Serum amyloid A level as a potential indicator of the status of endurance horses
Reasons for performing study: Changes in serum levels of acute phase proteins (APPs) reflect the acute phase reaction, a rapid and nonspecific response to any tissue damage. Serum amyloid A (SAA) is the main APP in horses, which increases in various diseases, surgical injuries and after long distance endurance rides; however, this nonspecific parameter has not been investigated as an indicator of subclinical disorders, which may result in elimination during endurance competitions.
Objectives: To evaluate the serum concentration of SAA as a potential indicator for the status of horses prepared for long distance endurance rides (120 and 160 km).
Materials and methods: Twenty Arabian horses were tested and 12 were eliminated during the ride and 8 completed the distances. Routine haematological and biochemical tests and measurement of serum concentrations of SAA were carried out before and after the competition. Results were compared using the Mann Whitney U test.
Results: Before the competition all haematological and biochemical parameters varied within reference ranges with no differences between the eliminated horses and the ones that successfully finished the competition. After the rides creatine phosphokinase activity and neutrophil: lymphocyte ratio reflecting exercise-induced leukogram changes increased (P<0.05) in both groups. Before the competition, the concentration of SAA remained within reference ranges; however, it was significantly (P<0.05) lower in horses that successfully finished the competition than in eliminated ones (411.7 ± 144 ng/ml vs. 5809.5 ± 2242.7 ng/ml). After the ride SAA levels increased (P<0.05) and were similar in both groups (13 833.8 ± 1354.3 ng/ml and 12 831.2 ± 1317.6 ng/ml).
Conclusions: Serum SAA level was the only laboratory parameter that indicated most (66.6%) of the eliminated horses before entering the competition. None of the horses with SAA level higher than 1000 ng/ml completed the distance. Thus, it may be postulated that serum SAA concentration may indicate a poor status of a horse, resulting in elimination during a competition.
Acute phase response (APR) is a rapid, nonspecific reaction to any form of tissue damage including infection, trauma, neoplasia or immunological disorders (Heegaard 2000; Fallon 2001; Petersen et al. 2004; Gruys et al. 2005). In man, analogous reaction has also been reported after prolonged exercise and it was postulated to be related to skeletal muscle damage (Fallon 2001). Acute phase response is promoted by the release of cytokines including interleukin-1, tumour necrosis factor-α and interleukin-6. This stimulation leads to a wide variety of systemic changes considered to enhance the defence and adaptive mechanisms of the body, including hormonal changes and the production of acute phase proteins (APPs) in the liver (Eckersall 2000; Petersen et al. 2004). Changes in serum APPs concentrations closely reflect the course of APR and clinical conditions. In man and domestic animals, the evaluation of C-reactive protein, serum amyloid A (SAA) and haptoglobin levels have been described as useful for assessing health status, including detection of subclinical infections and evaluation of the severity of infection and convalescence period (Hulten et al. 1999b; Fallon 2001; Hulten and Demmers 2002; Petersen et al. 2004; Gruys et al. 2005; Hobo et al. 2007). However, there are variations in the pattern of APPs production in response to inflammation and other pathological conditions and large differences among species (Eckersall 2000; Petersen et al. 2004).
Serum amyloid A (SAA) is the most sensitive APP in horses, compared with other APPs (Pepys et al. 1989; Hulten and Demmers 2002; Petersen et al. 2004; Hobo et al. 2007). Its concentration changes more rapidly than other APPs and it may increase 100–1000 times as a response to tissue damage (Pepys et al. 1989; Hulten and Demmers 2002; Pollock et al. 2005). In healthy horses, SAA is present at trace levels only (Hulten et al. 1999a; Petersen et al. 2004), but it increases quickly to over 20 000 ng/ml in response to acute inflammation (Hinchcliff et al. 2004). High SAA concentrations have been reported in aseptic inflammation, surgical trauma (Pepys et al. 1989; Hulten et al. 1999a; Hulten and Demmers 2002; Pollock et al. 2005) and during infections with Rhodococcus equi, Equine influenza serotype A2 (H3N8), Equine herpes virus serotype 1 and Streptococcus equi (Pepys et al. 1989; Hulten et al. 1999b; Hulten and Demmers 2002; Petersen et al. 2004). Therefore, it is useful in the management of bacterial and viral infections in horses as large scale monitoring in stables and as a prognostic factor in relation to clinical severity and the recovery of individual horses (Pepys et al. 1989; Hulten et al. 1999b; Petersen et al. 2004). Even as early as before the exact diagnosis is provided, the SAA level serves as a helpful tool for deciding what further testing should be performed and whether a horse is fit for training (Petersen et al. 2004).
