Short-term Evaluation of Serum Amyloid A after Exercise in Clinically Healthy Horses

Serum amyloid A (SAA), the major equine acute-phase protein, is often measured after the race to investigate whether poor performances could depend on inflammation. The aim of this study was to assess whether there is an increase in concentration of SAA in serum samples collected from 12 clinically healthy Standardbred horses 1 hour after a standard race. Exercise induced an increase in red blood cells, hematocrit, and total proteins but not in SAA. However, a two- to threefold increase of SAA concentration as compared with prerace values was found in three horses. In conclusion, the concentration of SAA in most of the samples collected 1 hour after the race remains unchanged as compared with prerace samples. However, individual variability in response to exercise exists. The evaluation of SAA immediately after the race is not clinically useful.     

Age Effects on Blood Gas,Spirometry, Airway Reactivity,and Bronchoalveolar Lavage Fluid Cytology in Clinically Healthy Horses

Background

Despite the increasing number of geriatric horses attended by veterinarians, there is a lack of understanding of aging‐related changes on the respiratory system of horses.

Objective

To identify aging‐related changes on the respiratory function and bronchoalveolar lavage fluid (BALF) cytology of horses.

Animals

Fifteen healthy young adult (2–11 years) and 16 healthy aged (≥20 years) horses.

Methods

The respiratory system was examined by measurement of arterial blood gases (ABG), use of respiratory inductive plethysmography (RIP) for assessment of breathing pattern and ventilatory parameters, histamine bronchoprovocation, and BALF cytology.

Results

No significant differences were detected with regard to values obtained by ABG or bronchoprovocation of young adult and aged healthy horses. In aged horses, there were significant differences in mean ± SD of the following parameters when compared to young horses: prolonged expiratory time (Te) measured by RIP (3.9 ± 1.5 s versus 3.0 ± 0.6 s), decreased percentage of alveolar macrophages (40.6 ± 11.3% versus 53.5 ± 9.6%), and increased percentage of lymphocytes (53.4 ± 9.5% versus 43.9 ± 11.0%). No correlations between airway reactivity and ventilatory parameters, ABG, or BALF cytology were found in this asymptomatic population.

Conclusions

These results suggest that aging does not cause changes in the results obtained by ABG, most RIP‐derived variables, and bronchoprovocation in the horse. A decreased percentage of macrophage and an increased percentage of lymphocytes in the BALF cytology may be expected in the asymptomatic geriatric horse and may be a result of aging.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.     

Serum Amyloid A (SAA) Concentration after Vaccination in Horses and Mules

Serum amyloid A (SAA) is a sensitive acute-phase response (APR) marker in equids. Prominent APRs with elevations of SAA concentrations ([SAA]) have been reported after vaccination. The authors hypothesized that vaccination with an inactivated EHV-1/-4 vaccine would cause increase in [SAA] and antibody responses and that higher [SAA] would be positively correlated with the antibody titer in both equids. Twelve Haflinger horses and 12 mules were included in this longitudinal prospective study. All horses and mules were vaccinated with a commercially available EHV-1/-4 vaccine. Blood was sampled before and after vaccination to measure [SAA] and virus-neutralizing response (VN-T). In horses and mules, significantly higher [SAA] were measured on days 1, 3, and 5 after EHV-1/-4 vaccination; [SAA] on day 1 after vaccination were only measured in animals that developed fever, where mean [SAA] were significantly higher in horses than in mules (horses: 1,365.75 ± 87.64 mg/L, mules: 615.5 ± 153.444 mg/L) (P > .05). Four horses and 2 mules developed fever after vaccination, lasting for ≤24 hours. Increased antibody responses (VN-T) on days 7 and 14 after vaccination were observed in all animals, whereas mules showed higher overall antibody responses. Nevertheless, [SAA] did not correlate with the intensity of the antibody responses (VN-T) stimulated by the vaccine (P < .05). EHV-1/-4 vaccination caused a prominent APR, higher in horses than in mules, but [SAA] did not correlate with antibody responses. Measuring [SAA] after vaccination could help identify severe APRs that may require longer resting intervals before training or competition.     

Plasma Procalcitonin Concentration in Healthy Horses and Horses Affected by Systemic Inflammatory Response Syndrome

Background

The diseases most frequent associated with SIRS in adult horses are those involving the gastrointestinal tract. An early diagnosis should be the goal in the management of horses with SIRS.

Objective

The objective of this study was to evaluate the plasma procalcitonin (PCT) concentration in healthy and SIRS horses to assess differences between the two groups.

Animals

Seventy‐eight horses (30 healthy and 48 SIRS).

