WO2000057163A1 - Early detection of inflammation and infection using infrared thermography - Google Patents

Abstract

The present invention provides a method for the detection of inflammation in animals using infrared thermography. The invention also provides a method for the detection of diseases or disorders that induce inflammation using infrared thermography. The present invention further provides a method for the detection of infections in animals using infrared thermography.

Description

EARLY DETECTION OF INFLAMMATION AND INFECTION USING INFRARED THERMOGRAPHY

This is a continuation-in-part o U S patent application Serial No 09/274,032. filed _. March 22, 1999, which is incorporated herein, by relerence, in its entirety

1. FIELD OF THE INVENTION

The invention relates to the use ol intrared thermography imaging in animals for the early detection of inflammation The invention further relates to the use ol intrared ι thermography in animals for the early detection ot infection

2. BACKGROUND OF THE INVENTION

Inflammation plays a fundamental role in host defenses and the progression of immune-mediated diseases The inflammatory response is initiated in response to tissue j5 injury (e g , trauma, ischemia, and toreign particles) and infection by a complex cascade of events, including chemical mediators (e g , cytokines and prostaglandins) and inflammatory cells (e g , leukocytes) The inflammatory response is characterized by increased blood flow, increased capillary permeability, and the influx ot phagocytic cells These events result in swelling, redness, warmth (altered heat patterns), and pus lormation at the site ol

20 ^mi^uf

A delicate well-balanced interplay between the humoral and cellular immune elements in the inflammatory response enables the elimination ot harmful agents and the initiation ot the repair ol damaged tissue When this delicately balanced interplay is disrupted, the inflammatory response may result in considerable damage to normal tissue and

25 may be more harmful than the original insult that initiated the reaction In these cases of uncontrolled inflammatory responses, clinical intervention is needed to prevent tissue damage and organ dysfunction Diseases such as Rheumatoid Arthritis. Osteoarthπtis. Crohn s disease, psoriasis, or inflammatory bowel disease, are characterized by chronic inflammation

3Q Early detection and localization of inflammation is a critical step in the implementation ol appropriate treatment ol a subject However, non-invasive techniques for the detection ol inflammation remain elusive A variety of techniques including computed tomography (CT), magnetic resonance imaging (MRI). ultrasonography, and scintigraphic imaging are used to attempt to image secondary effects or markers ot inflammation

25 However, CT. MRI. and ultrasonography rely on anatomical changes that result Irom inflammation, which occur late in the inflammatory response (van der Laken. C J . et al , 1998, European Journal of Nuclear Medicine 25 535-546) Therefore, these techniques are not useful for detecting the early phase in the development of inflammation Scintigraphic imaging is a non-invasive method ot scanning the entire body using radiopharmaceuticals (e g , radio labeled receptor-specific small proteins and peptides), which specifically bind to receptors abundant in the area of inflammation The use ot radiopharmaceuticals for imaging inflammation is limiting because it reqimes (1) that the radiopharmaceutical specifically interacts with its receptor, (n) that the radiopharmaceutical has a high affinity tor its receptor, (in) that the radiopharmaceutical specifically localizes to the site of inflammation, which is dependent on the receptor expression in the inflammatory response; (iv) that the receptor is accessible to the radiopharmaceutical. (v) that the radiopharmaceutical has high and early uptake, (vi) that the radiopharmaceutical is rapidly cleared, (vu) that the radiopharmaceutical does not accumulate in non-targeted tissues and result in high background, and (vin) that the radiopharmaceutical is not toxic (van der Laken, C J , et al , 1998, European Journal of Nuclear Medicine 25" 535-546) The induction of a biological response by a radiopharmaceutical is a maior drawback ot using scintigraphic imaging In addition to these technologies, inflammation may also be detected by teehng or visual observance of the site of miury or pain However, this method is only useful for detecting the late stages in the development of inflammation

The inability to diagnose and image inflammation in vivo continues to be a maior obstacle to the successful treatment of inflammatory disorders Currently, the only viable method for diagnosing inflammatory disorders, such as fibrosis, is by biopsy This method is invasive and often results in an amount of healthy tissue being removed along with the tissue suspected ot being affected by inflammation Therefore, a great need exists for an accurate, non-invasive, rapid, and inexpensive method for detecting inflammation

2.1. INFECTIOUS DISEASES

Viral and bacterial infections typically result in the development of local or systemic inflammation and catabolism of tissues at the site of infection The inflammatory response to an infection whether acute or chronic is often tissue or organ centered and as such is characterized by increased blood flow and white blood cell activity (i e , phagocytic cell activity) in affected areas The appearance of localized swelling, discoloration and tissue debris are often apparent and significant tissue damage can result

Early detection of viral and bacterial infections is important not only for the implementation of appropriate treatment ot a subject but also for the prevention of the spread ot the infections A variety ot methods are available for the detection and clinical diagnosis of viral and bacterial infections, including lmmunologic methods, which detect the presence ot viral or microbial antigens or antibodies specific to a virus or microbe A variety ot

9 - immunological assays are available for detecting viral or microbial antigens or antibodies specific to a virus or microbe, including ELISAs (enzyme linked immunosorbent assays), solid-state radioimmunoassays, and immunofluorescent assays. However, immunological assays for detecting viral or bacterial infections require a laboratory and someone with technical expertise to perform the assays. Further, the biological samples required to perform immunological assays are not easily obtained from an animal. Additionally, the immunological assays are too costly for individual or sporadic infections and are generally not performed until clinical symptoms have manifested. Therefore, a need exists for a simple, rapid, non-invasive and inexpensive diagnostic technique for the early detection of viral and microbial infections.

2.2 MASTITIS

Mastitis is an inflammation of the mammary gland normally caused by a bacterial or mycotic pathogen. The disease is of great concern in the dairy industry, where significant economic loss can occur due to the requirement to not use the affected milk for human consumption and due to the shortened milking life of the affected animals. The etiology of the disease is well described in the literature pertaining to this topic, e.g., see, Siegmund et al., 1973, The Merk Veterinary Manual 4^th ed., Merck and Comp. Rathway. N.J.; Blood et al.,

1983, Veterinary Medicine 6^th ed., Bailliere Tindall , London. The successful treatment of mastitis is possible using a variety of animal management, milking hygiene and antibiotic agents. However, given the expense and labour for the treatment of mastitis, treatment is usually not initiated until the condition is diagnosed clinically.

Numerous mastitis tests have also been proposed, including most recently the use of electrical conductivity of the milk (Notsuki et al, 1983, Proceedings of the World

Conference on Animal Production Vol 2., 891-892; Datta et al., 1984, Transactions of the American Society of Agriculture Engineers 27: 1204-1210; Batra, T.R. and McAllister, A.J.,

1984, Canadian J. Anim. Sci. 64:305-312; Maatje, K. and Rossing, W., 1991, Mastitis Newsletter 16:6-7; Lake et al., 1991. J. Dairy Sci. 59: 11-19: Biagetti, D.R., 1992. Rivista- di-Ingegneria Agraria 23:200-207; Nielsen et al., 1992, J of Dairy Sci. 75: 606-614; Tongel et al., 1994, Proceedings 3^rd International Dairy Housing Conference. Orlando. Florida, 257- 262). In addition to electrical conductivity, the use of milk components have been suggested as good indicators of mastitis, including such elements as sodium, chloride, potassium, lactose and bovine serum albumin (BSA) (Fernando et al., 1985, J. Dairy Sci. 68: 449-456), milk temperature (Datta et al., 1984, Transactions of the American Society of Agriculture Engineers 27: 1204-1210; Rossing et al.. 1984. Proceedings of the National Conference

- 3 - American Society of Agricultural Engineers. Chicago. 606-613: Jarman et al., 1986, J. Dairy Sci. 69:(suppl 1.) 178), milk pH (Mijnen et al, 1983. Netherlands Milk and Dairy Journal 37:65-77), milk anti-trypsin (Manila et al. 1985. J. Dairy Sci. 68: 114-122) as well as general milking information such as volume or yield (Nielsen et al. 1994, Veterinary Research 25:285-289). Numerous patents have been issued describing the methods of mastitis detection, particularly for the use of electrodes or a variety of electrical conductivity tests for milk (U.S. Patent No. 3.989,009: U.S. Patent No. 3,968.774: U.S. Patent No. 4,156.179; Australian Patent Application AU A178 553/81 ; U.S. Patent No. 5,302,903; U.S. Patent No. 5,416,417). All of these aforementioned procedures can be useful. However, none are particularly effective at early detection (e.g., within the first few hours) of mastitis onset and, as described by Batra and McAllister (1984), these aforementioned procedures often have an unacceptably high percentage of false negatives (i.e.. failure to identify an infected cow). For example, electrical conductivity is reported to have a 29.4% false negative value and is also shown to be unreliable unless selective milk samples are used (Noksuki et al., 1983, Proceedings of the World Conference on Animal Production Vol 2., 891-892).

Mastitis is currently detected predominantly by the use of inflammatory tests such as the "Wisconsin Mastitis Test'^' or CMT, which as described by Siegmund (1973, page 817) is a rather time consuming laboratory type diagnostic method which will indicate the relative leukocyte or somatic cell count in the milk of cows suspected of having mastitis.

Unfortunately, these types of tests are not particularly effective in detecting the earliest onset or subclinical cases of mastitis. Furthermore, the need to capture the animal and collect milk samples complicates the use of this method. These factors are important in that the earlier the mastitis condition can be detected, the earlier treatments can begin and the higher the likelihood of successful treatment in a shorter period of time.

As mentioned previously, these tests have in common the requirement of collecting and analyzing milk samples from animals suspected of having mastitis. Clinical diagnosis of the infected animal is also routinely conducted. However, clinical signs of mastitis usually do not occur until the animal has progressed well into the disease state. Furthermore, some diagnostic tools, such as rectal temperature, while usually efficacious, are often not as sensitive as would be desired or are simply impractical. Again, it should be noted that the earlier a diagnosis can be performed, the earlier treatment can be initiated, which results in a lower treatment cost and a more successful outcome. Therefore, there remains a need for an accurate, inexpensive, non-invasive, rapid method for predicting early mastitis onset in dairy animals. 2.3. BOVINE VIRAL DIARRHEA

Bovine virus diarrhea (BVD) virus is a pestivirus that is characterized by erosions and hemorrhages of the alimentary tract (Siegmund. O.H., 1973, The Merck Veterinary Manual. Merck and Co. Inc. Rathway. NJ: and Blood et al., 1983, Veterinary Medicine.

