ARTICLE IN PRESS Research in Veterinary Science ■■ (2015) ■■–■■

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Post-exercise dynamics of serum amyloid A blood concentration in thoroughbred horses classified as injured and non-injured after the race A. Turlo a,*, A. Cywinska a, M. Czopowicz b, L. Witkowski b, E. Szarska c, A. Winnicka b a

Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland c Military Institute of Hygiene and Epidemiology, Warsaw, Poland b

A R T I C L E

I N F O

Article history: Received 16 December 2014 Accepted 13 April 2015 Keywords: Serum amyloid A Racehorse Orthopedic injury Acute phase response

A B S T R A C T

The aim of this study was to evaluate serum amyloid A (SAA) concentration in horses with orthopedic injuries acquired during racing and in healthy ones after completing the race. Injuries of bone and tendon did not cause radical increase in SAA concentration observed in other inflammatory conditions. SAA concentration correlated positively with white blood cell count (WBC) on the 3rd–4th days after race being significantly higher in the injured horses than in the control group in that time. It was suggested that racing effort may cause increase in SAA level, more pronounced in horses manifesting clinical signs of orthopedic injury after the race. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Acute phase response (APR) is a primary systemic reaction induced by tissue injuries of various types. It is manifested by dramatic increase in the blood concentration of acute phase proteins (APPs), in horses mainly serum amyloid A (SAA). Numerous experimental and clinical studies display 10 to 1000-fold increase in SAA blood level within 24 to 48 h after tissue damage and elevated SAA concentrations in various infectious and non-infectious diseases (Coutinho da Silva et al., 2014; Hobo et al., 2007; Hulten and Demmers, 2002; Jacobsen et al., 2006). Nevertheless, little is known about the dynamics of SAA concentration in horses with injuries of bones and tendons, especially related to sport training. Bone fractures of various severity have been reported to elicit significant increase in APP expression in human and mice (Bauza et al., 2011; Buttenschoen et al., 2000). In horses, the inflammatory-like response is triggered by microdamages of musculoskeletal system after strenuous exercise (Cywin´ska et al., 2012; Horohov et al., 2012; Lamprecht et al., 2008). However, there is no clear evidence of systemic reaction in horses with orthopedic injuries acquired during exercise. Thus, the aim of this study was to evaluate the blood SAA profile in horses with racing-related orthopedic injuries in comparison to healthy ones subjected to races.

* Corresponding author. Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland. Tel.: +48 697 028 484; fax: +48 22 593 60 27. E-mail address: [email protected] (A. Turlo).

The study involved 25 Thoroughbred racehorses, 2–5 years old, stabled and trained in one racing facility. All procedures were approved by the local ethical committee, the owners and the trainers. The injured (experimental) group (n = 15) consisted of horses with clinical signs of orthopedic injury following the race and were recruited during one racing season. Racing distances varied appropriately for the age and experience of the horse (1200 to 2400 m). Peripheral blood samples were collected daily beginning from the day when the clinical signs of the injury (lameness, heat or swelling) were observed, up to the 6th day after the race. Diagnosis was based on clinical examination and diagnostic imaging (radiographs and ultrasound) performed by the equine veterinary practitioner. Horses from the examined group were subjected to stall rest or light exercise (30 min in horse walker) depending on veterinary recommendations. Control group (n = 10) consisted of horses without signs of orthopedic disorder during the week following the race. Blood samples in the control group were collected every day beginning with the first day after race. Horses from the control group were exercised 30 min daily in horse walker. Four control horses left the racing facility before the last sampling due to the end of racing season. Blood samples were acquired by jugular venipuncture into 20 ml syringes and transferred immediately into K2-EDTA tubes for hematological tests and plain tubes for SAA analysis. EDTA samples were kept in +4°C and analyzed within 5 h from collection for routine hematological parameters including the total number of white blood cells (WBC) counted with an automated hematology analyzer (ABC Vet, Horiba ABX). Plain tubes were centrifuged at 4380 g for 5 minutes; the collected serum was frozen and stored at −20 °C for

http://dx.doi.org/10.1016/j.rvsc.2015.04.008 0034-5288/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: A. Turlo, et al., Post-exercise dynamics of serum amyloid A blood concentration in thoroughbred horses classified as injured and non-injured after the race, Research in Veterinary Science (2015), doi: 10.1016/j.rvsc.2015.04.008