Methods

Prospective in vivo multicentric study. Horses were classified as SIRS if at least 2 of the following criteria were met: abnormal leukocyte count or distribution, hyperthermia or hypothermia, tachycardia, tachypnea. Healthy horses showed no clinical or laboratory signs of SIRS. Plasma PCT concentrations were measured with a commercial ELISA assay for equine species. Results were expressed as mean±standard deviation. T‐test for unpaired data was performed between healthy and SIRS group. SIRS group was divided in 4 subgroups and t‐test was performed between healthy versus each subgroup.

Results

PCT concentrations in healthy and SIRS horses were 18.28 ± 20.32 and 197.0 ± 117.0 pg/mL, respectively. T‐test showed statistical differences between healthy versus SIRS group and between healthy versus all subgroups.

Conclusions and Clinical Importance

Results showed an increase in PCT concentration in SIRS horses as previously reported in humans and dogs. PCT could be used as a single assay in equine practice for detection of SIRS.     

Synovial Fluid Cytokines and Eicosanoids as Markers of Joint Disease in Horses

OBJECTIVE: To evaluate the value of various synovial fluid cytokines and eicosanoids to diagnose joint disease or categories of joint disease. STUDY DESIGN: Prospective acquisition of clinicopathologic data. ANIMALS OR SAMPLE POPULATION: Client-owned or donated horses: 50 joints with no evidence of disease; 28 joints with acute disease; 32 joints with chronic disease; 9 joints with cartilage damage and no other signs of joint disease. METHODS: Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), prostaglandin F1-alpha (PGF(1)-alpha), and leukotriene B(4) (LTB(4)), were measured in equine synovial fluid by immunoassay and categorized according to duration and degree of joint disease. Any test value for a given category that was different from normal was further analyzed for sensitivity (S), specificity (Sp), and operating point (most valid test cutoff value). Likelihood ratios and predictive values were calculated at the operating point. Mediator concentrations were correlated to synovial fluid white blood cell count. Tests were reported as poor, fair, good, or excellent based on predictive values of <.25,.25-.5,.5-.75, or >.75, respectively. RESULTS: TNF synovial fluid concentration as a predictor of joint disease was good, and the value of TNF (maximum S and Sp) indicating joint disease was >36 pg/mL. IL-1beta as a predictor of joint disease was good, and the value of IL-1beta indicating joint disease was >4.5 pg/mL. IL-6 concentration was an excellent predictor of joint disease. Any IL-6 in synovial fluid indicated joint disease and correlated highly with synovial fluid white blood cell count (P <.0001). PGE(2) was a good-excellent predictor of disease (positive predictive value [PPV] = 0.75), and the concentration indicating joint disease was >22.5 pg/mL. The diagnostic PGF(1)-alpha concentration indicating severe chronic joint disease was identified to be >16.5 pg/mL with very high sensitivity (S = 1) and specificity (Sp =.89). PGF(1)-alpha concentrations > 9.5 pg/mL had a good PPV (.69) and NPV (.6) for any joint disease. TBX(2) concentrations below 31.5 pg/mL (S =.57; Sp =.61) were a very good predictor of joint disease (PPV =.72). LTB(4) concentration appeared to be greater in severe acute joint disease than normal joints; this was not significant (P =.15) and correlated highly with synovial fluid white blood cell count (P =.0001). CONCLUSIONS: The ability of a single value from a joint in an adult horse predicting the presence of joint disease was often good (.5-.75), and was excellent (> or =.75) for IL-6 and PGE(2). TNF-alpha and IL-1beta were no more effective than white blood cell count in screening for joint disease. IL-6 was the most sensitive and specific for joint disease and could be an excellent screening test for the presence of joint disease when lameness is difficult to identify or is intermittent. PGE(2) would be a functional screening test for the presence of any joint disease and offers a differentiating feature because values were not influenced by white blood cell count. PGF(1)-alpha values > 16.5 pg/mL identified chronic severe joint disease and may be clinically useful when there are minimal radiographic changes but substantial articular cartilage degradation.     

Secretoglobin and Transferrin Expression in Bronchoalveolar Lavage Fluid of Horses with Chronic Respiratory Disease

Background

Lower expression of secretoglobin and transferrin has been found in the bronchoalveolar lavage fluid (BALF) of a small number of horses with experimentally induced signs of recurrent airway obstruction (RAO) compared to healthy controls.

Hypothesis/Objectives

Secretoglobin and transferrin BALF expression will be similarly decreased in horses with naturally occurring clinical signs of RAO and in horses with experimentally induced clinical signs of RAO as compared to healthy controls and intermediate in horses with inflammatory airway disease (IAD).

