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The use of digital infrared thermography and measurement of oxidative stress biomarkers as tools to diagnose foot lesions in sheep
Small Ruminant Research 127 (2015) 80–85
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
The use of digital infrared thermography and measurement of oxidative stress biomarkers as tools to diagnose foot lesions in sheep Saranika Talukder a , Gianfranco Gabai b , Pietro Celi a,∗ a b
Faculty of Veterinary Science, The University of Sydney, Narellan, NSW 2567, Australia Dipartimento di Scienze Sperimentali Veterinarie, Universita ‘di Padova, Italy
a r t i c l e
i n f o
Article history: Received 18 September 2014 Received in revised form 23 February 2015 Accepted 11 April 2015 Available online 23 April 2015 Keywords: Infrared thermography Oxidative stress Footrot Sheep
a b s t r a c t This study reports preliminary data on the use of digital infrared thermography (IRT) and biomarkers of oxidative stress (OS) to diagnose foot lesions in sheep. Interdigital space skin temperatures were obtained from crossbred rams with healthy (n = 9) and with foot lesions (n = 6) with a FLIR T620 series infrared camera. Interdigital space lesions were scored using a five point scoring system (0–4). Blood was sampled from all rams and plasma was analysed for reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP), and advanced oxidation protein products (AOPP). The degree of OS was estimated by the ratio of ROMs/BAP (U Carr/mol/L) multiplied by 100 to give an OS index (OSI). Footrot scores were used to stratify the rams in two groups: healthy group (HG; n = 9) or foot lesion (FL; n = 6), if the lesions were recorded as absent or present, respectively. Differences in OS biomarkers and IRT temperatures between the two groups were analysed by a linear mixed model. A significant (P < 0.05) increase of IRT temperature was observed in rams with foot lesions compared with healthy rams. Rams that presented foot lesions had significantly higher values of ROMs (P < 0.05) and OSI (P < 0.001) and lower concentration of BAP than healthy rams (P < 0.05). In conclusion, IRT and biomarkers of oxidative stress were able to identify sheep with foot lesions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lameness is a common cause of welfare and economic concerns in sheep-keeping countries (Winter, 2008). The most important infectious agents that can induce foot lesions and lameness in sheep are footrot and interdigital dermatitis (Egerton et al., 1989). Dichelobacter nodosus is the essential organism for causing footrot, while interdigital dermatitis is caused by Fusobacterium necrophorum, which is also thought to play an important role in the
∗ Corresponding author. Tel.: +61 2 9351 1782; fax: +61 2 4655 2374. E-mail address: [email protected] (P. Celi). http://dx.doi.org/10.1016/j.smallrumres.2015.04.006 0921-4488/© 2015 Elsevier B.V. All rights reserved.
pathogenesis of footrot (Egerton et al., 1989). Pathogenesis, clinical appearance, differential diagnosis and control measures of interdigital dermatitis and footrot have been recently reviewed (Winter, 2008; Raadsma and Egerton, 2013; Allworth, 2014). While methods of treatment and control of these diseases are readily available, the need to make a rapid and simple diagnosis remains a key feature in treating and controlling lameness in sheep flocks. For example, a rapid and sensitive competitive real-time PCR method for early detection of D. nodosus directly from interdigital swabs of sheep has been developed (Stäuble et al., 2014). Prevention of lameness should be considered a priority economically as the control cost comprises only 3% of the total expenses for an outbreak. Moreover, the response
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Fig. 1. Infrared camera images of interdigital space imaged from healthy (A) or with foot lesions (affected) rams (B). The polygonal shapes indicate the area of interdigital space along with the areas’ maximum temperature.
rate to treatment is higher when lameness is treated at a very early stage (Winter, 2009). Currently, the most common method employed by researchers and clinicians in detection of interdigital dermatitis and footrot is the visual observation of interdigital space. Visual diagnosis involves the use of a system to score the severity of the footrot lesion. A commonly used system to score footrot in small ruminants is an Australian system with five ordinal scores (Egerton and Roberts, 1971). However, this approach can be time consuming, subjective, depends on the experience of the observer and requires regular observation that is both logistically and economically challenging. Therefore, reliable, practical and non-invasive methods to monitor frequently and rapidly the presence of foot lesions are needed. In this circumstance, infrared thermography (IRT) might be useful for rapid screening of foot lesions in sheep. In veterinary medicine, IRT has been successfully used in various applications such as for the detection of bovine viral diarrhoea (Schaefer et al., 2004), bovine respiratory disease (Schaefer et al., 2007, 2012), foot-and-mouth disease (RainwaterLovett et al., 2009), bluetongue (Pérez de Diego et al., 2013) and clinical mastitis (Hovinen et al., 2008; Polat et al., 2010; Martins et al., 2013) and to test for detection of oestrus in dairy cows (Talukder et al., 2014b). Infrared thermography has also been applied to diagnose hoof and foot lesions in horses and cattle (Alsaaod and Büscher, 2012; Main et al., 2012; Stokes et al., 2012; Alsaaod et al., 2014). It has been reported the IRT is able to detect circadian changes of foot temperature in sheep (D’Alterio et al., 2011) and that it can measure differences between hooves temperature of healthy and footrot affected herds (Lehugeur, 2012), however in the same study, Lehugeur was not able to detect difference in temperature of different foot scores. Therefore, further studies are needed to evaluate IRT as a tool to diagnose foot lesions in sheep. Oxidative stress, the result of an imbalance between pro-oxidants and antioxidants, is an active field of research in ruminant medicine and has been implicated in numerous disease processes including sepsis, mastitis, acidosis, ketosis, enteritis, pneumonia, respiratory, and joint diseases (Lykkesfeldt and Svendsen, 2007; Celi, 2010, 2011b).
