Patent ID: 12196656

The invention is described herein by way of example and not limitation, by reference to the accompanying drawings. Many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. All documents cited herein are expressly incorporated by reference.

EXAMPLES

Example 1

1. Introduction

Racing thoroughbred horses have been selectively bred to produce optimal performances of speed and endurance on the race track. In order to achieve athletic excellence the horse must undergo a rigorous exercise programme. Just as human athletes strive to find the right balance between training hard enough to maximise performance but not so hard that stress induces either injury or a compromised immune system, so too with the horse trainers [1]. Since clinical symptoms in horses may only appear when over-stressing has already occurred, methods to determine imminent problems at sub-clinical stages are at a premium. Current methods of detecting when health is becoming compromised focus on blood biomarkers. Of three current measures, red blood cell counts, white blood cell counts and blood biochemistry, the most commonly used is total white cell count, usually coupled with estimates of the relative abundance of the five main types of white cell, the neutrophils, lymphocytes, monocytes, eosinophils and basophils. White cell counts can change rapidly in response to adverse health but the changes tend to be transient and to differ depending on the stimuli. For example the total white cell count may decrease to below normal in response to acute inflammation or virus attack but may increase in response to prolonged inflammation or bacterial infection [2]. Similarly, neutrophils, which normally make up 60% of the total white cells, may decrease quickly in response to acute stress but increase quickly when fighting acute infection [3]. Nonetheless, neutrophil and lymphocyte counts can be used to diagnose airway inflammation disease and recurrent airway obstruction [4] using bronchoalveolar lavage.

Although the various white cell counts have the potential to indicate a range of common conditions, there are a number of important issues. First, and most importantly, changes in white cell numbers can occur for reasons other than disease or injury, such as being agitated at the time of blood collection. Second, base levels are rather variable, with younger thoroughbreds in particular differing greatly in their white cell counts from one week to another without any evidence of infection or inflammation [5]. Third, the fact that cell number can go down as well as up may cloud the interpretation of tests where multiple opposing stimuli are present. For these reasons, trainers often treat white blood cells with scepticism as being too difficult to understand and too variable to provide a reliable indicator of a horse's overall health profile.

A more reliable tool should aim to reflect specifically the changes in blood biochemistry that occur at the onset of stress. When an animal suffers tissue injury, acute phase proteins are produced in the liver and released into the bloodstream and the result is localised inflammation. Similar responses are noted for a wide range of conditions including trauma, arthritis, surgery or bacterial, viral and parasitic infection [6,7,8] indicating that the acute phase response is generic and may be mounted to any form of tissue damage. Acute phase proteins thus appear a logical target for an improved test for stress-related injury during training. Two promising candidate proteins are fibrinogen, which has been the most commonly measured acute phase protein for some time, and serum amyloid A (SAA), which is becoming increasingly popular as a diagnostic of acute infection.

Fibrinogen is a plasma glycoprotein synthesised by the liver and is converted by thrombin into fibrin during blood coagulation. Fibrinogen is normally present at between 2-4 mg/ml but this rises following inflammation regardless of the cause. Indeed, fibrinogen may be the sole indicator of inflammation [9,10,11,]. Elevated levels of fibrinogen may indicate chronic inflammation or reflect the progression of an infection [12]. Novel inflammation causes the level of fibrinogen to increase above normal within 24-48 hours and in proportion to the degree of inflammation, and remain elevated for up to 10 days [13]. This relatively rapid response means that fibrinogen elevation may occur before clinical symptoms of illness [14, 15].

Serum Amyloid A (SAA) is a second acute phase protein that is also produced in the liver. Normal levels in healthy horses are very low but increase rapidly to peak 24-48 hours after infection [16]. Circulating SAA concentrations may increase up to 100 fold in response to an infection but it disappears rapidly after the infection has abated [17], making it an excellent ‘real time’ diagnostic tool for tracking progression and recovery. Previous studies have shown that elevated SAA may also be used for detecting the presence of inflammatory disease of the airways [6], gut and musculoskeletal system [7, 19]. As with fibrinogen, the severity of the inflammation is reflected in the degree of elevation of SAA.

The purpose of the current study is to investigate the relationship between classic white cell counts and the two indicators of an inflammatory response, fibrinogen and SAA across a large sample of thoroughbred horses in training. We find evidence that WBC, fibrinogen and SAA capture different aspects of a horse's physiology. WBC counts fluctuate across a rather narrow range and correlate well with parallel changes in many elements of blood chemistry, suggesting that they track normal homeostatic fluctuation. In contrast, fibrinogen and SAA tend to vary little except in a small subset of horses where both markers tend to show markedly elevated levels.

2. Materials and Methods

A population of thoroughbred horses bred for flat racing were screened at two random dates, once at the beginning of the racing season (1-2 May 2012 n=105) and once at the end of the racing season (2-3 Sep. 2012 n=118). The horses were a random mixture of males and females, a mixture of grades, ranging in age from 2 to 5 year old and had raced a maximum of 5 times each. All horses are managed in the same way with individual boxes, photoperiod of 4:30 am to 9 pm, a natural indoor temperature (18 to 20° C.) and the same feeding and training schedules. The horses underwent one workout of approximately 20-30 minutes per day between the hours of 6 am and 10 am. The horses were allowed to rest for a period of 4-7 hours post exercise before blood draw. Detailed veterinary analysis of each horse immediately post sampling would be desirable but was beyond the scope of the current study. The horses names, existing injuries, illnesses and medications were not recorded, however it was noted by the veterinarian that all horses were fit for work. A large degree of overlap between the 2 sets of horses tested is expected. The complete blood count consists of the red cell series (Red Blood Cell (RBC) Count, Hemoglobin (Hgb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), platelets (Plt) and the white cell series (Total White Blood Cells, Neutrophils (Neut), Lymphocytes (Lymph), Monocytes (Mono), Eosinophils (Eosin) and Basophils (Baso)). The red series and the white cells were assayed using a calibrated Advia 2120 (Abbott) analyser.

In addition to cell counts we also monitored a range of blood chemistry components: Fibrinogen, Serum Amyloid A, Creatine Kinase (CK), Aspartate Amino Transferase (AST), Urea, Creatine (Creat), Total Protein (TotP), Glutamate Dehydrogenase (GLDH), Gamma-Glutamyl Transaminase (GGT), Alkaline Phosphatase (ALP), Lactose Dehydrogenase (LDH), Globulin (Glob) and Albumin (ALB). The fibrinogen was measured using a calibrated ACL Elite analyser from Instrumentation Laboratory. SAA was measured using a calibrated Konelab 20 instrument from Thermo Scientific with the ‘Eiken’ Serum Amyloid A test reagents supplied by Mast Diagnostic. The Eiken assay is a human immunoturbido metric method which has been previously validated in horses [20]. According to the manufacturer the range of the test is 5-500 μg/ml with a coefficient of variation for less than 10% and an accuracy of 85-115% when a known concentration is measured. The measurement of 57 samples reported a correlation coefficient (r) as r=0.981 and the regression line as y=0.971x+2 [21].