5 Baillere. Tindall. London). Type land type 2 strains as well as subgroups of BVD virus have been identified. Animals infected with BVD virus typically exhibit anorectic conditions, rumen stasis, temperature elevations and diarrhea between days 4 and 10 postinfection. Type 2 BVD virus is associated with higher levels of gastrointestinal tract hemorrhage, morbidity and mortality than type 1BVD virus.

10 BVD is readily transmitted by oral contact and is present in the bovine populations of most countries. BVD is a significant problem in North American cattle populations, causing high morbidity and mortality especially in veal, dairy and beef populations (Cortese et al., 1998, J. Am. Vet. Med. Association 213: 1312-1319). Further, the ability to obtain a reliable vaccine has remained elusive (Cortese et al., 1998, supra).

15 BVD is currently detected and diagnosed by immunological assays such as serum neutralization assays and serum immuno-diffusion assays. A clinical scoring test is also frequently used to describe or rank the severity of the disease progression and symptoms (see. e.g., Blood et al, 1983, supra: and Cortese et al, 1998, supra). The immunological assays are laborious, time consuming and expensive, and require the collection of a

20 biological sample. Thus, there remains a need for an inexpensive, non-invasive, accurate and rapid method for the detection of an infectious disease such as BVD.

2.4. INFRARED THERMOGRAPHY

Infrared thermography is a non-invasive technique that enables temperatures to be 25 monitored and recorded. Unsuccessful attempts have been made to use infrared thermography in human medicine as a diagnostic aid for a variety of conditions, such as tumor detection and cardiovascular disease (Clark, J.A. and Cena, K., 1972, J. of Mammalogy 54:1003-1007). Infrared thermography has been attempted in veterinary medicine to detect and diagnosis a variety of conditions, such as podotrochlosis in horses (Turner,T.A., 1983, Am. J. Vet. Res. 30 44:535-539) and clinical damage in an udder (Tsykalo, NL. et al., 1982, USSR (7):49-50) . The early infrared thermography detection systems were bulky, complex, and required frequent recharging with liquid nitrogen. Furthermore, the spatial resolution was poor, the exposure time was long, and the minimum resolvable temperature difference was large for the infrared thermography systems. Reliable detection of inflammation was not achieved. In 35 addition, many physicians and veterinarians were not adequately trained to interpret the data from the infrared imagery and there was a high false positive rate. Thus, the infrared

5 - thermography was branded as a failure and has not been explored much by the medical or veterinary communities for the past three decades.

3. SUMMARY OF THE INVENTION The present invention provides a method using infrared thermography for the detection of inflammation in animals. The invention also provides a method using infrared thermography for the diagnosis of diseases or disorders that induce inflammation such as inflammatory disorders, allergies, and viral or bacterial infection. The invention further provides a method using infrared thermography for the detection of an infection in an animal. In particular, the present invention provides for the detection of an infection in an animal by measuring temperature changes resulting from the animal's immune response to the infection using infrared thermography. The catabolism of tissue and the inflammatory response induced in response to an infection in an animal both generate temperature changes which can be measured using infrared thermography. The present invention is based, in part, on the surprising discovery that temperature differences less than 1 °C are clinically significant. This discovery was made possible by employing induction models of mastitis and BVD that allowed the Applicants to evaluate inflammation or infection resulting from known etiologies and to compare the infrared characteristics obtained using an infrared camera with outcomes obtained with other diagnostic procedures. Accordingly, Applicants' discovered that temperature differences less than 1 °C indicate early or subclinical inflammation or infection, and that temperature differences greater than 1 °C indicate later stages of development of inflammation or clinical infection.

4. DESCRIPTION OF THE FIGURES

Figure 1 is a graph of rectal temperature and udder infrared thermography values for milking dairy cows having mastitis induced in the left distal quadrant (n=20). Data for both the left and right distal quarters of the udder are shown.

Figure 2 is a graph of Nagase (N-acetyl-beta-D-glucosaminidase) and udder infrared thermography values for milking dairy cows having mastitis induced in the left distal quadrant (n=20). Data for both the left and right distal quarters of the udder are shown.

Figure 3 is a graph of BSA (Bovine Serum Albumin) and udder infrared thermography values for milking dairy cows having mastitis induced in the left distal quadrant (n=20). Data for both the left and right distal quarters of the udder are shown. Figure 4 is a graph of somatic cell count and udder infrared thermography values for milking dairy cows having mastitis induced in the left distal quadrant (n=20). Data for both the left and right distal quarters of the udder are shown.

Figure 5 is a graph of image area (pixels) for the left and right distal quarters of the udder in milking dairy cows having mastitis induced in the left distal quadrant (n=20).

Figure 6 is a graph is of rectal temperature and udder infrared thermography values for a milking dairy cow (n=l) having mastitis induced in the left distal quarter. Data for both the left and right distal quarters of the udder are shown.

Figure 7 is a graph of NAGase and udder infrared thermography values for the animal of Figure 6. Data for both the left and right distal quarters of the udder are shown.

Figure 8 is a graph of BSA and udder infrared thermography values for the animal of Figures 6 and 7. Data for both the left and right distal quarters of the udder are shown.

Figure 9 is a graph of total temperature values (mean udder temperature x image area) for milking dairy cows having mastitis induced in the left distal quadrant (n=20). Data for both the left and right distal quarters of the udder are shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of infrared thermography for the early or subclinical detection of inflammation in animals. The present invention also relates to the use of infrared thermography in the diagnosis of diseases or disorders that induce inflammation and/or induce the catabolism of tissues. The present invention provides methods for detecting inflammation of an anatomical structure of an animal, preferably a mammal and more preferably a non-human animal. The present invention further provides methods for detecting infection of an anatomical structure of an animal, preferably a mammal. In one embodiment, the present invention provides methods for detecting infection of an anatomical structure in a non-human animal. In yet another embodiment, the present invention provides methods for detecting infection in humans. The term "anatomical structure'^' used herein refers to any definable area of an animal, preferably a tissue or a joint of an animal, that radiates infrared energy and which may or may not be symmetrical. The invention provides methods for detecting inflammation of all anatomical structures of animals, except the joints. The present invention also provides methods for detecting inflammation of the joints of all mammals, except humans. The invention also provides methods for detecting inflammation or infection in all non-human mammals, including but not limited to pigs, horses, cows (e.g.. Bos taunts and Bos indicus), dogs and cats. The present invention also provides methods for detecting local or systemic infection in animals, preferably a mammals. Further, the present invention also provides methods for detecting acute or chronic infection in animals, preferably a mammals.

The invention provides a method for detecting inflammation of an anatomical structure of an animal, comprising the following steps: (i) obtaining an infrared thermographic image of an anatomical structure of an animal: (ii) determining the mean temperature of the infrared thermographic image; and (ϋi) detecting early or subclinical inflammation of an anatomical structure of an animal if there is a change in the mean temperature of less than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of the same animal or a population of animals of the same species obtained from infrared thermographic images taken when there was no inflammation of the anatomical structure. The term "'subclinical" as used herein refers to inflammation of an anatomical structure of an animal that has not manifested itself clinically.

The invention also provides a method for detecting inflammation of an anatomical structure of an animal, comprising the following steps: (i) obtaining an infrared thermographic image of an anatomical structure of an animal; (ϋ) determining the mean temperature of the infrared thermographic image; and (ϋi) detecting late stage development of inflammation of an anatomical structure of an animal if there is a change in the mean temperature of greater than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of the same animal or a population of animals of the same species obtained from infrared thermographic images taken when there was no inflammation of the anatomical structure.

The invention also provides a method for detecting inflammation of an anatomical structure of an animal, comprising the following steps: (i) obtaining an infrared thermographic image of an anatomical structure of an animal after an event: (ϋ) comparing the infrared thermographic image obtained to infrared thermographic images of the same anatomical structure of the same animal or a population of animals of the same species prior to the event; and (ϋi) detecting mflammation of the anatomical structure of the animal if there is a relative difference in the temperature of the anatomical structure of the animal. The term "event" as used herein refers to any activity that may result in inflammation of an anatomical structure of an animal, including surgery.

The present invention provides a method for detecting inflammation of an anatomical structure of an animal, comprising the following steps: (i) obtaining an infrared thermographic image of an anatomical structure of an animal: (ϋ) obtaining an infrared thermographic image of the symmetrical anatomical structure of the animal: (ϋi) determining the total temperature of the infrared thermographic images for the symmetrical anatomical structures: and (iv) detecting mflammation of an anatomical structure if the total temperature

8 - ot the symmetrical anatomical structures differ by greater than a predetermmed amount The term "symmetrical anatomical structure^"' as used herein refers to an anatomical structure that has symmetry to another anatomical structure of an anmial (e g , one leg compared to another leg of an animal). The mvention also provides a method for detectmg mflammation ot an anatomical structure of an animal, compnsmg the following steps: (1) obtaining an mfrared thermographic image ot the anatomical structure of an anmial: (11) obtammg an mfrared thermographic image ot the symmetrical anatomical structure ot the animal; (in) comparmg the infrared thermographic image obtamed to an mfrared thermographic image of the symmetrical anatomical structure ot the animal; and (iv) detectmg mflammation of the anatomical structure of the animal if there is a relative difference in the temperature between the anatomical structure and the symmetrical anatomical structure of the animal.

The present mvention also provides a method for detectmg when a clinical treatment for treatmg mflammation of an anatomical structure ot an anmial was successful, comprising the following steps: (I) obtammg an mfrared thermographic image ot the anatomical structure of the animal; (u) determining the mean temperature of the mfrared thermographic image; and (m) detectmg the successful treatment of mflammation of the anatomical structure by comparmg the mean temperature ot the anatomical structure with the mean temperature of the same anatomical structure obtamed from the same animal or a population of animals ot the species when healthy.