In veterinary medicine the most investigated causes of excessive free radical production, which then results in oxidative stress, are represented by metabolic and environmental cues (Celi, 2010, 2011b) and inflammatory events (Sordillo and Aitken, 2009). Selenium is an essential element in the diet of animals and is important in host antioxidant defense (Rivera et al., 2005). Recently, it has been shown that whole-blood selenium is decreased in footrot-affected sheep and that parenteral selenium supplementation accelerates recovery from footrot (Hall et al., 2009). Therefore, it seems that footrot could induce oxidative stress, leading to increase in formation of free radicals. We hypothesised that rams affected by foot lesions will have higher interdigital space temperatures and higher oxidative stress compared to healthy rams. Therefore, the aim of this study was to assess the potential use of IRT and oxidative stress biomarkers as tools to diagnose foot lesions in sheep. 2. Materials and methods 2.1. Animals and classification of feet lesions In this study 15 cross bred (Merino × Dorset) rams (3 ± 1 years) were used from a farm located near Camden, NSW (34.0544◦ S, 150.6958◦ E). This region has been identified as an area with medium-high rainfall (764 mm/year) which is thought to be a predisposing factor for footrot and interdigital dermatitis. Rams were kept in a single paddock were they grazed native pasture and had ad libitum access to water. Rams were inspected and awarded a score of 0 (healthy) to 4 (severe footrot) for each hoof, as per the scoring system described by Egerton and Roberts (1971). All scoring was carried out by the same technician. Examples of the clinical scoring system can be found in Raadsma and Egerton (2013). This scoring and classification system has been successfully used for describing differences between animals in terms of relative susceptibility (Raadsma et al., 1991; Conington et al., 2008) and it was considered as the gold standard (reference test) for detection of hoof lesions. The scores of each foot were averaged for each ram so that they could be allocated into a group, healthy (HG; n = 9) or foot lesion (FL; n = 6), respectively, if the lesions were recorded as absent (feet without foot lesion; all rams in the HG group had no foot lesions with the exception of three rams who were scored 0.5 for one foot and 0 for the remaining three) or present (feet with foot lesions). 2.2. Infrared thermography A hand held portable infrared camera (FLIR, 620 series; FLIR Systems Co. Ltd., St Leonards, NSW, Australia.) was used to collect the thermal
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images on three different days at the same time of day (9.00 am) to avoid any circadian changes of foot temperature (D’Alterio et al., 2011). The average (±SEM) temperature and humidity observed at the time of measurement were 14.3 ± 0.5 ◦ C and 72 ± 2.8%, respectively. The emissivity value was set to 0.98 and thermograph resolution was calibrated to ambient temperature and humidity as per manufacturer’s recommendation. Thermal imaging was performed at approximately 0.5 m from the animal, in a dorsal–palmar 45◦ oblique direction, ensuring that the interdigital space of the left front foot, left rear foot, right front foot and right rear foot appeared clearly in the centre of the images for each foot (Fig. 1). Rams were restrained in a standing position and each limb was lifted to expose the interdigital space; feet were not scrubbed or washed. Images were stored on a memory card and then transferred to a laptop for analysis of the image using ThermaCAM Researcher Professional 2.9. Using this software the maximum, minimum and average temperatures can be defined from an area defined on the image by drawing a free hand polygonal shape that covered the interdigital space used for temperature measurement. 2.3. Blood collection and oxidative stress assays Blood samples were collected by jugular venepuncture into plain lithium heparinised (1× 10 ml) vacutainers (BD, North Ryde, NSW, Australia) and placed immediately on ice. Plasma was separated by centrifugation and stored at −20 ◦ C pending analysis. The amount of free oxygen radicals in plasma samples was determined by measuring the concentrations of ROMs, using a colorimetric assay according to kit instructions (d-ROMS Test, Diacron International, Grosseto, Italy). The results are expressed in Carratelli units (Carr U), where 1 Carr U corresponds to 0.08 mg/100 mL of hydrogen peroxide. The concentrations of antioxidants were measured using the BAP test according to kit instructions (Diacron International, Grosseto, Italy). The results of BAP are expressed in mol/L of reduced iron. Plasma concentrations of ROMs and BAP were determined in a dedicated spectrophotometer (FREE Carpe Diem, Diacron International). The extent of oxidative stress was expressed as an oxidative stress index (OSI), which was estimated using the ratio dROM:BAP × 100, as the combination of d-ROM and BAP results provides a more accurate representation of oxidative stress status (Celi, 2011a). Advanced oxidation protein products were measured according to the methods of Witko-Sarsat et al. (1998). Plasma concentrations of albumin were determined in a spectrophotometer (FLUOstar Optima, BMG Labtech) using commercial kits supplied by BioAssay Systems (albumin). 2.4. Statistical analysis
Table 1 Interdigital space skin temperature and foot rot score in healthy rams or with foot lesions.
◦
Maximum temp ( C) Minimum temp (◦ C) Average temp (◦ C) Footrot score
Healthy group
Foot lesion
SEM
P-value
35.7 30 33.7 0.06
37.0 31.2 35.1 2.2
0.7 0.5 0.7 0.1
0.04 0.02 0.05 0.01
Values are means ± SEM.
