Tumor necrosis factor activity in serum from neonatal foals with presumed septicemia.

A study was performed to determine prevalence of tumor necrosis factor (TNF) activity in serum of equine neonates with presumed sepsis and to determine correlation between serum TNF activity and severity and outcome of disease. Twenty foals less than 21 days old were considered suitable for inclusion in this study by satisfying clinical and laboratory criteria suggestive of septicemia. At admission, blood samples were collected from all foals for determination of serum TNF activity, then clinical course and outcome of disease were recorded. Thirty-one clinically normal foals less than 21 days old served as controls for serum TNF activity. Serum TNF activity was estimated by use of an in vitro cytotoxicity bioassay and WEHI 164 clone-13 murine fibrosarcoma cells. Of the 20 foals with presumed sepsis, 5 had high serum TNF activity. Mean heart rate (P less than 0.005), mucosal petechial hemorrhages (P = 0.06), and death rate (P = 0.06) were greater in the group of foals with high serum TNF activity. These foals also had a lower mean neutrophil count (P less than 0.001), greater band-to-segmented neutrophil ratio (P less than 0.0001), and more prevalent neutrophil toxic changes (P = 0.07) than did foals without serum TNF activity (P = 0.02). Joint swelling was more prevalent in foals without serum TNF activity. Results of the study indicate that serum TNF activity is correlated with clinical criteria of sepsis in equine neonates. An association was apparent between disease severity and serum TNF activity in this group of foals with presumed septicemia.     

Immunodeficiency manifested by oral candidiasis and bacterial septicemia in foals

Oral candidiasis and bacterial septicemia were diagnosed in 8 foals that had laboratory and/or pathologic evidence of immunodeficiency. Two foals suffered solely from complete failure of passive transfer of colostal immunoglobulins. Six foals had evidence of immune defects but did not meet the criteria for diagnosis of any of the currently recognized primary equine immunodeficiency syndromes. All six of these foals died or were euthanatized due to bacterial infections. One foal with failure of passive transfer recovered and the other died of a mesenteric torsion before the effect of treatment could be evaluated.     

Fibrin deposits and organ failure in newborn foals with severe septicemia

Background: Septicemia in human neonates frequently is complicated by activation of the coagulation system, disseminated intravascular coagulation (DIC) and multiple organ failure syndrome, which may contribute to high mortality. In adult horses with DIC, the lung has been the organ most frequently affected by fibrin deposits. In addition, in vivo studies suggest that hemostatic mechanisms may be immature in foals <1‐day old. Hypothesis: Newborn foals with severe septicemia have fibrin deposits in their tissues independently of their age, and these fibrin deposits are associated with organ failure. Animals: Thirty‐two septic and 4 nonseptic newborn foals euthanized for poor prognosis. Methods: Tissue samples (kidney, lung, and liver) collected on postmortem examination were stained with phosphotungstic acid hematoxylin (PTAH) and immunohistochemistry (IHC) for blind histologic examination. A fibrin score (grades 0–4) was established for each tissue sample and for each foal. Medical records were reviewed for assessing clinical evidence of organ failure during hospitalization. Results: Fibrin deposits were found in most septic foals (28/32 when using IHC and 21/32 when using PTAH), independently of the age of the foal. The lung was the most affected tissue (97% of the septic foals). Additionally, organ failure was diagnosed in 18/32 septic foals (8 with respiratory failure, 14 with renal failure), although a statistical association with severe fibrin deposition was not identified. Conclusions and Clinical Importance: Nonsurviving septic foals have fibrin deposits in their tissues, a finding consistent with capillary microthrombosis and DIC.     

Plasma pharmacokinetics, pulmonary distribution, and in vitro activity of gamithromycin in foals

The objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Rhodococcus equi. A single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. Concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils were determined using HPLC with tandem mass spectrometry detection. The minimum inhibitory concentration of gamithromycin required for growth inhibition of 90% of R. equi and S. zooepidemicus isolates (MIC(90)) was determined. Additionally, the activity of gamithromycin against intracellular R. equi was measured. Mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35±1.77 μg/mL) and BAL cells (8.91±1.65 μg/mL) compared with PELF (2.15±2.78 μg/mL) and plasma (0.33±0.12 μg/mL). Mean terminal half-lives in neutrophils (78.6 h), BAL cells (70.3 h), and PELF (63.6 h) were significantly longer than those in plasma (39.1 h). The MIC(90) for S. zooepidemicus isolates was 0.125 μg/mL. The MIC of gamithromycin for macrolide-resistant R. equi isolates (MIC(90)=128 μg/mL) was significantly higher than that for macrolide-susceptible isolates (1.0 μg/mL). The activity of gamithromycin against intracellular R. equi was similar to that of azithromycin and erythromycin. Intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain PELF concentrations above the MIC(90) for S. zooepidemicus and phagocytic cell concentrations above the MIC(90) for R. equi for approximately 7 days.     