Animals

Recurrent airway obstruction‐affected and control horses were subjected to an experimental hay exposure trial to induce signs of RAO. Client‐owned horses with a presumptive diagnosis of RAO and controls from the same stable environments were recruited.

Methods

Pulmonary function and BALF were evaluated from control and RAO‐affected research horses during an experimental hay exposure trial (n = 5 in each group) and from client‐owned horses (RAO‐affected horses, n = 17; IAD‐affected horses, n = 19; healthy controls, n = 5). The concentrations of secretoglobin and transferrin in BALF were assessed using Western blots.

Results

Naturally occurring and experimentally induced RAO horses had similar decreases in BALF transferrin expression, but secretoglobin expression was most decreased in naturally occurring RAO. Secretoglobin and transferrin expression were both lower in BALF of RAO‐affected horses than in IAD‐affected and control horses.

Conclusions and Clinical Importance

Secretoglobin and transferrin expression is decreased in BALF of RAO‐affected horses after both experimental and natural exposure. Secretoglobin and transferrin likely play clinically relevant roles in the pathophysiology of RAO, and may thus be used as biomarkers of the disease.     

Glucocorticoid Receptor Density and Binding Affinity in Healthy Horses and Horses with Systemic Inflammatory Response Syndrome

Background

Dysregulation of the hypothalamic‐pituitary‐adrenal (HPA) axis occurs in horses with systemic inflammatory response syndrome (SIRS). Peripheral resistance to glucocorticoids has not been investigated in horses.

Objective

To determine if glucocorticoid receptor (GR) function in horses can be measured using flow cytometry, and to use this information to evaluate HPA axis dynamics.

Animals

Eleven healthy adult horses in parts 1 and 2. Ten horses with SIRS and 10 age and sex matched controls in part 3.

Methods

Flow cytometry was used to evaluate GR density and binding affinity (BA) in 3 healthy horses in part 1. In part 2, exogenous ACTH was administered to eight healthy horses. Their cortisol response and GR properties were measured. In part 3, CBC, serum biochemistry, cortisol and ACTH, and GR properties were compared between controls without SIRS (n = 10) and horses with SIRS (n = 10), and between survivors and nonsurvivors (n = 4 and n = 6 respectively).

Results

Flow cytometry can be used to measure GR properties in equine PBMCs. No correlation was observed between plasma cortisol concentration and GR density or BA in healthy horses (r = −0.145, P = .428 and r = 0.046, P = .802 respectively). Nonsurvivors with SIRS had significantly decreased GR BA (P = .008). Horses with triglyceride concentration > 28.5 mg/dL had increased odds of nonsurvival (OR=117; 95% CI, 1.94–7,060). GR BA <35.79% was associated with nonsurvival (OR = 30.33; 95% CI, 0.96–960.5).

Conclusions and Clinical Importance

Tissue resistance to glucocorticoids contributes to HPA axis dysfunction in adult horses with SIRS. These horses might benefit from treatment with exogenous glucocorticoids.     

Cytokine Concentrations in Bronchoalveolar Lavage Fluid from Horses with Neutrophilic Inflammatory Airway Disease

Background

Multiple cytological patterns occur in bronchoalveolar lavage fluid (BALF) of horses with inflammatory airway disease (IAD). Only few data on BALF cytokine profiles are available for horses with IAD, and are limited to mRNA expression.

Hypothesis/Objective

Cytological profiles of IAD are associated with different BALF immunological pathways. To investigate BALF cytokine concentrations in a large number of horses with neutrophilic IAD.

Animals

One hundred and thirty‐eight client‐owned Standardbred racehorses in active training.

Methods

Prospective observational study. BALF samples were obtained from left and right lungs. Interleukin (IL)‐4, interferon (IFN)‐γ, and tumor necrosis factor (TNF)‐α concentrations were determined by ELISA.

Results

Fourteen horses had normal BALF cytological profiles and 56 exhibited evidence of bilateral neutrophilic IAD. Twenty‐four horses showed BALF with, respectively, IAD‐ and CTL consistent cytology and were excluded; as were 44 horses because of evidence of pulmonary hemorrhage. TNF‐α (56 ± 115 pg/mL; P = .034) and IFN‐γ concentrations (104 ± 247 pg/mL; P = .044) were significantly higher for IAD horses, compared with controls (respectively 19 ± 41 and 80 ± 116 pg/mL). Horses with ‘neutrophil’ subtype had significantly higher IFN‐γ concentrations (110 ± 154 pg/mL), than ‘neutrophil/metachromatic’ (56 ± 54 pg/mL; P = .028) and ‘neutrophil/metachromatic/eosinophil’ subtypes (44 ± 23 pg/mL; P = .012).