All tests were performed by the suitably qualified in-house lab technician. To minimise the impact of circadian fluctuations and to allow for horses to return to the resting state, blood was drawn between 2 μm and 3 pm according to in-house procedures and veterinary recommendation by the in-house vet. The blood was drawn into blood tubes appropriate for the parameters to be tested. The results for each of the parameters under analysis in this study for each of the 223 horses were compiled and analysed using Microsoft excel.

3. Results

The three primary measures obtainable from blood that we were most interested in were the classical total white cell count and two proteins associated with the inflammatory response, fibrinogen and SAA. We began by asking whether, across the entire range of observed values, there was a general tendency for high and low values in one measure to be associated with high and low values in another. Since several of the trait value distributions were strongly non-normal we used non-parametric rank correlation tests rather than a standard Pearson correlation.

Rank correlations between our three primary measures and all other traits are presented in Table 1. Among the three primary measures, the two indicators of inflammation correlate positively and highly significantly with each other, but there is no association between either of these and WBC. As might be expected, WBC counts are positively correlated with many of the other sub-classes of blood cell counts, particularly neutrophils, lymphocytes and red blood cells. Among the blood chemistry measures, WBC is associated with GLDH and ALP, while the inflammation proteins both correlate with total protein and globulin, but also exhibit weak correlations with several others. Red blood cells are interesting, since they correlate positively with WBC and SAA but negatively with fibrinogen.

From the point of view of diagnosing imminent health issues, weak correlations between two or more measures across all horses may or may not be biologically relevant. For example, mild dehydration might result in transiently higher protein concentrations across many/most molecules, and this could drive correlations even across a sample of equally healthy animals.

More clinically relevant, therefore is the tendency for measures to show concordance when levels have risen outside what might be considered the ‘normal’ range of values. We first explored published tables giving the ‘normal ranges’ for different classes of horse (e.g. ‘thoroughbreds’ or ‘2 year olds in training’) but several traits in these systems were in contradiction with one another and routinely yielded values outside the expected ranges depending on which reference method was applied. Consequently, we turned to a more unbiased approach. We arbitrarily assumed that the highest 15% of observed values for each parameter were ‘elevated’ and used simple chi-squared tests to ask whether these elevated values at our three focal variables tended to be associated with elevated values in each of the other traits. 15% was chosen as a balance between a lower fraction that would have too little statistical power and a higher fraction that might be deemed unrealistic. This method therefore bypasses the need to predefine ‘normal’ and ‘abnormal’.

The chi-squared tests for concordance of high values are summarised in Table 2. With (overly) stringent full Bonferroni correction for conducting 63 tests, four tests are significant experiment-wide: Fib v SAA (X2=43.7, 1df, P=1.4×10−11), WBC v Neutrophils (X2=27.9, 1df, P=1.3×10−7), WBC v Lymph (X2=13, 1df, P=3.2×10−4) and WBC v ALP (X2=11.4, 1df, P=7.5×10−4). In addition, a number of other combinations yield significance at P=0.05 uncorrected, noticeably Total Protein, Neutrophils and Globulin, which all show associations with all three of our primary measures. It is reassuring that the strongest association, using both statistical models, by some way is the one between fibrinogen and SAA, the two measures of the inflammatory response. In all cases the associations are positive, in that the highest values for one trait occur disproportionately frequently with high values at another trait.

TABLE 1Correlation between values in diverse blood assays in 224 thoroughbredracehorses. Two proteins associated with the inflammatory response,Serum Amyloid A and Fibrinogen, and total White Cell Count arecompared against each other and against 20 other cell count/protein assays.In each case a non-parametric Spearman rank correlation is performed.Values presented are the resulting P-values. Values significant at P < 0.05are indicated with one asterisk, those significant experiment-wide areindicated with two asterisks. Assay abbreviations are found in methods.FibrinogeSAAWBCSAA1.1 × 10−09**WBC0.950.21Neut0.920.413.8 × 10−28**Lymph0.005*0.01*3.0 × 10−08**Mono0.080.091.3 × 10−04**Eosin0.760.490.28Plt0.03*0.390.01*Baso0.02*0.03*0.41RBC0.02*2.69 × 10−03**7.8 × 10−07**Hgb0.450.03*3.1 × 10−05**Hct0.460.03*1.1 × 10−04**TotP3.9 × 10−05**0.02*0.10Creat0.310.170.38Urea0.02*0.450.26GGT0.090.820.27AST0.330.02*0.03*CK0.009*0.03*0.13LDH0.04*0.04*0.09GLDH0.060.427.5 × 10−05**ALP0.100.091.1 × 10−12**ALB0.500.180.41Glob2.2 × 10−07**2.5 × 10−03*0.18

TABLE 2Concordance of occurrence of extreme values amongassays. Two diverse blood proteins associated with theinflammatory response, Serum Amyloid A andFibrinogen, and total White Cell Count arecompared against each other and against 20 othercell count/protein assays. In each case a simple2 × 2 test of homogeneity is conducted to test for anassociation between the top 15% of values observed.Values presented are interpreted with one degree offreedom. Values significant at P < 0.05 are indicatedwith one asterisk, those significant experiment-wideare indicated with two asterisks. Abbreviations ofassays are found in methods.FibrinogenSAAWBCSAA45.7**WBC1.14.2*Neut6.1*7.2*27.9**Lymph2.91.813.0**Mono2.11.46.2*Eosin0.00.00.0Plat0.00.00.5Baso4.4*1.30.0RBC2.90.70.9Hgb3.41.00.5Hct1.71.00.0TotP3.9*5.6*8.7*Creat0.40.90.8Urea5.0*0.00.0GGT0.40.10.1AST0.52.50.2CK0.00.10.0LDH2.32.10.5GLDH0.26.5*1.5ALP2.31.911.4**ALB1.10.91.2Glob3.9*9.4*8.7*
4. Discussion

We explored the relationship between a number of standard blood parameters in a sample of thoroughbred racehorses in training. Our data reveal that while the most commonly used indicator of health, total white cell count, correlates broadly with both individual cell sub-type counts and several elements of blood chemistry, there is relatively poor agreement between horses with the highest white cell counts and the highest values in other measures such as the inflammatory markers SAA and fibrinogen. In contrast, two components of the inflammatory response, SAA and fibrinogen, correlate relatively weakly with WBC and blood chemistry but show excellent agreement with one another when it comes to high values. Furthermore, by application of two separate statistical models of analysis, similar trends can be observed demonstrating that this study group was indeed a random sample population of thoroughbred racehorses and may not have been overly influenced by particularly ‘extreme’ individuals.

