Acute Phase Proteins in Racehorses with Inflammatory Airway Disease

Background

Systemic inflammation is observed in horses with heaves and could also be present in horses with a lesser degree of pulmonary inflammation.

Hypothesis/Objectives

It was hypothesized that racehorses with inflammatory airway disease (IAD) have increased concentration of circulating acute phase proteins. The objective of this study was to compare serum acute phase proteins of racehorses with and without lower airway inflammation.

Animals

Serum from 21 client‐owned Standardbred racehorses with exercise intolerance and lower airway inflammation and serum from 10 client‐owned Standardbred racehorses with exercise intolerance without lower airway inflammation.

Methods

In a case–control study, serum samples from previously characterized horses presented for exercise intolerance with or without lower airway inflammation based on bronchoalveolar lavage fluid cytology were analyzed for serum amyloid A protein (SAA), C‐reactive protein (CRP), and haptoglobin using commercial ELISAs.

Results

There was no significant differences between groups for SAA (non‐IAD versus IAD, median (range): 3.47 (0.06–34.94) versus 6.33 (0.06–80) μg/mL, P = .49), CRP (10.87 (2.05–29.03) versus 4.63 (0.02–31.81) μg/mL, P = .23) or haptoglobin (900.36 (607.99–2018.84) versus 749.54 (530.81–1076.95) μg/mL, P = .09).

Conclusions and Clinical Importance

In this population of poorly performing racehorses in training, serum SAA, CRP, and haptoglobin were not helpful in distinguishing between horses with IAD from horses with exercise intolerance from other causes.     

Selected Biochemical Indicators of Equine Rhabdomyolysis in Arabian Horses: Acute Phase Proteins and Trace Elements

Although creatine kinase (CK), aspartate transaminase (AST), cytokines, and oxidative stress parameters were shown to be useful biomarkers for diagnosis of equine rhabdomyolysis, additional biomarkers of the disease may be of interest to indicate prognosis of the disease. Therefore, the present study investigated acute phase proteins and trace elements as additional biomarkers of ER. Sixty male horses (4-6 years old) of 2 equal groups were used. Horses of the first group were clinically healthy and served as a control group, whereas horses of the second group were ER-diseased animals. Harvested sera were used for estimation of the activities of CK, AST, lactate dehydrogenase (LDH), serum amyloid A (SAA), haptoglobin (Hp), ceruloplasmin (Cp), copper, iron, and zinc, whereas plasma samples were used for determination of fibrinogen. The present findings revealed a significant (P ≤ .05) increase in values of CK, AST, and LDH in diseased horses compared to those in control values. In addition, a significant (P ≤ .05) increase in SAA (162.6 ± 5.32 mg/L), Hp (3.6 ± 0.54 g/L), Cp (39.32 ± 2.31 mg/L), and copper (28.36 ± 1.23 μmol/L) along with a significant (P ≤ .05) reduction in levels of iron (9.32 ± 0.23 μmol/L) and zinc (8.65 ± 1.02 μmol/L) was recorded in diseased horses compare to that in controls (11.3 ± 2.2 mg/L, 0.8 ± 0.2 g/L, 24.23 ± 1.32 mg/L, 18.41 ± 1.03 μmol/L, and 14.2 ± 0.42 μmol/L, respectively). In conclusion, SAA, Hp, Cp, copper, and zinc were useful prognostic biomarkers for the diagnosis of ER in Arabian horses.     