ARTICLE IN PRESS A. Turlo et al./Research in Veterinary Science ■■ (2015) ■■–■■

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Table 1 Serum amyloid A concentration in compared groups of horses. Day

1st–2nd 3rd–4th 5th–6th a

Racing group

Injured group

n

Median (interquartile range) [ng/ml]

N

Median (interquartile range) [ng/ml]

10 10 6

1357 (889 to 10965) 973 (663 to 1585)a 638 (549 to 1511)

11 15 15

2215 (1232 to 14852) 4664 (882 to 24106)a 1381 (560 to 5322)

Mann–Whitney U test p-value

0.607 0.026 0.407

Significant difference between the control and the injured group.

SAA analysis. SAA concentrations were measured using an enzyme linked immunosorbent assay (PHASE SAA Assay, Tridelta Ltd). Sample dilution was 1:1000 instead of 1:2000, recommended by the manufacturer’s protocol, and the results appropriately recalculated. The test was previously validated for determination of SAA concentrations in horses (Coutinho da Silva et al., 2014; Cywin´ska et al., 2012; Pollock et al., 2005). SAA concentrations in groups were presented as medians and interquartile ranges in parentheses. Comparisons between groups were made with a Mann–Whitney U test. Correlation between SAA concentration and WBC in both groups was measured using Pearson product-moment correlation coefficient. A two-tailed p-value below 0.05 was considered significant. Data were analyzed using STATISTICA 10 (StatSoft). The following injuries were diagnosed in experimental group: distal bone fracture (including incomplete fractures of proximal phalanx, third metacarapal and carpal bones) (n = 6), bucked shins syndrome (n = 4), tendonitis (n = 4) and mixed injury (n = 1). Eleven horses were identified as injured within the first 24 to 48 h after race, whereas in 4 horses injuries were recognized between 48 to 72 h. SAA concentration tended to decrease during the first 6 days after race in all horses; however, the median SAA concentration was significantly higher in the injured group only at 3rd–4th days after race (p = 0.026) (Table 1, Fig. 1). At the same time, a significant positive correlation between the concentration of SAA and total WBC in both injured and control groups (r = 0.577 and r = 0.785, respectively) occurred. For SAA concentration, area under receiver operating characteristic curve (AUC) was 0.68 (95% CI: 0.46, 0.90) compared to the AUC = 0.49 (95% CI: 0.25, 0.73) for WBC count. At the cut-off of 2000 ng/ml, sensitivity and specificity of the SAA assay were 67.7% (CI 95%: 41.7%, 84.8%) and 80.0% (CI 95%: 49.0%, 94.3%), respectively. This study determined for the first time blood SAA concentrations in racehorses after clinical orthopedic injury related to the

intensive exercise. Statistical confirmation of the differences between injured and non-injured group was hampered by large variations in SAA levels among individuals. The results showed that in the injured horses, SAA levels were not as high as described in various inflammatory diseases (>50 000 ng/ml; Coutinho da Silva et al., 2014; Hobo et al., 2007; Hulten and Demmers, 2002; Jacobsen et al., 2006). Median SAA concentration was the highest at 3rd–4th days after injury and slightly exceeded the upper reference limit for horses (20 000 ng/ml). The possible cause of minor SAA changes in our study may be the relatively small mass and vascularization of damaged tissues. This hypothesis is incompatible with the studies in other species, indicating intensive release of APPs in bone fractures (Bauza et al., 2011; Buttenschoen et al., 2000). Similar intensity and course of the SAA response was previously reported by Pollock et al. (2005) in horses undergoing elective and non-elective surgery. The mean peak concentration of SAA observed after surgery approximated the highest median concentration reported in our study. Injuries, however, cannot be foreseen to measure the pre-injury SAA values in examined horses. It precludes complete evaluation of SAA response after injury, as the magnitude of the change is important for interpretation of APPs level. The recent study on thoroughbred racehorses confirms that blood SAA concentration appears a more sensitive indicator of health issues than total WBC (Anhold et al., 2014). Similar observation was made in the neonatal foals with infectious diseases (Hulten and Demmers, 2002). In our study, the median concentration of SAA was significantly higher in injured than in healthy horses at 3rd–4th days after the race and therefore may be used to confirm trauma detection. Despite positive correlation with WBC, there was a tendency for SAA to be a more accurate indicator of injury. This appears in line with the previous findings and confirms important role of SAA as a useful marker in the diagnosis of equine diseases. References