The present mvention also provides a method for detectmg an infection m animal compnsmg the following steps: (1) obtammg an infrared thermographic miage ot the anatomical structure or a portion thereof of the animal: and (u) detectmg early or subclinical infection of said animal if there is a change in the mean temperature of less than 1 °C relative to the mean temperature of the same anatomical structure m the same anmial pre-mfection or relative to the mean temperature of the same anatomical structure m a population of unmfected animals of the same species, background and class. In preferred embodiments of the present mvention, the anatomical structure of an animal imaged to detect infection is the eye or the nose (i.e.. a smus) The present mvention also provides a method for detecting an infection in an animal compnsmg the following steps: (I) obtammg an mtrared thermographic image of the anatomical structure or a portion thereof ot the anmial. and (n) detectmg clinical mfection of said animal if there is a change m the mean temperature of greater than 1 °C relative to the mean temperature ot the same anatomical structure in the same anmial pre-infection or relative to the mean temperature of the same anatomical structure m a population of unmfected animals ot the same species, background and class

- 9 - The present invention also provides a method for detectmg when a clinical treatment for treating an mfection in an animal was successful, comprising the following steps: (i) obtaining an infrared thermographic image of the anatomical structure of the anmial: and (ϋ) detecting the successful treatment of the infection by comparing the mean temperature of the anatomical structure of the animal to the mean temperature of the same anatomical structure of the same animal preinfection or a population of uninfected animals of the same species.

The present invention provides a method for detecting a local infection of an anatomical structure of an animal, comprising the following steps: (i) obtaining an infrared thermographic ϋnage of an anatomical structure of an animal: (ϋ) obta iing an mfrared thermographic image of the symmetrical anatomical structure of the animal; (ϋi) determining the total temperature of the mfrared thermographic images for the symmetrical anatomical structures: and (iv) detecting a local mfection of an anatomical structure if the total temperature of the symmetrical anatomical structures differ by greater than a predetermined amount. The invention also provides a method for detecting a local mfection of an anatomical structure of an animal, comprising the following steps: (i) obtammg an infrared thermographic image of the anatomical structure of an animal; (ϋ) obtaining an mfrared thermographic image of the symmetrical anatomical structure of the animal; (ϋi) comparing the infrared thermographic image obtained to an infrared thermographic image of the symmetrical anatomical structure of the animal; and (iv) detecting infection of the anatomical structure of the animal if there is a relative dϋ erence in the temperature between the anatomical structure and the symmetrical anatomical structure of the animal.

5.1 INDUCTION MODEL OF MASTITIS The present invention is based upon the surprising discovery that temperature differences less than 1 °C are clinically signϋicant. This discovery was made possible, in part, by employing an induction model of mastitis, which displays a known etiology, such that infrared thermal expression could be compared to known outcomes. The use of the induction model has many advantages including: (i) the inflammatory agent is known both in quantitative and quaϋtative terms; (ϋ) the exact time of the onset of mflammation is known; and (ϋi) the exact stage or progression of the inflammation is known. Furthermore, due to the unique anatomy of the udder of a cow, the progression of an infected quarter can be compared to a non-infected quarter. The udder of a dairy cow is unique in that all four quarters are essentially independent in terms of their vascular supply (Sisson. S., The Anatomy of the Domestic Animal. W.B. Saunders Comp.. Phϋadelphia. 4^th ed. Revised by J.D. Grossman, page 618). such that inflammation induced in one quarter of the udder through the use of a mastitis induction model does not affect any other quarter of the udder. Hence, the animal can act as its own control.

Briefly, in achieving the mvention, one quarter of the udder of a test population of lactating dairy cattle was infected with Escherichia coli (E. coli) endotoxin and the time course of the resulting inflammation was followed for several days using a variety of analytical tools, including infrared thermography. Over a 72 hour time course, milk samples were obtained from the left (induced) and right (non-induced) distal (hind) quarters of^" the udder and analyzed for objective indicators of mflammation by conventional analytical procedures. Contemporaneously with the milk samples, mfrared thermographic images of the Q cows were obtamed, so that the infrared thermal expression of^" the animal could be monitored over the course of the induced mflammation.

It was found that within hours after induction of inflammation, significant changes in the thermal expression of^" the cows could be detected with infrared thermography. This was surprising, in that, as discussed previously, conventional thought would dictate that any temperature changes occurring in subclinical cases of mastitis would be too subtle to detect. Moreover, these changes in thermal expression were observed in all cows in which mflammation was induced, indicating that altered thermal expression, as detected by infrared thermography, is a reϋable indicator of inflammation. Significant changes in infrared thermal expression included: (i) a temperature increase; (ϋ) a more rapid rate of temperature change; 0 and (ϋi) swelling of the affected quarter of the udder, resulting in a reduction in the symmetry of the thermal expression between the udder quarters with the affected quarter being both hotter and larger. In the present mvention, one or more of these changes, detected by mfrared thermography, is used to diagnose mflammation.

In one embodiment of the present invention, mastitis in a mammal is detected by: 5 (i) obtaining an infrared thermographic miage of^" a mammary gland of^" said mammal, said infrared thermographic image providing temperature information about said mammary gland; and, (ϋ) identϋying said mammal as having a high probabϋity of having mastitis if a measure of said temperature information is greater than a predetermined value by at least a predetermined amount. In another embodiment of the present invention, mastitis in a mammal 0 having an udder is detected by: (i) obtaining an infrared thermographic ϋnage of one quarter of the udder of^" said mammal at time 0. said mfrared thermographic image providing temperature information about said udder quarter of said mammal; (ϋ) obtaining an infrared thermographic image of the same quarter of the udder of said mammal at a later time, said infrared thermographic image providing temperature information about said udder of said 5 mammal: (ϋi) determining a total temperature for a first image, said first image correspondϋig to said quarter of the udder of said mammal at time 0: (iv) determinϋig a total temperature for a second ϋnage, said second image correspondϋig to said quarter of the udder of^" said mammal at a later time: and (v) identifying said mammal as having a high probabϋity of having mastitis if the total temperature for said first image differs from the total temperature for said second image by greater than a predetermmed amount. In yet another embodiment of the present invention, mastitis in a mammal having an udder is detected by: (i) obtaining images of the two frontal quarters or two rear quarters of the udder of^" said mammal; (ϋ) determinϋig the total temperature of a first image, said first image corresponding to one frontal quarter or one rear quarter of^" the udder of^" said mammal; (ϋi) determining the total temperature of a second image, said second ϋnage correspondϋig to the other frontal quarter or the other rear quarter of the udder of said mammal; and (iv) identifying said mammal as having a high probabϋity of having mastitis if the total temperature of said first image differs from the total temperature of said second image by greater than a predetermined amount.

5.2. INDUCTION MODEL OF BVD VIRUS TYPE 2 The present mvention is based, in part, on the surprising discovery that mean temperatures less than 1 °C obtamed using mfrared thermography are indicative of an infection. This discovery was made possible by employing an induction model of a viral infection displaying a known etiology such that infrared thermographic expression could be compared to known outcomes. Briefly, a population of BVD and infectious respiratory disease (IBR) seronegative calves were infected intranasally with BVD type 2 virus (2xl0⁷ TCID₅₀ of type 2 strain 24515) and the t ne course of the resulting mfection was followed for approximately three weeks. A variety of laboratory tests and clinical scoring procedures were used including infrared thermography. In addition, a group of contemporary, uninfected calves were studied simultaneously. Biological samples (i.e., blood and sahva samples) and mfrared images were obtamed from infected and uninfected calves about every second day postinfection.

StatisticaUy significant changes in the mean temperature of anatomical structures (e.g., the eye and nose) of BVD virus infected animals were detected using mfrared thermography as early as 1 day post-infection and that such changes were often of^" magnitudes less than 1 °C. Further, the BVD virus mfection was detected using infrared thermography several days to one week before it was detected using laboratory tests for objective measurements of, for example, acute phase proteins such as haptoglobin, and before it was detected using conventional clinical scores.

In one embodiment of the present mvention, infection in an animal is detected by: (i) obtaining an mfrared thermographic image of an anatomical structure of said animal, said infrared thermographic miage providing temperature information about said anatomical structure; and (ϋ) identifying said animal as having a high probabϋity of having an infection if a measure of said temperature information is greater than a predetermmed value by at least a predetermined amount. In another embodiment of the present invention, mfection in an animal is detected by: (i) obtaining an mfrared thermographic image of an anatomical structure of said animal, said mfrared thermographic image providing temperature information about said anatomical structure; (ϋ) obtammg an infrared thermographic image of^" the same anatomical structure of^" an uninfected animal or a population of uninfected animals, said mfrared thermographic image(s) providing temperature information about said anatomical structure; and (ϋ) identifying the animal in step (i) as having a high probabϋity of having an infection if a measure of the temperature information in step (i) is greater than the temperature information in step (ϋ).

5.3. INFRARED THERMOGRAPHIC CAMERA

Capturing reϋable infrared data from ϋve animals is a technical and operational challenge. Moving conscious animals to designated analytical or assessment rooms where equipment, monitors and data collection are fixed permanently, is not always possible. In fact, handling and management procedures by themselves can be stressful to animals resulting in non-steady state or aberrant temperature profiles. Hence, the animal technician or camera operator is often required to move to the animal's environment. Therefore, utϋizing a user friendly infrared camera that is installed in the animal's environment or that is portable is an advantage when capturing infrared data from ϋve animals.

In one embodiment of the invention, the infrared thermographic camera is held and operated with one hand, which is a significant advantage when obtaining infrared thermographic images of an anatomical structure of animals. In a preferred embodiment, the portable, hand held camera is Hght enough to be managed easϋy. In another embodiment, the infrared thermographic camera is installed in the animal's environment (e.g., a barn). In another preferred embodiment of the mvention, the mfrared thermographic camera: (i) is designed to operate and function optimally within the range of temperatures normaUy anticipated in animals displaying inflammation (25 °C to 35 °C) without recaϋbration; (ϋ) is capable of^" resolving temperature differences of less than 1 °C; (ϋi) has a lens focal length that is optimal for use in closer ranges with animals (e.g., focal length = 6 centimeters to infinity); (iv) has a wavelength range of^" 5 to 14 μm; (v) is encased in a hardened, water resistant case, which is compatible for the capture of data in anmial environments; (vi) has a flip out display for accurate viewing of the image; and (vϋ) is capable of compact data storage in the instrument and/or linkage to peripheral monitors. In the examples described, the Inframetrics

- 13 - 760 broadband camera (Inframetrics Co. North Billercia, MA) was used to obtain the mfrared thermographic images.