Blood chemistry and white cell counts are both used routinely as indicators of health however readings in healthy horses are far from constant and vary with levels of hydration and other factors. For this reason, measurements are generally conducted in as standardised a way as possible, at the same time of day and the same time relative to feeding and exercise. Nonetheless, variation still seems likely due to factors such as individual-specific patterns in urination, environmental temperature and anxiety, and this appears to be reflected in the way most of the white cell counts and blood chemistry measures exhibit some degree of cross-correlation.

To understand which part of the range of observed values of a given trait are associated with ill-health as opposed to natural daily and hourly variation in homeostasis would involve tracking the fate of horses that were trained at a constant level until clinical symptoms developed. However, such an experiment is largely precluded by the need to act pre-emptively so as to maximise horse welfare. Instead, therefore, we focused entirely on correlations between the various blood analytes in general (Table 1), comparing these with the level of concordance seen between high value readings for the same measurements (Table 2). In this way we can see the extent to which different measurements co-vary across their entire range, a pattern that would suggest correlation with some other factor such as diurnal variation in hydration, as opposed to a specific tendency for high values at one measure to be associated with high values at another, a pattern that tends to identify an unusual subset of horses. We presume that such subsets represent horses with, in this case, an on-going inflammatory response.

Our argument is that, from experience, a small but unknown subset of our number of horses in training are likely to have incipient health issues. If these horses can be detected, they should be contributing unusually high trait values. Moreover, if two or more traits are useful as indicators, these should show good agreement in their highest values. When we interrogate our data in this way we find a reversal, with WBC showing weaker correlations among the highest 15% of values compared with fibrinogen and SAA. By implication, fibrinogen and SAA show agreement in identifying a subset of horses with unusual readings, most parsimoniously explained by these horses currently suffering some level of injury or illness involving the inflammatory response. The apparent lack of specificity of WBC counts likely reflects the large diversity of factors that can affect them, many of which are not directly related to health.

Our results raise questions both about what WBC are detecting and what they are expected to detect as a pre-performance assay. Cell counts undoubtedly fluctuate in a biologically meaningful way, but there are two complications. First, the correlation between WBC and many of the blood chemistry measures suggests that the majority of the variation in our sample is due to normal variation in blood concentration rather than specific responses to a particular challenge. Second, the range of stimuli capable of impacting WBC is wide, diverse and some may even depress cell counts. Consequently, a single WBC is unlikely to tell us much about incipient problems. Better would be a monitoring programme based on repeated measures so that sudden changes could be better identified, but even here the meaning of such changes may be difficult.

In comparison with WBC, fibrinogen and SAA appear to have considerably better discriminatory power, both largely agreeing with each other about a subset of horses with clearly elevated readings. The implication is that these horses may have an otherwise undetected health problem. From a diagnostic perspective, this brings both positive and negative aspects. The negative aspect is that SAA and fibrinogen will not identify horses suffering from problems that are not currently causing an inflammatory response. The positive side is that these two blood proteins, in contrast to WBC, appear to identify a relatively specific state, that of horses exhibiting an inflammatory response.

5. Conclusions

We conclude that fibrinogen and SAA have excellent potential as biomarkers and are likely to be more informative about conditions relevant to horses in training compared with the widely used WBC.

References for Example 1

[1] Parry-Billings M, Budget R, Koutedakis Y, Blomstrand E, Brooks S, Williams C, et al. Plasma amino acid concentrations in the overtraining syndrome: possible effects on the immune system. Med Sci Sports Excer 1992; 24:1353-1358.[2] Rickets S W. Hematologic and Biochemical abnormalities in athletic horses. In: Hinchcliff K W, Keneps A J, Geor R J, editors. Equine Sports Medicine and Surgery, Philadelphia: W.B Saunders; 2004, p. 952.[3] Welles E G. Interpretation of Equine Leukocyte Responses. In: Weiss D J, Wardrop K J. Schalm's Veterinary Hematology, 6.ed, lowa: Wiley-Blackwell; 2010, p. 317 [4] Couetil L L, Hoffman A M, Hodgson J, Buechner-Maxwell V, Viel L, Wood J L N, et al. Inflammatory Airway Disease of Horses. J Vet Intern 2007; 21:356-361.[5] Grondin T M, Dewitt S F, Normal hematology of the horse and donkey. In: Weiss D K, Wardrop K J. Schalm's veterinary hematology, 6.ed, lowa: Wiley-Blackwell, 2010; p. 821-828.[6] Hultén C, Sandgren B, Skioldebrand E, Klingeborn B, Marhaug G, Forsberg M. The acute phase protein serum amyloid A (SAA) as an inflammatory marker in equine influenza virus infection. Acta Vet Scand 1999; 40:323-333.[7] Hulten C, Gronlund U, Hirvonen J, Tulamo R M, Suominen M M, Marhaug G, et al. Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins during induced non-infectious arthritis in the horse. Equine Vet J 2002; 34:699-704.[8] Pepys M B, Baltz M L, Tennent G A. Serum amyloid A (SAA) in horses: objective measurement of the acute phase response. Equine Vet J 1989; 21:106-109.[9] Jacobsen S, Nielsen J V, Kjelgaard Hansen M, Toelboell T, Fjeldborg J, Halling Thomsen M, et al. Acute phase response to surgery of varying intensity in horses: a preliminary study. Vet Surg 2009; 38:762-769.[10] Pusterla N J, Watson J L, Wilson W D. Diagnostic approach to infectious respiratory disorders. Clin Tech Eq Pract 2006; 5:174-186.[11] Allen B V, Kold S E. Fibrinogen response to surgical tissue trauma in the horse, Equine Vet J 1988; 20:441-443.[12] Burrows G E. Dose-response of ponies to parenteralEscherichia coliendotoxin. Can J Comp Med 1981; 45:207-210.[13] Crisman M V, Scarratt W K, Zimmerman K L. Blood Proteins and Inflammation in the horse. Vet Clin Equine Practice 2008; 24:285-297.[14] Heidman P, Madigan J E, Watson J L.Rhodococcus equiPneumonia: Clinical Findings, Diagnosis, Treatment and Prevention. Clin Tech Eq Pract 2006; 5:203-210.[15] Takizawa Y, Hobo S J. Usefulness of plasma fibrinogen concentration measurement in diagnosis of respiratory disorders in thoroughbred horses. Equine Sci 2006; 2:22-37.[16] Satue K, Calvo A, Gardon J. Factors Influencing Serum Amyloid Type A (Saa) Concentrations in Horses. Open Journal of Veterinary Medicine 2013 3:58-66.[17] Tape C, Kisilevsky R. Apolipoprotein A-I and apolipoprotein SAA half-lives during acute inflammation and amyloidogenesis. Biochem Biophys Acta 1990; 1043:295-300.[18] Vandenplas M L, Moore J N, Barton M H, Roussel A J, Cohen N D. Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic. Am J Vet Res 2005; 66:1509-1516.[19] Jacobsen S, Thomsen H, Nanni S. Concentrations of serum amyloid A in serum and synovial fluid from healthy horses and horses with joint disease, Am J Vet Research 2006; 67:1738-1742.[20] Jacobsen S, Kjelgaard-Hansen M, Petersen H, Jensen A L. Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations. Vet J 2006; 172 (2): 315-319.[21] Mast Group (No publication date)Eiken Serum Amyloid A(SAA) [Online] Merseyside, Mast Group LtdAvailable: http://www.mastgrp.com/Eiken/InfoSheet/SAA % 20reagents.pdf [Accessed 18 Sep. 2013].

