogs to a Simulated Avalanche Search and Rescue mission (SASR). Seventeen avalanche dogs from the Italian Military Force Guardia di Finanza (SAGF dogs) were monitored during a simulated search for a buried operator in an artificial avalanche area (SASR). Heart rate (HR), body temperature (RBT) and blood samples were collected at rest the day before the trial (T0), immediately after helicopter transport at the onset of the SASR (T1), after the discovery of the buried operator (T2) and 2h later (T3). Heart rate (HR), rectal body temperature (RBT), cortisol, aspartate aminotransferase (AST), creatine kinase (CK), non-esterified fatty acids (NEFA) and lactate dehydrogenase (LDH) were measured. During the search mission the behaviour of each SAGF dog was measured by focal animal sampling and qualitatively assessed by its handler and two observers. Inter-rater agreement was evaluated. Snow and environmental variables were also measured. All dogs successfully completed their search for the buried, simulated victim within 10min. The SASR was shown to exert significant increases on RBT, NEFA and cortisol (P<0.001), CK and HR (P<0.01), AST and LDH (P<0.05). These indicate the activation of a response to stress probably induced by the addition of factors such as helicopter transport, disembarking, and the search and rescue exercise. However, changes were moderate and limited over time, progressively decreasing with complete recovery at T3 except for sera cortisol that showed a slightly slower decline. More time walking within the search was related to lower RBT, conversely to walking. Standing still with head up and exploring with head-up were inversely related with HR. Agreement between handler and observers' opinions on a dog's search mission ability was found only for motivation, signalling behaviour, signs of stress and possessive reward playing. More time signalling was related to shorter search time. In conclusion, despite extreme environmental and training conditions only temporary physiological and behavioural changes were recorded in the avalanche dogs. Their excellent performance in successful simulated SASR may be attributable to extensive training and good dog-handler relationships. Simulated SASR did not seem to impair SAGF dogs' performance or welfare.