5.4. PROTOCOL FOR INFRARED THERMOGRAPHIC IMAGING For predicting the early onset of inflammation, each animal or animals suspected of presenting inflammation in a population are scanned from about 1-3 meters away. For detection of inflammation due to mastitis, the preferred range is 175 cm. Infrared thermographic images of all non-human animals are coUected preferentially from the distal (hind) view showing a clear display of the back two quarters. However, other images such as the ventral or lateral view would also have utiϋty.

Environmental factors such as motion, extraneous radiant energy, and ambient temperature must be controUed when using infrared thermography to detect inflammation. Motion, for example, can be controlled by immobϋizing the animal (e:g., a cow can be tied with a neck chain). Preferably, the animals should be at rest when the mfrared images are obtained and should not be experiencing the thermal effects resulting from the digestion of^" food when the infrared images are obtained. Infrared thermographic images should be obtained under cover and shielded from the sun. Preferably, the ambient temperature of the environment should be within the animals thermal neutral zone, which is typicaUy between 20°C and 30°C. Artϋacts such as debris on the surface of the animal, scar tissue, irregular patterns of hair length, liniment and wraps should be eliminated to avoid interference with the infrared thermographic image(s). The anmial also should be acclimated to the site of^" the examination for at least ten minutes prior to the examination. In a preferred embodiment, the infrared images should be obtamed at the same tune of day such that circadian and diurnal rhythm is taken into account.

5.5. INTERPRETATION OF INFRARED THERMOGRAPHIC IMAGES

The thermal expression of^" an animal is determined by obtaining mfrared thermographic images. As used herein, the term "infrared thermographic image" is meant to include a scan output in the form of^" either or both a visual image and corresponding thermal or temperature data. The output from infrared cameras used for infrared thermography typically provides an image comprising a pluraϋty of pixel data points, each pixel providing a temperature data point that can be further processed by computer software to generate, for example, mean temperature for the image, or for a discrete area of the image, by averagϋig the data points over the number of pixels. It wϋl be appreciated by those of skill in the art that an infrared thermographic image, comprising a pluraϋty of pixels, provides a large number of temperature data points. Therefore, before comparmg the temperature information to a predetermined value, determinϋig a rate of temperature change, or determmmg a dϋ^'ference in total temperature, it is useful to obtain some measure that is representative of the entirety of the temperature information provided by an infrared thermographic miage or a part thereof. Selected measures for the temperature information derived from each infrared thermographic ϋnage for the subject animal are determined by statistical techniques known in the art. Preferred measures include measures of central tendency, measures of dispersion, and measures of total temperature.

The term "measure of central tendency" as used herein is a statistical measure of a

10 point near the center of^" a group of data points: without limitation, the term includes the mean, median, and mode. The term "measure of dispersion" as used herein is meant to include statistical measures of^" spread from the measure of central tendency for the group, and include, without limitation, variance, standard deviation and coefficient of^" variation. Definitions of^" these statistical terms may be found in standard statistics texts, such as Steel and Torrie

15 (1960) R.G.D. Steel and J.H. Torrie. McGraw Hill Company, Inc., NY. which definitions are incorporated herein by reference. As used herein, the term "total temperature" means a measure of^" the central tendency for the temperature information from an infrared thermographic image x image area or image volume expressed in pixels (e.g., if^" the mean temperature = 20 °C and the image is equal to 200 pixels, then the total temperature = 20 °C x

20 200 pixels = 4000 pixels).

An uncaϋbrated, digitized thermographic image may consist of^", for example, 135 X 256 pixels. In analyzing the thermographic image, the relative radiant surface temperature represented by each pixel of the uncaϋbrated image may be represented by assigning each pixel a numerical value in the range from, for instance. 0 to 255. The pixel values are

25 mapped to actual Celsius temperature by relating them to the maximum and minimum temperature settings of the infrared camera through the following formula:

Actual Temperature = (max temp setting-min temp setting) X pixel value

256

To assist a human operator in viewing the mfrared thermographic images on a

30 computer monitor, pseudo colours can be generated by assigning a specific colour to aU pixels with temperature values within a certain range.

The entire thermographic ϋnage may be processed. In a preferred embodiment, only data for a part of the image corresponding to the area of interest of^" the animal is analyzed. Known computer analysis procedures, such as piano metry. can be used to restrict the image

35 analysis to the selected area of interest of the animal (e.g., a fixed "box^" area can be appϋed around the eyes for a group of animals of mterest). For each infrared thermographic image obtamed for an anmial. the image area and the selected image temperature statistics are calculated Selected statistical measures ot the temperature mformation (each pixel m the mfrared thermographic miage providmg a temperature data pomt), such as the mean, median, mode, standard deviation, variance, and coefficient of variation can be determmed by well- known statistical techniques such as those described by Steel and Torrie (1980) Suitable software tor analyzmg the thermographic images include Thermogram™ image software (Inframetrics. Inc, North Bϋlercia, MA) and Viewscan™ Software (Viewscan Ltd , Concord, ON ) Mathematical models usmg such analytical approaches as neural nets can also utilized to analvze the thermographic image In one embodiment of the present mvention. temperature differences between symmetrical anatomical structures are compared to detect inflammation For example, the lack ot symmetry between affected and non-affected quarters of an cow s udder can be used to detect mastitis In a preferred embodiment, the area or volume mformation is combmed with the mfrared thermographic temperature to better discern the lack of symmetry between the affected and the non- affected anatomical structure The area or volume represented by selected portions ot the mfrared thermographic miages can be determmed by known techniques

In an embodiment ol the present mvention, inflammation ot an anatomical structure ot an animal is detected if a measure of temperature mformation for an mfrared thermographic image of an anatomical structure of the animal dϋfers by at least a predetermmed amount or a statistically significant amount from a predetermmed value In another embodiment of the present mvention, mfection an animal can is detected ϋ a measure of temperature mformation ot an anatomical structure differs by at least a predetermmed amount or a statistically significant amount from a predetermmed value The predetermmed value may represent pubϋshed conventional temperature data representmg animals ot the same species as the subiect animal, which can be adjusted to reflect mfrared thermographic temperature values Alternatively, the predetermmed value may be an arbitrary value, the value having been determmed through trial and error to be useful for detectmg inflammation or mfection of an anatomical structure of an animal Preferably, the predetermmed value represents an equivalent measure of temperature mformation tor intrared thermographic images of the particular anatomical structure obtamed for members of a population ot the same species ot anmial bemg exammed when there was no mflammation or mfection of the anatomical structure More preferably, the predetermmed value represents an equivalent measure of temperature mformation for one or more mfrared thermographic miages ot the anmial obtamed at a time when there was no mflammation or infection ot the anatomical structure of the animal, and more preferably, when the anmial was healthy In a preferred embodiment, a change m the mean temperature of less than 1 °C of an anatomical structure relative to the mean temperature ot the same anatomical structure of the same animal or a population of animals of the same species obtamed from mtrared thermographic miages taken when there was no mflammation of the anatomical structure indicates early or subclinical inflammation In another preferred embodmient. a change m the mean temperature of greater than 1 °C of an anatomical structure relative to the mean temperature ot the same anatomical structure of the same animal or a population of animals of the same species obtamed from mfrared thermographic images mdicates late stage development of mflammation. In another preferred embodiment, inflammation of an anatomical structure of an animal is detected if the mean of the temperature information obtamed from the mfrared thermographic image is preferably greater than 0.2°C. more preferably greater than 0.1 °C the mean of the temperature information for previously obtamed mfrared thermographic miages of^" the same animal when there was no mflammation of the anatomical structure. In yet another preferred embodmient. mflammation of an anatomical structure ot an animal is detected if the mean of the temperature mtormation obtamed from the mtrared thermographic miage is preferably greater than 0.2 °C. more preferably greater than 0.1 °C the mean temperature obtamed from infrared thermographic images for the same anatomical structure ot the same species ot animal when there was no mflammation of the anatomical structure. In a preferred embodiment, a change m the mean temperature of less than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of the same animal premtection mdicates early or subclinical infection In a preferred embodiment, a change m the mean temperature of less than 1 °C ot an anatomical structure relative to the mean temperature of the same anatomical structure ot one or more unmfected animals of the same species mdicates early or subclinical mfection In a preferred embodmient. a change m the mean temperature greater than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of the same animal premfection mdicates clinical mfection. In yet another preferred embodmient, a change m the mean temperature greater than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of one or more unmfected animals ot the same species mdicates clinical mfection.

In another embodiment, the rate ot change m temperature (not the absolute value per se) ol an anatomical structure of an animal relative to the rate of change m temperature of the same anatomical structure m the animal premfection mdicates mfection In another embodiment, the rate of change m temperature (not the absolute value per se) of an anatomical structure of an anmial relative to the rate ot change in temperature of the same anatomical structure of^" one or more uninfected animals of the same species indicates infection. In another embodiment, infection of^" an anatomical structure of an animal is detected if the mean of the temperature ϋiformation obtained from the infrared thermographic image is preferably greater than ().2°C, more preferably greater than 0.1 °C the mean of the temperature information for previously obtained mfrared thermographic images of the same anatomical structure of the same animal preinfection. In yet another embodiment, infection of an anatomical structure of an animal is detected if the mean of the temperature information obtained from the infrared thermographic image is preferably greater than 0.2°C, more preferably greater than 0.1 °C the mean temperature obtained from infrared thermographic images for the same anatomical structure of one or more uninfected animals of^" the same species.

In another embodiment of the present invention, inflammation or mfection of an anatomical structure is detected ϋ^" a measure of temperature information for an infrared thermographic ϋnage of an anatomical structure of the animal is equivalent to or greater than the predetermmed value for the anatomical structure of the anmial. Preferably, the predetermined value represents the mean temperature obtained from infrared thermographic images of the same anatomical structure in members of the same species of an animal when there is inflammation or an mfection.

In another embodiment of the present invention, mflammation or infection of an anatomical structure of an animal is detected if the change in temperature obtained by successive infrared images of the same anatomical structure of^" the same animal is greater than a predetermined rate, preferably greater than a rate of 0.1 °C/hour. Preferably, successive infrared images of^" an anatomical structure of an animal are taken every 10, 30 or 60 minutes.

In a further embodmient of^" the present invention, inflammation of an anatomical structure of an animal is detected ϋ^" the total temperature of^" a section of an infrared thermographic image correspondϋig to one anatomical structure of the animal dϋfers by more than a predetermined amount, preferably 10%, from the total temperature of a section of the infrared thermographic image corresponding to the symmetrical anatomical structure of the animal. The total temperature preferably represents the area or volume of the relevant image section, which can be represented as a number of pixels, multipϋed by the mean pixel temperature.

