Cochet-Bonnet aesthesiometer-determined corneal sensitivity in neonatal foals and adult horses

Corneal touch threshold (CTT) was measured in sick neonatal foals, healthy foals, and healthy adult horses with a Cochet-Bonnet aesthesiometer. The mean overall CTT for the adult horses, sick foals, and healthy foals was 4.82 +/- 0.87 cm, 3.21 +/- 0.24 cm, and 5.01 +/- 0.61 cm, respectively. The central cornea of adult horses was more sensitive than the limbal cornea. Corneal sensitivity was significantly reduced in sick neonatal foals compared to adults. The mean Schirmer I tear test values were significantly lower in foals than adults, and were 14.2 +/- 1.0 mm, 12.8 +/- 2.4 mm, and 18.3 +/- 2.1 mm wetting in sick neonatal foals, normal neonatal foals, and adult horses, respectively. Reduced corneal sensation and lower tear production may be associated with ulcerative keratitis and slow corneal healing in some foals.     

Lung surfactant function and composition in neonatal foals and adult horses

BACKGROUND: Lung surfactant function and composition are varied and adapted to the specific respiratory physiology of all mammalian species. HYPOTHESIS: Lung surfactant function and composition are different in neonatal foals as compared to adult horses. ANIMALS: Six adult horses, 7 term foals (<24 hours old), and 4 premature foals were used. Animals were part of the Auburn University teaching herd except for 3 client-owned premature foals. METHODS: Bronchoalveolar lavage fluid (BALF) was obtained from all animals. Ultracentrifugation of cell-free BALF separated surfactant into crude surfactant pellets (CSP) and supernatant. Both fractions were analyzed for phospholipid and protein content with the Bartlett and bicinchoninic acid method, respectively. Phospholipid composition of the CSP was determined by using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of the CSP was measured with a pulsating bubble surfactometer. Results from term foals (<24 hours old) were compared statistically to those from adult horses. Values of P < .05 were considered significant. RESULTS: BALF phospholipid content was similar between adult horses and term foals, but BALF protein content was significantly decreased in term foals. Phosphatidylglycerol was significantly decreased, phosphatidylinositol was significantly increased, and the minimum surface tension was significantly increased in the CSP from term foals compared to adult horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Surface tension and phospholipid composition of surfactant in neonatal foals are significantly different compared to adult horses. These changes may influence biophysical and immunologic functions of surfactant.     

Liver biopsy techniques for adult horses and neonatal foals to assess copper status

Objective To evaluate standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals, as techniques for obtaining tissue for assessment of copper status. The techniques were evaluated with respect to ease of use and effect on the animal.
Procedure Twenty of 24 Thoroughbred mares and 21 of their foals were biopsied. The animals were part of a larger study of the effect of copper supplementation on copper status and the prevalence of developmental orthopaedic disease. Livers were also collected from unrelated horses and sampled to investigate the variability in the distribution of copper in liver tissue.
Result The biopsy technique caused no lasting effect on the mares, but there was an increased risk of viscus penetration associated with taking multiple biopsy cores. The use of ultrasonography to scan the target area for the liver identified four cases that were not appropriate candidates for liver biopsy, because of large intestine being located in the biopsy area. In the foals there were no serious postoperative adverse effects, nor was there any evidence of problems caused by the procedure when the abdomen was examined post-mortem at 5 months of age. In livers collected to investigate the variability of copper concentration, copper appeared to be relatively evenly distributed through the liver.
Conclusion Standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals are convenient procedures for obtaining liver tissue for assessing copper status in horses. The use of ultrasound to identify liver tissue is recommended, especially in older mares.     