Peritoneal d-Dimer Concentration for Assessing Peritoneal Fibrinolytic Activity in Horses with Colic

Background: Plasma d -dimer concentration is a useful marker to assess systemic coagulation and fibrinolytic activities in humans, dogs, and horses. Peritoneal fibrinolytic activity increases in horses with colic, especially in horses with endotoxin in the peritoneal fluid.
Hypothesis/Objectives: Peritoneal d -dimer concentration can be used to assess peritoneal fibrinolytic activity in horses with severe gastrointestinal (GI) disorders and altered peritoneal fluid.
Animals: Two hundred and twenty-one colic horses and 15 control horses.
Methods: Prospective observational clinical study. Blood and peritoneal fluid were collected on admission. Horses were grouped according to diagnosis, peritoneal fluid analysis, and outcome. Peritoneal d -dimer concentration was determined, together with peritoneal tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) activities. Plasma d -dimer concentration also was measured.
Results: Peritoneal d -dimer concentration was significantly higher in all colic groups compared with controls, and in horses with enteritis, peritonitis, and ischemic disorders compared with horses with large intestinal obstructions. Peritoneal d -dimer concentration was significantly higher in horses with altered peritoneal fluid (modified transudate and exudate) compared with horses with normal peritoneal fluid analysis. Plasma d -dimer concentration also was significantly higher in the peritonitis group, and in horses with altered peritoneal fluid analysis. Peritoneal and plasma d -dimer concentrations also were significantly higher in nonsurvivors. Peritoneal d -dimer concentration was significantly correlated with decreased peritoneal t-PA activity and increased peritoneal PAI-1 activity.
Conclusions and Clinical Importance: Peritoneal d -dimer concentration is markedly higher in severe GI disorders, and it can be used to assess peritoneal fibrinolytic activity in horses with colic.     

Effects of Blood Contamination on Peritoneal D-Dimer Concentration in Horses with Colic

Background: Peritoneal D-Dimer concentration can be determined to assess peritoneal fibrinolysis activity in horses with gastrointestinal disorders. However, blood contamination of peritoneal fluid may occur during collection and could alter peritoneal D-Dimer concentration.
Hypothesis/Objectives: Blood contamination in peritoneal fluid does not affect interpretation of peritoneal D-Dimer concentration in horses with colic.
Animals: Thirty-four horses with colic and 4 healthy horses.
Methods: Peritoneal fluid and blood samples were simultaneously collected upon admission. Then, peritoneal fluid was serially contaminated with the horse's own blood; final contaminations corresponded to 1, 5, 10, and 20% of blood in peritoneal fluid. D-Dimer concentration was determined in blood, peritoneal fluid, and contaminated peritoneal fluid samples. Data were analyzed using a longitudinal linear model and a generalized estimating equations analysis to assess the quantitative and qualitative variations of the effect of blood contamination on peritoneal D-Dimer concentration.
Results: Peritoneal D-Dimer concentration was only quantitatively affected when peritoneal fluid was contaminated at 20% of blood. However, when using increasing cut-off values of peritoneal D-Dimer concentration (100, 2,000, 8,000, and 16,000 ng/mL), this effect disappeared at the highest cut-off values (8,000 and 16,000 ng/mL). When peritoneal fluid contamination was grouped as "minimally contaminated" (≤1% of blood) and "highly contaminated" (≥5% of blood), no significant differences on D-Dimer concentration between both groups at each cut-off value were observed.
Conclusions and Clinical Importance: Although quantitative results of peritoneal D-Dimer concentration could be affected by high levels of blood contamination (≥20%), interpretation of increased peritoneal fibrinolytic activity was not significantly affected.     

Influence of Fluid Therapy on Gentamicin Pharmacokinetics in Colic Horses

The aminoglycoside antibiotic gentamicin is commonly used in equine medicine for the prevention and treatment of Gram-negative and staphylococcal bacteria in surgically treated colic patients. The pharmacokinetics of gentamicin in these patients might be altered by the disease status, and/or under the influence of fluid therapy. The purpose of this study was to investigate the effect of intravenous fluid treatment on gentamicin kinetics in colic patients. Colic patients subjected to laparotomy were given fluid infusions according to clinical status. Following gentamicin administration, blood samples were taken for gentamicin analysis at different time points, and the main pharmacokinetic parameters including Vc, Vss, t(1/2) and MRT were calculated. Horses undergoing fluid therapy showed a significantly different t(1/2), clearance and MRT as compared to non-infused patients. However, taking into account the clinical status of the patients receiving fluid support, the data suggest that endotoxaemia, rather than fluid therapy, influence gentamicin pharmacokinetics following laparotomy.     