average IRT temperature was greater in FL rams compared to their healthy flock mates. The ability of IRT in detecting foot rot was further compared with the foot scoring system (as a reference test) using ROC curves. The cut-off value, sensitivity, specificity, and area under the ROC curve for IRT were 36.38 ◦ C, 83.3%, 77.8%, and 0.91, respectively (Fig. 2). Rams that presented foot lesions had significantly higher values of both ROMs (P < 0.05) and OSI (P < 0.05), while the concentration of BAP was significantly lower in ram with foot lesions (P < 0.05); no differences were observed for AOPP, albumin and their ratio (Table 2). Relative to the control group, plasma concentrations of d-ROMs on average, increased by 30% in rams with foot lesions, while BAP concentrations decreased by 15%; consequently OSI increased by 54% in rams with foot lesions. Pearson correlation coefficients for the parameters measured in this study are reported in Table 3. Plasma ROMs were positively correlated with average, maximum, minimum IRT temperature and with footrot score (r = 0.6754; P < 0.01, r = 0.7193; P < 0.01, r = 0.6503; P < 0.01 and r = 0.7429; P < 0.001, respectively). Similarly, OSI was positively correlated with average, maximum, minimum IRT temperature and with footrot score (r = 0.583; P < 0.05, r = 0.6228; P < 0.01, r = 0.5236; P < 0.05 and r = 0.8092;
All the variables were initially assessed using descriptive statistics and variables that had skewed distributions were logarithmically transformed. Thermal data and oxidative stress biomarker profiles were compared between the two groups (HG and FL) by linear mixed models using GenStat 14th Edition (VSN International, Hertfordshire, UK) including sheep identification number as a random effect in each of these models following the approach mentioned in Talukder et al. (2014a). Average maximum temperature of all four feet (left front foot, left rear foot, right front foot and right rear foot) was considered in the analysis in line with previous studies in dairy cattle (Talukder et al., 2014b, 2015). For logarithmically transformed variables, the back transformed means were used for presentation of results. In addition, a receiver operating characteristic (ROC) analysis was used to compare the diagnostic performance of IRT temperatures and foot lesions scores (as a reference test) and to calculate the optimal efficiency threshold values by estimating differences of the area under the ROC curves using the statistical package software MedCalc (Version 11.6, 2011; MedCalc Software, http://www.medcalc.org). The P value of ≤0.05 was considered as significant. Pearson correlation coefficients were used to determine associations between the measurements evaluated in this study.
3. Results In the current study, significant (P < 0.05) differences between maximum IRT temperatures were observed between HG and FL groups (Table 1, Fig. 1). The highest maximum IRT temperature noted in rams with foot lesions was 1.3 ◦ C greater than that of healthy rams. In addition,
Fig. 2. Receiver operating characteristic (ROC) curve for infrared thermography (IRT) hoof temperature for foot rot. The true positive rate (sensitivity) is plotted in function of the false positive rate (100specificity). The cut-off value and area under the ROC curve for IRT were 36.38 ◦ C and 0.91 (z-value = 5.24, P ≤ 0.0001).
0.004 0.004 0.001
17.6 36.56 0.48
18.9 35.01 0.54
1.6 1.28 0.04
0.604 0.349 0.381
AOPP
ROMs, reactive oxygen metabolites; BAP, biological antioxidant potential; OSI, oxidative stress index; AOPP, advanced oxidative protein products. Values are expressed as means ± SEM.
OSI
P < 0.001, respectively). Finally, footrot score was positively correlated with average, maximum and minimum IRT temperature (r = 0.5739; P < 0.05; r = 0.6218; P < 0.01; r = 0.5725; P < 0.05, respectively) while it was negatively correlated with BAP (r = −0.6074; P < 0.01).
–
P-value
6 125 0.21
– −0.3944
SEM
154 3627 4.28
– −0.0254 0.9273
Foot lesion
116 4179 2.77
– −0.0594 −0.3099 0.0785
Healthy group
Albumin
ROMs (U Carr) BAP (mol/L) OSI (arbitrary units) AOPP (mol/L) Albumin (g/L) AOPP/albumin (mol/g)
AOPP/albumin
Table 2 Oxidative stress biomarkers in healthy rams or with foot lesions.
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– −0.6923** 0.0838 0.4436 −0.0989 – −0.3568 0.9176*** −0.04 −0.1179 0.0262 – 0.7429*** −0.6074** 0.8092*** 0.1488 −0.1871 0.2275 – 0.5725* 0.6503** −0.0515 0.5236* −0.164 −0.1318 −0.1063 – 0.8693*** 0.6218** 0.7193** −0.2041 0.6228** −0.2601 −0.0872 −0.1954
ROMs Footrot score IRT min IRT max IRT AVG
– 0.9639*** 0.9113*** 0.5739* 0.6754** −0.1802 0.583* −0.3069 −0.0294 −0.2698 IRT AVG IRT max IRT min Footrot score ROMs BAP OSI AOPP Albumin AOPP/albumin
Table 3 Pearson correlation coefficients of oxidative stress biomarkers, IRT temperatures and footrot scores.
In the current study, interdigital space temperature was significantly higher in rams with foot lesions compared to rams without any foot lesions; IRT was able to detect elevated temperature associated with foot lesions. These finding are in agreement with those of Alsaaod and Büscher (2012), Main et al. (2012), Stokes et al. (2012) and Alsaaod et al. (2014) who observed foot temperature difference between infected and healthy cattle. Stokes et al. (2012) used the maximum foot temperature to differentiate cows with or without different feet lesions. The ROC curve presented in the current study demonstrates the presence of a strong agreement between IRT temperature thresholds for foot lesion and the scoring system used as gold standard for their diagnosis. When using a temperature threshold of 36.38 ◦ C as a critical cut-off between feet with and without lesions (Fig. 2), the observed high sensitivity (0.83) of the IRT suggests that this technique might be able to identify a considerable percentage of true positives. On the other hand, the relatively lower specificity (0.78) suggests that the temperature threshold identified a number of healthy hooves as having lesions (false positives). The positive correlation between IRT temperatures and footrot score bring further support to our approach. Therefore, IRT has potential for detecting temperature changes associated with the presence of foot lesions in rams. In this respect, the development of highly specific and sensitive cut-offs IRT temperatures is required to aid in the diagnosis, monitoring and treatment of foot lesions in sheep. The observed increase in ROMs and decrease in BAP concentration indicates the presence of increased oxidative stress in rams affected by foot lesions. Indeed, OSI was more than double in rams with foot lesions compared to healthy rams and both OSI and ROMs were positively correlated with IRT temperatures and footrot score. Plasma levels of ROMs are considered an indicator of free radical production (Miller et al., 1993) which can lead to an increase in lipid peroxidation (Halliwell, 2009). Although specific biomarkers of lipid peroxidation, such as malondialdehyde (MDA) or thiobarbituric acid reactive substances (TBARS),
BAP
4. Discussion
IRT AVG, average IRT temperature; IRT max, maximum IRT temperature; IRT min, minimum IRT temperature; ROMs, reactive oxygen metabolites; BAP, biological antioxidant potential; OSI, oxidative stress index; AOPP, advanced oxidative protein products. * P< 0.05. ** P< 0.01. *** P< 0.001.
