Schaefer, A. L., Cook, N., Tessaro, S. V., Deregt, D., Desroches, G., Dubeski, P. L., Tong, A. K. W. and Godson, D. L. 2004. Early detection and prediction of ...
Early detection and prediction of infection using infrared thermography1 A. L. Schaefer2, N. Cook2, S. V. Tessaro3, D. Deregt3, G. Desroches4, P. L. Dubeski2, A. K. W. Tong2, and D. L. Godson5 2Lacombe
Research Centre, Agriculture and Agri-Food Canada, PO Box 6000, C and E Trail, Lacombe, Alberta, Canada T4L 1W1; 3Lethbridge Laboratory (Animal Diseases Research Institute), Canadian Food Inspection Agency, PO Box 640, Lethbridge, Alberta, Canada T1J 3Z4; 4Public Works and Government Services Canada. #1000, 9700 Jasper Ave., Edmonton, Alberta, Canada T5J4E2; and 5Department of Veterinary Microbiology, Western College of Veterinary Medicine, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4. Contribution no. 1018, received 14 November 2002, accepted 24 October 2003. Schaefer, A. L., Cook, N., Tessaro, S. V., Deregt, D., Desroches, G., Dubeski, P. L., Tong, A. K. W. and Godson, D. L. 2004. Early detection and prediction of infection using infrared thermography. Can. J. Anim. Sci. 84: 73–80. Early detection and/or prediction of disease in an animal is the first step towards its successful treatment. The objective of this study was to investigate the capability of infrared thermography as a non-invasive, early detection method for identifying animals with a systemic infection. A viral infection model was adopted using 15 seronegative calves whose body weight averaged 172 kg. Ten of these calves were inoculated with Type 2 bovine viral diarrhoea virus (strain 24515) and five were separately housed and served as uninfected controls. A simultaneous comparison of infrared characteristics in both infected and control animals was conducted over approximately 15 d. In addition, measures of blood and saliva cortisol, immunoglobulin A, blood haptoglobin and clinical scores were obtained. Infrared temperatures, especially for facial scans, increased by 1.5°C to over 4°C (P < 0.01) several days to 1 wk before clinical scores or serum concentrations of acute phase protein indicated illness in the infected calves. The data suggest that infrared thermal measurements can be used in developing an early prediction index for infection in calves. Key words: Infection, early detection, infrared thermography, cattle Schaefer, A. L., Cook, N., Tessaro, S. V., Deregt, D., Desroches, G., Dubeski, P. L., Tong, A. K. W. et Godson, D. L. 2004. Dépistage et prévision précoces des infections par thermographie infrarouge. Can. J. Anim. Sci. 84: 73–80. Le dépistage ou la prévision rapide de la maladie chez un animal est la première étape vers la guérison. La présente étude devait vérifier l’utilité de la thermographie infrarouge comme méthode de dépistage précoce non invasive en vue de l’identification des animaux atteints d’une infection systémique. Pour cela, les auteurs ont recouru à un modèle d’infection virale constitué de 15 veaux séronégatifs de 172 kg en moyenne. Ils ont inoculé le virus de type 2 de la diarrhée des bovins (souche 24515) à dix veaux et gardé les cinq autres à part comme témoins. Ensuite, ils ont simultanément comparé les paramètres infrarouges des sujets infectés et sains pendant une quinzaine de jours. Parallèlement, ils ont dosé le cortisol dans le sang et la salive, l’immunoglobine A, l’haptoglobine sérique et noté les signes cliniques. La température infrarouge, de la face surtout, passe de 1,5°C à plus de 4°C (P < 0.01) plusieurs jours à une semaine avant qu’apparaissent les signes cliniques ou que les concentrations sériques attribuables à la phase aiguë de production des protéines virales n’indiquent l’existence de la maladie chez les veaux infectés. Les résultats donnent à penser qu’on pourrait se servir des relevés thermiques infrarouges pour mettre au point un indice de prévision rapide de l’infection chez les veaux. Mots clés: Infection, thermographie infrarouge, bovins