Acute phase proteins as indicators of calf herd health

The potential for using acute phase proteins (APPs) in the assessment of herd health was studied by examining the levels of serum haptoglobin, serum amyloid A (SAA) and plasma fibrinogen in relation to clinical findings and leukocyte counts in calves. Two groups of calves from conventional dairy farms were studied. The animals were examined 10 times during the first six weeks after introduction into a new environment. Haptoglobin, SAA and fibrinogen were analysed and weight gain, disease symptoms and treatments were recorded. Analysis of antibodies against viral infections was performed. An acute phase reaction (APR) score was established at each sampling by combining the APP results and total leukocyte counts. The health status differed between the two groups, although no manipulation of health had been performed, except that the group with a higher incidence of disease had a concurrent experimental infection with lungworm as part of another study. In the group with a higher incidence of disease, the mean weight gain was significantly lower, and the number of sampling days with elevated serum concentrations of APPs, and the mean maximum concentrations of haptoglobin and fibrinogen were significantly higher compared to the healthier group. The APR score was significantly higher at days 4 and 8 of the study in the group with a higher incidence of disease. The results indicate that measurement of APPs could be a useful tool for evaluation of health in calf herds.     

Serum Amyloid A and Haptoglobin Concentrations and Liver Fat Percentage in Lactating Dairy Cows with Abomasal Displacement

Background: There has been increased interest in measuring the serum concentration of acute phase reactants such as serum amyloid A [SAA] and haptoglobin [haptoglobin] in periparturient cattle in order to provide a method for detecting the presence of inflammation or bacterial infection.
Objectives: To determine whether [SAA] and [haptoglobin] are increased in cows with displaced abomasum as compared with healthy dairy cows.
Animals: Fifty-four adult dairy cows in early lactation that had left displaced abomasum (LDA, n = 34), right displaced abomasum or abomasal volvulus (RDA/AV, n = 11), or were healthy on physical examination (control, n = 9).
Materials and Methods: Inflammatory diseases or bacterial infections such as mastitis, metritis, or pneumonia were not clinically apparent in any animal. Jugular venous blood was obtained from all cows and analyzed. Liver samples were obtained by biopsy in cattle with abomasal displacement.
Results: [SAA] and [haptoglobin] concentrations were increased in cows with LDA or RDA/AV as compared with healthy controls. Cows with displaced abomasum had mild to moderate hepatic lipidosis, based on liver fat percentages of 9.3 ± 5.3% (mean ± SD, LDA) and 10.8 ± 7.7% (RDA/AV). [SAA] and [haptoglobin] were most strongly associated with liver fat percentage, r _s=+0.55 ( P < .0001) and r _s=+0.42 ( P = .0041), respectively.
Conclusions and Clinical Importance: An increase in [SAA] or [haptoglobin] in postparturient dairy cows with LDA or RDA/AV is not specific for inflammation or bacterial infection. An increase in [SAA] or [haptoglobin] may indicate the presence of hepatic lipidosis in cattle with abomasal displacement.     

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.     

Acute phase proteins,proinflammatory cytokines and oxidative stress biomarkers in sheep,goats and she-camels with Coxiella burnetii infection-induced abortion