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were not measured in this study, studies in dairy cows have observed an increase in TBARS level in lame cows compared to healthy herd mates (Al-Qudah and Ismail, 2012). Plasma antioxidant status is the result of the interaction of many different compounds and systemic metabolic interactions and it was measured with the BAP test in this study. The BAP test provides a global measurement of many antioxidants, including uric acid, ascorbic acid, proteins, ␣-tocopherol, and bilirubin (Benzie and Strain, 1996). As a single measure, the BAP test provides relevant information that may effectively describe the dynamic equilibrium between pro-oxidants and antioxidants in the plasma compartment. In sheep, it has been shown that heat stress decreases plasma BAP concentrations (Chauhan et al., 2014). The observed decrease in BAP concentration in rams with foot lesions is probably the consequence of the concomitant increase in ROMs levels. Indeed, changes in the components of antioxidants systems are often not the cause, but the consequence of the oxidative stress induced by higher free radical activity (Venditti and Meo, 2006). The observed negative correlation between BAP concentration and footrot score brings further support to the above statement. The calculated OSI values clearly indicate that rams with foot lesions had higher levels of pro-oxidants (ROMs) and decreased antioxidant capacity (BAP). These data indicate that oxidative damage, as assessed by an individual animal’s OSI (pro-oxidants/antioxidants), is indicative of their health status. Indeed, when the OSI is high (prooxidants > antioxidants) it would be reasonable to expect decreased health and wellbeing conditions for the individual animal that will result in decreased adaptation to environmental challenges. For example, when oxidative stress was monitored in dairy cows by means of ROMs and BAP, the information on oxidative stress level was more accurate when combining the ROMs and BAP data in the calculated OSI than using them separately (Pedernera et al., 2010). Moreover, increased OSI values have been observed in foals affected by respiratory diseases (Crowley et al., 2013; Po et al., 2013). Pasture-based systems can improve the oxidative status of ruminants due to the elevated antioxidant content of green grass and by providing ruminants with health benefits from certain vitamins and minerals. However, as free radicals and antioxidants are involved in several physiological functions it might be beneficial to supplement sheep with antioxidants (Celi, 2010). For example, the effectiveness of selenium in reducing oxidative stress and the severity of inflammatory events in dairy cattle has been reported (Sordillo, 2013). Selenium is an essential element in ruminant’s diet which provides a significant source of antioxidant defence and plays an important role in optimising both the immune response and disease resistance (Spears and Weiss, 2008). Indeed, it has been demonstrated that footrot affected sheep have lower blood selenium concentration and that selenium supplementation resulted in a more rapid improvement of foot lesions (Hall et al., 2009). Albumin is a free radical scavenger but it is also the predominant oxidised protein contributing to AOPP formation (Celi, 2011b). Indeed, concentrations of AOPP can be associated with embryonic losses and are considered an indicator of inflammation and oxidative stress in dairy cows (Celi
et al., 2011, 2012). The lack of differences in albumin and AOPP concentrations between HG and FL affected rams may reflect the mild level of lesions observed in this study. 5. Conclusion The data gathered in this study suggest that measuring IRT interdigital space temperatures and oxidative stress biomarkers in sheep may help in the diagnosis of foot lesions on endemic farms, however its potential as diagnostic tool needs further evaluation before the practical benefits and on-farm adoption of this technology can be considered. Longitudinal studies investigating the progression or regression of foot lesions in a larger population of affected sheep are required to determine in greater detail the association between changes in interdigital space temperatures and oxidative stress biomarkers and the severity of foot lesions. Finally, further studies should investigate the reliability of IRT among sheep kept under various environmental conditions such as air temperature, velocity, and humidity. Conflict of interest statement None of the authors are personally or professionally affiliated with other people or organisations that could inappropriately influence the content of the paper. Acknowledgments The authors are grateful to Craig Kristo for assistance in the scoring and classification of foot lesions and to the University of Sydney Poultry Research Foundation for providing the use of the infrared camera. The authors wish to acknowledge the Honda Foundation for providing financial support to purchase the FREE Carpe Diem. References Al-Qudah, K.M., Ismail, Z.B., 2012. The relationship between serum biotin and oxidant/antioxidant activities in bovine lameness. Res. Vet. Sci. 92, 138–141. Allworth, M.B., 2014. Challenges in ovine footrot control. Small Rumin. Res. 118, 110–113. Alsaaod, M., Büscher, W., 2012. Detection of hoof lesions using digital infrared thermography in dairy cows. J. Dairy Sci. 95, 735–742. Alsaaod, M., Syring, C., Dietrich, J., Doherr, M.G., Gujan, T., Steiner, A., 2014. A field trial of infrared thermography as a non-invasive diagnostic tool for early detection of digital dermatitis in dairy cows. Vet. J. 199, 281–285. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Celi, P., 2010. The role of oxidative stress in small ruminants’ health and production. Rev. Bras. de Zootec. 39, 348–363. Celi, P., 2011a. Biomarkers of oxidative stress in ruminant medicine. Immunopharmacol. Immunotoxicol. 33, 233–240. Celi, P., 2011b. Oxidative stress in ruminants. In: Mandelker, L., Vajdovich, P. (Eds.), Studies on Veterinary Medicine. Humana Press, New York, pp. 191–231. Celi, P., Merlo, M., Barbato, O., Gabai, G., 2012. Relationship between oxidative stress and the success of artificial insemination in dairy cows in a pasture-based system. Vet. J. 193, 498–502. Celi, P., Merlo, M., Da Dalt, L., Stefani, A., Barbato, O., Gabai, G., 2011. Relationship between late embryonic mortality and the increase in plasma advanced oxidised protein products (AOPP) in dairy cows. Reprod. Fert. Dev. 23, 527–533.