The condition of endurance horses and the efficiency of training is routinely monitored on the basis of heart rate and blood analysis including haematocrit (HCT), red blood cell count (RBC), haemoglobin concentration (HGB), erythrocyte sedimentation rate (ESR) and creatine phosphokinase (CPK) activity (Szarska 2003; Hinchcliff et al. 2004; Fielding et al. 2009). Before entering the competition the horses undergo subjective physical examination by a qualified veterinarian, so they are believed to be healthy and prepared for the ride. According to the authors’ knowledge, the concentrations of APPs in the horses have not been investigated in the context of preparation for endurance competition. It has been reported, however, that SAA levels markedly increased after long distance rides but remained unchanged after short and moderate distances (Cywinska et al. 2008). This fact suggests that long and heavy exertion stimulates the reaction analogous to APR also in the horse. It is not clear if an increase in APPs may reflect overtraining or other causes of poor condition of endurance horses that may result in elimination during long distance competitions. Nevertheless, this hypothesis is probable, as SAA level may serve as an indicator of subclinical disorders.
Therefore, the aim of this study was to evaluate the serum concentration of SAA as a potential indicator for the status of horses prepared for long distance endurance rides (120 and 160 km).
Materials and methods
Horses and competitions
Twenty Arabian horses participating in 120 and 160 km endurance rides in Poland (CEI 3* competitions in Kuznia Nowowiejska and Warka) were included in this study. The owners and the Veterinary Commission agreed to the procedures. The competitions were held in June and September, in good weather (temperatures 22–24°C) and similar terrain conditions. All the horses were clinically healthy, dewormed and vaccinated at a similar time and did not receive medication or suffer from any infection in the preceding 3 weeks (according to their owners’ knowledge). Eight of the horses successfully completed the distance and passed all veterinary checks, and 12 were eliminated due to lameness or for metabolic reasons (Table 1).
Table 1. Horses included in the study
| 1||8 m||120||Finish||20.4||13,344.7|
| 2||7 g||120||Finish||31.6||16,157.5|
| 3||10 g||120||Finish||434.3||14,281.3|
| 4||8 g||120||Finish||33.2||14,892.5|
| 5||9 g||160||Finish||940.5||7,719.5|
| 6||14 g||160||Finish||898.8||15,155.2|
| 7||11 g||160||Finish||154.4||19,830.9|
| 8||12 s||160||Finish||780.5||9,288.9|
| 9||9 s||120||Elim. lame (1 vet gate)||4,734.1||10,862.9|
|10||11 s||120||Elim. lame (2 vet gate)||255.2||10,521.9|
|11||8 m||120||Elim. lame (3 vet gate)||1,299.5||13,290.6|
|12||13 g||120||Elim. lame (3 vet gate)||12,144.6||13,412.1|
|13||10 g||120||Elim. metabol., poor recovery (3 vet gate)||283.5||10,698.1|
|14||11 g||120||Elim. lame (4 vet gate)||11,655.3||13,048.4|
|15||11 g||160||Elim. lame (1 vet gate)||10,665.3||13,700.4|
|16||9 m||160||Elim. lame (1 vet gate)||368.4||6,125.2|
|17||12 m||160||Elim. lame (1 vet gate)||1,379.8||11,612.0|
|18||12 g||160||Elim. metabol., poor recovery (3 vet gate)||1,315.2||11,729.7|
|19||9 m||160||Elim. lame (3 vet gate)||25,372.3||25,722.8|
|20||10 s||160||Elim. metabol., poor recovery (4 vet gate)||240.5||13,251.6|
Peripheral blood samples were obtained by jugular venipuncture before and after the competition. Samples were aspirated into 20 ml syringes and immediately transferred into sterile EDTA tubes for haematological tests and into plain tubes for serum analyses. EDTA samples were analysed immediately for routine haematological parameters: haematocrit (HCT), haemoglobin concentration (HGB), the number of red blood cells (RBC), the number of platelets (PLT), total number of leukocytes (WBC), counted with an automated haematology analyser (Abacus) and differential counts were determined manually from smears by counting 100 cells. Plain tubes were centrifuged at 4380 g for 5 min, collected serum was immediately frozen and kept at −20°C until analysed Serum samples were used for the measurement of creatine phosphokinase (CPK) activity and serum amyloid A concentration. Creatine phosphokinase activity was assayed by kinetic method using the reagent kit (Pointe Scientific). Serum amyloid A levels were measured in triplicate using an enzyme linked immunosorbent assay (PHASE Serum Amyloid A Assay1) according to the manufacturer's protocol.
Statistical procedures, mean and s.e. were computed using the STATISTICA 6.0 for Windows. Results are expressed as mean (standard errors of mean [s.e.]). The differences between the group of eliminated horses and the horses that completed the distance and the differences between the values before and after the ride in both groups were tested for statistical significance using the Mann Whitney U test, P≤0.05 was considered significant.
