Background

Despite increasing interest in the area, there are still surprisingly few reliable and resource-effective indicators of stress in dogs to monitor their welfare and wellbeing. Existing measures are time-consuming to collect, need relatively complex and costly equipment or can be ambiguous and difficult to interpret. Physiological measures have been criticized for being expensive whilst lacking stability and specificity. However, most concerning is their inability to differentiate between emotional states of contrasting valence (i.e. positive/negative) – a key requirement for effective welfare assessment. For example, increased cortisol/creatinine ratio might be observed when a dog experiences a new environment for the first time, this shows increased arousal. However, it does not reveal whether that experience was perceived as positive or negative by the dog. This traditional assessment normally requires the saliva samples being sent to a specialist laboratory for analysis, which is both costly and takes a turn-around time of around 2 to 5 days for results. There is therefore an urgent requirement for the identification of a physiological measure that is not subject to these limitations, being simple, quick, and cheap to record, and allowing differentiation between states of positive and negative valence.

Technology Overview

Dogs being carnivores do not normally have the starch (sugar) degrading enzyme amylase present at biologically useful levels in oral saliva. The inventors have produced data to evidence that dogs suffering from significant chronic stress have highly elevated amylase level in their saliva, but levels are only very mildly increased above basal levels with moderate acute stress.

The inventors have data comparing cortisol levels (the current gold standard) with amylase levels, and amylase correlates as a good indicator of severe stress unlike cortisol that is elevated by moderate and both positive and negative stresses.

Methodology and Potential Product:

The researcher’s proposed “stress” test will involve swabbing the mouth of the dog to take saliva sample for analysis. The amount of saliva sampled will be standardised. The swab will then be diluted, and a standardised fixed volume applied to a Lateral Flow Device (LFD). Presence of elevated amylase will be indicated by the presence of a positive line. Potentially the device could have two positive lines with two different sensitivities indicating moderate and high stress being detected.

Currently, no LFD is available or being marketed for the detection of dog amylase. Lateral flow kits do exist for the detection of human and fungal amylase, but these have low levels of cross-reactivity to dog amylase. The antibodies required for the detection of dog amylase have been validated in commercial ELISA kits, which would potentially allow transposing of these reagents to LFDs.

Stage of Development

The following opportunity of using amylase concent rations/level as a stress marker for dogs has been demonstrated to a post-proof-of-concept stage by the inventors and is currently at the Technology Readiness Level (TRL) 4 (i.e. technology/component of technology has been validated in a laboratory environment). To put in context a market ready product is at TRL 9.

Technical - Evidence for Amylase as a biomarker for negative arousal in dogs

• Dogs subjected to mild negative arousal conditions produced a significantly higher amylase concentration in the saliva than mild positive arousal, evidencing that amylase is a marker for negative arousal, unlike the current “gold standard” test using cortisol which increases under both negative and positive stress.
• Negative Arousal condition had a 5-fold higher amylase concentration (mean of 8.1ng/ml) in the saliva than mild negative arousal condition, suggesting that amylase may be a useful measure of negative arousal levels.
• Amylase levels directly correlated with behavioural measures of negative arousal and those of owner questionnaires (p≤0.05).
• These findings suggest that amylase is an excellent biomarker for negative arousal in dogs. This would potentially allow, for the first time, the rapid identification of negatively arousal in dogs in real-time, using rapid lateral flow testing to identify dogs with amylase levels outside the normal reference range.
• One of the potential limitations of the amylase biomarker is that a saliva sample has to be taken from the dogs mouth/lips. To overcome this potential limitation, the researchers have devised a method using tennis balls (as most dogs are happy to at least briefly hold, if not play with a tennis ball), and use this as the method with greater than 85% of efficiency for biomarker levels (both amylase and cortisol) with balls when compared to the direct mouth sample for the same dog (less than 15% sample loss), which the researchers think is rather amazing and well within acceptable sampling variation.
• The researchers have been able to calculate a normal reference range of salivary amylase in dogs. Under normal conditions they found the mean value to be 1.41ng/ml, with a standard deviation of 0.78ng/ml. This produces a normal non-stress reference range of 0 to 3.76ng/ml, and they propose provision cut-off levels of 0-3.9ng/ml as normal, and 4.0+ng/ml as negatively aroused.
• Analysis of baseline (non-aroused) dogs, found no significant variation in the amount amylase present in the saliva for common dogs breeds such as Labradors, spaniels, etc.
• Analysis of baseline (non-aroused) dogs, found no significant variation in the amount amylase present in the saliva for age (dog studied ranged from 1 year to 14 years old) and no significant difference between sexes.

Summary: There is now initial evidence that the researchers can effectively collect saliva using tennis balls, and that the amylase test could be applicable to all major breeds or dogs, regardless of age or sex.

Next Steps: Independently and in partnership with potential licensees

• Identification of saliva amylase levels in cohorts of dogs in real-life environments, to determine the minimum and maximum levels occurring in different environments, including working dogs, rehoming centres, etc. This will provide the absolute boundaries for “normal” ranges.
• Case studies of dogs undergoing rehoming. Analysis of dog saliva amylase levels in real-life situations, and use of the amylase levels as predictors of dog arousal/stress. This will be directly compared to traditional analysis (animal body movements, vocalisation, etc), and to monitor if welfare interventions (behaviour modification) is improving the dogs’ quality of life.
• Biochemical testing of levels of amylase that could be measured using alternative test methods such as lateral flow analysis, and determination if the sensitivity levels would be suitable for determining cut-off thresholds for dog salvia amylase analysis.

Benefits

1. Cost – Current physiological measures require expensive tests performed in specialist laboratories.
2. Time – Following further development (outlined above), amylase tests will be able to be performed on-site using a simple test kit.
3. Specificity – Unlike current measures, amylase assesses negative arousal, and will not require additional measures for accurate interpretation.
4. Quick, easy to obtain, non-invasive saliva sample used as the test sample.

Applications

The inventors believe that salivary α-amylase is such a measure and would have widespread appeal for those involved in canine welfare, including: staff at rescue shelters and/or boarding kennels wishing to monitor the welfare of their dogs; veterinarians assessing dog health; military and police for assessing working dog welfare; working dog charities such as guide dogs; clinical animal behaviourists; and animal welfare scientists. This new measure has the potential to transform canine welfare assessment, as well as being important for evaluating the success of interventions and management/husbandry initiatives designed to improve dog welfare.

Opportunity

The University is looking for development partners for the Lateral Flow Test applications of the technology and potential licensees.