Example 2

Introduction and Methods

Clinical symptoms in horses may only appear when over-stressing has already occurred, and there is an unmet need to provide methods to determine imminent problems at sub-clinical stages.

The SAA levels of a group of thoroughbred horses bred for flat racing were recorded over a three month period (April to June and n=61) as part of a routine biochemical panel for pre-performance testing. Of the 61 horses tested 25 ran during the testing period. The horses were managed in the same way under the same training schedule. SAA levels were determined using a two-step lateral flow immunoassay and a lateral flow reader (LFR101) by the suitably qualified in-house laboratory technician. The names of the horses and the events in which they participated were recorded but not reported. The data was observed and three relevant ranges for horses in training became apparent. Horses with a SAA concentration below 7.5 μg/ml are clinically well, free from subclinical infection and with the exception of those conditions which do not invoke a SAA response, are fit and healthy horses. Horses that ran with SAA levels of 15 μg/ml and over performed below expectation, particularly those with SAA levels in excess of 30 μg/ml. Horses with a SAA of greater than 200 μg/ml are clinically unwell with visible symptoms. The SAA levels of 16 horses in the study group was tested more than once to monitor recovery and/or to further impaired performance.

Results and Discussion

In the group of horses that tested with a SAA concentration greater than 200 μg/ml (n=5) (Table 1), 3 of the group had infections confirmed by either clinical examination or further by diagnostic investigation. One record for a recent inoculation of the equine herpes virus is reported. One horse of the group ran during the study and did not perform to expectation. The horse was assumed to have virus and subsequent testing at a later date identified mucus and neutrophils in a tracheal wash (Table 2).

TABLE 1Levels above 200 μg/ml-Elevated SAA post vaccination and clinicalconfirmation of infection in 3 of the 4 horses who were not vaccinated.Subsequent testing of the remaining horse confirmed the presenceof bacterial or viral infectionSAACommentsμg/mlInfectionon PerformanceTrainers Comments641.7n/an/aEquine herpes virusvaccination539.6Confirmed byn/apresence of bacteriain lung wash331.7Confirmed byn/aInfected leg woundclinical examination ofwound243.4Confirmed byn/aCracked heelstracheal wash234.72Not confirmedPerformedPotential virus, visiblythan expectationsout of form lower

TABLE 2A horse with an elevated SAA >200 μg/ml was monitored post-racefollowing a poor performance. Neutrophils were subsequently identifiedin a tracheal wash. SAA had returned to normal levels after antibiotictreatment.DateSAA μg/mlTrainer Comments18.04.13234.72Performed below expectation - suspected virus25.04.1363.461.05.13128.47Mucus and neutrophils in tracheal wash07.05.131.38Post antibiotic treatment

SAA concentrations between 30 μg/ml and 200 μg/ml were recorded for 15 (Table 3) horses in the study group. 6 of the 16 ran with an elevated SAA and all performed lower than the expected standard according to comments recorded at the time of testing. Post-race testing revealed mucus and blood in the tracheal wash of one of the 6 runners while another with a known elevated SAA concentration had been diagnosed with a bacterial lung infection 10 days prior to the race. In this instance SAA was monitored and levels had decreased but remained elevated on the day of racing and performance was recorded as below expectations.

SAA levels were monitored over the course of 9 days for one horse in the group of 6 under-performing horses (Table 4). 0 μg/ml was recorded on the day of racing however SAA had increased to 32.46 μg/ml and 34.01 μg/ml on day 2 and 3 respectively with no clinical symptoms were detected. SAA levels had returned to 0 μg/ml by day 9 of testing. A knee injury was sustained by one of the runners during the race and in addition a suspected viral infection was recorded for the same horse. SAA levels, post-performance were measured at 175 μg/ml which supports the likelihood of a pre-existing of viral infection. SAA was the sole indicator of a subclinical challenge for 3 of the 6 underperformers.

Of the horses who did not run within this range no clinical symptoms were recorded at the time of testing for two of the group however the remaining 8 horses, 4 were being monitored, 1 had an abnormal tracheal wash without confirmation of infection and 2 had no clinical symptoms. Comments were not recorded for one horse with an elevated SAA.

TABLE 3SAA concentrations above 30 μg/ml but below 200 μg/ml (wheresymptoms are visible) indicate the presence of an underlying issue.Horses which appear twice within the table are marked withas asterisk (*).SAAPerformanceμg/mlInfectionCommentsTrainer Comments196.92No clinical symptoms189.17No clinical symptoms175.39PerformedKnee injury sustainedbelowduring race. Suspectedexpectationvirus.*128.47Confirmed by thepresence ofmucus andneutrophils intracheal wash*119.42PerformedNo clinical symptomsbelowexpectation*108.23ConfirmedPost antibiotic treatmentbacterial infectionin lungs*97.90Confirmed bybacteria intracheal wash93.59Confirmed bybacteria intracheal wash79.82PerformedNo clinical symptomsbelowexpectation65.18Confirmed bywhite blood cellsin tracheal wash*65.18Confirmed byPerformedbacterial infectionbelowin lungsexpectation*63.46Confirmed by theSAA is decreasingpresence ofmucus andneutrophils intracheal wash55.71*52.26Confirmed byPost antibiotic treatmentbacteria intracheal wash52.26Confirmed byPerformedBlood in tracheal washmucus inbelowtracheal washexpectation46.4146.24PerformedNo clinical symptomsbelowA very consistentexpectationflat horse*34.01*32.4631.60Not confirmedAbnormal tracheal wash

TABLE 4A horse was monitored was 9 days following a poor performance,SAA levels had returned to normal by day 9 of testing. There were noclinical symptoms.DateSAA μg/mlTrainers Comments21.05.0Performed below expectation22.05.32.46—23.05.34.01—30.05.0—

Seven horses were recorded with an SAA concentration between 15 μg/ml and 30 μg/ml (Table 5). 3 of the 7 raced during the duration of the study and 2 performed below expectations. There were no visible symptoms of illness observed in the runners and further investigation revealed only an elevated AST concentration for one of the pair. Of the remaining horses that did not run, all were under investigation for previous poor performance and SAA levels were elevating or decreasing when recorded.