Before the ride the erythrogram and CPK activity in all the horses varied within normal ranges for endurance horses (Hinchcliff et al. 2004). Regardless of the distance the horses were prepared for, the values were similar, so they were analysed as mean values calculated for eliminated horses and the ones that completed the ride. There were no differences between these groups and only slight differences among individuals. According to the norm for endurance horses, slight neutrophilic leukocytosis was detected in 4 horses which had completed the distance and in 7 eliminated ones; however, the values remained within the normal range for Arabian horses (Feldman et al. 2000; Hinchcliff et al. 2004). Mean leukogram values for both groups did not differ significantly. Exercise-induced leukogram changes (Marlin and Nankervis 2002) were observed in eliminated horses and the ones that completed the distance, with no differences between the groups. These changes include the significant increase in the total number of white blood cells, the number of neutrophils, neutrophil to lymphocyte ratio (N:L), as well as a significant decrease in the numbers of eosinophils and lymphocytes. Similarly, the creatine phosphokinase activity increased significantly after the ride, with no differences between the groups (Table 2).
Table 2. Haematological and biochemical parameters
|Horses that have completed the distance|| || |
| RBC (×1012/l)||9.17 ± 0.33||10.21 ± 0.52|
| HCT (%)||37.71 ± 0.99||42.16 ± 1.85|
| HGB (g/dl)||13.41 ± 0.45||14.7 ± 0.53|
| WBC (×109/l)||10.22 ± 0.82||16.44 ± 1.73b|
| Neutrophils (×109/l)||6.73 ± 0.61||14.18 ± 1.32b|
| Lymphocytes (×109/l)||3.11 ± 0.47||1.83 ± 0.19a|
| Eosinophils (×109/l)||0.22 ± 0.07||0.02 ± 0.01b|
| N:L||2.49 ± 0.46||8.71 ± 1.56b|
| CPK (u/l)||147.42 ± 22.29||3352.06 ± 1410.92b|
| SAA (ng/ml)||411.7 ± 144||13,833.8 ± 1354.3a|
|Eliminated horses|| || |
| RBC (×1012/l)||8.92 ± 0.33||9.64 ± 0.44|
| HCT (%)||38.08 ± 1.19||40.97 ± 1.72|
| HGB (g/dl)||13.19 ± 0.35||14.25 ± 0.51|
| WBC (×109/l)||11.4 ± 0.85||18.09 ± 2.19b|
| Neutrophils (×109/l)||7.59 ± 0.61||15.81 ± 2.17b|
| Lymphocytes (×109/l)||3.23 ± 0.38||1.8 ± 0.26b|
| Eosinophils (×109/l)||0.37 ± 0.09||0.07 ± 0.04b|
| N:L||2.72 ± 0.43||9.86 ± 1.4c|
| CPK (u/l)||181.28 ± 11.49||1673.09 ± 479.32b|
| SAA (ng/ml)||5809.5 ± 2242.7*||12,831.2 ± 1317.6a|
The concentrations of serum amyloid A remained within normal ranges for equine species only when very wide ranges were taken (Rossdale & Partners Vetinary Surgeons 2010). The mean precompetition SAA level in eliminated horses was significantly higher than in the horses that completed the distance (Table 2.). After the ride SAA levels increased in all horses, with no significant differences between the groups. In the horses that completed the distance the SAA concentrations never exceeded 1000 ng/ml. The group of eliminated horses was heterogeneous. High (more than 1000 ng/ml) SAA concentrations were determined in 8 horses (7 eliminated due to lameness and 1 for metabolic reasons), but in 4 horses (2 eliminated due to lameness and 2 for metabolic reasons) the SAA levels were low, similar to the values obtained in the horses that completed the distance (Table 1). Eliminated horses were carefully examined by a veterinarian; they rested and eventually received supportive therapy. None of the horses needed surgery or aggressive treatment.
Proper preparation for long, arduous exertion is critical for success in endurance competition. Welfare of the horse is paramount, so horses competing are serially examined throughout the ride by veterinarians and removed from the competition when they are deemed unfit to continue. Horses are eliminated under the categories of lameness or metabolic, including poor cardiopulmonary recovery, synchronous diaphragmatic flutter, colic and exertional myopathy (Carlson et al. 1976; Hinchcliff et al. 2004; Fielding et al. 2009).
In our study, most of the horses (9 individuals) were eliminated due to lameness, which is, in general, the most common reason for elimination from competition. The most common lameness problems in endurance horses include suspensory desmitis, tendinitis, muscle pain, sore feet, degenerative joint disease, interference injuries and trauma. Sometimes the reason for lameness is not diagnosed, especially when horses fully recover in a few days (Hinchcliff et al. 2004). Certainly proper care, particularly shoeing and precision during training and competition are critical to prevent injury and lameness. However, it is very hard to completely avoid injuries and early detection of problems is essential for minimising layoff and maintenance of health. In our study, all horses were sound when entering the ride and none of the clinical or haematological parameters suggested otherwise. High precompetition concentrations of SAA were observed in 7 (out of 9) horses eliminated due to lameness. We postulate that this fact may have suggested a slight pre-existing pathology with no clinical signs, that worsen with exertion, leading to lameness. The reason for this is unknown, but overtraining or slight injury cannot be excluded, because the horses are continuously subjected to a high concussive load.
