Device and Methods

Abstract

The present invention relates generally to methods and materials pertaining to assays, for example immunoassays, for biomarkers in body fluids e.g. blood. The invention also relates to diagnostic or screening methods for infections, and methods of differentiating between infectious and non-infectious conditions in mammals, particularly equines, for monitoring response to anti-infective/antibiotic therapy. The invention further relates to a test fluid collection system adapted to permit dilution and analysis of the collected test fluid. The invention further relates to monitoring exertional rhabdomyolysis in equines, and assay devices for all these things.

Claims

1.-20. (canceled)

21. A method of predicting whether a mammal exhibiting an abnormal symptom or behaviour will have an improved benefit from administration of an anti-infective, a period of rest, or a combination thereof, the method comprising: determining whether a body fluid sample obtained from the mammal has a concentration of Serum Amyloid A above about 15 μg/ml; and predicting the mammal to have an improved benefit from administration of an anti-infective, a period of rest, or a combination thereof based on the concentration of Serum Amyloid A being above about 15 μg/ml, the concentration indicating that the abnormal symptom or behaviour results from an infectious disease.

22. The method as claimed in claim 21, wherein the infectious disease is one caused by a bacterial infection or a viral infection or a protozoal infection.

23. The method as claimed in claim 21, wherein the anti-infective is an antibiotic.

24. The method as claimed in claim 21, wherein the concentration of Serum Amyloid A is determined using an analytical device.

25. The method as claimed in claim 21, wherein the concentration of Serum Amyloid A is determined using an analytical device and wherein the analytical device is a portable lateral flow device comprising: (i) a housing; and (ii) a flow path leading from a sample well to a viewing window, through which the body fluid sample can flow by capillary action, wherein the presence of Serum Amyloid A produces a line in the viewing window which indicates a Serum Amyloid A concentration of at or above 15 μg/ml in the body fluid sample, and wherein the device measures a Serum Amyloid A concentration over at least the range of 7.5 to 3000 μg/ml, and wherein the assay result within the concentration range 7.5 to 1000 μg/ml can be visually interpreted with the naked eye based on the intensity of the line, optionally by comparison to a reference showing different line intensities.

26. The method as claimed in claim 25, wherein the flow path has an analyte-detection zone comprising a conjugate release zone and a detection zone, and wherein the line reveals the amount of Serum Amyloid A in the sample.

27. The method as claimed in claim 25, wherein the portable lateral flow device further comprises a control zone positioned upstream or downstream of the analyte-detecting zone, capable of indicating the assay has been successfully run.

28. The method as claimed in claim 25, wherein the portable lateral flow device utilises a sandwich format and consists of a nitrocellulose membrane upon which an anti-Serum Amyloid A antibody and a control antibody have been immobilized.

29. A method of monitoring a mammal's response to a treatment, the method comprising: determining whether a concentration of Serum Amyloid A in a body fluid sample of said mammal is above a specified level after treatment has been administered, wherein if the concentration of Serum Amyloid A is above the specified level it indicates that the treatment is to be administered again, and wherein if the concentration of Serum Amyloid A is below the specified level it indicates that the treatment can be withdrawn, wherein the treatment to the mammal comprises a period of rest for the mammal and/or administration of an anti-infective to the mammal, and wherein said specified level is 15 μg/ml.

30. The method of claim 29, wherein the anti-infective is an antibiotic.

31. The method of claim 29, wherein the concentration of Serum Amyloid A is determined using an analytical device.

32. The method as claimed in claim 29, wherein the concentration of Serum Amyloid A is determined using an analytical device and wherein the analytical device is a portable lateral flow device comprising: (i) a housing; and (ii) a flow path leading from a sample well to a viewing window, through which the body fluid sample can flow by capillary action, wherein the presence of Serum Amyloid A produces a line in the viewing window which indicates a Serum Amyloid A concentration of at or above 15 μg/ml in the body fluid sample, and wherein the device measures a Serum Amyloid A concentration over at least the range of 7.5 to 3000 μg/ml, and wherein the assay result within the concentration range 7.5 to 1000 μg/ml can be visually interpreted with the naked eye based on the intensity of the line, optionally by comparison to a reference showing different line intensities.