TABLE 5SAA concentrations between 15 ug/ml and 30 ug/ml.SAAPerformanceμg/mlInfectionCommentsTrainer Comments*29.88Fibrinogen elevated28.16Confirmed byAST elevatedpresence ofbacteria intracheal wash28.16Performed belowNo clinical symptomsexpectation*25.92Performed asBlood in tracheal washexpected24.71Not confirmedAbnormal tracheal washAntibiotic treatment*19.5518.68Performed belowAST elevatedexpectation*17.8215.24

3 SAA concentrations were recorded for horses between 7.5 μg/ml and 15 μg/ml. One horse of the group ran performing as expected. The two remaining horses did not run and were being monitored following an injury and an abnormal tracheal wash (Table 6). Horses within this range may be in the initial stages of an SAA elevation or they may be recovering from an infection where concentrations have dropped significantly. For this reason it is difficult to realise the potential of SAA when used within this range. A more reliable range begins at 15 μg/ml above which poor performance is likely when considering this set of data.

TABLE 6SAA concentrations between 7.5 μg/ml and 15 μg/mlSAAPerformanceTrainersDateμg/mlInfectionCommentsComments09.0514.38NotBlood in trachealconfirmedwash18.0511.80Knee injured19.047.75Performed asexpected

69 SAA levels were recorded below 7.5 μg/ml for 55 horses. SAA levels were being monitored for 14 of the group. 13 ran during the study period and with the exception of one horse that later measured with an elevated SAA, all performed as expected. Outside of 5 those horses being monitored only one confirmed infection was reported.

TABLE 7SAA concentrations below 7.5 μg/mlSAAPerformanceTrainerμg/mlInfectionCommentsComments7.49Performed asexpected7.494.05Performed asexpected3.19Chronic lung problems3.19Performed asexpected1.81Performed asexpected1.38Post antibiotics treatment0.60Confirmed bypresence ofmucus andneutrophils intracheal wash0.430Abnormal profile0Performed asexpected0Out of form0Performed asexpected00PerformedbelowexpectationBlood in tracheal wash00Fibrinogen elevated0Fibrinogen elevated00Injured knee0Performed asexpected000000Lung allergy0000Post antibiotic treatment0Blood in tracheal wash0Lung allergy0Lung allergy0Performed asexpected0Performed asexpectedAbnormal tracheal wash0Lung allergy0Blood in tracheal wash000Performed asexpected0InfectionBlood and mucus in tracheal washconfirmed bymucus intracheal wash00Performed asexpectedPerformed asexpected000Post antibiotic treatment0Confirmed byneutrophils intracheal wash0Elevated AST

The SAA levels of a number of horses were recorded on more than one occasion during the study in order to monitor recovery. Those horses for which four or more data points were recorded are reported here (Table 8). The data indicates that SAA levels resolve before the traditional WBC profile returns to normal. In addition SAA can be used to determine the efficacy of a treatment by monitoring the response of the protein post administration.

TABLE 814 horses were monitored to access SAA as a tool for monitoringrecovery during the study.SAATrainerDateNameμg/mlComments24.0428.16Mucus and bacteria in trachealwash26.040WBC blood profile is stillabnormal30.04002.05021.050Performed below expectation22.0532.4623.0534.0130.050SAA normal26.0493.59Abnormal tracheal wash02.0524.71On antibiotics14.050Post antibiotic treatment24.04343.29Bacteria in tracheal wash,cracked heels30.04009.05011.06501.72Bacterial infection in lungs11.06108.23Post antibiotic treatment21.0665.18Performed below expectation.09.0514.38Increased blood in tracheal wash14.050SAA normal22.0552.26Performed below expectation30.050SAA normal04.06394.95Infected leg wound11.060SAA normal

The vast majority of horses are physiologically healthy and this is reflected in the data. As SAA concentration exceeds 7.5 μg/ml the performance of the horses in the study decreased with few exceptions making SAA a very relevant biomarker for horses in training. The data for the performance of horses between 7.5 μg/ml and 15 μg/ml is rather inconclusive and performance is hard to predict. Above 15 μg/ml a decline in performance persists however other indicators of a reduced physiological health status do not always accompany the measurement. A particular decline in performance is noted above 30 μg/ml and elevated SAA is more often accompanied by other indications of infection most notably a tracheal wash elevated neutrophil count. As SAA concentrations further increase clinically visible symptoms of illness become apparent and above 200 μg/ml, all records were accompanied by visible illness and/or additional abnormal test results.

Three relevant ranges have emerged, <7.5 mg/ml, 15 μg/ml and >200 μg/ml. Since a decline in performance is most notable above 15 μg/ml, a useful tool for determining SAA does not indicate the presence of the protein until levels have reached or exceeded this value to facilitate ease of interpretation. An increase in the test signal should correspond to an increase in SAA levels and be present in the form of a single band. Such a testing format makes the user immediately aware that SAA in present in a concentration which may require attention when a signal appears. The absence of a signal indicates to the user that the horse is in a healthy state. The SAA concentration can then be semi-quantitatively determined with the use of a reference card demonstrating levels of intensity to which the test signal can be compared or a quantitative reading can be determined with the use of an electronic reader.

Example 3

A case study was conducted to assess the efficacy of SAA as a tool to monitor and manage the recovery of horses. A previous study had indicated that SAA concentrations above 200 μg/ml are accompanied by clinical symptoms and therefore SAA may be used monitor recovery and response to treatment; however the range above 200 μg/ml was not fully investigated due to the limitation of elevated samples.

For this reason an additional case study was performed to specifically evaluate SAA as a tool for monitoring recovery. Clinical comments were provided by a veterinarian at the time of testing. The name of the horse was recorded but is not reported.

The SAA levels of a horse that presented with travel sickness (pleuropneumonia) were measured daily for 13 days following a 6689 kilometre transportation (Table 1,FIG.11). Clinical examination, SAA testing and scanning were carried out by an ambulatory veterinarian. The horse was treated with Ceftiofur (antibiotic), Marbofloxacin (antibiotic), Flunixin (anti-inflammatory) Metronidazole (antibiotic) and Gastrogard (treatment/prevention of equine ulcers). The rapid increase on day 7 corresponds with a neck injury from treatment administration.

TABLE 1SAA measurements and clinical assessment of a horse withtravel sickness.SAADateμg/mlVeterinarian Comments21.06379.5Temp 102, thin, dull. Fluid on right hand sidechest & consolidated lung.22.063493Improved condition, temp 101. Lungconsolidation.23.063285Temp normal24.063384Temp normal, consolidation resolving25.063112Temp normal, consolidation resolving26.062640Temp normal, consolidation resolving, reducedflunixin27.063960Temp normal, consolidation resolving, reducedflunixin28.062755Gas pocket in neck, changed ceftiofur tocefquinome29.06838Scan much improved30.06475Temp consistent 99.801.07199Temp consistent 99.802.07101Temp consistent 99.4, stop flunixin03.0739Scan improved, small comet tails,stop marbocyl
Discussion

The data from the case study indicate that SAA can be used to efficiently monitor the recovery of the horse. The study particularly demonstrates the use of SAA to determine the biochemical efficiency of the course of treatment and SAA elevations and decreases were in agreement with clinical examination.

