Calcium Regulating Hormones and Serum Calcium and Magnesium Concentrations in Septic and Critically Ill Foals and their Association with Survival

Background: Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival.
Hypothesis: Blood concentrations of ionized calcium (Ca²⁺) and magnesium (Mg²⁺) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival.
Animals: Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of ≤7 days were included.
Methods: Prospective, observational study. Blood was collected at initial examination for analysis. Foals with positive blood culture or sepsis score ≥14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays.
Results: Septic foals had decreased Ca²⁺ (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg²⁺, PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82).
Conclusions and Clinical Importance: Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.     

Hemostatic and Fibrinolytic Indices in Neonatal Foals with Presumed Septicemia

Thirteen coagulation tests evaluating hemostatic and fibrinolytic indices and serum cytokine and plasma endotoxin concentrations were obtained in 34 foals with a positive sepsis score (septic group) and 46 age-matched healthy foals. Compared to healthy foals, the prothrombin, activated partial thromboplastin, and whole blood recalcification times were significantly longer in septic foals. The fibrinogen and fibrin degradation products concentrations, percent plasminogen, alpha-2 antiplasmin, and plasminogen activator inhibitor activities, and tumor necrosis factor and interleukin-6 activities were greater in septic foals. Protein C antigen and antithrombin III activity were significantly lower in septic foals. Blood cultures were positive for growth and endotoxin was detected in 19 of 29 and 15 of 30 septic foals, respectively. In septicemic foals with detectable endotoxin in the plasma, the prothrombin and activated partial thromboplastin times were significantly longer and the plasminogen and antithrombin III activities were significantly less than in septic foals in which endotoxin was not detected. Twenty-three of the 34 septic foals did not survive. Septic foals that did not survive were most likely to have a positive blood culture in which a gram-negative organism was isolated. Histopathologic evidence of hemorrhage was evident in 11 foals at postmortem examination and thrombosis was identified in 2 foals. The prothrombin time was significantly longer in foals that had multisite hemorrhage at postmortem examination. The results of this study indicate that clinically relevant alternations in hemostatic and fibrinolytic indices occur in neonatal foals with septicemia and that derangements can be correlated with the presence of endotoxin in plasma. Derangements in hemostatic or fibrinolytic indices were helpful in identification of septic foals with increased risk of coagulopathy, but were not helpful in predicting hemorrhage as compared to thrombus formation. Survival of septicemic foals was correlated with gram-negative bacteremia, but not with the presence of endotoxin or coagulopathy.     

Plasma Adrenomedullin Concentrations in Critically Ill Neonatal Foals

Background

Bacterial sepsis remains a leading cause of morbidity and mortality in neonatal foals, but accurate diagnostic and prognostic markers are lacking. Adrenomedullin (AM) is a polypeptide with diverse biologic effects on the cardiovascular system that increases in septic humans and laboratory animals.

Hypotheses

Plasma AM concentration (p[AM]) is increased in septic neonatal foals compared to sick nonseptic and healthy control foals, and p[AM] is predictive of survival in septic neonatal foals.

Animals

Ninety critically ill (42 septic, 48 sick nonseptic) and 61 healthy foals <1 week of age.

Methods

A prospective observational clinical study was performed. Venous blood was collected from critically ill foals at admission and from healthy foals at 24 hours of age. Critically ill foals were categorized as septic or sick nonseptic based on blood culture results and sepsis score. Plasma [AM] was measured by using a commercially available ELISA for horses. Data were analyzed by using the Mann‐Whitney U‐test and P < .05 was considered significant.

Results

Plasma [AM] was not significantly different between septic and sick nonseptic foals (P = .71), but critically ill foals had significantly increased p[AM] compared to healthy controls (P < .0001). In critically ill foals, p[AM] was not predictive of survival (P = .051). A p[AM] cutoff concentration of 0.041 ng/mL provided a test sensitivity of 91% and specificity of 54% to predict illness.

Conclusions and Clinical Relevance

Plasma [AM] shows promise as a marker of health in neonatal foals, but p[AM] increases nonspecifically during perinatal illnesses and is not necessarily associated with sepsis.     