33. The method as claimed in claim 32, wherein the flow path has an analyte-detection zone comprising a conjugate release zone and a detection zone, and wherein the line reveals the amount of Serum Amyloid A in the sample.

34. The method as claimed in claim 32, wherein the portable lateral flow device further comprises a control zone positioned upstream or downstream of the analyte-detecting zone, capable of indicating the assay has been successfully run.

35. A method of monitoring disease progression in a mammal, the method comprising: determining whether a concentration of Serum Amyloid A in a body fluid sample of said mammal is above a specified level, wherein if the concentration of Serum Amyloid A is above the specified level it indicates that the mammal requires a treatment to treat the disease; administering the treatment to the mammal; and determining whether the concentration of Serum Amyloid A in a body fluid sample of said mammal is above or below the specified level following treatment, wherein if the concentration of Serum Amyloid A is below the specified level it indicates that the disease is responding to the treatment and the mammal is improving and the treatment can be withdrawn, or if the concentration of Serum Amyloid A is above the specified level it indicates that the disease is progressing and the treatment can be continued, wherein the treatment to the mammal comprises a period of rest for the mammal; and/or administration of an anti-infective to the mammal, and wherein said specified level is 15 μg/ml.

36. The method of claim 35, wherein the anti-infective is an antibiotic.

37. The method of claim 35, wherein the concentration of Serum Amyloid A is determined using an analytical device.

38. The method as claimed in claim 35, wherein the concentration of Serum Amyloid A is determined using an analytical device and wherein the analytical device is a portable lateral flow device comprising: (i) a housing; and (ii) a flow path leading from a sample well to a viewing window, through which the body fluid sample can flow by capillary action, wherein the presence of Serum Amyloid A produces a line in the viewing window which indicates a Serum Amyloid A concentration of at or above 15 μg/ml in the body fluid sample, and wherein the device measures a Serum Amyloid A concentration over at least the range of 7.5 to 3000 μg/ml, and wherein the assay result within the concentration range 7.5 to 1000 μg/ml can be visually interpreted with the naked eye based on the intensity of the line, optionally by comparison to a reference showing different line intensities.

39. The method as claimed in claim 38, wherein the flow path has an analyte-detection zone comprising a conjugate release zone and a detection zone, and wherein the line reveals the amount of Serum Amyloid A in the sample.

40. The method as claimed in claim 38, wherein the portable lateral flow device further comprises a control zone positioned upstream or downstream of the analyte-detecting zone, capable of indicating the assay has been successfully run.

Description

FIGURES

[0301] FIG. 1: Lateral flow assay device with integrated body fluid collector

[0302] FIG. 2: Lateral flow assay device with integrated body fluid collector

[0303] FIG. 3: Lateral flow assay device with a traffic light system of go, caution and stop

[0304] FIG. 4: Correlation of elevated fibrinogen and elevated SAA to each other

[0305] FIG. 5: Relationship between elevated Fibrinogen, elevated SAA and abnormal total white blood cell count

[0306] FIG. 6: Elevated Fibrinogen and Total White Blood Cell Count Correlation

[0307] FIG. 7: Highly Elevated Fibrinogen (>5 mg/ml) and Total White Blood Cell Count Correlation

[0308] FIG. 8: Correlation between normal fibrinogen and low total white blood cells

[0309] FIG. 9: Correlation between normal fibrinogen and high total white blood cells

[0310] FIG. 10: Relationship between elevated SAA and normal total white blood cells

[0311] FIG. 11: Correlation between SAA levels and horses which performed as expected and those that did not. All but one horse performed as expected when SAA was measured at 7.5 μg/ml or below.

[0312] FIG. 12: SAA concentrations during the recovery of a horse diagnosed with travel sickness.

[0313] FIG. 13: Example reference card for determining SAA levels.