In an embodiment of^" the present invention, area or volume information alone, independent from temperature information, can be used to detect inflammation of an anatomical structure of an animal. Inflammation of an anatomical structure of an animal is detected ϋ^" the area or volume of a section of an infrared thermographic image correspondϋig to one anatomical structure of the animal dϋfers by more than a predetermmed amount, preferably 10%, from the area or volume of a section of the infrared thermographic image corresponding to the symmetrical anatomical structure of the animal.

The infrared thermographic temperature mformation can be normalized or standardized by compensatmg the temperature ϋiformation to account for one or more of the foUowing: (i) the state of lactation of the animal; (ϋ) the state of parity of the animal; (ϋi) the circadian temperature variation: (iv) the diurnal temperature variation: (v) the animal breed; (vi) the animal housing conditions: or (vϋ) the geographic location. An adjustment for the state of lactation of an animal would be useful for normalization because animals in early lactation typicaUy have a higher milk production and hence larger udders. An adjustment for the state of parity of an animal would also be useful for normalization because cows, for example, typically in their third or fourth parity wϋl have larger udders than cows in their first parity. Adjustments to normalize the mfrared thermographic data depending on when an anmial is observed during the day should be performed because an animal^'s normal temperature wϋl fluctuate over a 24 hour period. The temperature change during the day will also vary with the time of day a cow is milked, hence, a normalization scale would be useful. Adjustments to normalize mfrared thermographic data obtained from dϋf^'erent breeds of animals should be performed because of dU^'ferences in their anatomical structures. Furthermore, adjustments to normalize the mfrared thermographic data obtamed from animals housed differently (e.g., in barns with concrete floors versus in barns with rubber matts) and in different geographic locations (e.g., Edmonton versus Orlando) should be performed.

5.6. INFLAMMATORY DISORDERS AND INFECTIOUS DISEASES

In one embodmient, inflammatory diseases in an animal, preferably a mammal and most preferably a human are detected using infrared thermography. Examples of inflammatory diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, acute respiratory distress syndrome, asthma, osteoporosis, Crohn's disease, reactive arthritis, Lyme disease, multiple sclerosis, contact dermatitis, psoriasis, graft rejection, graft versus host disease, and sarcoidosis. In another embodiment, diseases or disorders that induce an inflammatory response in an animal are detected by mfrared thermography. Examples of such diseases and disorders include, but are not limited to, allergic rhinitis, gastrointestinal allergies, food aUergies, eosinophiϋa, conjunctivitis, and glomerular nephritis.

In another embodmient. infectious diseases in an animal, preferably a mammal and most preferably a human are detected using infrared thermography. Infectious diseases include diseases associated with yeast, fungal, viral and bacterial infections. Viruses causing viral infections include, but are ϋmited to. BVD virus, herpes simplex virus (HSV), hepatitis B virus (HBV). hepatitis C virus (HCV). human T-cell lympho trophic virus (HTLV) type land II. human immuno deficiency virus (HIV), cytomegalovirus. papϋloma virus, polyoma viruses, adenoviruses. Epstein-Barr virus, poxviruses. influenza virus, measles virus, rabies virus. Sendai virus, poliomyelitis virus, coxsackieviruses, rhinoviruses. reoviruses. and rubeϋa virus. Bacterial pathogens causing mfections include, but are not limited to, Streptococcus pyogenes, Streptococcus, pneumoniae. Neisseria gonorrhoea, Neisseria meningitidis. Corynebacterium diphtheriae , Clostridium botulinum, Clostridium perfringens. Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis , Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa. Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus. Edwardsiella tarda. Yersinia enterocolitica, Yersinia pestis, Yersiniά' pseudotitberculosis. Shigella dysenteriae. Shigella flexneri, Shigella sonnei, Salmonella typhimurium. Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii. Borrelia burgdorferi. Leptospira icterohemorrhagiae, Mycobacterium tuberculosis. Toxoplasma gondii. Pneumocystis carinii. Francisella tularensis, Brucella abortus, Brucella suis. Brucella melitensis. Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugiimushi, Chlamydia spp., and Helicobacter pylori.

6. EXAMPLE: DETECTION OF MASTITIS USING

INFRARED THERMOGRAPHY

In order that the invention described herein may be more fuUy understood, the foUowϋig examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

MATERIAL & METHODS

Twenty mature lactatmg Holstein cows at 120 days post-partum were housed at the Agriculture and Agri-Food Canada Dairy Research Unit at Lennoxvϋle, Quebec, and were managed in a manner consistent with and representative of the dairy industry in North America, and in compϋance with the Canadian Councϋ of Anmial Care Guideϋnes. The left distal quarter of the udder of each animal was infused with 10 μg of E. coli endotoxin (serotype 055:B5, Sigma-Aldrich Co.) in 10 ml of sterile saline.

20 - Fϋteen ot the cows were additionally treated with expermiental inflammation inhibitors The twenty cows were divided into tour treatment groups ot five animals each as follows: (1) control, no prophylactic treatment: (n) amino guanidine introduced mto the cistern ot the infected teat; (m) arginine methyl ester introduced mto the cistern of the infected teat: and (iv) dexamethasone introduced mto the cistern ot the mtected teat. The treatments were appned m an ettort to attenuate the mastitis response.

Milk samples from the control (right distal) and induced (left distal) quarters ot each animal were coUected at 13 hours and 1 hour pre-mduction and also at 2. 6, 9, 12, 24. 36, 48, 60 and 72 hours post-mduction. The milk samples were analyzed tor obiective mdicators of mastitis by conventional analytical procedures as discussed hereinafter. Infrared thermographic miages of both distal quarters were simultaneously taken at these times and at 0.5, 1. 1.5, 2 and 2.5 hours post-mduction. An Inframetrics 760™ broadband camera (Intrametrics Inc., North Bϋlenca, MA) fitted with a 0 5 X lens was used to coUect the mfrared images Workmg indoors, images of the posterior surface ot the udder ot each anmial were obtamed from a distance of 2. lm. The Images were recorded on videotape with a videocassette recorder. The analog Images were captured and digitized using a computer equipped with a Matrox Meteor™ video card (Matrox Electronic Systems Ltd.. Montreal, Quebec. Canada) The images were saved as bitmap files usmg Corel Draw™ (Corel Corporation. Ontario, Canada). The bitmap images were calibrated and the udder manuaϋy traced to identify the left and right halves of the udder The image area m number ot pixels, and the minimum, maximum and average temperatures, and the standard deviation of the average temperature were recorded and tabulated Analysis of the data was performed usmg the computer programs Excel™ (Microsoft Corp., Redmond, Washmgton, USA) and SAS™ (SAS Institute Inc., Cary, North Carolina, USA) The progression of mastitis development was obiectively monitored usmg conventionaϋy known tests such as the somatic ceϋ count m the milk samples (Batra. T.R. and McAllister. A.J., 1984, J. Amm. Sci. 64: 305-312), BSA (Fernando. R.S. et al., 1985, J. Dairy Sci 449-456), body temperature (Maatje, K. and Rossmg, W., 1991, Mastitis Newsletter 16: 6-7), and presence of the enzyme N-acetyl-beta-D-glucosaminidase (NAGase) in the milk samples. NAGase is a lysosomal enzyme secreted m the mammary gland during mflammation. The presence of NAGase m milk is an indication of tissue damage (Perdigon. G. et al. 1986, J Dairy Sci. 69: 27-31; Fang, W et al., 1995. J. Dairy Sci. 79- 76-82. Losnedahl, K.J. et al , 1996, Illinois Dairy Report 1-4: Fang, W and Pyorala, S., 1996. J Dairy Sci. 79:76-82). By simultaneously testmg standard indicators of mastitis and obtaining mfrared thermographic images, it was possible to monitor the precise change m mfrared characteristics paraUel to the standard test results

- 21 RESULTS

The results are presented in tabular form in Tables 1 and 2, and m graphical form in Figures 1-9. The treatments with experimental mflammation inhibitors were largely ineffective, and did not signϋϊcantly change the mastitis response. Therefore, the data in Tables 1 and 2. and Figures 1-9. is not presented separately for each of the anti-inflammation treatment groups. Figures 6-9 provide least square means of data for the 20 animals tested. Figures 6-8 show separately the results obtained from one of the 20 animals tested, the individual animal (reference no. 5029) showing a false-negative result for mastitis when measured by rectal temperature rather than by mfrared thermography. The same mfrared thermographic ("IRT") data is depicted in each of Figures 1-4, plotted along with data obtained from various known techniques for detecting mastitis. Figure 9 provides the IRT data presented in the form of^" total temperature (mean temperature x image area or volume).

The results are most readϋy understood with reference to the figures. Figure 1 shows the mean temperature of the infrared thermographic image of^" the left distal quarter of the udder (induced) and the mean temperature of the infrared thermographic ϋnage of the right distal quarter of the udder (control) plotted over a 24 hour time course, together with rectal temperature plotted over the same time frame. Based upon the results depicted in Figure 1, the IRT data for the left and right distal quarters of the udder is very simuar, although mastitis was induced only in the left distal quarter. One possible explanation for this is that the high heat transfer capacity through the water found in Hving ceϋs accounts for the even temperature distribution observed between the distal quarters of the udder. The results from Figure 1 also indicate that the absolute change in temperature detected by IRT is greater than that detected by measurement of^" rectal temperature, and that the rate of^" temperature change detected by IRT is greater than that detected by measurement of rectal temperature. The results in Table 1 indicate that the mfrared thermographic image of the udder detected a statisticaUy significant temperature dϋference (p < 0.05) by the 1 hour point after mastitis induction, whereas a sigmficant difference in rectal temperature was not detected untϋ much later (the 6 hour point after mastitis induction).