Assay Development

A two-step lateral flow immunoassay was developed for horses after the observation of data which supported the use of SAA as a pre-performance test and as a health management tool for monitoring recovery and/or response to treatment. During the course of the study it became apparent that fit and healthy horses were reported with SAA concentrations less than 7.5 μg/ml while impaired performance largely corresponded with levels above 15 μg/ml. Levels in excess of 200 μg/ml were accompanied by clinical symptoms, the resolution or exacerbation of which was reflected by the level of SAA.

The lateral flow assay was developed in the sandwich format and consists of a nitrocellulose membrane upon which an anti-SAA antibody and a control antibody have been immobilised. The membrane is assembled together on a backing material with a glass fibre conjugate pad, a cellulose sample pad and a cellulose absorbent pad. The conjugate pad contains the anti-SAA-colloidal gold complex which is required for the detection of SAA. The sample pad contains additional reagents which increase the stability and performance of the assay. The materials are assembled together into a plastic housing which consists of a sample well and a viewing window. Prior to sample application, the sample is diluted 1/800 in a running buffer (5 μl in 4 ml). The sample is applied to the sample pad via the sample well. The reagents within the sample and conjugate pad become mobile and move through the membrane to test line where a signal is raised if SAA is present at or above 15 μg/ml in the sample. The intensity of the test line visibly increases as the concentration of SAA increases up to a visible maximum 1000 μg/ml where the line becomes saturated to the eye. The range can be further extended up to 3000 μg/ml using an electronic reader. A semi-quantitative reading can be determined by use of a reference card upon which representations of the intensity of 15 μg/ml, 50 μg/ml, 200 μg/ml and 1000 μg/ml are available for comparison.

Example 4—SAA to Distinguish Infectious and Non-Infectious Disease or Syndromes

Introduction and Methods

A number of case studies were compiled from data generated through two Equine Veterinary Hospitals. SAA levels were determined by the in-house laboratory technicians and clinical comments were provided by Board Certified Internal Medicine Veterinarians

The levels of SAA were determined in horses diagnosed with infectious and non-infectious diseases and was observed to respond most rapidly and dramatically to bacterial and viral infections, while allergies, EIPH and other non-infectious inflammatory conditions showed little or no response. SAA levels were also observed to elevate during colic and post-colic surgery indicating SAA as a potent marker of infection and not a marker general inflammation.

Results and Discussion

Infectious

SAA PeakCommonNo. ofDiagnosisRangeSymptomsCaseBacterial Lung45-1028Bacteria in trach6Unidentified Viral16-1109Fever, filled legs10InfectionRhodococcus equi709-4936Mucus, Cough7Rotavirus1416-2763Diarrhea, loss of2Post-Colic Surgery100-1000+Discomfort,3InfectionSwellingPost-Gelding709-4453Swelling7Abscess14-4868Swelling, Heat.5Cellulitis4931Heat, Pain.2Encephalitis2838Fever1Osteomyelitis329-905Swelling, Lameness3Pneumonia141-5000+Cough, Fever5Peritonitis5000+Abdominal pain1
Non-infectious

SAA PeakCommonNo. ofDiagnosisRangeSymptomsCaseExercise Induced0-45Blood in trach wash3PulmonaryAllergy0Snorting, coughing.1Blood in trach wash.Colic100-1000+Abdominal pain3Inflammatory Bowel10Abdominal pain1DiseaseAirway2.2Cough, Mucus1Edema0Swelling1Exertional0Lameness,52RhabdomyolysisCramping.Heaves0Cough, Mucus1

The case studies compiled in Table 1 demonstrates the potential for serum amyloid a to be used as a method for differentiating between infectious and non-infectious conditions.

Levels of SAA detected for EIPH are considered to be from early stages of a secondary bacterial infection.

The benefits of such a method extend to diagnostic procedures where an infection can be confirmed before further investigation as well as allowing for the prompt initiation of a suitable treatment regime for sick horses based on whether they are being treated for an infectious disease such as those associated with micro-organisms or non infectious illness such as those associated with the environment or lifestyle or genetic factors. Furthermore SAA has been seen to elevate to a larger extend when the horse is challenged with a bacterial infection compared to a viral infection which creates scope for SAA to be used not only as a marker of differentiation between infectious and non infectious disease but also as a method of differentiating between the organism responsible which has implications for the type of therapy administered e.g. viral infections will not respond to antibiotic therapy.

Example 5—SAA as a Screening Tool in Newborn Foals

Introduction

Screening for SAA in newborn foals, under 10 hours old, has been shown to be an excellent risk-reduction method and can clearly identify foals susceptible to liver failure, diarrhea and other infectious conditions.

SAA (Serum Amyloid A) was adapted as part of a health screening test for newborn foals. A white blood cell count (WBC) was also conducted as part of the screening process. As WBC naturally fluctuate and can go down and up when challenged, SAA as a point of care screening tool is a more reliable indicator of a newborns changing health status.

Methods

Testing was conducted by the suitably qualified in-house laboratory technician and clinical comments were provided by a licensed Veterinarian within 24 hours of birth. Data was collected from 22 newborns in total and analysed to determine the potential for SAA as a screening tool for compromised newborns.

Results and Discussion

22 newborn foals were screened of which 15 tested SAA negative and 7 tested SAA positive (Table 1). WBC data for the newborns ranged from 7.1-17.9×103/μl.

TABLE 1Blood results and clinical notes from 22 newbornsCaseSAANo.(μg/ml)Clinical Notes116.5Healthy newborn, developed R.Equi onemonth later.20Healthy newborn37.5Healthy newborn, went on to developmultiple joint and bone infections.40Healthy newborn50Healthy newborn60Healthy newborn70Healthy newborn90Healthy newborn100Healthy newborn1181.5Healthy newborn120Healthy newborn130Healthy newborn141464.5Rotavirus diagnosed and sent to hospital150Healthy newborn160Healthy newborn17265Elevated BUN and creatine180Healthy newborn190Healthy newborn200Healthy newborn2135.5Healthy newborn, went on to develop22167.5Weak

Of the 7 SAA positive newborns, 1 was diagnosed within 24 hours of birth with a viral infection and transferred to hospital. Another displayed elevated BUN and creatine levels in addition to elevated SAA.

A third newborn was described as weak. Two newborns went on to develop health problems a later date, one within the first month of birth and one within two weeks.

The newborn in Case 1 (Table 1) was diagnosed withRhodococcus equione month after the SAA determination. The newborn was determined to be healthy on examination and had a mildly elevated SAA level.Rhodococcus equiis a bacterial infection and one of the most common causes of pneumonia in foals. Infected foals may remain lively and asymptomatic until late in the course of the disease. Infection has been recognized as endemic on some farms and costs related to illness and mortality may be high at these locationsRhodococcus equiis nearly ubiquitous in soil and while the infection is unlikely to have been contracted immediately after birth, SAA can be used in as a screening test for early identification of those newborns who may be at risk of developing the infection.