[0314] FIG. 14a: is a perspective view of the test fluid collection system according to the present invention. FIG. 14b is an exploded view of the test fluid collection system of FIG. 14a.

[0315] FIG. 15: Bar chart showing recurrence of “tying up” in horse population tested in Example 7.

[0316] FIG. 16a and FIG. 16b: Examples of devices for monitoring “tying up” by assay of CK and AST and CK and SGOT, respectively; while FIG. 16c and FIG. 16d are examples of devices for monitoring “tying up” by assay of CK and AST.

[0317] FIG. 17a, FIG. 17c and FIG. 17b: SAA concentrations determined in horses during infection (FIG. 17a), compared with other indicators (FIG. 17b and FIG. 17c). This demonstrates the significant utility of SAA as a prognostic or diagnostic marker.

[0318] 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

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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].

[0323] 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 [13] 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 [18] and musculoskeletal system [7, 19]. As with fibrinogen, the severity of the inflammation is reflected in the degree of elevation of SAA.

[0324] 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

[0325] A population of thoroughbred horses bred for flat racing were screened at two random dates, once at the beginning of the racing season (01-02 May 12 n=105) and once at the end of the racing season (02-03 Sep. 12 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.

[0326] 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 ug/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].

[0327] 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 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. 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

[0328] 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.

[0329] Rank correlations between our three primary measures and all other traits are presented in

[0330] 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.

[0331] 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.

[0332] ‘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’.

[0333] 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 (X.sup.2=43.7, 1df, P=1.4×10.sup.−11), WBC v Neutrophils (X.sup.2=27.9, 1df, P=1.3×10.sup.−7), WBC v Lymph (X.sup.2=13, 1df, P=3.2×10.sup.−4) and WBC v ALP (X.sup.2=11.4, 1df, P=7.5×10.sup.−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-US-00005 TABLE 1 Correlation between values in diverse blood assays in 224 thoroughbred racehorses. Two proteins associated with the inflammatory response, Serum Amyloid A and Fibrinogen, and total White Cell Count are compared 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 < 0.05 are indicated with one asterisk, those significant experiment-wide are indicated with two asterisks. Assay abbreviations are found in methods. Fibrinoge SAA WBC SAA 1.1 × 10− WB 0.95 0.21 Neut 0.92 0.41 3.8 × 10.sup.−28** Lym 0.005* 0.01* 3.0 × 10.sup.−08** ph 0.08 0.09 1.3 × 10.sup.−04** Eosi 0.76 0.49 0.28 Plt 0.03* 0.39 0.01* Bas 0.02* 0.03* 0.41 o 0.02* 2.69 × 10.sup.− 7.8 × 10.sup.−07** Hct 0.46 0.03* 1.1 × 10.sup.−04** TotP 3.9 × 10.sup.− 0.02* 0.10 Crea 0.31 0.17 0.38 Urea 0.02* 0.45 0.26 GGT 0.09 0.82 0.27 AST 0.33 0.02* 0.03* CK 0.009* 0.03* 0.13 LDH 0.04* 0.04* 0.09 GLD 0.06 0.42 7.5 × 10.sup.−5** ALP 0.10 0.09 .sup. 1.1 × 10.sup.−12** ALB 0.50 0.18 0.41 Glob 2.2 × 10.sup.− 2.5 × 10− 0.18 indicates data missing or illegible when filed