Pharmacokinetics of erythromycin in foals and in adult horses

The pharmacokinetic parameters of erythromycin in foals were determined following intravenous administration of 5.0 mg/kg to animals aged 1, 3, 5 and 7 weeks. The distribution of the drug was described by a two-compartment open model, and no significant differences were observed between coefficients on which the parameters were based. Pharmacokinetic values were also determined for four mares given 5.0 mg/kg intravenously and for six 10–12 week-old foals given 20.0 mg/kg intravenously. The half-life of erythromycin for all groups of animals (foals less than 7 weeks, mares, foals 10–12 weeks) was 1.0–1.1 h; the apparent volume of distribution was between 2.3 and 7.2 l/kg, and the clearance of the drug from the body was between 1.9 and 5.0 mg/kg/h. No drug could be detected in the serum following oral administration of 5.0 mg/kg erythromycin estolate; detectable levels were found for 5 h in mares given 12.5 mg/kg, and for 8 h in foals given 20.0 mg/kg orally. Peak levels in foals given the drug orally were 0.42 μg/ml at 120 min after administration. Foals given 10.0 mg/kg of erythromycin base intramuscularly had serum concentrations detectable 12 h later, the peak level achieved was 1.44 μg/ml serum 90 min after administration and concentrations exceeded 0.25 μg/ml for 6 h. In the mares the milk concentrations were approximately twice those in serum. Recommendations were made for drug dosage to be used in the treatment of Corynebacterium equi pneumonia of foals.     

Comparative pharmacokinetics of minocycline in foals and adult horses

The objective of this study was to compare the pharmacokinetics of minocycline in foals vs. adult horses. Minocycline was administered to six healthy 6‐ to 9‐week‐old foals and six adult horses at a dose of 4 mg/kg intragastrically (IG) and 2 mg/kg intravenously (i.v.) in a cross‐over design. Five additional oral doses were administered at 12‐h intervals in foals. A microbiologic assay was used to measure minocycline concentration in plasma, urine, synovial fluid, and cerebrospinal fluid (CSF). Liquid chromatography–tandem mass spectrometry was used to measure minocycline concentrations in pulmonary epithelial lining fluid (PELF) and bronchoalveolar (BAL) cells. After i.v. administration to foals, minocycline had a mean (±SD) elimination half‐life of 8.5 ± 2.1 h, a systemic clearance of 113.3 ± 26.1 mL/h/kg, and an apparent volume of distribution of 1.24 ± 0.19 L/kg. Pharmacokinetic variables determined after i.v. administration to adult horses were not significantly different from those determined in foals. Bioavailability was significantly higher in foals (57.8 ± 19.3%) than in adult horses (32.0 ± 18.0%). Minocycline concentrations in PELF were higher than in other body fluids. Oral minocycline dosed at 4 mg/kg every 12 h might be adequate for the treatment of susceptible bacterial infections in foals.     

Serum lactoferrin and immunoglobulin G concentrations in healthy or ill neonatal foals and healthy adult horses

BACKGROUND: Lactoferrin is a colostral glycoprotein with antimicrobial properties. HYPOTHESES: (1) Serum lactoferrin and immunoglobulin G (IgG) concentrations are correlated and increase in healthy foals after ingestion of colostrum; (2) compared to healthy foals, ill foals will have lower lactoferrin concentrations that correlate with their IgG concentration, neutrophil count, the diagnosis of sepsis, and survival; and (3) plasma concentrations of lactoferrin will be less than serum concentrations. ANIMALS: Healthy foals (n = 16), mature horses (n = 10), and ill foals 1-4 days old (n = 111) that were examined for suspected sepsis were used for blood collection. Colostrum was obtained from 10 healthy mares unrelated to the foals. METHODS: Blood was obtained from the healthy foals at birth and 1-3 days of age and from the ill foals at admission. Serum IgG was quantified by single radial immunodiffusion (SRID). Lactoferrin concentrations in colostrum and blood were determined by an enzyme-linked immunosorbant assay. The sepsis score, blood culture results, neutrophil counts, and survival were obtained on ill foals. RESULTS: The mean colostral lactoferrin concentration was 21.7 microg/mL. Compared to values at birth, serum IgG (18+/-2 versus 2,921+/-245 mg/dL, SEM) and lactoferrin (249+/-39 versus 445+/-63 ng/mL, SEM) concentrations were significantly greater in healthy foals 1-3 days old. Serum lactoferrin concentration in 1-3-day-old healthy foals was not different from mature horses or ill foals. IgG and lactoferrin concentrations were significantly correlated only in healthy foals. Serum lactoferrin concentrations were significantly lower in ill neutropenic foals. The serum IgG concentration was significantly lower in ill foals as compared to healthy foals. Only serum IgG was significantly less in ill foals with a positive sepsis score and in nonsurvivors, Plasma lactoferrin concentrations were lower than serum concentrations, although values were significantly correlated. CLINICAL IMPORTANCE: Although both serum IgG and lactoferrin concentrations increase in healthy foals after ingestion of colostrum, only serum IgG is significantly correlated with the sepsis score and outcome.     