Fig. 1. Serum amyloid A concentration in examined horses.

Anhold, A., Candon, R., Chan, D.-S., Amos, W., 2014. Comparison of elevated blood parameter values in a population of thoroughbred racehorses. Journal of Equine Veterinary Science 34, 651–655. Bauza, G., Miller, G., Kaseje, N., Wigner, N.A., Wang, Z., Gerstenfeld, L.C., et al., 2011. The effects of injury magnitude on the kinetics of acute phase response. The Journal of Trauma 70, 948–953. Buttenschoen, K., Fleishmann, W., Haupt, U., Kinzl, L., Buttenschoen, D.C., 2000. Translocation of endotoxin and acute-phase proteins in malleolar fractures. The Journal of Trauma 48, 241–245. Coutinho da Silva, M.A., Canisso, I.F., MacPherson, M.L., Johnson, A.E., Divers, T.J., 2014. Serum amyloid A concentration in healthy periparturient mares and mares with ascending placentitis. Equine Veterinary Journal 45, 619–624. Cywin´ska, A., Szarska, E., Górecka, R., Witkowski, L., Hecold, M., Bereznowski, A., et al., 2012. Acute phase protein concentrations after limited and long distance endurance rides in horses. Research in Veterinary Science 93, 1402–1406. Hobo, S., Niwa, H., Anzai, T., 2007. Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia. The Journal of Veterinary Medical Science/The Japanese Society of Veterinary Science 69, 827–830. Horohov, D.W., Sinatra, S.D., Chopra, R.K., Jankowitz, S., Betancourt, A., Bloomer, R.J., 2012. The effect of exercise and nutritional supplementation on proinflammatory cytokine expression in young racehorses during training. Journal of Equine Veterinary Science 32, 805–815. Hulten, C., Demmers, S., 2002. Serum amyloid A (SAA) as an aid in the management of infectious disease in foal: comparison with total leukocyte count, neutrophil count and fibrinogen. Equine Veterinary Journal 34, 693–698.

Please cite this article in press as: A. Turlo, et al., Post-exercise dynamics of serum amyloid A blood concentration in thoroughbred horses classified as injured and non-injured after the race, Research in Veterinary Science (2015), doi: 10.1016/j.rvsc.2015.04.008

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Jacobsen, S., Niewold, T.A., Halling-Thomsen, N., Nanni, S., Olsen, E., Lindegaard, C., et al., 2006. Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis. Veterinary Immunology and Immunopathology 110, 325–330. Lamprecht, E.D., Bagnelli, C.A., Williams, C.A., 2008. Inflammatory responses to Three modes of intense exercise in Standardbred mares – a pilot study. Comparative Exercise Physiology 5, 115–125.

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Pollock, P.J., Prendergast, M., Schumacher, J., Bellenger, C.R., 2005. Effects of surgery on the acute phase response in clinically normal and diseased horses. The Veterinary Record 156, 538–542.

Please cite this article in press as: A. Turlo, et al., Post-exercise dynamics of serum amyloid A blood concentration in thoroughbred horses classified as injured and non-injured after the race, Research in Veterinary Science (2015), doi: 10.1016/j.rvsc.2015.04.008

Post-exercise dynamics of serum amyloid A blood concentration in thoroughbred horses classified as injured and non-injured after the race.

The aim of this study was to evaluate serum amyloid A (SAA) concentration in horses with orthopedic injuries acquired during racing and in healthy one...
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