Concentrations of amino acids in the plasma of neonatal foals with septicemia

Concentrations of amino acids in the plasma of 13 neonatal foals with septicemia were compared with the concentrations of amino acids in the plasma of 13 age-matched neonatal foals without septicemia. Analysis of the results revealed significantly lower concentrations of arginine, citrulline, isoleucine, proline, threonine, and valine in the plasma of foals with septicemia. The ratio of the plasma concentrations of the branched chain amino acids (isoleucine, leucine, and valine) to the aromatic amino acids (phenylalanine and tyrosine), was also significantly lower in the foals with septicemia. In addition, the concentrations of alanine, glycine, and phenylalanine were significantly higher in the plasma of foals with septicemia. Therefore, neonatal foals with septicemia had significant differences in the concentrations of several amino acids in their plasma, compared with concentrations from healthy foals. These differences were compatible with protein calorie inadequacy and may be related to an alteration in the intake, production, use, or clearance of amino acids from the plasma pool in sepsis.     

Pulmonary disposition of tilmicosin in foals and in vitro activity against Rhodococcus equi and other common equine bacterial pathogens

The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against Rhodococcus equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to seven healthy 5- to 8-week-old foals by the intramuscular route. Concentrations of tilmicosin were measured in serum, lung tissue, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils. Mean peak tilmicosin concentrations were significantly different between sampling sites with highest concentrations measured in blood neutrophils (66.01+/-15.97 microg/mL) followed by BAL cells (20.1+/-5.1 microg/mL), PELF (2.91+/-1.15 microg/mL), lung tissue (1.90+/-0.65 microg/mL), and serum (0.19+/-0.09 microg/mL). Harmonic mean terminal half-life in lung tissue (193.3 h) was significantly longer than that of PELF (73.3 h), bronchoalveolar cells (62.2 h), neutrophils (47.9 h), and serum (18.4 h). The MIC90 of 56 R. equi isolates was 32 microg/mL. Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus spp., Pseudomonas spp., and Enterobacteriaceae.     

Temporal changes in cytokine expression of foals during the first month of life

Foals are uniquely susceptible to a wide variety of opportunistic infections normally associated with immunodeficiencies. Little is understood about the immune system of foals during the neonatal period. An apparent age-related susceptibility predisposes neonatal foals to infectious diseases and hinders therapeutic and preventative interventions for these diseases. Cytokine expression is correlated with the type of immune response as well as the severity of a disease. In this study, we measured foal peripheral blood mononuclear cell (PBMC)-specific mRNA cytokine expression from 72 foals from three different farms during the first 4 weeks of life. Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) were cloned and transcribed in vitro to generate antisense probes for ribonuclease protection assays. Using linear mixed-effect models, we determined that IFN-gamma, TGF-beta1, and IL-1alpha increased significantly (P<0.05) with age.     

Detecting endotoxin activity in bovine serum using an automated testing system

The aim of the present study was to compare the ability of the commercially available portable test system (PTS^TM) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS^TM was not significantly different from that of the traditional LAL-based assays. The results using PTS^TM correlated with those using KT (r²=0.963, P<0.001) or KC assays (r²=0.982, P<0.001). Based on these findings, the PTS^TM could be applied as a simplified system to assess endotoxin activity in bovine serum.     

Dose-response relationship for the antipyretic effect of meloxicam in an endotoxin model in cats

The antipyretic efficacy of meloxicam was evaluated in a feline endotoxin model using a replicated change-over design. Twelve adult cats of both sexes were allocated at random to three experimental groups. At 30 min prior to the intravenous (i.v.) endotoxin challenge (0.5 µg/kg body weight(b.w.)), 2 animals in each group received an i.v. injection of 0.1, 0.3 or 0.5 mg meloxicam/kg b.w. and the two remaining animals in each group received physiological saline. In a second phase, 21 days later, the meloxicam/placebo treatment was exchanged within each group. The rectal temperature and scores for general demeanour were determined at 30-min intervals from before dosing to 300 min after the endotoxin challenge. Haematological parameters were analysed before and 60 min after administration of endotoxin. The results indicated a significant dose-dependent antipyretic response to meloxicam after endotoxin challenge. The antipyretic response in the medium- and high-dose meloxicam groups did not differ significantly, but both were significantly different from the low-dosage group. The individual effects of endotoxin on general demeanour were rather variable but meloxicam tended to have a beneficial effect. Endotoxin induced a reduction in the white blood cell count but this was not influenced by meloxicam. It was concluded that the pyretic endotoxin model is very suitable for studying new NSAIDs in cats and that the optimum single dose of meloxicam in this model was 0.3 mg/kg b.w.Abbreviations AUC area under the curve - b.w. body weight - i.v. intravenous - LPS lipopolysaccharide - MCV mean corpuscular volume - NSAID non-steroidal anti-inflammatory drug - WBC white blood cell count     