Comparison of cefepime pharmacokinetics in neonatal foals and adult dogs

The pharmacokinetics of cefepime, a new fourth generation cephalosporin with enhanced antibacterial activity, was examined in neonatal foals and adult dogs. Cefepime was administered intravenously (i.v.) at a dose of 14 mg/kg to five neonatal foals and six adult dogs. Blood samples were collected in both groups of animals and plasma cefepime concentrations measured by reverse-phase high-performance liquid chromatography (HPLC). Cefepime concentrations in both groups of animals were described by a two-compartment pharmacokinetic model with elimination half-lives of 1.65 and 1.09 h for the foal and dog, respectively. We tested whether or not pharmacokinetic parameters for cefepime could be scaled across species using principles of allometry. The parameters of elimination half-life (t(1/2)beta), apparent volume of distribution (VDarea), and systemic clearance (CL) were scaled linearly to body weight on a double logarithmic plot with allometric exponents for body weight of 0.26, 1.08 and 0.72, respectively. This study further determined doses for cefepime, a potentially useful antibiotic for neonatal foals and dogs, from the pharmacokinetic values. An i.v. dose of cefepime estimated from this study for treating sensitive bacteria was 11 mg/kg every 8 h for neonatal foals and 40 mg/kg every 6 h for dogs.     

Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1-1000ng/mL) for various times. Recombinant IL-10 (10-5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.     

Serotonin-containing cells in the gastrointestinal tract of newborn foals and adult horses

Serotonin (5-hydroxytryptamine, 5-HT), a regulatory amine of mucosal enterochromaffin cells plays an important role in the control of gastrointestinal smooth muscle contraction and epithelial secretion. Serotonin has also been associated with gastric ulcers, diarrhoea and abdominal pain. In spite of the high incidence of these gastrointestinal disorders in newborn foals and adult horses, no data are available regarding 5-HT immunoreactive cells (i.c.) in the gastrointestinal tract (GIT) of foals, and for adult horses, data are incomplete and contradictory. In this study, the distribution and relative frequency of 5-HT i.c. in the GIT of newborn foals and adult horses were determined immunohistochemically. In foals as in adults, a relatively large number of 5-HT i.c. were detected in all portions of the GIT. In foals, a significantly higher amount of cells was found in the pyloric region and margo plicatus of the stomach, as well as in the caecum and colon ascendens compared with adults. Our results provided rationale for further research concerning the role of 5-HT i.c. during the milk diet or in the regulation of gastrointestinal growth/cell proliferation, and in the pathogenesis of gastric ulcers, especially in newborn foals.     

Accuracy of indirect measurement of blood pressure in neonatal foals

The objectives of this study were to assess, in anesthetized neonatal foals, the accuracy of 2 automated indirect oscillometric monitors for measurement of mean arterial pressure (MAP), to determine the optimal site of cuff placement for MAP monitoring, and to determine the relationship between arterial blood pressure and cardiac output. Ten neonatal foals were anesthetized and instrumented with a catheter in the metatarsal artery for direct MAP monitoring and measurement of cardiac output by lithium dilution. Concurrent MAP measurements were obtained with Cardell and Dinamap oscillometric monitors with cuffs placed at 3 different sites (coccygeal, metatarsal, and median arteries). Blood pressure was manipulated by varying the depth of anesthesia and by administration of dobutamine or phenylephrine. A statistically significant (P = .025) interaction was found between the type of monitor and cuff placement site. With the Cardell monitor, placement of the cuff over the coccygeal artery resulted in a significantly lower bias than placement over the median or dorsal metatarsal artery (P < .0001 and P = .0149, respectively). No significant difference in bias was found with cuff placement site when using the Dinamap monitor. The correlation coefficient (r) between MAP and cardiac output was 0.47. Indirect oscillometry with a cuff placed over the coccygeal artery or dorsal metatarsal artery is an acceptable method for measuring MAP in foals. Blood pressure does not correlate well with cardiac output in anesthetized foals.     

