Evaluation of polymyxin B in an ex vivo model of endotoxemia in horses

OBJECTIVE: To evaluate effects of polymyxin B sulfate (PMB) on response of horses to endotoxin, using an ex vivo model. ANIMALS: 8 healthy horses. PROCEDURE: In a crossover design, 3 doses of PMB (100, 1,000, and 10,000 U/kg of body weight) and physiologic saline solution (control) were evaluated. Prior to and for 24 hours after administration of PMB, blood samples were collected into heparinized tubes for use in 2 assays. For the endotoxin-induced tumor necrosis factor (TNF) assay, blood samples were incubated (37 C for 4 h) with 1 ng of Escherichia coli or Salmonella Typhimurium endotoxin/ml of blood. Plasma was harvested and assayed. For the residual endotoxin activity assay, plasma was collected into sterile endotoxin-free borosilicate tubes, diluted 1:10 with pyrogen-free water, and incubated for 10 minutes at 70 C. Escherichia coli endotoxin (0.1 or 1 ng/ml of plasma) was added to the thawed samples prior to performing the limulus ameobocyte lysate assay. Serum creatinine concentrations were monitored for 1 week. RESULTS: Compared with baseline values, PMB caused a significant dose- and time-dependent decrease in endotoxin-induced TNF activity. Compared with baseline values, residual endotoxin activity was significantly reduced after administration of 10,000 U of PMB/kg. Compared with baseline values, 1,000 and 5,000 U of PMB/kg should inhibit 75% of endotoxin-induced TNF activity for 3 and 12 hours, respectively. Serum creatinine concentrations remained within the reference range. CONCLUSION AND CLINICAL RELEVANCE: Results of the study suggest that PMB is a safe, effective inhibitor of endotoxin-induced inflammation in healthy horses.     

Adverse effects of polymyxin B administration to healthy horses

BackgroundPolymyxin B (PolyB) is used to treat endotoxemia in horses; neurologic and nephrogenic adverse effects occur in humans.ObjectivesTo describe PolyB adverse effects in horses.AnimalsFive healthy horses (ataxia 0/5), 1 horse with cervical osteoarthritis (ataxia 1/5).MethodsProspective blinded randomized cross‐over trial; 3‐weeks wash out. Horses received PolyB (PolyB 6000 IU/kg IV, 7 doses q12h, n = 6) and PolyB/gentamicin (PolyB 6000 IU/kg IV, q12h 7 doses; gentamicin 10 mg/kg IV q24h 4 doses n = 4, or q12‐24 h 5 doses because of an additional erroneous dose, n = 2). Daily neurological examinations were video recorded, and ataxia graded by 3 observers. Urine status, urinary GGT/creatinine ratio, plasma creatinine, and urea were assessed every other day, EMG daily. Mixed model analysis was used to evaluate factors associated with ataxia grade and [PolyB].ResultsMedian ataxia score increased from 0/5 (range 0‐2/5) to 2/5 (range 1‐3/5) during administration and declined to 0.5/5 (range 0‐2/5) after cessation. Gentamicin co‐administration (P < .01, effect size: .8), number of PolyB doses (P < .001, effect size: .6), and time since last PolyB dose (P < .001, effect size: .5) had a significant effect on ataxia grades, while horse, day, [Genta], [PolyB], and [PolyB]_CSF did not. Gentamicin co‐administration and [Genta] C_peak had no effect on median [PolyB] C_peak (4.67 and 4.89 μg/ml for PolyB and PolyB/gentamicin, respectively). Urinary GGT/creatinine ratio was elevated in 3/6 horses receiving PolyB/gentamicin. The EMG remained unchanged.Conclusions and Clinical ImportancePolyB caused transient ataxia, worsening with cumulative PolyB doses and gentamicin co‐administration. Nephrotoxicity of PolyB was only evident when gentamicin was co‐administered.     