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
The use of digital infrared thermography and measurement of oxidative stress biomarkers as tools to diagnose foot lesions in sheep Saranika Talukder a , Gianfranco Gabai b , Pietro Celi a,∗ a b
Faculty of Veterinary Science, The University of Sydney, Narellan, NSW 2567, Australia Dipartimento di Scienze Sperimentali Veterinarie, Universita ‘di Padova, Italy
a r t i c l e
i n f o
Article history: Received 18 September 2014 Received in revised form 23 February 2015 Accepted 11 April 2015 Available online 23 April 2015 Keywords: Infrared thermography Oxidative stress Footrot Sheep
a b s t r a c t This study reports preliminary data on the use of digital infrared thermography (IRT) and biomarkers of oxidative stress (OS) to diagnose foot lesions in sheep. Interdigital space skin temperatures were obtained from crossbred rams with healthy (n = 9) and with foot lesions (n = 6) with a FLIR T620 series infrared camera. Interdigital space lesions were scored using a five point scoring system (0–4). Blood was sampled from all rams and plasma was analysed for reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP), and advanced oxidation protein products (AOPP). The degree of OS was estimated by the ratio of ROMs/BAP (U Carr/mol/L) multiplied by 100 to give an OS index (OSI). Footrot scores were used to stratify the rams in two groups: healthy group (HG; n = 9) or foot lesion (FL; n = 6), if the lesions were recorded as absent or present, respectively. Differences in OS biomarkers and IRT temperatures between the two groups were analysed by a linear mixed model. A significant (P < 0.05) increase of IRT temperature was observed in rams with foot lesions compared with healthy rams. Rams that presented foot lesions had significantly higher values of ROMs (P < 0.05) and OSI (P < 0.001) and lower concentration of BAP than healthy rams (P < 0.05). In conclusion, IRT and biomarkers of oxidative stress were able to identify sheep with foot lesions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lameness is a common cause of welfare and economic concerns in sheep-keeping countries (Winter, 2008). The most important infectious agents that can induce foot lesions and lameness in sheep are footrot and interdigital dermatitis (Egerton et al., 1989). Dichelobacter nodosus is the essential organism for causing footrot, while interdigital dermatitis is caused by Fusobacterium necrophorum, which is also thought to play an important role in the
∗ Corresponding author. Tel.: +61 2 9351 1782; fax: +61 2 4655 2374. E-mail address: [email protected] (P. Celi). http://dx.doi.org/10.1016/j.smallrumres.2015.04.006 0921-4488/© 2015 Elsevier B.V. All rights reserved.
pathogenesis of footrot (Egerton et al., 1989). Pathogenesis, clinical appearance, differential diagnosis and control measures of interdigital dermatitis and footrot have been recently reviewed (Winter, 2008; Raadsma and Egerton, 2013; Allworth, 2014). While methods of treatment and control of these diseases are readily available, the need to make a rapid and simple diagnosis remains a key feature in treating and controlling lameness in sheep flocks. For example, a rapid and sensitive competitive real-time PCR method for early detection of D. nodosus directly from interdigital swabs of sheep has been developed (Stäuble et al., 2014). Prevention of lameness should be considered a priority economically as the control cost comprises only 3% of the total expenses for an outbreak. Moreover, the response
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Fig. 1. Infrared camera images of interdigital space imaged from healthy (A) or with foot lesions (affected) rams (B). The polygonal shapes indicate the area of interdigital space along with the areas’ maximum temperature.
rate to treatment is higher when lameness is treated at a very early stage (Winter, 2009). Currently, the most common method employed by researchers and clinicians in detection of interdigital dermatitis and footrot is the visual observation of interdigital space. Visual diagnosis involves the use of a system to score the severity of the footrot lesion. A commonly used system to score footrot in small ruminants is an Australian system with five ordinal scores (Egerton and Roberts, 1971). However, this approach can be time consuming, subjective, depends on the experience of the observer and requires regular observation that is both logistically and economically challenging. Therefore, reliable, practical and non-invasive methods to monitor frequently and rapidly the presence of foot lesions are needed. In this circumstance, infrared thermography (IRT) might be useful for rapid screening of foot lesions in sheep. In veterinary medicine, IRT has been successfully used in various applications such as for the detection of bovine viral diarrhoea (Schaefer et al., 2004), bovine respiratory disease (Schaefer et al., 2007, 2012), foot-and-mouth disease (RainwaterLovett et al., 2009), bluetongue (Pérez de Diego et al., 2013) and clinical mastitis (Hovinen et al., 2008; Polat et al., 2010; Martins et al., 2013) and to test for detection of oestrus in dairy cows (Talukder et al., 2014b). Infrared thermography has also been applied to diagnose hoof and foot lesions in horses and cattle (Alsaaod and Büscher, 2012; Main et al., 2012; Stokes et al., 2012; Alsaaod et al., 2014). It has been reported the IRT is able to detect circadian changes of foot temperature in sheep (D’Alterio et al., 2011) and that it can measure differences between hooves temperature of healthy and footrot affected herds (Lehugeur, 2012), however in the same study, Lehugeur was not able to detect difference in temperature of different foot scores. Therefore, further studies are needed to evaluate IRT as a tool to diagnose foot lesions in sheep. Oxidative stress, the result of an imbalance between pro-oxidants and antioxidants, is an active field of research in ruminant medicine and has been implicated in numerous disease processes including sepsis, mastitis, acidosis, ketosis, enteritis, pneumonia, respiratory, and joint diseases (Lykkesfeldt and Svendsen, 2007; Celi, 2010, 2011b).