TABLE-US-00006 TABLE 2 Concordance of occurrence of extreme values among assays. Two diverse blood proteins associated with the inflammatory response, Serum Amyloid A and Fibrinogen, and total White Cell Count are compared against each other and against 20 other cell count/protein assays. In each case a simple 2 × 2 test of homogeneity is conducted to test for an association between the top 15% of values observed. Values presented are interpreted with one degree of freedom. Values significant at P < 0.05 are indicated with one asterisk, those significant experiment-wide are indicated with two asterisks. Abbreviations of assays are found in methods. Fibrinogen SAA WBC SAA 45.7** WB 1.1 4.2* Neut 6.1* 7.2* 27.9** Lym 2.9 1.8 13.0** Mon 2.1 1.4 6.2* Eosi 0.0 0.0 0.0 Plat 0.0 0.0 0.5 Bas 4.4* 1.3 0.0 RBC 2.9 0.7 0.9 Hgb 3.4 1.0 0.5 Hct 1.7 1.0 0.0 TotP 3.9* 5.6* 8.7* Crea 0.4 0.9 0.8 Ure 5.0* 0.0 0.0 GGT 0.4 0.1 0.1 AST 0.5 2.5 0.2 CK 0.0 0.1 0.0 LDH 2.3 2.1 0.5 GLD 0.2 6.5* 1.5 ALP 2.3 1.9 11.4** ALB 1.1 0.9 1.2 Glo 3.9* 9.4* 8.7* b

4. Discussion

[0334] 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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

[0340] 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

[0341] [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. [0342] [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. [0343] [3] Welles E G. Interpretation of Equine Leukocyte Responses. In: Weiss D J, Wardrop K J. Schalm's Veterinary Hematology, 6.ed, Iowa: Wiley-Blackwell; 2010, p. 317 [0344] [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. [0345] [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, Iowa: Wiley-Blackwell, 2010; p. 821-828. [0346] [6] Hulten 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. [0347] [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. [0348] [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. [0349] [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. [0350] [10] Pusterla N J, Watson J L, Wilson W D. Diagnostic approach to infectious respiratory disorders. Clin Tech Eq Pract 2006; 5:174-186. [0351] [11] Allen B V, Kold S E. Fibrinogen response to surgical tissue trauma in the horse, Equine Vet J 1988; 20: 441-443. [0352] [12] Burrows G E. Dose-response of ponies to parenteral Escherichia coli endotoxin. Can J Comp Med 1981; 45:207-210. [0353] [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. [0354] [14] Heidman P, Madigan J E, Watson J L. Rhodococcus equi Pneumonia: Clinical Findings, Diagnosis, Treatment and Prevention. Clin Tech Eq Pract 2006; 5:203-210. [0355] [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. [0356] [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. [0357] [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. [0358] [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. [0359] [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. [0360] [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. [0361] [21] Mast Group (No publication date) Eiken Serum Amyloid A (SAA)[Online] Merseyside, Mast Group Ltd Available: http://www.mastgrp.com/Eiken/InfoSheet/SAA %20reagents. pdf [Accessed 18 Sep. 2013].

Example 2

[0362] Introduction and Methods

[0363] 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.

[0364] 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 ug/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.

[0365] Results and Discussion

[0366] 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-US-00007 TABLE 1 Levels above 200 μg/ml-Elevated SAA post vaccination and clinical confirmation of infection in 3 of the 4 horses who were not vaccinated. Subsequent testing of the remaining horse confirmed the presence of bacterial or viral infection SAA Infection μg/ml on Performance Comments Trainers Comments 641.7 n/a n/a Equine herpes virus vaccination 539.6 Confirmed by n/a presence of bacteria in lung wash 331.7 Confirmed by n/a Infected leg wound clinical examination of wound 243.4 Confirmed by n/a Cracked heels tracheal wash 234.72 Not confirmed Performed Potential virus, visibly out of form lower than expectations

TABLE-US-00008 TABLE 2 A horse with an elevated SAA >200 μg/ml was monitored post-race following a poor performance. Neutrophils were subsequently identified in a tracheal wash. SAA had returned to normal levels after antibiotic treatment. Date SAA μg/ml Trainer Comments 18.04.13 234.72 Performed below expectation-suspected virus 25.04.13 63.46  1.05.13 128.47 Mucus and neutrophils in tracheal wash 07.05.13 1.38 Post antibiotic treatment

[0367] 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.

[0368] 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.