Reduced efficacy of anthelmintics in young compared with adult horses

Studies on a Thoroughbred breeding farm in Ohio from 1982 to 1988 demonstrated the value of three anthelmintic pastes (ivermectin, oxibendazole, pyrantel pamoate) in controlling benzimidazole resistant cyathostomes (small strongyles) in adult horses. However, a comparison of drug efficacy in suppressing faecal egg counts for the full period between treatments showed a significant reduction in efficacy of all drugs in yearling horses compared with adults. Mean faecal egg counts of adult horses were generally kept below 100 eggs per gram (epg) of faeces when using oxibendazole or pyrantel pamoate at four to five week intervals and ivermectin at eight week intervals. By contrast, mean counts of young horses rose as high as 655 epg (oxibendazole), 729 epg (pyrantel pamoate) and 852 epg (ivermectin) within the same time period after treatment. Individual counts of treated yearlings sometimes exceeded 3,000 epg. Three distinct mechanisms appeared to be involved in the poor results in young horses. These were 1) anthelmintic refuge, 2) anthelmintic resistance, and 3) anthelmintic avoidance.     

Association of adrenocorticotrophin and cortisol concentrations with peripheral blood leukocyte cytokine gene expression in septic and nonseptic neonatal foals

Background

The hypothalamic‐pituitary‐adrenal (HPA) is influenced by the proinflammatory cytokines IL‐6, IL‐1β, and TNF‐α in critically ill humans. Information about the association of cytokines with the HPA axis in neonatal foals is lacking.

Hypothesis/Objectives

The objectives were to describe for hospitalized septic and nonseptic foals (1) temporal changes in blood concentrations of ACTH, and cortisol, and leukocyte cytokine gene expression, and (2) coassociation of these HPA axis hormones with blood leukocyte cytokine gene expression.

Animals

Hospitalized septic foals (N = 15) and hospitalized nonseptic foals (N = 11).

Methods

Blood samples, obtained from study foals at admission (T = 0), and 24 (T = 1), 48 (T = 2), 72 (T = 3), and 96 (T = 4) hours after admission, were processed to isolate RNA from leukocytes and to harvest plasma and serum for hormone assays. Plasma ACTH and serum cortisol concentrations were determined by radioimmunoassay. Leukocyte mRNA expression of IL‐1β IL‐6, IL‐8, IL‐10, and TNF‐α was determined using RT‐PCR.

Results

Cortisol concentrations were greater (P < .05) in foals at admission than at other time points. The expressions of IL‐8 and IL‐10 mRNA were lower (P < .05) at each time point in septic than in nonseptic foals. Among septic foals, ACTH was positively associated (P = .0026) with IL‐6 mRNA expression.

Conclusions

Sepsis influences secretion of the HPA axis hormones and expression of cytokines in foals. A positive association with the HPA axis and IL‐6 expression was detected. The clinical importance of these findings requires additional study.     

Mean platelet component as an indicator of platelet activation in foals and adult horses

BACKGROUND: Mean platelet component (MPC) is a new platelet variable, measured by modern commercial complete blood count analyzers, that is reduced during platelet activation in humans and small animals. HYPOTHESIS: MPC decreases in horses with clinical conditions that cause platelet activation and disseminated intravascular coagulation (DIC). ANIMALS: We obtained 418 CBCs from 100 sick and 20 healthy neonates and 178 sick and 45 sound adult horses. Sick neonates were classified into septic and nonseptic, and DIC and non-DIC groups. Adults were grouped by diagnoses (systemic inflammatory disorders, gastrointestinal problems, and thrombocytopenia). METHODS: MPC together with platelet count, mean platelet volume, platelet distribution width, and platelet component distribution width were measured with a commercial analyzer and compared between the different disease and control groups in neonates and in adults. RESULTS: MPC values were significantly lower in the septic and nonseptic neonates (24.0 +/- 3.5 g/dL and 26.6 +/- 2.6 g/dL, respectively) than in the control group (28.1 +/- 1.7 g/dL). Neonates with DIC had the lowest MPC values (23.8 +/- 6.3 g/dL). MPC values in adult horses were significantly lower in the inflammatory (23.5 +/- 4.7 g/dL), gastrointestinal obstruction (23.0 +/- 5.0 g/dL), enteritis (23.6 +/- 4.6 g/dL), ischemic (23.9 +/- 5.1 g/dL), and thrombocytopenia (20.2 +/- 5.7 g/dL) groups when compared with control horses (26.2 +/- 3.5 g/dL). Other platelet variables were not different between the control and the disease groups. CONCLUSION AND CLINICAL IMPORTANCE: MPC might be a useful variable for quickly and easily detecting platelet activation in sick neonates and adult horses.     

Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR

A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.     

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.     

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.