Sick Neonatal Foals Do Not Demonstrate Evidence of Oxidative Stress

Critical illness in humans is associated with alterations in oxidative stress and the concentration of antioxidant molecules; however, this association has not been examined in equine neonates. The purpose was to determine the concentration of various antioxidant molecules, as well as a marker of oxidative stress, in the serum and plasma of normal and sick neonatal foals and their dams. Results demonstrated that the concentration of selenium was less (61.71 vs. 77.93 ng/mL; P = .002) in sick versus healthy neonates, whereas the concentration of vitamin E was slightly higher in sick compared with healthy foals (4.36 vs. 3.17 mg/mL); however, this did not achieve statistical significance (P = .31). The vitamin E concentration was greater (5.37 vs. 3.43 mg/mL; P = .01) and serum selenium was less in sick mares (129.50 vs. 184.78 ng/mL; P = .0001) compared with healthy mares. In addition, the serum concentration of selenium is lower in neonates than in their dams in the perinatal period (70.10 vs. 173.34 ng/mL; P = .0001). Glutathione peroxidase activity was less in sick foals (7.04 nmol/min/mL) compared with healthy foals (9.13 nmol/min/mL) (P = .19), and 3-nitrotyrosine (3-NT) concentration/mg protein was less in sick foals versus healthy foals (geometric mean, 1.24 vs. 2.07 nmol 3-NT/mg protein). This difference did not achieve statistical significance (P = .09); however, when a subset of critically ill foals was examined, the assayed concentration of 3-NT/mg protein was even less (0.99 nmol 3-NT/mg protein) and did statistically differ from healthy foals (P = .03).     

Blood arginine vasopressin, adrenocorticotropin hormone, and cortisol concentrations at admission in septic and critically ill foals and their association with survival

BACKGROUND: Sepsis is an important cause for neonatal foal mortality. The hypothalamic-pituitary-adrenal axis (HPAA) responses to sepsis are well documented in critically ill humans, but limited data exist in foals. The purpose of this study was to evaluate the HPAA response to sepsis in foals, and to associate these endocrine changes with survival. HYPOTHESIS: Blood concentrations of arginine vasopressin (AVP), adrenocorticotropin hormone (ACTH), and cortisol will be higher in septic foals as compared with sick nonseptic and healthy foals. The magnitude of increase in hormone concentration will be negatively associated with survival. ANIMALS: Fifty-one septic, 29 sick nonseptic, and 31 healthy foals of < or =7 days of age were included. METHODS: Blood was collected at admission for analysis. Foals with positive blood culture or sepsis score > or =14 were considered septic. Foals admitted with disease other than sepsis and healthy foals were used as controls. AVP, ACTH, and cortisol concentrations were measured using validated immunoassays. RESULTS: AVP, ACTH, and cortisol concentrations were increased in septic foals. Septic nonsurvivor foals (n = 26/51) had higher plasma ACTH and AVP concentrations than did survivors (n = 25/51). Some septic foals had normal or low cortisol concentrations despite increased ACTH, suggesting relative adrenal insufficiency. AVP, ACTH, and cortisol concentrations were higher in sick nonseptic foals compared with healthy foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased plasma AVP and ACTH concentrations in septic foals were associated with mortality. Several septic foals had increased AVP : ACTH and ACTH : cortisol ratios, which indicates relative adenohypophyseal and adrenal insufficiency.     

Vaccination Response of Young Foals to Keyhole Limpet Hemocyanin: Evidence of Effective Priming in the Presence of Maternal Antibodies

The ability to produce antibodies is essential for protection from infectious disease; however, in the neonate, maternal antibodies have been proposed to interfere with the foal's ability to respond to vaccination. In species other than the equid, keyhole limpet hemocyanin, a high-molecular weight protein, is used in vivo as an experimental vaccine component because of its high intrinsic immunogenicity. In this study, we show that young foals are able to produce a primary antibody response to vaccination at an early age. Thus, foals, like human infants, are capable of responding to antigenic exposure to a novel antigen (keyhole limpet hemocyanin) during the neonatal period. Although vaccinating foals in the presence of maternal antibodies failed to induce a primary serological response, priming occurred as comparable anamnestic responses were detected upon subsequent exposure to the antigen. There was no evidence of tolerance induction.     

