Anna Cywińska

Acute phase protein concentrations after limited distance and long distance endurance rides in horses

Acute phase proteins (APP) have been described as useful for assessing health in human and animal patients, as they closely reflect the acute phase reaction (APR). In humans and dogs a reaction analogous to APR has also been described after prolonged or strenuous exercise. The aim of this study was to determine, if similar reactions occur in endurance horses after limited and long distance rides. Seventeen horses that successfully completed various distance competitions were tested. Routine haematological and biochemical tests were performed and the concentrations of serum amyloid A (SAA), C-reactive protein (CRP) and haptoglobin were measured. Typical endurance exercise-induced haematological and biochemical changes were observed in all horses, regardless the distance. After long distance rides, the level of SAA markedly increased, but CRP and haptoglobin concentrations remained unchanged. After limited distance rides no changes in the levels of APPs were noted. Exercise-induced APR in horses occurred only after prolonged, strenuous exertion, and differed from APR in inflammation in that only SAA concentration was increased.

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.

Serum amyloid A (SAA) concentration after training sessions in Arabian race and endurance horses

BackgroundSerum amyloid A (SAA) is the major acute phase protein in horses. Its concentration increases in various pathologies but also in response to prolonged, strenuous effort. The purpose of this study was to establish whether routine race and endurance training produces changes in the SAA level in Arabian horses. Additionally, the differences between SAA response in experienced endurance horses and endurance horses that were beginning their career were investigated.ResultsThere were no changes in SAA concentrations after race training and endurance training in experienced horses. In horses that were beginning their endurance training, exercise produced an increase in SAA level as compared with rest level.ConclusionIn Arabians, the SAA concentration seems to be a good indicator of endurance training but is useless in race training. The routine training of experienced horses, which were prepared for long distance rides, did not promote any changes in the SAA level. In contrast, a significant increase in the SAA concentration was observed in horses that were beginning their endurance training and were only prepared for moderate distance rides and underwent the same effort. Further research is needed to elucidate whether this difference reflects too heavy training or adaptation to an increasing workload. Additionally, the adaptation to long distance rides in Arabians may include a reduced acute phase response.

Fasciola hepatica - the pilot study of in vitro assessing immune response against native and recombinant antigens of the fluke.

Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.

Changes in salivary and plasma cortisol levels in Purebred Arabian horses during race training session

Physical activity and stress both cause an increase in cortisol release ratio. The aim of this study was to evaluate the use of saliva samples for the determination of cortisol concentrations indicating the work-load level in horses during race training. Twelve Purebred Arabian horses aged 3-5 years were studied during the routine training session. After the warm-up, the horses galloped on the 800 m sand track at a speed of 12.8 m/s. Three saliva samples, and three blood samples were collected from each horse. Both types of samples were taken at rest, immediately after return from the track and after 30 min restitution. The concentrations of blood lactic acid (LA), and cortisol in saliva and plasma samples were measured and analyzed. Blood LA, plasma and salivary cortisol levels increased significantly after exercise (P < 0.05). Salivary cortisol concentration determined 30 min after the exercise correlated significantly with plasma cortisol level obtained immediately after exercise (P < 0.05) as well as measured 30 min after the end of exercise (P < 0.05). The determination of cortisol concentration in saliva samples taken from racehorses 30 min after the end of exercise can be recommended to use in field conditions to estimate the work-load in racehorses.

Multiple etiologies of equine recurrent uveitis – A natural model for human autoimmune uveitis: A brief review

Equine recurrent uveitis (ERU) has various etiologies, with Leptospira infection and genetic predisposition being the leading risk factors. Regardless of etiology, expression of ocular proteins associated with maintenance of the blood-ocular barrier is impaired in ERU. The recurring-remitting cycle of ERU repeatedly disrupts the blood-ocular barrier, allowing the previously immune-privileged ocular environment to become the site of a progressive local autoimmune pathology that ultimately results in tissue destruction and vision loss. The immune-mediated process involves humoral and cellular mechanisms. Intraocular antibodies either produced in the eye or that leak through the blood-ocular barrier, are often present at higher levels than in serum and react with antigens in ocular tissue of horses with ERU. Ocular infiltration of auto-aggressive lymphocytes occurs with each uveitis episode and is the most crucial contributor to inflammation and eye damage. Recurring uveitis episodes may be initiated when epitopes of an ocular antigen become visible to the immune system (intramolecular spreading) or another autoantigen (intermolecular spreading), resulting in a new inflammatory reaction.

