Heart rate variability analysis in sheep affected by transmissible spongiform encephalopathies
© Crown; licensee BioMed Central Ltd. 2011
Received: 12 October 2011
Accepted: 14 December 2011
Published: 14 December 2011
The function of the autonomic nervous system can be assessed by determining heart rate variability (HRV), which is impaired in some brainstem diseases in humans. Transmissible spongiform encephalopathies (TSEs) in sheep are diseases characterised by accumulation of disease-associated prion protein in the brainstem, including nuclei of the parasympathetic nervous system. This study was undertaken to assess whether analysis of HRV can be used as an aid in the diagnosis of TSEs in clinically affected, naturally or experimentally infected sheep.
When HRV indices were compared between 41 clinical TSE cases (18 sheep infected with scrapie and 23 sheep infected with bovine spongiform encephalopathy), 11 control sheep and six sheep reported as scrapie suspects or dosed with BSE brain homogenate, which were not confirmed as TSE cases by postmortem tests, no significant differences were found between the groups. Median heart rate was significantly different but only when sheep were grouped by gender: it was higher in female TSE cases than in control sheep and higher in female than castrated male ovine classical BSE cases.
HRV analysis was not useful as a diagnostic aid for TSEs of sheep.
Transmissible spongiform encephalopathies (TSEs) in sheep, such as scrapie or experimental bovine spongiform encephalopathy (BSE), are characterised by the accumulation of disease-associated prion protein (PrPsc) in the brainstem, particularly in the parasympathetic nucleus of the vagus nerve [1, 2], which is used for the confirmatory immunohistochemical diagnosis of these prion diseases. The axons of neurons from this nucleus contribute to the formation of the motor component of the vagus nerve , which - together with the nucleus ambiguus in the brainstem - provide parasympathetic innervation of the cardiac sinoatrial node . The function of the parasympathetic and sympathetic nervous system can be assessed by measuring heart rate variability (HRV), either by calculation of indices using statistical methods on R-R intervals (time domain analysis) or by spectral analysis of an array of R-R intervals (frequency domain analysis) derived from an electrocardiogram (ECG) . Brainstem lesions have been shown to alter HRV in humans [6, 7]. If PrPsc accumulation in brainstem nuclei had any effect on their function one would expect detectable changes in HRV. We have previously been unsuccessful in using this method to aid the diagnosis of TSEs in cattle  but results in a pilot study in sheep suggested that HRV assessment may be useful as pre-clinical test for TSE infection . As a result of the pilot study we evaluated whether clinically affected TSE-positive sheep could be distinguished from TSE-negative sheep by HRV analysis. Some of the data used had been presented at a conference at the European Society of Veterinary Neurology .
Animals and disease confirmation
All procedures involving animals were carried out in accordance with the Animal (Scientific Procedures) Act 1986, under licence from the United Kingdom Government Home Office, which was granted following an internal ethical review process within the Veterinary Laboratories Agency (VLA).
The TSE status of each sheep was confirmed by postmortem tests, which included immunohistochemical examination (IHC) with monoclonal antibodies (mAbs) R145 (for all sheep except for eight sheep of New Zealand origin), 6H4 or P4 (for ovine BSE cases, to distinguish them from scrapie) according to established methods  and - for those sheep that were negative by pathological examination and the eight New Zealand-derived sheep -discriminatory Western immunoblot (Hybrid technique ).
Details of sheep with recorded electrocardiograms
Number of animals
Gender: female, castrated male
Age range in months: min-max (median)
Cheviot (5), Romney (3), Suffolk (3)
Romney (8), Suffolk (5), Charollais (1), Cheviot (1), Mule (1), Welsh Mountain (1),
Romney (11), Suffolk (8), Poll Dorset (3), Cheviot (1)
Inoculated with BSE/reported scrapie suspects, not confirmed4
Romney (4), Suffolk (1), Welsh Mountain (1),
Heart rate monitoring
Heart rate monitoring was carried out as described recently for cattle  with disposable skin-adhesive electrodes (Unilect, Unomedical Ltd. Stonehouse, UK) and added gel (Lectron II, Pharmaceutical Innovations, Newark, USA) to improve conductivity. The base-apex lead was chosen: the negative electrode was placed at the caudal angle of the left scapula, the positive electrode was placed at the intercostal space caudal to the left olecranon and the ground electrode was placed at the left paralumbar fossa. Recordings were made whilst the animal was restrained in a pen and lasted for at least 310 seconds. In the majority of cases, the ECG was recorded at the day of cull (49 sheep, 84%) or one day prior to cull (5 sheep, 9%); in two (3%) clinically affected sheep with classical BSE the recording was taken at 6 days and 21 days prior to cull respectively and in two sheep (3%) orally challenged with classical BSE brain but without disease confirmation the recording took place 1506 days prior to cull.