Evaluation of autogeneic and allogeneic cortical chip grafting in a feline tibial nonunion model

A feline tibial nonunion model was used to evaluate the efficacy of fresh autogeneic and deep-frozen allogeneic cortical chip grafts for bone repair. Rigidly fixated tibial ostectomies with a segmental defect length of 1 cm were grafted with 3 mm X 2 mm chips of cortical bone in 12 skeletally mature cats. Six cats were given fresh autogeneic bone (group A) and 6 were given deep-frozen allogeneic bone (group B). Bone healing was evaluated radiographically and histologically over a 12-week period. Consolidating bony callus and palpable stability of the tibia after bone plate removal were evident in all group A cats and 4 of the 6 group B cats by 12 weeks after surgery. Bone remodeling had progressed to intermediate restoration of diaphyseal structure in 2 cats of each group. Fibrous replacement of graft chips, absence of bony callus, and instability of the tibia after bone plate removal were evident in 1 group B cat at 12 weeks. The remaining group B cat was withdrawn from the study at 6 weeks due to loosening of the bone plate and screws.     

Serum butyrylcholinesterase and paraoxonase 1 in a canine model of endotoxemia: effects of choline administration

Butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) are two serum enzymes synthesized by the liver that are related with inflammation. The main objectives of this study were to determine changes in serum BChE and PON1 by using a canine model of endotoxemia, and to evaluate whether choline alters BChE and PON1 activities during inflammation. For this purpose, a total of 20 mongrel dogs were divided into four groups: control, choline (C), lipopolysaccharide (LPS), and LPS+C. Dogs in the control group were injected with 0.9% NaCl (0.2 ml/kg, i.v.). Dogs in C and LPS+C groups received choline chloride (20 mg/kg, i.v., three times with 4 h intervals). Endotoxin was injected (0.02 mg/kg, i.v., once) to the dogs of LPS and LPS+C groups. Statistically significant decreases in BChE and PON1 activities in LPS group were detected 24 and 48 h post injection, respectively. No statistically significant changes in BChE and PON1 activities at different times were detected in control, C, or LPS+C groups. In conclusion, the data obtained in present study revealed a decrease in serum BChE and PON1 activities in dogs during experimentally induced endotoxemia and that choline administration attenuates these changes.     

The antipyretic effect of polymyxin B in endotoxin-induced fever

LPSS. typhimurium (0.3 g per kg) injected intravenously into conscious goats induced an inhibition of the extrinsic ruminal contractions in association with pyrexia and changes in heart rate. Pretreatment with polymyxin B (3 mg per kg i.v.) antagonized the endotoxin-induced symptoms in this species. In rabbits, the intravenous administration of LPSE. coli (0.1 g per kg) resulted in a febrile response, which could be markedly reduced by pretreatment with polymyxin B. Unlike endotoxins, the febrile response to leucocytic pyrogen was not inhibited after pretreatment with this antibiotic. Moreover, polymyxin B caused only a minimal decline in the febrile response due to sodium nucleinate from yeast (10 mg per kg). These results strengthen the view that the antipyretic activity of polymyxin B is due to an interaction of this cationic macromolecule with the anionic endotoxin molecule.     

Pharmacokinetics of polymyxin B administered via the bovine mammary gland

Polymyxin B was infused into normal, chronically inflamed, and acutely inflamed quarters of the mammary gland of lactating cows at dosages ranging between 1 and 2 million units (100–200 mg) per quarter. Samples of milk from treated and non-treated quarters, jugular venous blood, subcutaneous abdominal (mammary) venous blood, and urine were collected at intervals after treatment and were assayed using microbiological test methods for polymyxin B concentrations. The drug was not absorbed from normal and chronically inflamed quarters; more than 90% of the infused dose was recovered in milk within 24 h after treatment, and drug residues were detected up to the ninth milking. Drug concentrations in milk from acutely inflamed quarters were significantly lower than in milk from normal quarters; 55% of the infused dose was recovered in the milk within 24 h after treatment. The drug was detected in milk from non-treated quarters, in blood from the subcutaneous abdominal vein, and in the urine during 36–48 h after acutely inflamed quarters were infused with the drug. These data indicate that polymyxin B is well distributed throughout, and is absorbed to a significant degree into the systemic circulation from the acutely inflamed udder.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.     