Infectious diseases are a significant concern to the livestock industry from the standpoint of both animal welfare and economic loss (McNeill et al. 1996). For example, it has been estimated that losses from bovine viral diarrhoea virus (BVDV) alone amount to US$10–40 million per million calvings (Houe 1999). Early detection of disease is paramount in implementing effective therapy and disease control measures, especially where animals are maintained at high stocking densities. Traditional clinical or serological examination of large numbers of cattle, as in a feedlot situation, is logistically and economically challenging, and visual surveillance alone is unlikely to be the most sensitive
means of detecting early disease. Furthermore, many of these techniques are invasive in that they require the capture and handling of animals and collection of clinical samples. Infrared thermography is a non-invasive technique for monitoring temperatures of bodies above absolute zero. This technology typically measures radiated electromagnetic energy in the 3–12 µm wavelength range. Infrared thermography has been used non-invasively to detect live animals likely to produce poor meat quality (Jones et al. 1995; Tong et al. 1995, 1997), reveal tissue composition characteristics (Schaefer and Tong 1998) and differentiate stress susceptibility in pigs of various genotypes (Schaefer
1Preliminary
Abbreviations: ADRI, Animal Disease Research Institute; dpi, days post-inoculation; HPA, hypothalamic-pituitaryadrenal; IRT, infrared thermography images
results have previously been reported in abstract form (Schaefer et al. 2000). 73
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Table 1. Clinical scores used in the current BVD induction model Clinical sign
Score
Lethargy
0 = none 1 = mild anorexia or listlessness 2 = moderate lethargy, slow to rise, anorectic 3 = recumbent 4 = death
Haemorrhage
0 = none 1 = few petechiae on mucous membranes or sclera 2 = moderate or severe petechiation or hematomas >1 cm 3 = large hematomas > 5 cm 4 = bloody diarrhea or epistaxis
Respiratory signs
0 = none 1 = clear nasal discharge or slight cough 2 = mucopurulent discharge or severe cough, slight increase in lung sounds 3 = severe pneumonia
Diarrhoea
0 = none 1 = mild or slight, < 5% dehydrated 2 = moderate, 5 to 10 % dehydrated 3 = severe and profuse, > 10% dehydrated
Modified from Cortese et al. (1998).
et al. 1989). Infrared thermography has also been used on humans and animals as a non-invasive diagnostic method for measuring physiological or pathological changes in skin temperature resulting from administration of pharmaceutical compounds, surgical procedures, changes in vascularity or blood flow, and both systemic (fever) and local (inflammatory) responses to disease conditions (Clark 1977; Schaefer et al. 1988; Spire et al. 1999; Cockcroft et al. 2000; Scott et al. 2000; Eddy et al. 2001; Heath et al. 2001). Ongoing improvements in infrared sensing technology suggest that thermography may have broader diagnostic applications but, as Head and Dyson (2001) have pointed out, more published data are needed from controlled, baseline studies on the reproducibility of thermography results. The objective of the current study was to assess the sensitivity of thermography against other clinical and diagnostic methods for the early detection of disease in calves under highly controlled conditions. The disease model used was acute BVD caused by strain 24515, a highly virulent type 2 BVDV (Carman et al. 1998). Bovine viral diarrhoea was a relevant choice because it is a significant disease problem for the cattle industry in North America and globally, and because this experimental model of acute BVD was well characterised (Cortese et al. 1998). BVDV infection can be comparatively difficult to treat but, if discovered early enough, supportive therapy can be of some benefit (Blood et al. 1983). Perhaps more importantly, the early detection of diseases such as BVD could be used in practice to isolate infected individuals prior to virus shedding, so the technology would have utility as a herd health tool. Therefore, research attempts to find ways of early detection are certainly relevant to a disease such as BVDV.