Conclusions and Clinical Importance

Cytokine concentrations in BALF suggested that neutrophilic IAD is associated with activation of the innate immune system and a possible T‐helper (Th)‐1 polarized response. This study also suggested that immunological pathways vary according to cytological IAD subtypes.     

Administration of Perioperative Penicillin Reduces Postoperative Serum Amyloid A Response in Horses Being Castrated Standing

Objectives: To compare postoperative inflammatory responses in horses administered perioperative procaine penicillin and those not administered penicillin using acute phase protein serum amyloid A (SAA) as a marker of inflammation. Study Design: Randomized clinical trial. Animals: Stallions (n=50) castrated under field conditions. Methods: SAA concentrations were determined on days 0, 3, and 8. Six horses were subsequently excluded because of elevated SAA concentrations on day 0. Of the remaining 50 horses, 26 were administered nonsteroidal anti‐inflammatory drug (NSAID) therapy and 24 were administered NSAID and 25,000 U/kg procaine penicillin on day 0, 1, and 2. Results: SAA concentrations increased significantly from preoperative levels in both groups, and on day 8 concentrations were significantly (P<.02) higher in horses administered only NSAID than in those administered procaine penicillin and NSAID. Infectious complications occurred more frequently (P<.01) in horses with preoperatively elevated SAA concentrations (the excluded horses) than in horses with normal preoperative SAA concentrations (the included horses). Conclusions: Perioperative antimicrobial therapy reduced the postoperative SAA response, suggesting that bacteria were present in the surgical wound and contributed to inflammation after castration. Horses with elevated preoperative SAA concentrations developed infectious complications more often than horses with normal preoperative SAA concentrations. Clinical Relevance: Administration of antimicrobials may be important in horses being castrated standing under field conditions. Increased SAA concentrations seem to be an indicator of increased surgical risk in horses and may be useful before elective surgery for planning.     

Effect of Four Antimicrobial Lavage Solutions on the Tarsocrural Joint of Horses

Three concentrations of povidone-iodine (0.1% w/v, 0.2% w/v, 0.5% w/v) and one concentration of chlorhexidine (0.5% w/v) were selected as antimicrobial joint lavage solutions. Through-and-through joint lavage was performed with one of these antimicrobial solutions on a tarsocrural joint of 12 horses. The contralateral tarsocrural joints (control limbs) were lavaged with a balanced electrolyte solution (BES). The effect of the lavage solution on the joints was evaluated with respect to lameness, foot flight pattern, soreness to joint palpation, articular and periarticular enlargement, and synovial fluid composition on Day 1,4, and 8 postlavage. On Day 8 postlavage, all horses were euthanized and the tarsocrural joints were examined.
All solutions induced a synovitis. Based on clinical assessment, synovial fluid protein levels, color, clarity, mucin clot forming ability, gross appearance of the joint at necropsy, and synovial membrane histologic evaluation, a similar, mild, transient, synovitis was induced by the BES and 0.1% povidone-iodine (PI) solution. The 0.2% PI solution induced a more prolonged neutrophilic response and poorer mucin clot forming ability in the synovial fluid as compared to the BES.
The 0.5% PI and 0.5% chlorhexidine solutions produced severe lameness, soreness to joint palpation, and limb enlargement. The elevated synovial fluid total protein content persisted significantly longer (p < 0.05) than the corresponding control (BES) solution. Histologic evaluation of the synovial membrane confirmed the presence of a moderate to severe neutrophilic synovitis in these treatment groups.     

Studies on the Physiological Chemistry of the Tibio-Tarsal Synovial Fluid of Healthy Bovines

The physical properties, chemical composition and cytological content of the synovial fluid of healthy native breed cattle, obtained by tibio-tarsal arthrocentesis, was studied.     

Analysis of Circulating Immune Complexes in the Serum of Clinically Healthy Horses

The aim of this study was to determine the level of circulating immune complexes (CIC) in equine serum and the electrophoretic properties of the protein fraction thereof. The study was carried out using 42 clinically healthy breeding horses of noble half-blood. The average extinction value of CIC's in equine serum was E450 nm = 0.278 ± 0.09 nm. Generally, 23 protein fractions were identified in the molecular mass range between 294 and 25 kDa. Eight fractions whose molecular masses were 137, 72, 65, 52, 42, 32, 28, and 26 kDa, respectively, appeared in all analyzed samples. In 20 of the analyzed serum an additional nine protein fractions whose molecular masses were 260, 110, 84, 78, 67, 62, 57, 45, and 39 kDa, respectively, were identified. Circulating immune complexes are a source of information about immunologic processes that take place in mammals. Determining the average values of CIC levels and identifying the electrophoretic profile of proteins in clinically healthy horses could be useful in research and diagnostics.     