Efficacy and Safety of a Commercial Fresh-Frozen Hyperimmune Plasma in Foals With Failure of Passive Transfer of Immunity

In foals more than 12 hours old, the only effective therapy for the treatment of failure of passive transfer (FPT) of immunity is transfusion of equine plasma. Use and efficacy of equine plasma for prophylaxis and treatment of sepsis, a condition primarily associated with FPT, are widely reported. However, plasma- and recipient-related factors associated with extent of IgG transfer and catabolism are not completely defined. Efficacy and safety of transfusion of a commercial fresh-frozen hyperimmune plasma were evaluated in hospitalized foals younger than 7 days of age with total or partial FPT. Sixty-two foals, classified as affected by FPT only, septic (infection plus systemic inflammatory response syndrome [SIRS]), and nonseptic sick, were included, and serum IgG concentration was measured at admission and 24 hours after plasma transfusion. In 25/62 foals, IgG level after 72 hours was also determined. The impact of different classification criteria for septic foals on IgG transfer was evaluated. Serum IgG measured 24 hours and 72 hours after plasma transfusion was significantly greater than at admission, but no significant difference was found in transfer efficacy (TE) between FPT, FPT septic, and FPT nonseptic foals and no significant difference was found in IgG concentration comparing foals with total and partial FPT or survivors and nonsurvivors. No significant difference was found comparing IgG concentration between bacteremic and nonbacteremic foals and foals with or without SIRS. No foal experienced adverse reactions to plasma transfusion. IgG TE and catabolism did not result significantly affected by the presence of sepsis or illness or by the outcome.     

Plasma C‐Reactive Protein and Haptoglobin Concentrations in Critically Ill Neonatal Foals

Background

Accurate diagnostic markers for sepsis in neonatal foals are needed. Plasma C‐reactive protein concentration (p[CRP]) and haptoglobin concentration (p[Hp]) are well‐established biomarkers of infection in humans, but studies are lacking in foals.

Hypotheses

p[CRP]) and p[Hp] are increased in septic foals compared to sick nonseptic and healthy control foals, and are predictive of survival.

Animals

Eighty critically ill foals (40 septic, 40 sick nonseptic) and 39 healthy control foals <1 week of age.

Methods

Multicenter, prospective observational clinical study. Venous blood was collected at admission from septic and sick nonseptic foals and from clinically healthy foals at 24 h of age. A diagnosis of sepsis was made based on positive blood culture or a sepsis score >11, and p[CRP] and p[Hp] were measured by using ELISA tests. Data were analyzed by using the Mann‐Whitney U‐test and forward stepwise multivariable linear regression. P < .05 was considered significant.

Results

Plasma [CRP] was positively associated with age, serum globulin, adrenomedullin, and bilirubin concentrations, aspartate aminotransferase activity, glutamyl‐transferase activity, band neutrophil count, and rectal temperature, and was increased in foals with toxic neutrophils, enterocolitis, colic, rib fractures and septic arthritis. Surprisingly, p[Hp] was lower in septic foals than in sick nonseptic foals. Neither p[CRP] or p[Hp] was predictive of survival in critically ill foals.

Conclusions and Clinical Importance

Plasma [CRP] increases with inflammation in neonatal foals but is not indicative of sepsis. Single time point, admission sampling of p[CRP] and p[Hp] do not appear to be useful biomarkers for sepsis in foals.     

Linear and Temporal Kinematics of the Walk in Warmblood Foals

Kinematic variables of the walk in adult horses have been well described in the literature, but few studies have investigated growth-associated changes in these parameters. The objective of this study was to quantify linear and temporal walk patterns in Warmblood foals during the preweaning growth period. Nine foals were videotaped at the walk at 3, 11, and 21 weeks of age. Repeated-measures analyses were used to compare trait means between age groups. No significant effects owing to gender were found. Although stride length and stride duration increased as foals aged, neither differed across age groups when adjusted for wither height or velocity. Most kinematic variables did not differ across age groups when adjusted for Froude number. Overstride distance decreased by more than 40% in a linear manner from 3 to 21 weeks, and had an inverse relationship with distance between diagonal limbs during stance phase. Diagonal stance duration was greater than lateral stance duration for all age groups, indicating foals did not achieve an even, four-beat rhythm by the end of the study period. Changes in walk kinematics over time were independent of differences in velocity and increasing height during growth, and may indicate the need to account for body length or other morphometrics when assessing gait parameters in growing animals. Further research is needed during postweaning growth to determine when kinematic variables become consistent with those of adult horses.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Changes in Hemostatic Indices in Foals Naturally Infected With Strongylus vulgaris