Recordings were amplified, digitised and processed using a micro 1401 MK II data acquisition unit and computer software Spike2 version 4 (CED, Cambridge, UK). Each R-peak of a QRS complex was marked. The HRV was determined from tachygrams of instantaneous heart rate, which were produced by plotting the length of the time between successive R peaks of a 5-minute ECG segment against cumulative time. Fast Fourier transform was performed on each tachygram, which separates the heart rate signal into its frequency components and quantifies them in terms of their relative intensity as power , using 1024 points to calculate the power spectrum. The range of the low frequency (LF) power band, representative of sympathetic and parasympathetic activity, was 0.04-0.15 Hz. The range for the high frequency (HF) power band, representative of parasympathetic activity at the respiratory frequency, was 0.15-1 Hz, which was based on a validation study of heart rate variability indices in sheep . HF and LF power were expressed as absolute values and in normalised units (proportion of LF or HF power contributing to the total power minus the very low frequency power component below 0.04 Hz). In addition, the LF:HF power ratio was determined as a measure of sympathovagal balance . Further variables determined were the mean heart rate (HR), the deviation of the mean R-R interval (in %) as a measure of sinus arrhythmia and the vasovagal tonus index (VVTI, natural logarithm of the variance in the RR-intervals  based on 300 R-R intervals).
Comparison of data and discussion
Time and frequency domain indices in the three different groups
Control sheep (N = 11)
TSE-positive sheep (N = 41)
TSE-negative suspects/dosed sheep (N = 6)
LF power [ms2]
HF power [ms2]
LF:HF power ratio
Mean heart rate [bpm]
Deviation from mean interval [%]
Our study included one sheep with atypical scrapie, which is characterised by far less PrPsc accumulation in the brainstem compared to classical scrapie and, in the former, PrPsc is predominantly found in the spinal tract nucleus of the trigeminal nerve [15, 25]. However, all of the HRV indices obtained from this sheep were within the range established for the other (classical) scrapie cases.
There is some variation in the band widths for low and high frequency power used by different researchers. Whilst the ranges in the current study were based on a previous study in lambs , a recent review suggested using a high frequency band of 0.20-0.40 Hz for sheep and goats, corresponding to a respiratory rate of 12-24 breaths per minute . However, a study of breathing frequency in ruminants recorded a rate of 54 breaths per minute , equivalent to 0.9 Hz, which is within the reference range for adult sheep of 12-72 breaths per minute . Others used a low frequency range of 0.032-0.138 Hz and 0.15-0.5 Hz for the high frequency band . When we used similar ranges in the current study (limited by the equipment to 0.03-0.14 Hz for the low frequency band), the results did not differ (data not shown).
HRV analysis was not able to distinguish TSE-affected sheep from healthy sheep or TSE-negative sheep (by postmortem tests) that were orally dosed with classical BSE brain homogenate or scrapie suspects. Separation by gender, however, revealed significant differences for the median heart rate, which was higher in female TSE-affected sheep than in female control sheep and - for sheep affected by classical BSE - higher in female than in castrated male sheep.
We are grateful to present and past members of staff at former VLA in the Pathology Unit and the former Department of Molecular Pathogenesis and Genetics at VLA for the postmortem examination and diagnosis of the sheep in this study. We particularly thank Susan Bellworthy, Dr Martin Jeffrey, Dr Marion Simmons and Dr Hugh Simmons for the management of the sheep studies and former Animal Health staff for referral of scrapie cases to VLA Weybridge. The sheep studies were funded by the UK Department for Environment, Food and Rural Affairs (project codes SE0230, SE1846, SE1860, SE1931, SE1945, SE1946) and the EU (through funding of the EU TSE Reference Laboratory).
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