[0369] 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-US-00009 TABLE 3 SAA concentrations above 30 μg/ml but below 200 μg/ml (where symptoms are visible) indicate the presence of an underlying issue. Horses which appear twice within the table are marked with as asterisk (*). SAA Performance μg/ml Infection Comments Trainer Comments 196.92 No clinical symptoms 189.17 No clinical symptoms 175.39 Performed Knee injury sustained below during race. Suspected expectation virus. *128.47 Confirmed by the presence of mucus and neutrophils in tracheal wash *119.42 Performed No clinical symptoms below expectation *108.23 Confirmed Post antibiotic treatment bacterial infection in lungs *97.90 Confirmed by bacteria in tracheal wash 93.59 Confirmed by bacteria in tracheal wash 79.82 Performed No clinical symptoms below expectation 65.18 Confirmed by white blood cells in tracheal wash *65.18 Confirmed by Performed bacterial infection below in lungs expectation *63.46 Confirmed by the SAA is decreasing presence of mucus and neutrophils in tracheal wash 55.71 *52.26 Confirmed by Post antibiotic treatment bacteria in tracheal wash 52.26 Confirmed by Performed Blood in tracheal wash mucus in below tracheal wash expectation 46.41 46.24 Performed No clinical symptoms below A very consistent expectation flat horse *34.01 *32.46 31.60 Not confirmed Abnormal tracheal wash

TABLE-US-00010 TABLE 4 A horse was monitored was 9 days following a poor performance, SAA levels had returned to normal by day 9 of testing. There were no clinical symptoms. Date SAA μg/ml Trainers Comments 21.05. 0 Performed below expectation 22.05. 32.46 — 23.05. 34.01 — 30.05. 0 —

[0370] 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-US-00011 TABLE 5 SAA concentrations between 15 ug/ml and 30 ug/ml. SAA Performance μg/ml Infection Comments Trainer Comments *29.88 Fibrinogen elevated 28.16 Confirmed by AST elevated presence of bacteria in tracheal wash 28.16 Performed below No clinical symptoms expectation *25.92 Performed as Blood in tracheal wash expected 24.71 Not confirmed Abnormal tracheal wash Antibiotic treatment *19.55 18.68 Performed below AST elevated expectation *17.82 15.24

[0371] 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-US-00012 TABLE 6 SAA concentrations between 7.5 μg/ml and 15 μg/ml SAA Performance Trainers Date μg/ml Infection Comments Comments 09.05 14.38 Not Blood in tracheal confirmed wash 18.05 11.80 Knee injured 19.04 7.75 Performed as expected

[0372] 69 SAA levels were recorded below 7.5 ug/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-US-00013 TABLE 7 SAA concentrations below 7.5 μg/ml SAA Performance Trainer μg/ml Infection Comments Comments 7.49 Performed as expected 7.49 4.05 Performed as expected 3.19 Chronic lung problems 3.19 Performed as expected 1.81 Performed as expected 1.38 Post antibiotics treatment 0.60 Confirmed by presence of mucus and neutrophils in tracheal wash 0.43 0 Abnormal profile 0 Performed as expected 0 Out of form 0 Performed as expected 0 0 Performed below expectation Blood in tracheal wash 0 0 Fibrinogen elevated 0 Fibrinogen elevated 0 0 Injured knee 0 Performed as expected 0 0 0 0 0 0 Lung allergy 0 0 0 0 Post antibiotic treatment 0 Blood in tracheal wash 0 Lung allergy 0 Lung allergy 0 Performed as expected 0 Performed as expected Abnormal tracheal wash 0 Lung allergy 0 Blood in tracheal wash 0 0 0 Performed as expected 0 Infection Blood and mucus in tracheal wash confirmed by mucus in tracheal wash 0 0 Performed as expected Performed as expected 0 0 0 Post antibiotic treatment 0 Confirmed by neutrophils in tracheal wash 0 Elevated AST