Synovial Fluid and Clinical Changes After Arthroscopic Partial Synovectomy of the Equine Middle Carpal Joint

Changes in synovial fluid and clinical variables after arthroscopic partial synovectomy of the middle carpal joint were studied in 12 normal horses. A 7 mm motorized synovial resector was inserted into each middle carpal joint; one middle carpal joint of each horse was randomly selected to have arthroscopic synovectomy (treated) and the opposite joint was lavaged (control). Lameness examinations and synovial fluid analyses were performed before operation and at 8, 14, 21, and 28 days after operation. Lameness variables did not differ between treated and control legs. Middle carpal and carpometacarpal joint circumference measurements were increased for 4 weeks. Synovial fluid specific gravity, pH, total protein, albumin concentration, and alpha-1-, beta- and gamma-globulin concentrations, at 8 and 14 days were significantly higher than before operation in both treated and control middle carpal joints. No significant differences were found between treated and control middle carpal joints at any time for color, clarity, pH, mucin clot formation, total protein, albumin, and globulin fractions. Arthroscopic partial synovectomy and lavage did not cause significant lameness and resulted in a synovitis indistinguishable from synovitis related to arthroscopic lavage alone.     

Effect of Repeated Arthrocentesis on Cytologic Analysis of Synovial Fluid in Dogs

Background: Serial arthrocentesis and synovial fluid examination can be used to monitor treatment efficacy in immune-mediated polyarthritis (IMPA), but whether this procedure induces inflammation that interferes with test result interpretation is unknown.
Objectives: The aim of this study was to determine the effect of repeated arthrocentesis on synovial fluid cytology in healthy dogs.
Animals: Nine healthy client-owned dogs.
Methods: Prospective study. Arthrocentesis was performed under sedation on 4 joints (both carpi, 1 tarsus, 1 stifle) on each dog every 3 weeks, a total of 4 times. Automated cell counts were done on stifle fluid, smears were made, and differential cell counts done on smears from all joints. Slides were evaluated microscopically for erythrocyte numbers, total nucleated cell count, differential cell count, and cell morphology. Data were analyzed by 2-way analysis of variance.
Results: A total of 144 synovial fluid samples were examined. Repeated arthrocentesis was not associated with increases in synovial fluid neutrophil numbers. Mild mononuclear inflammation was detected in 13 samples from 6 dogs.
Conclusions and Clinical Importance: Serial arthrocentesis at 3-week intervals can rarely be associated with mild mononuclear joint inflammation, but does not appear to induce neutrophilic inflammation, at least in healthy dogs, and can be useful to monitor treatment response in canine IMPA.     

奶牛跗关节的关节内窥镜径路及滑膜形态

应用关节内窥镜对奶牛跗关节的进镜部位和滑膜形态进行了研究。结果表明 ,跗关节可选 3个进镜部位 :背内侧囊在关节前方较突出部位 ,靠近趾内侧伸肌健 ,向外为跖背侧静脉和腓浅神经 ;跖内侧囊和跖外侧囊一般选在最突出的部位 ,跖外侧囊尽量靠前以避开足底外侧静脉 ,跖内侧囊不要靠后以防伤及深屈肌腱。关节不同部位的滑膜绒毛也不同 ,关节囊小且突起小的部位 ,关节滑膜绒毛一般呈短柱状或丝状 ;关节囊大的部位 ,一般呈菜花状或膜状 ,也有长丝状绒毛。在背内侧囊见有从未报道的拱桥状绒毛。靠近关节软骨处和关节固有韧带里面没有绒毛     

Morphological Effects of Arthroscopic Partial Synovectomy in Horses

Gross and microscopic effects of arthroscopic partial synovectomy on synovium and articular cartilage of middle carpal joints were studied in 15 horses. A 7-mm diameter motorized synovial resector was inserted into each middle carpal joint and arthroscopic partial synovectomy and lavage or arthroscopic lavage alone was performed. Study periods were 0 (three horses), 16 (three horses), and 30 days (six horses). No gross evidence of degenerative joint disease was observed at day 16 or 30. At 30 days, resected areas lacked villi and there was deposition of fibrin on the synovial surface with varying amounts of newly formed fibrovascular tissue. Thirty days after arthroscopic synovectomy, normal synovium had not formed in equine middle carpal joints.