The relationship between heavy exertion and the increase in SAA concentration has been confirmed in all horses included in our study. Post competition, SAA levels were high in all horses and it was accompanied by an increase in CPK activity. Therefore, and as suggested in man (Fallon 2001), the reaction analogous to APR might occur due to skeletal muscle damage. However, it has been shown that short and moderate distance rides do not have such an effect on SAA levels (Cywinska et al. 2008), so proper training should not either. Serum amyloid A levels in the horses prepared for the competition should be low, unless overtraining or injury take place. In our study, all horses were prepared for the long distance ride and veterinary examination before the competition did not show any abnormalities. However, none of the horses with precompetition SAA levels higher than 1000 ng/ml completed the distance. We postulate that serum SAA concentrations may indicate a subclinical state that may worsen and result in elimination from the competition. The question is what SAA level should be taken into consideration as a suggestion of a poor condition of the horse?
The physiological normal range for serum SAA concentrations in horses is poorly defined. The authors present various results, probably due to various methods of SAA measurement and heterogeneity of tested horses. Beaufort Cottage Laboratories recommend a very wide range from 0–20 000 ng/ml; however, they show 1300 ng/ml as a mean for adult Thoroughbred horses in training and at stud as well as for non-Thorougbred ones. Other authors reported that SAA concentration in healthy horses is close to zero and increases to 20 000 ng/ml or more in inflammation (Hinchcliff et al. 2004), so that high values indicate disease rather than a normal state. Moreover, individual variations in SAA peak concentration has been described in horses subjected to the same stimulus (Hulten et al. 1999a). Hulten et al. (1999a) determined the reference range for adult horses at below 7000 ng/ml. Other authors recommend lower values. Jacobsen et al. (2006) determined serum SAA ranges in healthy horses as 480–2300 ng/ml; however, they used a turbidometric immunoassay developed for human serum and did not confirm the values by the method specific for equine species. Pollock et al. (2005), who used the Tridelta Phase series, reported that baseline values of SAA concentration in the horses subjected to elective surgery were at the level of 146 ± 60 ng/ml. In our study, the mean precompetition SAA concentration in the horses that successfully finished the ride was 411.7 ± 144 ng/ml. None of the horses with an SAA level higher than 1000 ng/ml completed the distance. However, not all eliminated horses had high precompetition SAA levels. It may be probable that 1000 ng/ml of SAA may be considered as suggesting poor condition in endurance horses. On the other hand, the precompetition SAA level in 2 of the horses that finished the ride was close to 1000 ng/ml (940.5 ng/ml and 898.8 ng/ml) and the precompetition SAA levels in eliminated horses were much higher (1299.5 ng/ml or more). Thus, the value discriminating the horses in good and insufficient condition is hard to estimate precisely (it probably lies between 1000 and 1300 ng/ml). It is also very likely that large individual differences exist and for endurance horses such a value should be estimated individually. This problem requires the study of a larger group.
Only 2 of the horses eliminated due to lameness had low precompetition SAA levels. They might have undergone trauma during the ride.
Three of the horses were eliminated for metabolic reasons (poor recovery in all cases). Only one of them had a high precompetition SAA concentration. Endurance horses have been reported to develop various physical and biochemical changes including tachycardia and plasma electrolyte changes during competition (Carlson et al. 1976; Hinchcliff et al. 2004). It has been shown that horses eliminated for metabolic reasons developed only mild haematological and biochemical changes, that did not indicate severe problems (Fielding et al. 2009). Similarly, in our study haematological parameters remained within reference ranges and precompetition SAA concentrations were low in 2 out of 3 horses. However, this finding seems of limited value due to a small number of horses eliminated for metabolic reasons.
In conclusion, we observed a strong relation between high precompetition SAA levels and failure to complete the distance. We postulate that serum SAA concentration measured before entering the competition may serve as an indicator of subclinical disorders, overtraining or slight injuries that may worsen with exertion and lead to elimination from the ride. We recommend further studies during the training season which could determine the relation between SAA level, the training load and condition of the horses.
The authors would like to thank Mr Pawel Borowiecki for his assistance with data analysis and Ms Anna Dudek, Mr Peter Sowinski and Ms Magdalena Sereda for their suggestions and corrections.
Conflicts of interest
1 Tridelta Ltd. Ireland.