Figures 2. 3 and 4 plot the same IRT temperature information as in Figure 1, together with various standard measurements used in the detection of mastitis. Figure 2 shows the NAGase levels in the left and right distal udder quarters over the first 24 hours after induction of mastitis in the left distal quarter. As expected, the NAGase level in the left distal quarter increased sharply, indicative of mastitis, whϋe there was kttle change in the NAGase level in the right distal quarter. As discussed earϋer, given the separate vascular suppϋes of the quarters of^" the udder in cattle, an increase in NAGase level in the non-induced quarter would not be expected. Figures 8 and 9 depict simϋar results, showing, respectively, a significant increase in BSA level and somatic ceϋ count in the left distal udder quarter and Uttle or no change in the right distal quarter. Figures 1 , 8 and 9 indicate that the mastitis induction model was indeed successful in inducing mastitis in the treated udder quarter, detectable by objective identifiers of mastitis, and that mastitis was also detected by IRT. Figures 6, 7 and 8 emphasize the superior results that can be achieved by the methods of^" the invention over other temperature measurement techniques. These figures provide data for one of the test animals (anmial no. 5029), in which rectal temperature remained nearly unchanged over the first 24 hours after induction of mastitis, whereas mean udder temperature as measured by IRT, changed significantly (Figure 6). Hence, in an animal in which measurement of rectal temperature disclosed a false-negative result, IRT of the udder correctly detected induced mastitis. Confirmation of induction of^" mastitis in animal no 5029 is documented in Figures 12 and 13 which show, respectively, significantly increased NAGase and BSA levels in the left distal quarter (induced) relative to the right distal quarter (non- induced). Figure 5 shows the change in udder quarter area, as represented by number of pixels in an IRT image, for left (induced) and right (non-induced) distal udder quarters for 20 animals over the 24 hour period after mastitis induction. The data in Figure 5 is independent of temperature, and only refers to the number of pixels in a defined area of^" the image. It is apparent in Figure 5 that the swelling of the left distal quarter of the udder relative to the right distal quarter (resulting in a lack of symmetry) as a result of mastitis induction was readϋy detected from the IRT ϋnage.

Figure 9 combines IRT image area and mean image temperature as a total temperature (mean pixel temperature x number of^" pixels). In Figure 1 , there was a very close symmetry between the IRT temperature of the left distal quarter and that of the right distal quarter, presumably due to the high heat transfer capacity of^" ϋving cells. Conversely, in Figure 9, the left distal quarter (induced) exhibits a much higher total temperature than the right distal quarter (non- induced). The temperature information remains the same as in Figure 1, but the greater area of^" the portion of^" the image representative of the left distal quarter of the udder relative to the area of^" the right distal quarter (as a result of swelling in response to mastitis) is reflected in the total temperature measurement.

Referring again to Figure 1 and to Table 1 , it wϋl be appreciated that the mean IRT image temperature at the time - 1 h (1 hour before induction of mastitis) reflects the IRT image temperature of the udder when the animals do not have mastitis, and therefore acts as a control IRT temperature for the animals in a healthy state. In the period from 3 hours post- ϋiduction and 12 hours post-induction, the mean IRT temperature for both the left and right hind udder quarters for the 20 animals was less than 1 °C greater than the control value of^" 32.19°C. Hence, an IRT udder temperature less than 1 °C greater than a control value for an anmial in a healthy state is ϋidicative of mastitis in a subject mammal.

Figure 1 and Table 1 shows that, during the first 24 hours after induction of the mastitis model, mean IRT temperature for both the left and right distal udder quarters for the 20 animals tested changed at a rate of at least 0.1 °C per hour, whether increasing or decreasing. Hence, a rate of^" change of IRT temperature of at least 0.1 °C per hour is indicative of mastitis in a subject mammal.

Figure 5 shows that during the first 24 hours after induction of mastitis in the left distal quarter of^" the udder, the area of the portion of the image corresponding to the induced quarter is at least 10% greater than that of the non-induced (control) right distal quarter of the udder. Thus, if the area of^" a portion of^" the image corresponding to a first quarter of the udder of the animal differs from the area of a portion of^" the image corresponding to a second quarter of the udder of the animal by greater than 10%, this is indicative of mastitis in the animal. Simϋarly, referring to Figure 9 and Table 2. during the first 24 hours after induction of mastitis in the left distal quarter of the udder, the total temperature (mean pixel temperature x number of pixels) of the portion of the image corresponding to the ϋiduced quarter is at least 10% greater than that of the non- induced (control) right distal quarter of the udder. Thus, if the total temperature of a portion of the image correspondϋig to a first quarter of^" the udder of the animal differs from the total temperature of a portion of the image correspondϋig to a second quarter of the udder of the animal by greater than 10%, this is indicative of mastitis in the animal.

7. EXAMPLE: DETECTION OF INFECTION USING

INFRARED THERMOGRAPHY

Fifteen British cross heifers seronegative for BVD and IBR were weaned from the mam herd foUowing standard Animal Disease Research Institute (ADRI: Lethbridge, Alberta) protocol and allocated to one of two treatment groups balanced by weight, body condition, age, coat color and hair condition. The two treatment groups consisted of: (1) five control, uninfected animals; and (2) ten BVD virus infected animals. The calves were housed in groups of five in three separate environmentaϋy controUed rooms and the body weights of the calves were coUected before the study began and after the study ended.

All ol^" the calves were given a balanced aϋaU^'a and barley ration which was designed to 1.5 times maintenance based on NRC recommendations, and aϋ calves were given ad Ubitum access to fresh water. Further, a rubberized mat bedding was provided for the animals to lay upon. The three animal rooms used in the study were aϋ kept at a constant

- 24 - temperature and humidity (approximately 24° C and 28 % humidity) with barometric pressure held constant Lightmg was adiusted to simulate normal dayϋght and darkness (12 hours of ϋght and 12 hours of dark). The calves were weaned and placed into their isolation rooms for a period ot 14 days prior to infection with BVD virus to aUow the animals to acclimatize to the rooms, people and procedures

A hand held portable Inframetrics broadband 760 camera was used to coϋect mfrared thermographic images Intrared thermographic miages were captured for aU of the animals at a fixed time every day. Lateral eye images consistmg of the eye orbital socket plus approximately 1 cm around the socket were captured daϋy. Frontal nose mtrared thermographic images consistmg of approximately 3 cm² located immediately between and above the nostrϋs were obtamed daϋy. Frontal ear mfrared thermographic consistmg of an area approximately 2 cm² m the middle of^" the inner ear surface were obtamed daϋy Left side (lateral) infrared thermographic miages representmg about 20 % of the animal's total surface area were obtamed Whύe this lateral image does not contam many thermoregulatory sites the side is nonetheless, like the dorsal image, representative ot an average surface mtrared temperature. Dorsal mfrared thermographic images consistmg of a square area representmg approximately 35000 pixels or probably 15 % ot the anmials surface area were also obtamed daϋy

Anmials were not captured when imaged but mstead a technician walked around the animals to obtam the mtrared thermographic images. Further, two separate Inframetrics cameras were used to obtam the mfrared thermographic miages of the control anmials and the BVD virus infected anmials

In addition to the mfrared measurements taken, clinical and physiological measurements were taken on days 0, 3, 6. 9 and 12 postmfection. The calves were captured m a head gate and 10 ml ot blood was coUected m a vacutamer by venus puncture From this blood sample, measurements ot differential blood ceU counts, cortisol. IgA and basic hematology (CBC) were performed. Also, viral titer and serological assays were performed. Further, health clinical scores were monitored on aU of the calves daύy usmg the score system developed by ADRI (Table 3). Aϋ of the animals were humanely euthanized toward the end of the disease course.

Necropsy was performed on aU anmials usmg estabϋshed scormg procedures

RESULTS

Infrared Thermographic Images of the Eye of Calves As evident from the results m Table 4, eye mfrared temperatures in anmials mfected with BVD virus mcreased throughout the BVD disease course relative to the eye mfrared temperatures for the same animals premfection. StatisticaUy sigmficant increases in the eye temperature (0.9° C, p<0.01) were observed as early as one day postϋifection and a maximum separation of 2.6° C from controls was obtained. In contrast to the early changes detected using infrared thermography, the presence of virus specϋic antibody was not detectable untϋ 5 or 6 days postinfection and StatisticaUy sigmficant changes in clinical scores were not evident untϋ eight days postinfection (Table 5). Further, there was no StatisticaUy sigmficant evidence of changes in objective measures of disease such as haptoglobin untϋ 10 days postinfection.

In comparison to the control animals, the BVD virus infected calves did not show any consistent signs in eye temperature increase untϋ 4 days postinfection. The eye temperatures for the BVD virus infected animals obtained a maximum separation of over 2° C by day 10 postinfection. The increases in the mean temperatures of the eye of BVD virus infected anϋnals proved to be StatisticaUy sigmficant and were obtained several days before signϋicant differences in clinical scores were observed. Further, when clinical scores were high enough to verify the presence of BVD, a Spearman Ranking test indicated that anmials with the highest clinical scores were also the animals with the highest infrared eye temperature (P<0.05). Therefore, the eye temperatures obtained usmg infrared thermography indicate that infrared thermography can be used to detect infection several days to one week prior to detection using conventional subjective (clinical scores; Table 5) or objective measurements such as haptoglobin (Table 6).

Infrared Thermographic Images of the Nose of Calves

As evident from the results in Table 7, the nose mfrared temperatues for the BVD virus infected calves began to elevate significantly as early as 4 days postinfection. Compared to their control, premfection temperatures, the BVD virus infected animals displayed a change in temperature (i.e.. delta T value) of just under 4° C by 9 to 10 days postinfection. The BVD virus infected animals displayed a delta T value of^" 4.6° C compared to uninfected control animals. Sϋrϋlar to the eye temperatures, the delta T values obtained for the nose were StatisticaUy signϋicant compared to either the animals own initial preinfection temperature or to control animals on comparative days. Thus, the nose temperatures measured using mfrared thermography in the BVD virus infected animals demonstrate that temperature changes detected by infrared thermography paraUel the changes seen in the course of^" an infectious disease. Further, these results demonstrate that infrared thermography can be used to detect infection several days earϋer than clinical scores (Table 5) or objective biological measurements for infection such as haptoglobin (Table 6). Infrared Thermographic Images of the Ear of Calves

As evident from the results in Table 8, ear temperatures for the BVD infected animals started to increase as early as 1 to 2 days postinfection and a maximum delta T value of approximately 4° C for mean temperatures 10 days postinfection. This was one of the largest delta T values obtained for any of the anatomical structures measured. However, consistent with the fact that ears are known to be involved in more acute thermoregulation in a homeothermic animal, the ear temperatures obtamed were highly variable. The variation in the ear temperatures obtained was the greatest in the BVD virus infected animals. Despite the large, StatisticaUy significant delta T values obtained when comparing BVD virus infected anmial temperatures to preinfection, baseline temperatures, the delta T values obtamed when comparmg BVD virus infected anmial temperatures to control, unmfected anmial temperatures were not high. Further, unlike eye temperatures, the ranking statistics were not highly signϋicant for ear temperatures.