In veterinary medicine the most investigated causes of excessive free radical production, which then results in oxidative stress, are represented by metabolic and environmental cues (Celi, 2010, 2011b) and inflammatory events (Sordillo and Aitken, 2009). Selenium is an essential element in the diet of animals and is important in host antioxidant defense (Rivera et al., 2005). Recently, it has been shown that whole-blood selenium is decreased in footrot-affected sheep and that parenteral selenium supplementation accelerates recovery from footrot (Hall et al., 2009). Therefore, it seems that footrot could induce oxidative stress, leading to increase in formation of free radicals. We hypothesised that rams affected by foot lesions will have higher interdigital space temperatures and higher oxidative stress compared to healthy rams. Therefore, the aim of this study was to assess the potential use of IRT and oxidative stress biomarkers as tools to diagnose foot lesions in sheep. 2. Materials and methods 2.1. Animals and classification of feet lesions In this study 15 cross bred (Merino × Dorset) rams (3 ± 1 years) were used from a farm located near Camden, NSW (34.0544◦ S, 150.6958◦ E). This region has been identified as an area with medium-high rainfall (764 mm/year) which is thought to be a predisposing factor for footrot and interdigital dermatitis. Rams were kept in a single paddock were they grazed native pasture and had ad libitum access to water. Rams were inspected and awarded a score of 0 (healthy) to 4 (severe footrot) for each hoof, as per the scoring system described by Egerton and Roberts (1971). All scoring was carried out by the same technician. Examples of the clinical scoring system can be found in Raadsma and Egerton (2013). This scoring and classification system has been successfully used for describing differences between animals in terms of relative susceptibility (Raadsma et al., 1991; Conington et al., 2008) and it was considered as the gold standard (reference test) for detection of hoof lesions. The scores of each foot were averaged for each ram so that they could be allocated into a group, healthy (HG; n = 9) or foot lesion (FL; n = 6), respectively, if the lesions were recorded as absent (feet without foot lesion; all rams in the HG group had no foot lesions with the exception of three rams who were scored 0.5 for one foot and 0 for the remaining three) or present (feet with foot lesions). 2.2. Infrared thermography A hand held portable infrared camera (FLIR, 620 series; FLIR Systems Co. Ltd., St Leonards, NSW, Australia.) was used to collect the thermal
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images on three different days at the same time of day (9.00 am) to avoid any circadian changes of foot temperature (D’Alterio et al., 2011). The average (±SEM) temperature and humidity observed at the time of measurement were 14.3 ± 0.5 ◦ C and 72 ± 2.8%, respectively. The emissivity value was set to 0.98 and thermograph resolution was calibrated to ambient temperature and humidity as per manufacturer’s recommendation. Thermal imaging was performed at approximately 0.5 m from the animal, in a dorsal–palmar 45◦ oblique direction, ensuring that the interdigital space of the left front foot, left rear foot, right front foot and right rear foot appeared clearly in the centre of the images for each foot (Fig. 1). Rams were restrained in a standing position and each limb was lifted to expose the interdigital space; feet were not scrubbed or washed. Images were stored on a memory card and then transferred to a laptop for analysis of the image using ThermaCAM Researcher Professional 2.9. Using this software the maximum, minimum and average temperatures can be defined from an area defined on the image by drawing a free hand polygonal shape that covered the interdigital space used for temperature measurement. 2.3. Blood collection and oxidative stress assays Blood samples were collected by jugular venepuncture into plain lithium heparinised (1× 10 ml) vacutainers (BD, North Ryde, NSW, Australia) and placed immediately on ice. Plasma was separated by centrifugation and stored at −20 ◦ C pending analysis. The amount of free oxygen radicals in plasma samples was determined by measuring the concentrations of ROMs, using a colorimetric assay according to kit instructions (d-ROMS Test, Diacron International, Grosseto, Italy). The results are expressed in Carratelli units (Carr U), where 1 Carr U corresponds to 0.08 mg/100 mL of hydrogen peroxide. The concentrations of antioxidants were measured using the BAP test according to kit instructions (Diacron International, Grosseto, Italy). The results of BAP are expressed in mol/L of reduced iron. Plasma concentrations of ROMs and BAP were determined in a dedicated spectrophotometer (FREE Carpe Diem, Diacron International). The extent of oxidative stress was expressed as an oxidative stress index (OSI), which was estimated using the ratio dROM:BAP × 100, as the combination of d-ROM and BAP results provides a more accurate representation of oxidative stress status (Celi, 2011a). Advanced oxidation protein products were measured according to the methods of Witko-Sarsat et al. (1998). Plasma concentrations of albumin were determined in a spectrophotometer (FLUOstar Optima, BMG Labtech) using commercial kits supplied by BioAssay Systems (albumin). 2.4. Statistical analysis
Table 1 Interdigital space skin temperature and foot rot score in healthy rams or with foot lesions.
◦
Maximum temp ( C) Minimum temp (◦ C) Average temp (◦ C) Footrot score
Healthy group
Foot lesion
SEM
P-value
35.7 30 33.7 0.06
37.0 31.2 35.1 2.2
0.7 0.5 0.7 0.1
0.04 0.02 0.05 0.01
Values are means ± SEM.