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 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.

The Effect of Different Types of Musculoskeletal Injuries on Blood Concentration of Serum Amyloid A in Thoroughbred Racehorses

Background Training-induced muscle, skeletal and joint trauma may result in acute phase response reflected by the changes in the blood concentration of serum amyloid A (SAA) in racehorses. It remains yet unclear if such systemic reaction could be triggered by sport injuries and what is the impact of different types of musculoskeletal trauma on SAA concentrations in racehorses. This study aimed to determine changes in the SAA blood concentration in racehorses with different types of injuries of musculoskeletal system. Materials and Methods The study involved 28 racehorses diagnosed after the race with bone fractures (n = 7), dorsal metacarpal disease (n = 11), joint trauma (n = 4) or tendon and muscle trauma (n = 6) and 28 healthy control racehorses. Serum samples were collected twice, between 1 and 4 days of the injury or succesful completion of the race. SAA concentration was measured using the commercial ELISA kit. Differences between mean SAA concentration in respective groups were analyzed using ANOVA and Tukey post-hoc test. Results Mean SAA concentration within the first 4 days of the injury of muscle and tendon was significantly higher than in bone fractures, dorsal metacarpal disease, joint trauma or in the healthy horses (p<0,001). There were no significant differences between the other groups. Conclusions Strain injuries of muscle and tendons can cause a moderate increase in SAA blood concentration in racehorses, reflecting the occurrence of the acute phase response. Similar reaction is not observed in the stress-related bone injuries.

In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations– 1 μM (10−3 mol/m3), 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance–expansion of Treg cells and lymphocyte apoptosis–was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.

Reference intervals for selected hematological and biochemical variables in Hucul horses.

BACKGROUND Hucul horses are the unique, genetically distinct breed of Carpathian Mountains. Even though they are recognized as primitive breed, many morphological differences between them and other primitive horses have been reported. Neither hematological nor blood biochemical studies in this breed have been conducted so far. OBJECTIVES The aim of this study was to establish the reference intervals for basic hematological and selected biochemical variables and to compare them with other breeds. MATERIAL AND METHODS Blood samples were collected from 168 Hucul horses and the analyses were performed using routine methods. Mainly nonparametric method was used to establish reference intervals. RESULTS The following reference intervals have been established (rounded to two significant digits): RBC: 7.0-13×1012/l; HGB: 106.1-195.8 g/l; HCT: 0.3-0.6 l/l; MCV: 35-50 fl; MCH 11.9-17.1 pg; MCHC: 31.9-34.8 g/dl; WBC: 7.5-22×109/l, bands: 0-0.5×109/l; segmented neutrophils: 3.3-10×109/l; eosinophils: 0-1.1×109/l; basophils: 0-0.3×109/l; lymphocytes: 1.9-12×109/l; monocytes: 0-0.2×109/l; PLT 95-350×109/l; MPV 5.2-7.0; ALP: 98-425 U/l; AST: 220-470 U/l; GGT: 9.1-31 U/l; total bilirubin: 6.5-29 μmol/l; CPK: 120-640 U/l; triglycerides: 0.1-0.9 mmol/l; urea: 3.8-11 mmol/l; creatinine: 44 -140 μmol/l; serum amyloid A: 130-5200 μg/l. CONCLUSIONS Hematological and biochemical variables in Hucul horses were closer to hot-blooded then to cold-blooded and primitive horses or wild equidae. The reference intervals presented in this study pose clinically useful tool for evaluation of blood check-up in Hucul horses.