Listeria monocytogenes septicemia in an immunocompromised dog

An 11‐year‐old, male castrated, Boston Terrier was presented to the North Carolina State University College of Veterinary Medicine Small Animal Emergency Service with a 2‐day history of progressive ataxia, left‐sided head tilt, and anorexia. The dog had previously been diagnosed with chronic lymphoid leukemia and suspected immune‐mediated destruction of his bone marrow precursor cells, possibly due to therapy with immunosuppressive dosages of prednisone and azathioprine. During the physical examination, abnormal findings included an increased body temperature and horizontal nystagmus. Diagnostic investigations included a computed tomography (CT) scan, which confirmed bilateral otitis media, and a blood culture, which was positive for Listeria monocytogenes serotype 4b (epidemic clone 1). Upon treatment with ampicillin/sulbactam, enrofloxacin, and minocycline, the dog became normothermic and the neurologic signs improved. L monocytogenes serotype 4b (epidemic clone 1) has been associated with outbreaks of human listeriosis originating from food contamination. Although rare case reports of Listeria spp. infection in dogs exist, an actual infection with the epidemic clone 1 strain has never before been reported in a dog. It should be included in the differential diagnoses in immunocompromised dogs with clinical signs of septicemia.     

An in vitro biomechanical investigation of an intramedullary nailing technique for repair of third metacarpal and metatarsal fractures in neonates and foals

OBJECTIVE: To evaluate a dorsoproximal extra-articular approach for insertion of 8.25-mm, solid-titanium, intramedullary (IM) interlocking nails into ostectomized foal third metacarpal (MC3) and third metatarsal (MT3) bones; to compare the monotonic mechanical properties of IM nail constructs with paired intact bones; and to determine the effects of age, body weight, fore- or hindlimb, and left or right limb on the mechanical testing variables. ANIMAL OR SAMPLE POPULATION: Twenty bone pairs (10 MC3, 10 MT3) collected from 10 foals of various weights and ages. METHODS: One bone from each pair was randomly selected to be ostectomized and stabilized using an 8.25-mm, solid-titanium IM nail, and four 3.7-mm titanium interlocking screws (construct). Constructs and contralateral intact bone specimens were tested in axial compression and palmaro-/plantarodorsal (PD) 4-point bending. Monotonic mechanical properties were compared between intact specimens and constructs with an ANOVA; significance was set at P <.05. RESULTS: Nail insertion caused bone failure in 6 MC3 and 2 MT3. In general, mean mechanical testing values indicated that intact specimens were significantly stronger and stiffer than constructs for all age and weight ranges when tested in compression and PD 4-point bending (P <.05). Bone strength and stiffness of intact specimens tested in compression and bending tended to increase linearly with age and weight. CONCLUSION: IM interlocking nail fixation of gap-ostectomized MC3 and MT3 with 8.25-mm IM nails and 3.7-mm interlocking screws did not achieve sufficient strength or stiffness to be recommended as the sole means of repair for comminuted MC3 and MT3 fractures in young foals. CLINICAL RELEVANCE: IM interlocking nail fixation of foal cannon bone fractures may be useful to decrease soft-tissue disruption at the fracture site; however, there is a risk of bone failure associated with extra-articular insertion. This method should be combined with other forms of external coaptation for added stability in axial compression and PD bending.     

Complement activity and selected hematologic variables in newborn foals fed bovine colostrum

Serum complement activity and selected hematologic variables were evaluated in 5 newborn foals fed bovine colostrum (principal group) and 6 foals allowed to nurse their dam (control group). Also, bovine colostrum was evaluated for anti-equine antibodies. Precolostral serum hemolytic and conglutinating complement activities were low and increased similarly in foals of both groups to reach adult values between 1 and 3 weeks after birth. Bovine colostrum strongly agglutinated, but did not hemolyse principal foals' RBC and blood containing all known equine blood group alloantigens. Hemolysis was not detected after administration of bovine colostrum. Physiologic anemia developed in foals of principal and control groups during the first week of life. Erythrocyte osmotic fragility in foals of the principal group prior to and after the ingestion of colostrum remained unchanged. However, at 36 hours after birth, there was a significant decrease in erythrocyte osmotic fragility in foals fed homologous colostrum.     