METHODS Animals and Management Animals were managed in accordance with the guidelines established by the Canadian Council on Animal Care (1993). Fifteen Angus-Hereford cross heifers, approximately 6 mo of age, from the BVDV-free herd at the Animal Disease Research Institute (ADRI), Lethbridge, Alberta, were used. These calves were weaned from the main herd and allocated to infection and control groups (10 and 5 calves, respectively); experimental groups were blocked by weight. The calves were housed in groups of five in three separate, environmentally controlled rooms in the biosafety level 3 facility at ADRI. Body weights were collected on all calves 3 wk prior to and at the end of the study. The calves were given a balanced alfalfa cube-based diet designed to provide 1.5 times maintenance requirements based on National Research Council (1984) recommendations. All calves were given ad libitum access to fresh water. A rubberised bedding mat was provided for the animals to lie on. Any debris (straw or manure) on the animals’ coats was brushed off before infrared measurements were taken. The calves were placed in their isolation rooms for acclimatisation 3 wk prior to BVDV inoculation. The infection challenge procedure consisted of a total intranasal dose of 2 × 107 TCID50 of type 2 BVDV, strain 24515 (1 mL in each nostril). Control animals were inoculated intranasally with cell culture medium only. Calves were monitored daily for signs of clinical disease. Blood samples were collected and rectal temperatures were measured on days 0, 3, 7, 10, 14, 17 and 21 d post-inoculation (dpi). Normal biosafety level 3 procedures were maintained by all personnel involved in the study as directed by ADRI staff. The trials ran for 3 wk post-inoculation or, in the case of a few animals, until the severity of illness warranted euthanasia for humane reasons. The three animal rooms used in the study were all kept at a constant temperature (approximately 14°C), humidity (28%), and barometric pressure. The calves had been adapted to these conditions prior to the study and were in their thermal neutral zone. Lighting was adjusted to simulate normal daylight and darkness (12 h light/12 h dark). Normal cleaning and maintenance procedures for the rooms established by ADRI were followed. Data Collection Infrared thermography images (IRT) on all animals were collected as follows: A hand held portable Inframetrics broadband 760 infrared scanner (FLIR Inframetrics 760, Boston, MA, USA) was used to collect images on all animals at approximately 1000 daily. Two separate infrared cameras (same model and make) were needed, one for the non-infected or clean control room and one for use in the BVDV challenge rooms. Animals were not captured at this time, but instead the infrared camera technician walked around the animals until he was able to capture the infrared thermographic image. In addition to blood sampling days (0, 3, 7, 10, 14, 17 and 21 dpi), the hand-held camera was also used daily postinoculation to collect dorsal and selective facial, lateral, and distal images. The objective of the current study was to exam-
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Table 2. Clinical score data for control and BVD infected animals BVD Pen 1 Animals
BVD Pen 2 Animals
Control Animal Number
Day
5
12
21
25
96
10
14
19
44
46
38
40
51
74
79
0 1 2 3 4 5 6 7 8 9 10
0 0 0 0 0 0 0 0 3 4 2
0 0 0 0 0 0 0 0 0 4 5
0 0 0 0 0 0 0 1 1 2 2
0 0 0 0 0 0 0 1 1 2 2
0 0 0 0 0 0 1 1 2 3 8
0 0 0 0 0 0 0 0 0 2 3
0 0 0 0 0 0 0 0 1 1 1
0 0 0 0 0 0 0 0 1 1 1
0 0 0 0 0 0 0 0 0 1 1
0 0 0 0 0 0 0 0 0 2 4
0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
ine the capability of IRT to detect BVDV infection during the early and developing stages of the disease. As evident in the clinical score data, the infected calves displayed the highest clinical scores by 11 dpi. Therefore, the IRT and other data are presented for 0–12 dpi. To ensure integrity of the thermal data, the following conditions were standardised in the current study: All animals and/or specific animal images were scanned from the same distance, approximately 1–3 m. The following environmental factors were controlled: Extraneous sunlight (animals were scanned under cover); varying environmental temperatures (room temperature was held constant); exercise (the animals were at rest and not under the influence of recent exercise); feeding (the animals were scanned post-prandially); circadian