Thus, ear temperature measurements usmg infrared thermography in BVD infected animals paraUel the course of the disease. Further, ear temperature measurements using mfrared thermography are at least as indicative of^" illness as clinical scores. However, the high degree of variabϋity in ear temperatures suggests that infrared thermographic images of this particular anatomical structure would be less reϋable for early detection of an infectious disease.

Infrared Thermo raphic Images of the Left Side of Calves

The data presented in Table 9 mdicates that the lateral temperature changes obtained using lateral mfrared thermographic images are not as sensitive or revealing as the eye or nose temperature changes for the detection of infection. Further, the lateral mfrared data was somewhat variable compared to the eye or nose mfrared data obtained. Nonetheless, as with the eye mfrared data, the lateral images demonstrated that compared to their own control temperatures on the day of infection, the BVD virus infected cattle showed a StatisticaUy sigmficant increase in mfrared temperature as early as one day postinfection. StatisticaUy significant changes in the mean temperature as determined usmg mfrared thermography were detected as much as 5 to 7 days before signϋicant statistical changes in subjective clinical scores (Table 5) or objective biological scores such as haptoglobin (Table 6) were evident.

StatisticaUy signϋϊcant differences in the mean temperature in BVD virus infected cattle compared to the mean temperature in uninfected control animals was detected at about 8 days postinfection or about the time when the eariiest clinical symptoms were beginning to appear. Nonetheless, the lateral infrared data demonstrates that infrared thermography can be used to identϋy anmials with an mtectious disease at the very least as early as the eariiest clinical scores and m many cases, one to several days before the presence of clinical scores

Infrared Thermographic Images of the Dorsal Side of Calves The results m Table 10 illustrate that the BVD virus mfected animals begm to show

StatisticaUy signϋicant mcreases in dorsal temperatures about 6 to 7 days postinlection and reach a delta T value of 1.8° C compared to their own initial premfection temperatures.

Compared to unmfected controls. StatisticaUy signϋicantly delta T values of about 1.5° C were obtamed BVD virus mfected animals. Further, as with the other mfrared thermographic images, mcreases m dorsal temperatures observed m mfected animals comcided with or preceded by several days the changes detected m cϋnical scores (Table 5) and objective biological assays such as haptoglobin (Table 6)

28 - Table 1: T ne course for infrared temperature measured by infrared thermography, rectal temperature and milk analysis parameters in cows utϋized in a mastitis ϋiduction model (n=20). Data represent least squares means.

Time Rectal Temp Infrared Temp NAGase Somatic CeU BSA

(h) °F °C μg/ml Counts g/dl

-1 101.2a 32.19a 0.39a 504a 0.329a

0.5 101.3a 32.36ab

1 102.0a 32.77bc

2 102.0a 32.97cd

3 102.7a 33.76e 2.39b 2.86b

6 105.3b 34.441^" 5.66b 4.17b

9 102.2a 34.94d 5.15b 3.13b

12 96.7b 33.42d 4.58b 2.35b

24 100.9a 30.99 5.64b 2875b 2.76b

36 101.2 33.15 5.59b 2753b 1.50b

48 101.1 31.43 4.72b 1849b 0.87a

60 101.7 33.1 1 3.46b 1370b 1.03a

72 101.1 31.68 2.44a 933a 0.67a

a,b, - means with different letters withm columns are significantly different (P<0.05)

Table 2: Time course for mean total temperature values (infrared thermographic temperatures X udder area in pixels) for left, distal udder quarter (mastitis ϋiduced) and right, distal udder quarter (non-induced) in lactatϋig daϋ-y cows. Values represent least squares means for 20 cows.

Time Total Temperature Values for Left (induced) and Right (non-induced; control) Udders

(h) Left Right

-1 52755 a 51486 a X

3 77553 b P=0.001 62395 b P=0.002 Y

6 81294 b P=0.001 63998 b P=0.001 Y

9 79250 b P=0.001 66237 b P=0.00i Y

12 66017 b P=0.002 53782 a P=0.50 Y

24 56916 a P=0.23 50630 a P=0.81 Y

36 60989 b P=0.02 54157 a P=0.44 Y

48 59322 b P=0.06 54015 a P=0.47 X

60 61971 b P=0.008 55370 a P=0.26 X

72 56745 a P=0.25 55571 a P=0.24 X

a,b, - means with dϋferent letters within columns are sigmiicantly different (P<0.05)

X,Y, - means with different letters within rows are signϋicantly different (P<0.05). Left is the mastitis induced distal quarter, right is the distal, non- induced quarter (control).

30 - Table 3: Clinical Scores

Clinical Sign Score

Lethargy 0 = none

1 = mϋd anorexia or Ustlessness

2 = moderate lethargy, slow to rise, anorectic

3 = recumbent

4 = death

Hemmorhage 0 = none

1 = few petechiae on smucous membranes or sclera

2 = moderate or severe petechiation or heatomas > 1

3 = large hematomas > 5 cm

4 = bloody diarrhea or epstaxis

Respiratory Signs 0 = none

1 = clear nasal discharge or sϋght cough, no treatment required

2 = mucopurulent discharge or severe cough, sϋght increase in lung sounds

3 = severe pneumonia

Diarrhea 0 = none

1 = mϋd or sϋght, < 5 % dehydrated

2 = moderate. 5 to 10 % dehydrated

3 = severe or profuse, > 10 % dehydrated

Table 4: Eye Infrared Thermographic Values

Day Mean Temperature in BVD Virus Mean Temperature in Control Infected Calves Calves

0 31.22 ax 32.0 y

1 32.11 bx 30.54 y

2 32.26 b 33.62 y

3 32.40 b 32.64 y

4 32.54 bx 31.90 (0.3)

5 32.66 bx 31.54 y

6 32.89 bx 31.82 y

7 33.31 bx 31.33 y

8 33.51 bx 31.47 y

9 33.38 bx 30.57 y

10 33.79 bx 31.64 y

a,b means with different letters within columns are signϋicantly different P<0.01 using 2 taϋed paired T-test

x,y means with dϋferent letters within rows are signϋicantly dϋferent P<0.01 using 2 taϋed unpaired T-test

- 32 - Table 5: Summary of Clinical Scores

Day Mean Cϋnical Scores in BVD Mean Clinical Scores in Control

Virus Infected Calves Calves

0 0 0 1 0.1 0

2 0.1 0 3 0.1 0

0 0

5 0 0

6 0.1 0

7 0.3 0 ₈ 0.9 0.2

9 2.2 0.1

10 2.9 0

11 3.2 0 12 2.7 0 13 1.2 0

Table 6: Serum Haptoglobin in BVD Infected and Uninfected Calves

Day BVD Values (μg/ml) Control Values (μg/ml)

0 <15 (one animal at 567) <15

3 308 (increase due to 2 animals) <15

7 218 (ϋicrease due to 2 annuals) <15

10 803 (aU animals ϋicrease) <15

Table 7: Nose Infrared Thermographic Values

Day Mean Temperature in BVD Mean Temperature in

Virus Infected Calves Control Calves

0 29.1 a 28.51 1 29.61 ax 26.89 y 2 29.43 a 29.88 3 29.96 a 28.68 4 31.52 bx 29.9 y.06 5 31.03 bx 28.7 y.05 6 31.48 bx 28.7 . 7 32.48 bx 29.46 y ₈ 32.9 bx 29.74 y

9 33.14 bx 28.58 y 10 32.77 bx 29.48 y

a,b means with different letters within columns are signϋicantly different P<0.01 using 2 taϋed paired T-test

x,y means with dϋferent letters within rows are signϋicantly dϋferent P<0.01 usmg 2 taϋed unpaired T-test

Table 8: Ear Infrared Thermographic Values

Day Mean Temperature in BVD Mean Temperature in

Virus Infected Calves Control Ca

22.38 a 23.35

22.41 a 22.58

2 23.29 a 24.76 3 23.19 a 24.95 4 23.89 b.06 24.90 5 24.1 b.09x 23.13 6 24.6 b.02x 23.14 y.25 7 25.9 b.01 24.56 ₈ 25.4 b.04 23.9 9 23.93 a 22.2

10 26.3 b.04x 22.7 y.07

a,b means with different letters within columns are signϋicantly different P<0.05 using 2 taϋed paired T-test

x.y means with different letters within rows are signϋicantly different P<0.05 using 2 taϋed unpaired T-test

Table 9: Lateral Infrared Thermographic Values

Day Mean Temperature in BVD Mean Temperature in Virus Infected Calves Control Calves

0 21.87 ax 23.21 y.05 1 22.61 b 22.2

2 22.6 b.07x 23.95 y 3 22.55 a.1 22.38 y 4 22.89 b 23.16 y 5 22.58 b.07 22.43 y 6 22.71 b 22.62 y 7 23.2 bx 22.07 y.04 ₈ 23.39 bx 22.0 y.03

9 23.1 b.02x 21.66 y 10 23.84 bx 22.1 1 y

a,b means with dϋ^'ferent letters within columns are signϋicantly dϋ^'ferent P<0.01 usϋig 2 taϋed paired T-test

x,y means with different letters withm rows are signUicantly dϋferent P<0.01 using 2 taϋed unpaired T-test

Table 10: Dorsal Infrared Thermographic Values

Day Mean Temperature in BVD Mean Temperature in

Virus Infected Calves Control Q

0 22.29 ax 20.62 y

1 22.74 ax 21.26 y

2 23.04 b 23.90 3 22.44 a 22.48 4 23.08 b 22.65 5 22.70 a 22.23 6 22.75 a(0.056) 22.15 7 22.95 bx 21.59 y

23.54 bx 21.96 y

9 23.06 bx 21.47 y 10 24.08 bx 22.58 y

a,b means with dϋferent letters within columns are signϋicantly different P<0.05 using 2 taϋed paired T-test

x,y means with different letters within rows are signUicantly different P<0.05 usϋig 2 taϋed unpaired T-test

The present mvention is not to be limited in scope by the specific embodmients described here . which are mtended as smgle illustrations of mdividual aspects of the mvention Indeed, various modifications ot the mvention in addition to those shown and described herem wύl become apparent to those skϋled m the art from the foregoing description and accompanymg drawmgs. Such modϋications are mtended to faU withm the scope of the appended claims.