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.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

Serum copper and ceruloplasmin activity at the early growing stage in foals.

Serum concentrations of copper (Cu), zinc (Zn), manganese (Mn), calcium (Ca) and inorganic phosphorus (P), as well as antigenic ceruloplasmin (Cp) and oxidase activity as a functional index for copper metabolism, were measured in 10 foals (5 males and 5 females) and their dams. Samples were harvested from the foals within 1 wk after birth and monthly from 1 to 17 mo of age. Samples were collected from their dams in the perinatal period (monthly from 2 mo before delivery to 5 mo postpartum). Serum oxidase activity, antigenic Cp and Cu in foals were extremely low at 1 wk. Serum Cp had the lowest value of 17.0 +/- 8.0 (mean +/- SD) mg/dL within the 1st wk, then increased rapidly up to 43.7 +/- 5.8 mg/dL at 1 mo, and maintained this level until the 17th mo. Serum Zn in foals had the highest value of 73.2 +/- 13.1 micrograms/dL within 1 wk, then decreased to 38.3 +/- 5.9 micrograms/dL by 17 mo. Serum Mn, Ca and P in mares were almost stable and within established reference ranges for our laboratory in the perinatal period, and these values in foals were also in the normal range. Even on appropriate feeding, serum Cu, Cp and oxidase activity were quite low a few weeks after birth, while a higher proportion of Cp-binding copper was found in the foals. This might be caused by the limited synthesis of ceruloplasmin in this period. These data suggest that newborn foals are in a critical situation of marginal copper status in the early stage of growth.     

Neutrophil function of neonatal foals is enhanced in vitro by CpG oligodeoxynucleotide stimulation

Rhodococcus equi is an intracellular bacterium that causes pneumonia in foals and immunocompromised adult horses. Evidence exists that foals become infected with R. equi early in life, a period when innate immune responses are critically important for protection against infection. Neutrophils are innate immune cells that play a key role in defense against this bacterium. Enhancing neutrophil function during early life could thus help to protect foals against R. equi infection. The objective of our study was to determine whether in vitro incubation with the TLR9 agonist CpG 2142 would enhance degranulation and gene expression of cytokines and Toll-like receptor 9 (TLR9) by neutrophils collected from foals at 2, 14, and 56 days of life, and to determine whether these stimulated responses varied among ages. Neutrophil degranulation was enhanced at all ages by in vitro stimulation with either CpG alone, R. equi alone, or in combination with either R. equi or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (P<0.05), but not by in vitro stimulation with fMLP alone. There were no significant differences among ages in CpG-induced cytokine expression, except for IL-12p40, which was induced more at 56 days of age than on days 2 or 14. Collapsing data across ages, CpG 2142 significantly (P<0.05) increased IL-6 and IL-17 mRNA expression. We concluded that in vitro stimulation of foal neutrophils with CpG enhances their function by promoting degranulation and inducing mRNA expression of IL-6 and IL-17, regardless of age.     

Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates

Javsicas, LH., Giguère, S., Womble, AY. Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01151.x. The objectives of this study were to determine the serum and pulmonary disposition of telithromycin in foals and to determine the minimum inhibitory concentration (MIC) of telithromycin against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. A single dose of telithromycin (15 mg/kg of body weight) was administered to six healthy 6–10‐week‐old foals by the intragastric route. Activity of telithromycin was measured in serum, pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells using a microbiological assay. The broth macrodilution method was used to determine the MIC of telithromycin, azithromycin, clarithromycin and erythromycin against R. equi. Following intragastric administration, mean ± SD time to peak serum telithromycin activity (T_max) was 1.75 ± 0.76 h, maximum serum activity (C_max) was 1.43 ± 0.37 μg/mL, and terminal half‐life (t_½) was 3.81 ± 0.40 h. Telithromycin activity, 4 h postadministration was significantly higher in BAL cells (50.9 ± 14.5 μg/mL) than in PELF (5.07 ± 2.64 μg/mL), and plasma (0.84 ± 0.25 μg/mL). The MIC₉₀ of telithromycin for macrolide‐resistant R. equi isolates (8 μg/mL) was significantly higher than that of macrolide‐susceptible isolates (0.25 μg/mL). The MIC of telithromycin for macrolide‐resistant isolates (MIC₅₀ = 4.0 μg/mL) was significantly lower than that of clarithromycin (MIC₅₀ = 24.0 μg/mL), azithromycin (MIC₅₀ =256 μg/mL) and erythromycin (MIC₅₀ = 24 μg/mL).