Strongylus vulgaris has been found endemic in equine populations subject to parasite control by targeted selective anthelmintic therapy. This study investigated hemostasis in foals naturally infected with S. vulgaris and monitored this response over the course of progressing infection stages. The hemostatic indices D-dimer, antithrombin III (ATIII), fibrinogen, prothrombin time (PT), and activated partial thromboplastin time were evaluated in weekly blood samples for up to 50 weeks in 12 foals born into a herd with high prevalence of S. vulgaris. Results were compared with weekly S. vulgaris antibody enzyme-linked immunosorbent assay values in all foals using a linear mixed effects model with repeated measures and to total numbers of S. vulgaris larvae in nine foals at necropsy with Pearson linear correlation. In the first week of life, all evaluated indices of hemostasis were significantly different from those observed in the rest of the study weeks, corresponding to previously demonstrated aberrancies in neonates. Significant changes were seen for D-dimer in weeks 11–24, 26–27, 30, and 39 compared with week 2, for PT in weeks 12–13 compared with week 6, and for ATIII in week 15 compared with week 4. Strongylus vulgaris antibody levels were statistically associated with D-dimer (P = .0076) and fibrinogen (P = .0004) concentrations. Naturally acquired infection with S. vulgaris was associated with changes suggestive of mild activation of coagulation, fibrinolysis, and inflammation. The results of this study may help elucidate the pathogenesis of the endarteritis, thromboembolism, and nonstrangulating intestinal ischemia that is observed in horses with S. vulgaris infection.     

Prospective Evaluation of Coagulation in Critically Ill Neonatal Foals

Background: Coagulopathy is a potentially underrecognized complication of sepsis and septic shock in critically ill neonatal foals.
Hypothesis: Critically ill neonatal foals have abnormalities in coagulation that are associated with disease severity and outcome.
Animals: Foals <72 hours old admitted to a neonatal intensive care unit.
Methods: Prospective, observational study. Blood was collected at admission, 24, and 48 hours for platelet count, prothrombin time, activated partial thromboplastin time, antithrombin activity and concentrations of fibrin degradation products, and fibrinogen in plasma from all foals.
Results: Sixty-three foals were enrolled and classified as Septic Shock (12), Septic (28), and Other (23). At least 1 abnormal value was found in 18/28 (64%) samples from the Septic Shock group, 66/85 (78%) from the Septic group, and 30/59 (51%) from the Other group ( P = .01). Coagulopathy (3 or more abnormal values) was present in 7/28 (25%) samples in the Septic Shock group, 14/85 (16%) samples in the Septic group, and 3/59 (5%) samples in the Other group ( P = .0028). Clinically detectable bleeding occurred in 8/12 (67%) Septic Shock cases, 11/28 (39%) Septic cases, and 3/23 (13%) Other cases ( P = .009). Foals in Septic Shock were 12.7 times more likely to have clinical evidence of bleeding than those in the Other group (95% CI 2.3–70, P = .004). Treatment with fluids or plasma did not have a detectable effect on coagulation values.
Conclusions and Clinical Importance: Coagulopathy commonly occurs in critically ill neonatal foals, especially those with sepsis and septic shock.     