The newborn in case 3 was assessed as a healthy newborn after birth and had a low level of SAA. Within two weeks of initial SAA testing the foal had developed multiple joint and bone infections during which SAA levels rose in excess of 3000 μg/ml.

In addition to be being used as a screening tool to identify potentially compromised newborns SAA was also used in this instance to determine the point at which antibiotic treatment should be withdrawn by monitoring the decrease in SAA levels.

SAA has been shown to detect the presence of viral infections including rotavirus. Rotavirus is a highly contagious virus that affects foals and if left untreated can become life threatening due to severe dehydration and malnutrition. Case 14 provides a second example of the potential for SAA to be used as an initial screening tool and then as a tool to monitor the response of a foal to treatment. The newborn was diagnosed with a rotavirus infection within 24 hours of birth and sent to hospital. SAA levels were then recorded until they fell within the normal range.

Unlike adult horses, newborns may display levels of SAA elevation in the very early hours of life due to liver activity unrelated to infection, its assumed that this may be related to the transfer of functionality from the placenta to the newborn liver as can been observed in case 17 below, where markers of poor liver function are elevated. Nevertheless it can be seen that SAA is a useful marker of foals that may develop health issues within the first 48 hours after birth, which is when foals are at highest risk of fatality.

Example 6—Test Fluid Collection System

Some key features of the system are shown inFIG.14, and are explained in more detail below:

Multi-Purpose Sample Collection Tip

The sample collection tip has been designed such that different methods can be used to collect a sample. Firstly, the dimensions of the tip allow for the attachment of a luer end needle so that blood can be collected straight from the vein. Alternatively, a lancet can be used to produce a drop of blood from the patient and the BCS used to pick up a metred volume from the drop using capillary action. Similarly, the BCS can pick up blood from the end of a syringe or from a container.

Collection Port

The BCS collection port can be considered to be the most important part of the device. The collection port comprises of two closely aligned surfaces. The space between the two surfaces determines the volume of sample to be collected. Due to the design of the port and nature of the hydrophilic-treated surfaces, sample collection occurs by capillary action and is quick and accurate. This removes the requirement for aspirating sample with a pipette, which removes the risk of error by users who would not be familiar with using pipettes.

Dispensing Channel and Nozzle

The BCS is designed to fit into the neck of a bottle, with the collection port and the dispensing channel located inside the bottle. The BCS/bottle can then be inverted for mixing. Reduction in volume of the bottle then forces the diluted solution into the dispensing channel and through the dispensing nozzle.

Example 7—Monitoring of Exertional Rhabdomyolysis

Exertional Rhabdomyolysis, also known as Tying up, is a condition induced by exercise, characterized by stiffness, hardened muscles in the hind quarters and reluctance to move.

The detection of elevated CK and AST levels in horses can be used to diagnose tying up, but the condition can be easily identified by physical examination. The benefit of testing for CK and AST when a horse ties up is the ability to monitor disease progression. If interpreted correctly, CK and AST levels can tell you if the horse is just beginning to tie up, whether it is responding well to an intervention or if the horse is recovering.

Methods

A study conducted over a six-month period (May-November '13) tested approx. 200 Thoroughbred horses bred for flat racing. During monthly routine blood testing, each horse had a complete blood cell count and biochemistry panel conducted. Creatine kinase (CK) and aspartate aminotransferase (AST) were part of the biochemistry assessment. As well as scheduled blood testing, additional tests were conducted to monitor a horses CK and AST levels when tying up was observed.

Results

Over a period of 6 months, 52 out of 200 horses tied up, accounting for 26% of the population. Of the 52 horses, 14 experienced recurrent episodes of tying up, ranging from 2 to 5 cases in the six months. The data is tabulated below.

The incidence of recurrence is shown inFIG.15.

The horses that experienced recurrent cases of tying up accounted for 52.5% of the total 80 cases, indicating a requirement to monitor horses prone to the condition.

Managing Exertional Rhabdomyolysis

Some horses are more prone to tying up than others, sometimes experiencing episodes of tying up back-to-back, which can be frustrating to trainer, costing money and time. Over the duration of this study, 14 horses were observed to have repeated episodes of tying up.

CKASTClinical notes66132279Set fast12361156Set fast19892380Set fast42171444Set fast3142441Set fast59981505Tying up57004451Set fast19481173Set fast10611027Set fast37801345Set fast142911948Set fast44151042Set fast47811194Set fast24971590Set fast38753930Set fast126073457Set fast11982570Tying up19342740Tying up5282539Set fast30341043Set fast8231288Set fast120351196Set fast11563993Set fast91283984Set fast11323984Set fast41913881Set fast5773517Set fast19492386Tying up231662600Set fast86821720Set fast11332020Set fast68373090Set fastASTClinical notes23291674Set fast7431462Set fast1015923Set fastCKASTClinical notes57191286Set fast1013802Set fast28061632Set fast60685190Set fast79475458Set fast81956940Set fast20862946Set fast50893743Set fast30103055Set fast23072796Tying up44513181Tying up37551040Set fast7201430Set fast17511996Set fast21591885Set fast014821962Set fast05501280Tying upSAACKASTClinical notes02638688Set fast03692920Set fast019481139Set fast0133915191Set fast0166678549Tying up0614014479Tying up02154120872Tying up0110315327Set fast034611674Set fast026311046Set fast017691068Set fast01832909Set fast
Device for Managing Exertional Rhabdomyolysis

In one embodiment of the device (FIG.16(a)) the device is a LFD and CK and AST are determined in sequence according to the positioning of the detection reagents on the LFD.

In such a device sample is added to a single sample port and moves along a channel where, for example, it first encounters the reagents required to detect and determine levels of AST and subsequently encounters the reagents necessary to detect and measure CK. In such a device CK and AST must both be measured, as the sample must come into contact with the reagents for the detection of each as it moves through the channel. CK and AST are clearly indicated (labelled) by markings on the device to differentiate between the two (i.e “CK” is printed on the device at the test line for CK and “AST” is printed on the device at the test line for AST).

In one embodiment of the device (FIG.16(b)the markings beside the test line for CK appear as “CK” and the markings for AST appear as “SGOT” representing serum glutamic oxaloacetic transaminase by which AST is also known. In this embodiment the CK marking is above the SGOT marking.

In one embodiment of the device (FIG.16(c)) CK and AST are measured on a LFD in parallel to each other. Such a device contains two distinct sample ports, which run in parallel to each other and which are not in fluid communication with each other. Sample is added in separate steps to each sample port. The first of the two channels which make up this embodiment contains only the reagents necessary for the detection of CK. The second of the two channels contains only the reagents necessary for the detection of AST. Such a device allows for the analysis of CK/AST in parallel or individually. In the Figure the channel which detects and measures CK is visible on the left (front) side of the cartridge. The channel which detects and measures AST is visible on the right (front) side of the cartridge. The sample zones directly underneath the sample ports of the device may be treated with reagents which encourage the performance of the test.