rhythm effects (animals were scanned at the same time of day). In addition, any extraneous debris (such as straw) was removed from the animals and the images were taken from dry surfaces. The output from the infrared scanner was an uncalibrated, digitised image made up of pixel points (300 × 180), each of which contains temperature data. The digitised image was saved in a greyscale graphics file prior to analysis using image analysis software [NIH Image (Macintosh) and Scion ImageJ (PC), National Institutes of Health, Research Services Branch, Bethesda, MD, USA]. Images were calibrated so that each shade of grey on the digitised image corresponded to a specific temperature. The relative surface temperature represented by each pixel was assigned a numerical value ranging from 0 to 255. These pixel values were then mapped to degrees Celsius by relating them to the following formula: Actual temperature = [[(max temp setting – min temp setting) × pixel value] / 256] + minimum temperature setting For computer analysis and human interpretation, pseudo colours were generated and added to the pixels (Fig. 2). The pixel points of thermal information were processed by appropriate computer software to generate mean temperatures over a given area of an anatomical structure. An orbital image was obtained by tracing an oval image over the orbital area, which included the eyeball itself plus approxi-
mately 1 cm surrounding this area. A square image of the nose was obtained by tracing an area approximately 1 cm2 on the hairless frontal surface on the nose. A square image of the ear traced an area approximately 1 cm2 on the midventral surface of the ear. A rectangular image of the dorsal surface of the calf traced an area approximately 20 cm by 40 cm on the mid-back surface. Both area mean and maximum temperatures were used. In addition to the infrared measurements, clinical and physiological measurements were taken on these same “bleed” days (0, 3, 7, 10, 14, 17 and 21 dpi). Calves were captured in a head gate and 10 mL of blood was collected by venous puncture. These samples were used for determination of differential blood cell counts via blood smear staining and direct microscope examination, cortisol concentration was measured by radioimmunoassay (Cook et al. 1997), IgA concentrations was measured by ELISA (Bethyl labs. Montgomery, TX) as well as basic haematology parameters (Cell-Dyn 700 hematology analyser, Sequioir – Turner Corp. Mountain View, CA). Serum antibody levels to BVDV were detected by a serum neutralisation test (Deregt et al. 1992). Antibody titres were recorded as the reciprocal of the highest dilution of serum that completely neutralised 50 TCID50 as determined by immunoperoxidase staining. Virus in serum and leukocytes was detected essentially as described previously by an immunoperoxidase test (Tessaro et al. 1999; Deregt and Prins 1998). Clinical scores were given daily for all calves using a clinical scoring system which was modified from that of Cortese et al. (1998) (Table 1). Serum haptoglobin concentration was determined using a monoclonal antibody-based capture ELISA procedure (Godson et al. 1996). The sensitivity of this assay was 15 µg mL–1. In most healthy cattle, the serum haptoglobin concentration would be below this level. For the current trial, no antibiotic treatment was given to any calf. All animals were humanely euthanised toward the end of the disease course at a time determined by ADRI staff. Necropsies were performed on all animals. Statistics Conventional parametric analysis of both thermographic and biological data was conducted by least squares analysis
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Table 3. Orbital infrared and rectal temperatures in BVD-infected calves. Data for the mean and max orbital temperature are the least squares means ± SE of the least squares means Day post BVDV induction
BVDV mean IR temp.
BVDV mean SE
BVDV max IR temp.
BVDV max SE
0 1 2 3 4 5 6 7 8 9 10
31.22a 32.11b 32.26bc 32.30bc 32.54bc 32.66bc 32.79bc 33.31bc 33.51bc 33.35bc 33.79bc
0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15
34.77a 35.59b 35.59bc 35.67bc 35.75bc 36.02bc 36.10bc 36.86bc 37.05bc 36.70bc 37.20bc
0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34
BVDV rectal temp.
BVDV rectal temp. SD
39.12a
0.61
39.58b
0.61
40.36b
0.61
40.37b
0.61
a–c Means within columns with different letters are statistically different at P < 0.05.