average IRT temperature was greater in FL rams compared to their healthy flock mates. The ability of IRT in detecting foot rot was further compared with the foot scoring system (as a reference test) using ROC curves. The cut-off value, sensitivity, specificity, and area under the ROC curve for IRT were 36.38 ◦ C, 83.3%, 77.8%, and 0.91, respectively (Fig. 2). Rams that presented foot lesions had significantly higher values of both ROMs (P < 0.05) and OSI (P < 0.05), while the concentration of BAP was significantly lower in ram with foot lesions (P < 0.05); no differences were observed for AOPP, albumin and their ratio (Table 2). Relative to the control group, plasma concentrations of d-ROMs on average, increased by 30% in rams with foot lesions, while BAP concentrations decreased by 15%; consequently OSI increased by 54% in rams with foot lesions. Pearson correlation coefficients for the parameters measured in this study are reported in Table 3. Plasma ROMs were positively correlated with average, maximum, minimum IRT temperature and with footrot score (r = 0.6754; P < 0.01, r = 0.7193; P < 0.01, r = 0.6503; P < 0.01 and r = 0.7429; P < 0.001, respectively). Similarly, OSI was positively correlated with average, maximum, minimum IRT temperature and with footrot score (r = 0.583; P < 0.05, r = 0.6228; P < 0.01, r = 0.5236; P < 0.05 and r = 0.8092;
All the variables were initially assessed using descriptive statistics and variables that had skewed distributions were logarithmically transformed. Thermal data and oxidative stress biomarker profiles were compared between the two groups (HG and FL) by linear mixed models using GenStat 14th Edition (VSN International, Hertfordshire, UK) including sheep identification number as a random effect in each of these models following the approach mentioned in Talukder et al. (2014a). Average maximum temperature of all four feet (left front foot, left rear foot, right front foot and right rear foot) was considered in the analysis in line with previous studies in dairy cattle (Talukder et al., 2014b, 2015). For logarithmically transformed variables, the back transformed means were used for presentation of results. In addition, a receiver operating characteristic (ROC) analysis was used to compare the diagnostic performance of IRT temperatures and foot lesions scores (as a reference test) and to calculate the optimal efficiency threshold values by estimating differences of the area under the ROC curves using the statistical package software MedCalc (Version 11.6, 2011; MedCalc Software, http://www.medcalc.org). The P value of ≤0.05 was considered as significant. Pearson correlation coefficients were used to determine associations between the measurements evaluated in this study.
3. Results In the current study, significant (P < 0.05) differences between maximum IRT temperatures were observed between HG and FL groups (Table 1, Fig. 1). The highest maximum IRT temperature noted in rams with foot lesions was 1.3 ◦ C greater than that of healthy rams. In addition,
Fig. 2. Receiver operating characteristic (ROC) curve for infrared thermography (IRT) hoof temperature for foot rot. The true positive rate (sensitivity) is plotted in function of the false positive rate (100specificity). The cut-off value and area under the ROC curve for IRT were 36.38 ◦ C and 0.91 (z-value = 5.24, P ≤ 0.0001).
0.004 0.004 0.001
17.6 36.56 0.48
18.9 35.01 0.54
1.6 1.28 0.04
0.604 0.349 0.381
AOPP
ROMs, reactive oxygen metabolites; BAP, biological antioxidant potential; OSI, oxidative stress index; AOPP, advanced oxidative protein products. Values are expressed as means ± SEM.
OSI
P < 0.001, respectively). Finally, footrot score was positively correlated with average, maximum and minimum IRT temperature (r = 0.5739; P < 0.05; r = 0.6218; P < 0.01; r = 0.5725; P < 0.05, respectively) while it was negatively correlated with BAP (r = −0.6074; P < 0.01).
–
P-value
6 125 0.21
– −0.3944
SEM
154 3627 4.28
– −0.0254 0.9273
Foot lesion
116 4179 2.77
– −0.0594 −0.3099 0.0785
Healthy group
Albumin
ROMs (U Carr) BAP (mol/L) OSI (arbitrary units) AOPP (mol/L) Albumin (g/L) AOPP/albumin (mol/g)
AOPP/albumin
Table 2 Oxidative stress biomarkers in healthy rams or with foot lesions.
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– −0.6923** 0.0838 0.4436 −0.0989 – −0.3568 0.9176*** −0.04 −0.1179 0.0262 – 0.7429*** −0.6074** 0.8092*** 0.1488 −0.1871 0.2275 – 0.5725* 0.6503** −0.0515 0.5236* −0.164 −0.1318 −0.1063 – 0.8693*** 0.6218** 0.7193** −0.2041 0.6228** −0.2601 −0.0872 −0.1954
ROMs Footrot score IRT min IRT max IRT AVG
– 0.9639*** 0.9113*** 0.5739* 0.6754** −0.1802 0.583* −0.3069 −0.0294 −0.2698 IRT AVG IRT max IRT min Footrot score ROMs BAP OSI AOPP Albumin AOPP/albumin
Table 3 Pearson correlation coefficients of oxidative stress biomarkers, IRT temperatures and footrot scores.
In the current study, interdigital space temperature was significantly higher in rams with foot lesions compared to rams without any foot lesions; IRT was able to detect elevated temperature associated with foot lesions. These finding are in agreement with those of Alsaaod and Büscher (2012), Main et al. (2012), Stokes et al. (2012) and Alsaaod et al. (2014) who observed foot temperature difference between infected and healthy cattle. Stokes et al. (2012) used the maximum foot temperature to differentiate cows with or without different feet lesions. The ROC curve presented in the current study demonstrates the presence of a strong agreement between IRT temperature thresholds for foot lesion and the scoring system used as gold standard for their diagnosis. When using a temperature threshold of 36.38 ◦ C as a critical cut-off between feet with and without lesions (Fig. 2), the observed high sensitivity (0.83) of the IRT suggests that this technique might be able to identify a considerable percentage of true positives. On the other hand, the relatively lower specificity (0.78) suggests that the temperature threshold identified a number of healthy hooves as having lesions (false positives). The positive correlation between IRT temperatures and footrot score bring further support to our approach. Therefore, IRT has potential for detecting temperature changes associated with the presence of foot lesions in rams. In this respect, the development of highly specific and sensitive cut-offs IRT temperatures is required to aid in the diagnosis, monitoring and treatment of foot lesions in sheep. The observed increase in ROMs and decrease in BAP concentration indicates the presence of increased oxidative stress in rams affected by foot lesions. Indeed, OSI was more than double in rams with foot lesions compared to healthy rams and both OSI and ROMs were positively correlated with IRT temperatures and footrot score. Plasma levels of ROMs are considered an indicator of free radical production (Miller et al., 1993) which can lead to an increase in lipid peroxidation (Halliwell, 2009). Although specific biomarkers of lipid peroxidation, such as malondialdehyde (MDA) or thiobarbituric acid reactive substances (TBARS),
BAP
4. Discussion
IRT AVG, average IRT temperature; IRT max, maximum IRT temperature; IRT min, minimum IRT temperature; ROMs, reactive oxygen metabolites; BAP, biological antioxidant potential; OSI, oxidative stress index; AOPP, advanced oxidative protein products. * P< 0.05. ** P< 0.01. *** P< 0.001.