In one embodiment of the deviceFIG.16(d)) CK and AST are determined by the application of sample to a single sample port. The sample then separates and moves along two separate channels. The two channels are chemically treated in the same fashion as those described above whereby the antibody reagents for the detection and measurement of CK are present only in the channel visible to the front left of the cartridge and those for the detection of AST are present only in the channel is which is visible on the front right of the device. The sample application zone of the device may be chemically treated in such a way as to encourage the performance of the test.

It will be appreciated that the orientation (left\right) in the above embodiments is not essential.

Example 8—Presence of SAA in Healthy Horses

Introduction and Methods

A population study involving 105 thoroughbred horses was conducted in order to understand the normal level of serum amyloid A in healthy horses. The horses were a random mixture of males and females, a mixture of grades, ranging in age from 2- to 5-year-old and had raced a maximum of five times each. All horses were managed in the same way with individual boxes, photoperiod of 4:30 AM to 9 PM, a natural indoor temperature (18-C-20-C), and the same feeding and training schedules. Detailed veterinary analysis of each horse immediately after sampling was beyond the scope of the present study. However, it was noted by the veterinarian that all horses were fit for work.

All blood analysis was performed by a suitably qualified in-house laboratory technician. To minimize the impact of circadian fluctuations blood was drawn between 2 PM and 3 PM according to in-house procedures and veterinary recommendation by the in-house vet. The blood was drawn into blood tubes appropriate for the parameters to be tested.

SAA was measured using a calibrated Konelab 20 instrument from Thermo Scientific and the “Eiken” test reagents supplied by Mast Diagnostic. According to the manufacturer the range of the test is 5-500 μg/ml with a coefficient of variation<10% and an accuracy of 85-115%.

The results for each of the parameters under analysis in this study for each of the 105 horses were compiled and analyzed using Microsoft excel.

Results and Discussion

We considered the absolute presence or absence of serum amyloid A (SAA) in a population of 105 thoroughbred racehorses bred for racing. From 105 subjects only 9 subjects were found to have any detectable level of SAA. The lowest detected level was 5.4 μg/ml with 4 out of the 9 positive SAA results under 25 μg/ml indicating that the sensitivity of the method was capable of consistently determining concentrations in this range, and it was considered that absolute negative results (zero) truly reflect the practical absence of the protein.

The use of total white blood cell (WBC) counts, plasma fibrinogen (Fb) and SAA have been well reported for the diagnosis of inflammation in horses. The normal ranges for WBC in thoroughbred racehorse has been well reported by Allen et. al. in 1984 who give various acceptable ranges for fillies, colts and of different ages and stages of training. For the purposes of this study the normal ranges for WBC's were considered to be 6.0-9.9×109/L. Fb is an acute phase protein which is now well established as a marker of inflammation in horses, initial reports for the use of fibrinogen as a marker of acute inflammation in horses include van Wuijckhuise-Sjouke (1984) and Patterson et. al. (1988). Again various normal ranges have been reported and for the purposes of this study 2.0-5.0 mg/ml is considered to be the normal range. Pepys et. al. reported the first immunoassay for SAA in 1989 and concluded that SAA was present only at “trace” levels in healthy horses but elevated rapidly following tissue injury (surgery) infection and inflammation. It is not clear whether the particular immunoassay used in that work had the required sensitivity to establish if SAA was actually absent.

Thus the present invention represents the first report that SAA is absent in normal healthy horses.

96 out of 105 (91.5%) subjects gave absolute negative results for SAA indicating that the normal level of SAA is in fact none or zero. For each of the 9 positive results, the levels of WBC and Fb were also considered to report if an inflammatory response may have been occurring. It was also considered reasonable that in any population of racehorses in training that up to 10% of the population may be suffering from some kind of inflammatory process, albeit at the sub-clinical or mildly clinical stages.

5 of the subjects had highly elevated SAA levels (between 969-1868 μg/ml) in each of these cases the Fb level was highly elevated (5.7 mg/ml or higher).

Possibly most interesting is that the 2 subjects with SAA levels within 5-20 μg/ml had corresponding WBC levels that were lower than the normal range while the fibrinogen was either normal or mildly elevated. As SAA is understood to respond earlier than Fb, these two examples may reflect very early stages of an inflammatory response, where WBC's become depleted in an immune response upstream of new WBC production, and, whereby Fb had not yet elevated. Subject 105 had an SAA level of 21 μg/ml and corresponding elevated Fb level of 5.5 mg/ml. None of the 96 SAA-negative horses had an elevated Fb measurement higher than 5.0 mg/ml.

In summary, this example demonstrates that from a population of 105 racehorses in training SAA is not at all present in normal healthy horses and in horses where SAA is detected its likely that an inflammatory process had been triggered.

TABLE 1WBCFibrinogenHorse(109/L)(mg/ml)SAA(19.74.9028.73.8038.73.7049.33.5057.94.40611.63.80773.40874.6096.42.70109.33.50117.130127.92.90136.140146.72.701593.60168.32.40177.83.80187.330198.82.602010.240219.43.60227.430237.53.60248.63.30257.83.30268.930278.13.302811.13.302983.90305.740319.13.50328.1403394.30348.13.35.4358.52.90367.73.40378.32.60388.33.603911.23.20409.13.14110.73.50429.63.104383.304415.63.504510.23.60468.13.90478.42.504811.83.10497.530509.930518.13.40529.73.20537.83.40548.73.40557.93.50568.430579.43.50589.53.20599.33.20607.83.40617.93.20628.93.806383.10647.73.306510.13.506655.217.1675.54.50686.240696.63.80708.43.60719.940726.74.40736.13.40746.53.20758.82.90765.23.70777.14.20785.63.207911.37.61225805.73.50817.13.308210.23.60838.83.80846.73.50855.2408610.430875.33.48.6887.940899.93.60908.23.80918.940926.43.80938.26.61868949.93.30955.95.79699610.16.41010976.84.10986.34.50996.54.7010010.37.515551018.53.201029.14.20103103.901046.73.701059.45.521.1

References

Used in example 8:1. Leucocyte counts in the healthy English Thoroughbred in Training. Allen B V, Kane C E, Powell D G. Equine Veterinary Journal 1984; 16:207-209.2. Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response.Pepys M B, Baltz M L, Tennent G A, Kent J, Ousey J, Rossdale P D. Equine Vet J. 1989 March; 21 (2): 106-9.3. Plasma fibrinogen as a parameter of the presence and severity of inflammation in horses and cattle.van Wuijckhuise-Sjouke L A. Tijdschr Diergeneesku4. Acute phase response in the horse: plasma protein changes associated with adjuvant induced inflammation. Patterson S D, Auer D, Bell K. Biochem Int. 1988 August; 17 (2): 257-6