and t-tests (Steel and Torrie 1960). The authors would note that in the present study, one of the primary reasons for using an induction model was to know precisely when disease induction occurred. Thus, for many of the comparisons used in the present study, the animal was used as its own control. For the infrared thermal data pertaining to the orbital, nose, ear and dorsal surfaces, treatment means were compared using the GLM model (SAS Institute, Inc. 1990). RESULTS Infrared Thermography Thermographic scans were collected from several anatomical sites on the calves including the side (lateral), back (dorsal) and hooves, as well as several facial areas, such as the ears (midventral area of ear), nose (centre of hairless nose area) and eye, which included the eye per se, plus approximately 1 cm of surrounding skin to include the lachrymal gland plus the tear duct. The change of temperatures in these areas, or the delta T values, for the BVDV-inoculated calves over the first 10 d of the trial were as follows: nose, 3.5°C or 0.35°C per day; ear, 3.9°C or 0.4°C per day; side, 1.9°C or 0.19°C per day; dorsal, 1.8°C or 0.18°C per day. For all of the above temperature sites, the changes were significantly different (P < 0.05) from the preinoculation temperatures at approximately 4–5 dpi. All inoculated animals responded to the infection. Notable in the present study was the observation that the orbital temperatures in the BVDV-inoculated calves (Table 3) displayed the earliest, most consistent, least variable and sensitive increases in temperature over the first 10 d of the trial or until the clinical scores indicated a physical response to the disease. This pattern is shown in Fig. 1. Quantitatively, this amounted to a total temperature change of 2.6°C or 0.26°C per day. Of interest also was that these changes in orbital temperature were significantly different from the pre-inoculation levels as early as day 1 (P < 0.05). Furthermore, orbital temperatures tended to be less variable (lower SD) than temperatures from other anatomical areas (Tables 4 and 5). The rectal temperatures in the BVDV-inoculated calves (Table 3) also indicated fever due to infection. Similar changes did not occur in the control animals. Orbital mean delta T value for control animals from days 0 to 10 was
Fig. 1. BVD infected calves mean orbital infrared temperature (°C).
0.4°C (32.0°C on day 0 ± 0.53°C to 31.6°C on day 10 ± 0.61°C) (P > 0.05). For control animals, there were no clear patterns in orbital infrared temperature values among days. Numerically, and even statistically, some day-to-day variation was observed in control animal orbital temperature [day 0, 32.0a; day 1, 30.54b; day 2, 33.62b; day 3, 32.64b; day 4, 31.90a; day 5, 31.54a; day 6, 31.82a; day 7, 31.33a; day 8, 31.47a; day 9, 30.57b; day 10, 31.64a. (a, b means with different letters are significantly different at P < 0.05)]. However, the few statistically significant differences that occurred in control animal orbital radiated temperatures, unlike the BVD treatment animals, appeared to be random and inconsistent. Rectal temperatures for control animals were relatively constant over the course of the trial (39.13 ± 0.26°C). Clinical Scores, Serology and Endocrine Data The criteria used to establish clinical scores are shown in Table 1. As evident in Table 3, the BVDV-inoculated calves displayed elevated clinical scores at 8–9 dpi, which reached a maximum at 11 dpi. Virus was isolated from three BVDVinoculated calves on 3 dpi and from all BVDV-inoculated calves on 7 dpi. Neutralising antibodies against BVDV were first detected at 10 dpi. The control calves remained nega-
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Table 4. Infrared radiated temperatures for the nose, ear and dorsal areas of BVD-infected calves . Data are the least squares means ± SE of the least squares means Day postBVD induction
Nose mean IR temp
0 1 2 3 4 5 6 7 8 9 10
29.21a 29.34a 29.43a 29.96a 30.73a 30.88b 31.25b 32.69bc 32.90bc 32.83bc 32.77bc
Nose mean SE
Ear mean IR temp.
Ear mean SE
0.41 0.40 0.40 0.40 0.41 0.40 0.40 0.41 0.40 0.41 0.40
22.38a 22.42a 23.29a 23.19a 23.79a 24.11ab 24.60ab 24.87ab 25.37b 23.93ab 26.28b
0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96
Dorsal mean IR temp 22.29a 22.74a 23.04ab 22.44a 23.08ab 22.70a 22.75a 22.95ab 23.54b 23.06b 24.08b
Dorsal mean SE 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34
a–c Means within columns with different letters are statistically different at P < 0.05.
Table 5. Serum cortisol, salivary IgA and serum haptoglobin levels for control and BVD infected animals Cortisol (nmol L–1) Day 0 3 7 10
Salivary IgA (mg mL–1)
Haptoglobin (µg mL–1)
Control
BVD
Control
BVD
Control
BVD
85.7a 70.3 84.7 77.7
115.2b 93.7 96.0 83.7
5.90 1.24a 3.03 3.54
6.34 4.93b 6.25 5.42