Acute phase proteins (APPs) and oxidative stress are helpful markers in diagnosis of several infectious diseases. APPs, proinflammatory cytokines and oxidative stress markers were evaluated for their role in the diagnosis of naturally acquired Coxiella burnetii (Q fever) associated with abortion in sheep, goats and she-camels. Blood, aborted materials and vaginal swabs were collected from mixed herds in the Eastern Province of Saudi Arabia. Antioxidant biomarkers showed significant decline in cases of abortion compared to control animals at delivery time. The correlation between disease status and all parameters ranged from moderate to high. The APPs, cytokines and the oxidative stress marker malondialdehyde (MDA) displayed a high degree of distinction between aborted sheep and goat and normal delivered animals (AUC > 0.90). However, only MDA showed a high degree of differentiation (AUC > 0.90) between aborted she-camels and normal delivered controls. In conclusion, results from our study allow us to recommend using APPs, cytokines and oxidative stress markers as an additional tool for diagnosis of naturally occurring C. burnetii infection in sheep, goats and she-camels. However, it does not replace standard procedures for detection of C. burnetii.     

The use of receiver operating characteristic (ROC) analysis to assess the diagnostic value of serum amyloid A, haptoglobin and fibrinogen in traumatic reticuloperitonitis in cattle

Acute phase proteins (APPs) such as serum amyloid A (SAA), haptoglobin (Hp), and plasma fibrinogen (Fb) are diagnostic and prognostic biomarkers. This study determined the pattern of SAA in traumatic reticuloperitonitis (TRP) in cows, and compared the findings with Hp, and plasma Fb concentrations in the differential diagnosis of TRP using receiver operating characteristic (ROC) analysis. Blood samples were collected from 89 cows: 53 cases of TRP, 16 animals with other internal disorders and 20 healthy controls.SAA and Hp had a similar pattern and magnitude of changes in TRP. Using the ROC method, the optimal cut-off for the diagnosis of TRP was 68 μg/mL for SAA and 0.74 g/L for Hp, with 100% sensitivity and 86.1% specificity; either of these parameters may therefore be used as a sensitive and relatively specific tool for the differential diagnosis of TRP in cattle. The diagnostic accuracy of plasma Fb was significantly lower than either SAA or Hp (P < 0.01). The combined use of these APPs (whilst aware of their conditional interdependence) may not provide any additional diagnostic information than can be obtained using either SAA or Hp alone.     

Acute phase proteins response in cats naturally infected by hemotropic mycoplasmas

Information about the acute phase proteins (APP) response in cats naturally infected with hemoplasmas and in cats co-infected with different species of hemoplasmas is lacking.This study evaluated serum amyloid A (SAA), haptoglobin (Hp) and albumin in 48 cats naturally infected with hemoplasmas, including 25 with Candidatus Mycoplasma haemominutum and 23 co-infected with different hemoplasmas agents; and in 10 healthy control cats.Infected cats had significantly higher Hp and lower albumin than controls. Symptomatic cats had significantly higher SAA and Hp, and lower albumin than asymptomatic animals, and also than controls. Asymptomatic cats had significantly higher Hp than controls. Concentrations of APP were not significantly different between single infected and co-infected cats.According with these results, hemoplasmosis should be considered when alterations in APP are detected in diseased cats with compatible clinical signs. Furthermore, a subclinical infection should be considered in apparently healthy cats from endemic areas with increased Hp.     

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.     

Acute phase proteins in cattle after exposure to complex stress

Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin concentrations were obtained before (0-sample) and at 8, 24 and 48 hours after the start of transportation. Upon arrival the animals gave the impression of being anxious, and they appeared to have difficulty coping with isolation and with being tied on the slippery floors of the research stable. Serum concentrations of SAA and haptoglobin increased significantly in response to the stressors (P < 0.01 and 0.05 at 48 hours, respectively). Additionally, the animals had transient neutrophilia at 8 and 24 hours (P < 0.05). In conclusion, the results of the study suggest that SAA and haptoglobin may serve as markers of stress in adult cattle.     