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were not measured in this study, studies in dairy cows have observed an increase in TBARS level in lame cows compared to healthy herd mates (Al-Qudah and Ismail, 2012). Plasma antioxidant status is the result of the interaction of many different compounds and systemic metabolic interactions and it was measured with the BAP test in this study. The BAP test provides a global measurement of many antioxidants, including uric acid, ascorbic acid, proteins, ␣-tocopherol, and bilirubin (Benzie and Strain, 1996). As a single measure, the BAP test provides relevant information that may effectively describe the dynamic equilibrium between pro-oxidants and antioxidants in the plasma compartment. In sheep, it has been shown that heat stress decreases plasma BAP concentrations (Chauhan et al., 2014). The observed decrease in BAP concentration in rams with foot lesions is probably the consequence of the concomitant increase in ROMs levels. Indeed, changes in the components of antioxidants systems are often not the cause, but the consequence of the oxidative stress induced by higher free radical activity (Venditti and Meo, 2006). The observed negative correlation between BAP concentration and footrot score brings further support to the above statement. The calculated OSI values clearly indicate that rams with foot lesions had higher levels of pro-oxidants (ROMs) and decreased antioxidant capacity (BAP). These data indicate that oxidative damage, as assessed by an individual animal’s OSI (pro-oxidants/antioxidants), is indicative of their health status. Indeed, when the OSI is high (prooxidants > antioxidants) it would be reasonable to expect decreased health and wellbeing conditions for the individual animal that will result in decreased adaptation to environmental challenges. For example, when oxidative stress was monitored in dairy cows by means of ROMs and BAP, the information on oxidative stress level was more accurate when combining the ROMs and BAP data in the calculated OSI than using them separately (Pedernera et al., 2010). Moreover, increased OSI values have been observed in foals affected by respiratory diseases (Crowley et al., 2013; Po et al., 2013). Pasture-based systems can improve the oxidative status of ruminants due to the elevated antioxidant content of green grass and by providing ruminants with health benefits from certain vitamins and minerals. However, as free radicals and antioxidants are involved in several physiological functions it might be beneficial to supplement sheep with antioxidants (Celi, 2010). For example, the effectiveness of selenium in reducing oxidative stress and the severity of inflammatory events in dairy cattle has been reported (Sordillo, 2013). Selenium is an essential element in ruminant’s diet which provides a significant source of antioxidant defence and plays an important role in optimising both the immune response and disease resistance (Spears and Weiss, 2008). Indeed, it has been demonstrated that footrot affected sheep have lower blood selenium concentration and that selenium supplementation resulted in a more rapid improvement of foot lesions (Hall et al., 2009). Albumin is a free radical scavenger but it is also the predominant oxidised protein contributing to AOPP formation (Celi, 2011b). Indeed, concentrations of AOPP can be associated with embryonic losses and are considered an indicator of inflammation and oxidative stress in dairy cows (Celi
et al., 2011, 2012). The lack of differences in albumin and AOPP concentrations between HG and FL affected rams may reflect the mild level of lesions observed in this study. 5. Conclusion The data gathered in this study suggest that measuring IRT interdigital space temperatures and oxidative stress biomarkers in sheep may help in the diagnosis of foot lesions on endemic farms, however its potential as diagnostic tool needs further evaluation before the practical benefits and on-farm adoption of this technology can be considered. Longitudinal studies investigating the progression or regression of foot lesions in a larger population of affected sheep are required to determine in greater detail the association between changes in interdigital space temperatures and oxidative stress biomarkers and the severity of foot lesions. Finally, further studies should investigate the reliability of IRT among sheep kept under various environmental conditions such as air temperature, velocity, and humidity. Conflict of interest statement None of the authors are personally or professionally affiliated with other people or organisations that could inappropriately influence the content of the paper. Acknowledgments The authors are grateful to Craig Kristo for assistance in the scoring and classification of foot lesions and to the University of Sydney Poultry Research Foundation for providing the use of the infrared camera. The authors wish to acknowledge the Honda Foundation for providing financial support to purchase the FREE Carpe Diem. References Al-Qudah, K.M., Ismail, Z.B., 2012. The relationship between serum biotin and oxidant/antioxidant activities in bovine lameness. Res. Vet. Sci. 92, 138–141. Allworth, M.B., 2014. Challenges in ovine footrot control. Small Rumin. Res. 118, 110–113. Alsaaod, M., Büscher, W., 2012. Detection of hoof lesions using digital infrared thermography in dairy cows. J. Dairy Sci. 95, 735–742. Alsaaod, M., Syring, C., Dietrich, J., Doherr, M.G., Gujan, T., Steiner, A., 2014. A field trial of infrared thermography as a non-invasive diagnostic tool for early detection of digital dermatitis in dairy cows. Vet. J. 199, 281–285. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Celi, P., 2010. The role of oxidative stress in small ruminants’ health and production. Rev. Bras. de Zootec. 39, 348–363. Celi, P., 2011a. Biomarkers of oxidative stress in ruminant medicine. Immunopharmacol. Immunotoxicol. 33, 233–240. Celi, P., 2011b. Oxidative stress in ruminants. In: Mandelker, L., Vajdovich, P. (Eds.), Studies on Veterinary Medicine. Humana Press, New York, pp. 191–231. Celi, P., Merlo, M., Barbato, O., Gabai, G., 2012. Relationship between oxidative stress and the success of artificial insemination in dairy cows in a pasture-based system. Vet. J. 193, 498–502. Celi, P., Merlo, M., Da Dalt, L., Stefani, A., Barbato, O., Gabai, G., 2011. Relationship between late embryonic mortality and the increase in plasma advanced oxidised protein products (AOPP) in dairy cows. Reprod. Fert. Dev. 23, 527–533.
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