Hypothalamic-Pituitary-Adrenal Axis Dysfunction in Hospitalized Neonatal Foals

Background: Transient hypothalamic-pituitary-adrenal (HPA) axis dysfunction occurs frequently in critically ill humans and impacts survival. The prevalence and impact of HPA axis dysfunction in critically ill neonatal foals are not well characterized.
Hypotheses: (1) HPA axis dysfunction occurs in hospitalized neonatal foals, and is characterized by inappropriately low basal serum cortisol concentration or inadequate cortisol response to exogenous adrenocorticotropic hormone (ACTH); (2) hospitalized foals with HPA axis dysfunction have more severe disease and are less likely to survive than hospitalized foals with normal HPA axis function.
Animals: Seventy-two hospitalized foals and 23 healthy age-matched foals.
Methods: Basal ACTH and cortisol concentrations were measured and a paired low-dose (10 μg)/high-dose (100 μg) cosyntropin stimulation test was performed at admission in hospitalized foals. HPA axis dysfunction was defined as (1) an inappropriately low basal cortisol concentration or (2) an inadequate increase in cortisol concentration (delta cortisol) after administration of cosyntropin, with cut-off values for appropriate basal and delta cortisol concentrations determined from results obtained in healthy age-matched foals.
Results: Forty-six percent of hospitalized foals had an inappropriately low basal cortisol concentration and 52% had an inadequate delta cortisol concentration after administration of the 100 μg dose of cosyntropin. An inadequate delta cortisol response to the high (100 μg) dose of cosyntropin was significantly correlated with shock and multiple organ dysfunction syndrome in hospitalized foals, and with decreased survival in a subgroup of septic foals.
Conclusions and Clinical Importance: HPA axis dysfunction occurs frequently in hospitalized neonatal foals, and negatively impacts disease severity and survival.     

Association of Blood Lactate Concentration and Outcome in Foals

Background: Lactate concentration in blood or plasma ([LAC]) and change in [LAC] are associated with survival in sick foals. Hypothesis: [LAC] and change in [LAC] over time are associated with survival at 96 hours and discharge in neonatal foals. Furthermore [LAC] and change in [LAC] over time correlate with blood culture results and blood pressure at admission. Animals: Two hundred and twenty‐five foals consecutively admitted to a Neonatal Intensive Care Unit. Methods: Retrospective case review. Foals ≤30 days of age with [LAC] from arterial (190) or umbilical (35) blood gas analysis ([LAC]_BG) at admission, 24, and 48 hours. [LAC]_BG, blood pressure, blood culture status, and outcome (survival versus nonsurvival at 96 hours and discharge) were recorded. Change in [LAC]_BG over time ([LAC]_BGΔT) was calculated. Results: [LAC]_BG was lower in survivors (96 hours and discharge) at all times. [LAC]_BGΔT was larger for survivors (96 hours). Odds of survival (96 hours and discharge) decreased 18, 39, 53 and 22, 38, and 47%, respectively, at each sample time for every 1 mmol/L increment in [LAC]_BG and increased 156% for each 1.0/day increment in [LAC]_BGΔT from admission to 24 hours at 96 hours. Blood pressure and [LAC]_BG were not correlated (P= .196) until removal of selected foals (mean arterial pressure <60 mmHg, admission [LAC]_BG <5.5 mmol/L) (P < .001). Bacteremia was not associated with [LAC]_BG. Proposed admission [LAC]_BG cut‐points for future studies were 6.5 mmol/L (96 hours) and 5.5 mmol/L (discharge). Conclusions and Clinical Importance: Prospective studies evaluating [LAC], [LAC]_BGΔT, and cut‐points in sick foals are warranted.     

A Comparison Between Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay Methods for Detecting Leptospira in Equine Recurrent Uveitis

Leptospirosis is a widespread zoonotic disease that affects humans and many species of animals. Leptospiral organisms have long been presumed to be associated with the presence of equine recurrent uveitis (ERU). The connection between ERU and leptospirosis can be established using various techniques. In the current study, we used a polymerase chain reaction (PCR) assay to help establish a diagnosis of ERU caused by Leptospira infection and compared the results with those of enzyme-linked immune assay (ELISA). A total of 31 and 30 serum samples were taken from horses with ERU (based on clinical diagnosis) and horses that were healthy, respectively, between June 2007 and December 2008. The results showed that from 31 affected horses, a total of seven and five samples were positive for leptospiral infection using PCR and ELISA, respectively, whereas there was no evidence of infection with Leptospira spp. in 30 serum samples of healthy horses. All of the positive samples in ELISA were also positive by PCR, whereas PCR detected two positive cases that were negative by ELISA. Although there was no significant difference between these two methods, it is apparent that PCR may be a more reliable tool for detecting the presence of leptospires in ERU.