Inflammatory Response of Healthy Horses Subjected to Small Colon Enterotomy and Treated or Not With Heparin

The acute phase response is a response to injury and depends on the severity of the trauma. Heparin is routinely used for postsurgical treatment of horses to prevent abdominal adhesions; however, its effect on inflammation is unknown. This study aimed to assess systemic inflammatory response of horses subjected to small colon enterotomy and to evaluate heparin effects on postsurgical inflammation. Ten adult horses were subjected to small colon enterotomy and were assigned to a control or a treatment group. Both groups received prophylactic antibiotics and flunixin, and the treatment group received 150 IU/kg heparin subcutaneously after surgery and every 12 hours for five days. WBC counts, peritoneal fluid evaluation, determination of serum and peritoneal haptoglobin (Hp), and serum amyloid A (SAA) were performed before, 12 hours, and 1, 2, 4, 6, 10, and 14 days after enterotomy. Forty-eight hours after surgery, a significant increase in serum Hp was observed in the control group, and SAA concentrations increased significantly in the both groups between 24 hours, 48 hours, and 4 days after surgery. The SAA and serum Hp concentrations produced no significant differences between the groups. Peritoneal Hp increased significantly in the control group 4 days after surgery and was significantly higher in the control group than in the treated group 14 days after surgery. Serum Hp and SAA identified the acute phase response changes faster, however, were not able to identify differences between groups. Peritoneal Hp concentrations identified inflammatory differences between the groups 14 days after surgery; the difference suggests that heparin may act decreasing inflammation.     

Acute phase response in mice experimentally infected with Trypanosoma congolense: a molecular gauge of parasite-host interaction

Mice infected with Trypanosoma congolense developed a severe anaemia 1 week after infection, which persisted till treatment with diminazine aceturate when the packed cell volume (PCV) recovered to pre-infection levels. This was accompanied by a marked increase in the plasma levels of the acute phase proteins (APP), serum amyloid P-component (SAP) and haptoglobin (Hp). The initial peak levels of Hp and SAP were attained 7 and 12 days post-infection (DPI), respectively. Thereafter SAP levels decreased significantly to near pre-infection levels, but later increased even after treatment to give a second peak 34 DPI after which there was a decline till the study was terminated. The Hp levels on the other hand decreased to an intermediate level after the initial peak increasing to a second peak 22 DPI. Thereafter Hp decreased significantly following diminazine aceturate treatment to reach pre-infection levels within 5 days post-treatment. This indicates that T. congolense-infected mice develop severe anaemia accompanied by an acute phase response leading to an increase in SAP and Hp but that following treatment divergent responses occurred indicating differences in the pathways for stimulation of the APP. Haptoglobin was shown to be an earlier indicator of infection and a better marker in monitoring the response to treatment.     

Changes in blood biomarkers in Arabian horses with Clostridium difficile-induced enterocolitis

Clostridium difficile (CD) is considered a major health care problem both in developing and developed countries; frequently reported to be associated with enterocolitis and diarrhea in horses and other animals. In this study, we examined acute phase response (APR), cytokines response, neopterin (NP) procalcitonin (PCT) production and oxidative stress condition in horses and foals with C. difficile-induced enterocolitis (CDIE) and evaluated the effectiveness of these parameters as biomarkers for the disease. A total of 407 Arabian horses in 35 stables were examined between January 2017 to December 2018. Only 24 out of 407 horses showed two or more signs of CDIE. The blood level of serum amyloid A (SAA), haptoglobin (HP), proinflammatory cytokines (TNF-α, IL-6 and IL1-β), serum malondialdehyde (MDA), PCT and NPT in horses with CDIE were higher than in healthy horses. Nevertheless, the levels of nitric oxide (NO), superoxide dismutase (SOD) and total antioxidant concentration (TAC) were considerably lower in diseased horses compared to those that were healthy. The ROC curves for eleven selected blood parameters, both in healthy horses and horses with CDIE demonstrated that all examined blood markers had significant levels of differentiation between CDIE cases and healthy controls (AUC > 87.5). The data in this study suggest that the evaluation of acute-phase proteins, cytokines, PCT, NPT, and oxidative stress biomarkers may well be used as a tool for diagnosis and assessment of CDIE and in disease pathogenesis in Arabian horses.