[0373] 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-US-00014 TABLE 8 14 horses were monitored to access SAA as a tool for monitoring recovery during the study. SAA Trainer Date Name μg/ml Comments 24.04 28.16 Mucus and bacteria in tracheal wash 26.04 0 WBC blood profile is still abnormal 30.04 0 02.05 0 21.05 0 Performed below expectation 22.05 32.46 23.05 34.01 30.05 0 SAA normal 26.04 93.59 Abnormal tracheal wash 02.05 24.71 On antibiotics 14.05 0 Post antibiotic treatment 24.04 343.29 Bacteria in tracheal wash, cracked heels 30.04 0 09.05 0 11.06 501.72 Bacterial infection in lungs 11.06 108.23 Post antibiotic treatment 21.06 65.18 Performed below expectation. 09.05 14.38 Increased blood in tracheal wash 14.05 0 SAA normal 22.05 52.26 Performed below expectation 30.05 0 SAA normal 04.06 394.95 Infected leg wound 11.06 0 SAA normal

[0374] 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.

[0375] Three relevant ranges have emerged, <7.5 mg/ml, 15 ug/ml and >200 ug/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

[0376] 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 ug/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.

[0377] 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.

[0378] 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-US-00015 TABLE 1 SAA measurements and clinical assessment of a horse with travel sickness. SAA Date μg/ml Veterinarian Comments 21.06 379.5 Temp 102, thin, dull. Fluid on right hand side chest & consolidated lung. 22.06 3493 Improved condition, temp 101. Lung consolidation. 23.06 3285 Temp normal 24.06 3384 Temp normal, consolidation resolving 25.06 3112 Temp normal, consolidation resolving 26.06 2640 Temp normal, consolidation resolving, reduced flunixin 27.06 3960 Temp normal, consolidation resolving, reduced flunixin 28.06 2755 Gas pocket in neck, changed ceftiofur to cefquinome 29.06 838 Scan much improved 30.06 475 Temp consistent 99.8 01.07 199 Temp consistent 99.8 02.07 101 Temp consistent 99.4, stop flunixin 03.07 39 Scan improved, small comet tails, stop marbocyl

[0379] Discussion

[0380] 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.

[0381] Assay Development

[0382] 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 ug/ml were accompanied by clinical symptoms, the resolution or exacerbation of which was reflected by the level of SAA.

[0383] 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 ul 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

[0384] Introduction and Methods

[0385] 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

[0386] 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.

[0387] Results and Discussion

[0388] Infectious

TABLE-US-00016 SAA Peak Common No. of Diagnosis Range Symptoms Case Bacterial Lung  45-1028 Bacteria in trach 6 Unidentified Viral  16-1109 Fever, filled legs 10  Infection Rhodococcus equi  709-4936 Mucus, Cough 7 Rotavirus 1416-2763 Diarrhea, loss of 2 Post-Colic Surgery    100-1000+ Discomfort, 3 Infection Swelling Post-Gelding  709-4453 Swelling 7 Abscess  14-4868 Swelling, Heat. 5 Cellulitis 4931 Heat, Pain. 2 Encephalitis 2838 Fever 1 Osteomyelitis 329-905 Swelling, Lameness 3 Pneumonia    141-5000+ Cough, Fever 5 Peritonitis   5000+ Abdominal pain 1

[0389] Non-infectious

TABLE-US-00017 SAA Peak Common No. of Diagnosis Range Symptoms Case Exercise Induced 0-45 Blood in trach wash 3 Pulmonary Allergy 0 Snorting, coughing. 1 Blood in trach wash. Colic    100-1000+ Abdominal pain 3 Inflammatory Bowel 10 Abdominal pain 1 Disease Airway 2.2 Cough, Mucus 1 Edema 0 Swelling 1 Exertional 0 Lameness, 52  Rhabdomyolysis Cramping. Heaves 0 Cough, Mucus 1

[0390] 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.

[0391] Levels of SAA detected for EIPH are considered to be from early stages of a secondary bacterial infection.

[0392] 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

[0393] Introduction

[0394] 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.

[0395] 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.

[0396] Methods

[0397] 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.

[0398] Results and Discussion

[0399] 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 7.1-17.9×10.sup.3/μl.