AU references cited herem are mcorporated herem by reference m the entirety for aU purposes.

- 39 -

Claims

WHAT IS CLAIMED:

1. A method for the detection of inflammation of an anatomical structure of an animal, comprising: (a) obtaining an infrared thermographic image of an anatomical structure of an animal;

(b) determining the mean temperature of the infrared thermographic image; and

(c) detecting early or subclinical inflammation of an anatomical structure of^" an anmial if there is a change in the mean temperature of less than 1 °C of^" an anatomical structure relative to the mean temperature of^" the same anatomical structure of the same animal obtamed from mfrared thermographic images taken when there was no mflammation of the anatomical structure.

2. A method for the detection of inflammation of^" an anatomical structure of an animal, comprising:

(a) obtaining an infrared thermographic image of an anatomical structure of^" an animal;

(b) determinϋig the mean temperature of the mfrared thermographic miage; ^and

(c) detecting early or subclinical mflammation of^" an anatomical structure of^" an animal tf there is a change in the mean temperature of less than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of a population of animals of the same species obtained from infrared thermographic images taken when there was no inflammation of^" the anatomical structure.

3. A method for the detection of inflammation of an anatomical structure of an animal, comprising: (a) obtammg an infrared thermographic image of an anatomical structure of^" an animal;

(b) determining the mean temperature of the infrared thermographic image; and

(c) detecting late stage development of inflammation of an anatomical structure of an animal U^' there is a change in the mean temperature of greater than 1 °C of an anatomical structure relative to the mean temperature of the same anatomical structure of the same animal obtamed from infrared thermographic images taken when there was no inflammation of the anatomical structure.

4. A method for the detection of inflammation of an anatomical structure of^" an animal, comprising:

(a) obtaining an mfrared thermographic ϋnage of an anatomical structure of an animal;

(b) determining the mean temperature of the infrared thermographic ϋnage; ^and

(c) detecting late stage development of inflammation of an anatomical structure of^" an animal if there is a change in the mean temperature of^" greater than 1 °C of an anatomical structure relative to the mean temperature of a population of animals of^" the same species obtained from infrared thermographic images taken when there was no mflammation of the anatomical structure.

5. A method for the detection of inflammation of^" an anatomical structure of an animal, comprising:

(a) obtammg an infrared thermographic miage of an anatomical structure of an animal:

(b) obtaining an mfrared thermographic image of the symmetrical anatomical structure of the animal;

(c) determinϋig the total temperature of^" the infrared thermographic images for the symmetrical anatomical structure: and (d) detecting inflammation of an anatomical structure if the total temperature of the symmetrical anatomical structures differ by greater than a predetermmed amount.

6. The method of Claim 5, wherein the temperature image symmetry of the anatomical structure is more than 10% different from the symmetrical anatomical structure.

7. A method for the detection of inflammation of an anatomical structure of^" an animal, comprising:

(a) obtammg an infrared thermographic image(s) of^" an anatomical structure of an animal over time: and (b) assessing the mfrared thermographic miages tor changes of temperature, wherem the rate ol change ol temperature greater than 0 1 °C/h mdicates mflammation.

8 The method ot Claim 1. 2, 3. 4. 5. 6 or 7. wherem the anatomical structure is

9. The method of Clam 1. 2, 3. 4. 5, 6 or 7, wherem the anatomical structure is a pint ot a non-human animal.

0 10 The method of Clami l, 2, 3. 4, 5, 6 or 7, wherem the animal is a mammal.

1 1. The method of Claim 1, 2, 3. 4, 5, 6 or 7. wherem the anmial is a non-human mammal.

12. A method for detectmg mastitis m a mammal, compnsmg the steps of:

(a) obtammg an mtrared thermographic miage of a mammary gland of said mammal, said mtrared thermographic image providmg temperature mformation about said mammary gland: and,

(b) identϋ^'ymg said mammal as having a high probabϋity of havmg mastitis if a o measure ot said temperature mformation is greater than a predetermmed value by at least a predetermmed amount.

13. The method accordmg to Claim 12. wherem said predetermmed value comprises a correspondmg measure ot temperature mtormation tor a mammary gland ol said mammal when unaltected by mastitis, or a correspondmg measure ol mammary gland temperature mtormation for a population representative of said mammal, said population compnsmg individuals not havmg mastitis.

14 The method accordmg to Claim 12, wherem said predetermmed value is 0 obtamed by mfrared thermography.

15. The method accordmg to Clami 12. wherem said measure is a measure of central tendency

5 16. The method accordmg to Clami 15. wherem said measure of central tendency is a mean.

17 The method accordmg to Clami 12. wherem said predetermmed amount is at least 0.1 ° C.

18 A method for detectmg mastitis m a mammal, compnsmg the steps of: (a) obtammg a plurality of successive mtrared thermographic miages over tmie ot a mammary gland ot said mammal, said mfrared thermographic images providmg temperature mtormation about said mammary gland; and,

(b) identϋying said mammal as havmg a probabϋity of havmg mastitis ϋ^'the change over tmie of^" a measure of said temperature mtormation provided by said successive images is greater than a predetermmed rate.

19. The method accordmg to Claim 18, wherem said measure is a measure of central tendency.

5 20 The method accordmg to claim 18, wherem said predetermmed rate is 0.1 °C per hour.

21. A method for detectmg mastitis m a mammal havmg an udder compnsmg the steps of: o (a) obtammg an mfrared thermographic image ot one quarter of the udder of said mammal at tmie 0;

(b) obtammg an mfrared thermographic image of the same quarter of the udder of said mammal at a later time;

(c) determining a total temperature for a first miage, said first miage 5 correspondmg to said quarter of the udder ot said mammal at time 0;

(d) determmmg a total temperature for a second miage, said second image correspondmg to said quarter of the udder of said mammal at a later time; and

(e) identϋymg said mammal as havmg a high probabϋity of^" havmg mastitis if the total temperature for said first image differs from the total temperature for said 0 second image by greater than a predetermmed amount

22. The method accordmg to Claim 21. wherem the total temperature of said first miage is determmed by multiplymg the area represented by said first image by the mean of the temperature mformation provided by said first image, and the total temperature of said second 5 miage is determmed by multiplymg the area represented by said second image by the mean of the temperature mformation provided by said second miage

23. The method accordmg to Claim 21, wherein said predetermmed amount is 10%.

24. A method for detecting mastitis in a mammal havmg an udder, comprising the steps of^" :

(a) obtaining miages of the two frontal quarters or two rear quarters of the udder of said mammal:

(b) determining the total temperature of^" a first image, said first image corresponding to one frontal quarter or one rear quarter of the udder of^" said mammal; (c) determinϋig the total temperature of^" a second image, said second miage corresponding to the other frontal quarter or the other rear quarter of the udder of^" said mammal; and

(d) identifying said mammal as having a high probabϋity of^" having mastitis if the total temperature of said first image differs from the total temperature of^" said second image by greater than a predetermmed amount.

25. The method accordmg to claim 24, wherein said predetermined amount is 10%.

26. The method according to Clami 12, 13, 14 .15, 16, 17, 18, 19, 20, 21. 22, 23,

24 or 25, wherein said mammal is of the species Bos taunts or Bos indicus.

27. The method according to Claim 12, 13. 14 ,15, 16. 17. 18, 19 or 20, wherem said mammal is a pig, horse, dog or cat.

28. A method for detecting when a clinical treatment for treating inflammation of^" an anatomical structure of an animal was successful, comprising the steps of :

(a) obtaining an infrared thermographic image of^" the anatomical structure of the animal: (b) determinϋig the total temperature of the infrared thermographic image; and

(c) detectmg the successful treatment of inflammation of^" the anatomical structure by comparing the total temperature of the anatomical structure with the total temperature of the same anatomical structure obtained from the same animal or a population of^" animals of the species when healthy.

29 A method for detectmg mflammation ot an anatomical structure of an animal, compnsmg the steps of:

(a) obtammg an mfrared thermographic miage ot the anatomical structure of an animal after an event; (b) comparmg the mfrared thermographic miage obtamed to mfrared thermographic images of the same anatomical structure ot the same anmial prior to the event; and

(c) detectmg mflammation of the anatomical structure of the animal ϋ there is a relative dϋference in the temperature of the anatomical structure of the animal

30. A method for detectmg mflammation of an anatomical structure of an animal, compnsmg the steps of:

(a) obtammg an mfrared thermographic image of the anatomical structure ot an animal after an event, (b) comparmg the mfrared thermographic image obtamed to mfrared thermographic images of the same anatomical structure of a population ot animals of the same species prior to the event; and

(c) detectmg mflammation ot the anatomical structure of the animal if there is a relative dϋference m the temperature of the anatomical structure of the animal

31 The method of Clami 29 m which the event is surgery

32. A method for the detection of mfection of an animal, compnsmg:

(a) obtammg an mfrared thermographic miage of an anatomical structure of an animal: and

(b) detectmg early or subclinical mfection of said animal ϋ^' there is a change m the mean temperature of less than 1 °C ot an anatomical structure relative to the mean temperature ot the same anatomical structure of the same animal premfection.

33 A method for the detection of infection of an animal, compnsmg:

(a) obtammg an mfrared thermographic image ot an anatomical structure of an animal: and

(b) detectmg early or subclinical mfection of said animal ϋ there is a change m the mean temperature of less than 1 °C of an anatomical structure relative to the mean temperature ot the same anatomical structure m a population of unmfected animals ot the same species

34. A method for the detection of infection of^" an animal, comprising:

(a) obtammg an infrared thermographic image of^" an anatomical structure of an animal: and (b) detecting clinical infection of said animal U^" there is a change in the mean temperature greater than 1 °C of an anatomical structure relative to the mean temperature of^" the same anatomical structure of^" the same animal preinfection.

35. A method for the detection of^" infection of^" an animal, comprising: (a) obtaining an infrared thermographic image of an anatomical structure of an animal; and

(b) detectmg cϋnical mfection of said animal if there is a change in the mean temperature greater than 1 °C of^" an anatomical structure relative to the mean temperature of the same anatomical structure in a population of^" uninfected animals of^" the same species.