TABLE-US-00018 TABLE 1 Blood results and clinical notes from 22 newborns Case SAA No. (μg/ml) Clinical Notes 1 16.5 Healthy newborn, developed R.Equi one month later. 2 0 Healthy newborn 3 7.5 Healthy newborn, went on to develop multiple joint and bone infections. 4 0 Healthy newborn 5 0 Healthy newborn 6 0 Healthy newborn 7 0 Healthy newborn 9 0 Healthy newborn 10 0 Healthy newborn 11 81.5 Healthy newborn 12 0 Healthy newborn 13 0 Healthy newborn 14 1464.5 Rotavirus diagnosed and sent to hospital 15 0 Healthy newborn 16 0 Healthy newborn 17 265 Elevated BUN and creatine 18 0 Healthy newborn 19 0 Healthy newborn 20 0 Healthy newborn 21 35.5 Healthy newborn, went on to develop 22 167.5 Weak

[0400] 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.

[0401] 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.

[0402] The newborn in Case 1 (Table 1) was diagnosed with Rhodococcus equi one month after the SAA determination. The newborn was determined to be healthy on examination and had a mildly elevated SAA level. Rhodococcus equi is 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 locations Rhodococcus equi is 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.

[0403] 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.

[0404] 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.

[0405] 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.

[0406] 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

[0407] Some key features of the system are shown in FIG. 14, and are explained in more detail below:

[0408] Multi-Purpose Sample Collection Tip

[0409] 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.

[0410] Collection Port

[0411] 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.

[0412] Dispensing Channel and Nozzle

[0413] 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

[0414] 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.

[0415] 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.

[0416] Methods

[0417] 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.

[0418] Results

[0419] 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.

[0420] The incidence of recurrence is shown in FIG. 15.

[0421] 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.

[0422] Managing Exertional Rhabdomyolysis

[0423] 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.

TABLE-US-00019 CK AST Clinical notes 6613 2279 Set fast 1236 1156 Set fast 1989 2380 Set fast 4217 1444 Set fast 314 2441 Set fast 5998 1505 Tying up 5700 4451 Set fast 1948 1173 Set fast 1061 1027 Set fast 3780 1345 Set fast 14291 1948 Set fast 4415 1042 Set fast 4781 1194 Set fast 2497 1590 Set fast 3875 3930 Set fast 12607 3457 Set fast 1198 2570 Tying up 1934 2740 Tying up 528 2539 Set fast 3034 1043 Set fast 823 1288 Set fast 12035 1196 Set fast 11563 993 Set fast 9128 3984 Set fast 1132 3984 Set fast 4191 3881 Set fast 577 3517 Set fast 1949 2386 Tying up 23166 2600 Set fast 8682 1720 Set fast 1133 2020 Set fast 6837 3090 Set fast AST Clinical notes 2329 1674 Set fast 743 1462 Set fast 1015 923 Set fast CK AST Clinical notes 5719 1286 Set fast 1013 802 Set fast 2806 1632 Set fast 6068 5190 Set fast 7947 5458 Set fast 8195 6940 Set fast 2086 2946 Set fast 5089 3743 Set fast 3010 3055 Set fast 2307 2796 Tying up 4451 3181 Tying up 3755 1040 Set fast 720 1430 Set fast 1751 1996 Set fast 2159 1885 Set fast 0 1482 1962 Set fast 0 550 1280 Tying up SAA CK AST Clinical notes 0 2638 688 Set fast 0 3692 920 Set fast 0 1948 1139 Set fast 0 13391 5191 Set fast 0 16667 8549 Tying up 0 6140 14479 Tying up 0 21541 20872 Tying up 0 1103 15327 Set fast 0 346 11674 Set fast 0 263 11046 Set fast 0 1769 1068 Set fast 0 1832 909 Set fast

[0424] Device for Managing Exertional Rhabdomyolysis

[0425] 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.

[0426] 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).

[0427] 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.

[0428] 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.

[0429] In one embodiment of the device FIG. 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.

[0430] It will be appreciated that the orientation (left\right) in the above embodiments is not essential.

Example 8—Presence of SAA in Healthy Horses

[0431] Introduction and Methods

[0432] 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.

[0433] 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.

[0434] 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 ug/ml with a coefficient of variation <10% and an accuracy of 85-115%.

[0435] 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.

[0436] Results and Discussion

[0437] 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.

[0438] 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 6.0-9.9×10.sup.9/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.

[0439] Thus the present invention represents the first report that SAA is absent in normal healthy horses.

[0440] 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.

[0441] 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).

[0442] 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.

[0443] 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-US-00020 TABLE 1 WBC Fibrinogen Horse (10.sup.9/L) (mg/ml) SAA( 1 9.7 4.9 0 2 8.7 3.8 0 3 8.7 3.7 0 4 9.3 3.5 0 5 7.9 4.4 0 6 11.6 3.8 0 7 7 3.4 0 8 7 4.6 0 9 6.4 2.7 0 10 9.3 3.5 0 11 7.1 3 0 12 7.9 2.9 0 13 6.1 4 0 14 6.7 2.7 0 15 9 3.6 0 16 8.3 2.4 0 17 7.8 3.8 0 18 7.3 3 0 19 8.8 2.6 0 20 10.2 4 0 21 9.4 3.6 0 22 7.4 3 0 23 7.5 3.6 0 24 8.6 3.3 0 25 7.8 3.3 0 26 8.9 3 0 27 8.1 3.3 0 28 11.1 3.3 0 29 8 3.9 0 30 5.7 4 0 31 9.1 3.5 0 32 8.1 4 0 33 9 4.3 0 34 8.1 3.3 5.4 35 8.5 2.9 0 36 7.7 3.4 0 37 8.3 2.6 0 38 8.3 3.6 0 39 11.2 3.2 0 40 9.1 3.1 41 10.7 3.5 0 42 9.6 3.1 0 43 8 3.3 0 44 15.6 3.5 0 45 10.2 3.6 0 46 8.1 3.9 0 47 8.4 2.5 0 48 11.8 3.1 0 49 7.5 3 0 50 9.9 3 0 51 8.1 3.4 0 52 9.7 3.2 0 53 7.8 3.4 0 54 8.7 3.4 0 55 7.9 3.5 0 56 8.4 3 0 57 9.4 3.5 0 58 9.5 3.2 0 59 9.3 3.2 0 60 7.8 3.4 0 61 7.9 3.2 0 62 8.9 3.8 0 63 8 3.1 0 64 7.7 3.3 0 65 10.1 3.5 0 66 5 5.2 17.1 67 5.5 4.5 0 68 6.2 4 0 69 6.6 3.8 0 70 8.4 3.6 0 71 9.9 4 0 72 6.7 4.4 0 73 6.1 3.4 0 74 6.5 3.2 0 75 8.8 2.9 0 76 5.2 3.7 0 77 7.1 4.2 0 78 5.6 3.2 0 79 11.3 7.6 1225 80 5.7 3.5 0 81 7.1 3.3 0 82 10.2 3.6 0 83 8.8 3.8 0 84 6.7 3.5 0 85 5.2 4 0 86 10.4 3 0 87 5.3 3.4 8.6 88 7.9 4 0 89 9.9 3.6 0 90 8.2 3.8 0 91 8.9 4 0 92 6.4 3.8 0 93 8.2 6.6 1868 94 9.9 3.3 0 95 5.9 5.7 969 96 10.1 6.4 1010 97 6.8 4.1 0 98 6.3 4.5 0 99 6.5 4.7 0 100 10.3 7.5 1555 101 8.5 3.2 0 102 9.1 4.2 0 103 10 3.9 0 104 6.7 3.7 0 105 9.4 5.5 21.1

REFERENCES

[0444] Used in example 8: [0445] 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. [0446] 2. Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response. [0447] 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. [0448] 3. Plasma fibrinogen as a parameter of the presence and severity of inflammation in horses and cattle. van Wuijckhuise-Sjouke L A. Tijdschr Diergeneeskd. 1984 Nov. 1; 109(21):869-72. [0449] 4. 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