Evaluation of cytologic findings in feline conjunctivitis

Background

Cytologic examination of smears prepared from ocular swabs of conjunctiva from cats with conjunctivitis permits identification of the type of inflammation and possibly specific microorganisms. Results of studies of the diagnostic utility of cytology for detection of infectious causes of feline conjunctivitis have been inconsistent.

Objectives

The objectives of this study were to describe cytologic findings in cats with conjunctivitis and to compare those findings with results of PCR analysis for feline herpesvirus (FHV‐1), Chlamydophila felis (C felis), and Mycoplasma felis (M felis).

Methods

Conjunctival smears from 88 cats with conjunctivitis and 10 healthy control cats were stained with a Romanowsky stain and evaluated for the type of inflammation and evidence of an infectious agent. PCR analysis for FHV‐1, C felis, and M felis was performed.

Results

Infectious agents identified by PCR analysis were FHV‐1 in 9 cats (10%), C felis in 8 cats (9%), and M felis in 6 cats (7%). Inclusions interpreted as chlamydial inclusions were found in all cytologic smears from cats positive for C felis by PCR analysis and in 3 PCR‐negative cats. Inclusions interpreted as Mycoplasma organisms were found in 3 of 6 cats that were PCR‐positive for M felis and in 1 PCR‐negative cat. FHV‐1 inclusion bodies were not detected on cytologic examination.

Conclusions

Cytologic examination can be diagnostic for C felis infection when many typical inclusions are present. Cytologic examination was unreliable in diagnosing M felis infection, and viral inclusions of FHV‐1 were not found in specimens stained with Romanowsky stains.     

Evaluation of Key's hypothesis in the feline tibia: an experimental model for augmented bone healing studies

Key's hypothesis states that a segmental long bone defect 1.5 times the diaphyseal diameter exceeds the regenerative capacity of bone in skeletally mature dogs and results in nonunion. This hypothesis was evaluated in 5 adult cats with rigidly fixated segmental tibial ostectomies ranging from 1.25 to 1.52 times the diaphyseal diameter. Clinical, radiographic, and histologic data were obtained over a 12-week period. Healing was classified as mature bony union, clinical union, delayed union, or nonunion. Absence of a consolidating callus and instability after removal of fixation devices was found for all cats at 12 weeks. Scant formation of new bone within the gap was histologically evident for only 1 cat. In the remaining 4 cats, fibrous tissue and striated muscle predominated within the gap, and independent healing of the proximal and distal cut ends of the bone were observed. The occurrence of 4 nonunions and 1 equivocally delayed union indicated that Key's hypothesis overestimates the regenerative capacity of bone in the cat. Failure of this experimental model to produce clinical union within 12 weeks demonstrated that this model is a valid method of investigating augmented bone healing techniques that promote union within this period in the cat.     

Evaluation of a feline viral rhinotracheitis-feline calicivirus disease vaccine.

An attenuated respiratory disease vaccine against feline viral rhinotracheitis (FVR) and feline calicivirus (FCV) disease was evaluated for safety and efficacy in specific-pathogen-free cats. Twenty cats were vaccinated twice intramuscularly, with 28 days between vaccinations. Ten unvaccinated cats were used as contact controls. Adverse effects were not noticed after vaccination, and the vaccinal virus did not spread to contact controls. Arithmetical mean serum-neutralizing titers against vaccinal FCV strain F9 and challenge FCV strain 255 were 1:13 and 1:15 at 28 days after the 1st inoculation. These titers increased to 1:45 and 1:196 after the 2nd inoculation. After challenge exposure of vaccinated cats to virulent FCV 255 virus, mean titers increased to 1:129 and 1:865, respectively for F9 and 255 viruses. The F9 postchallenge mean titer for vaccinated cats was 21.5 times higher than that for the 8 contact controls that survived challenge exposure. The arithmetical mean serum neutralizing titer for FVR was low (1:2) after the 1st vaccination, but increased to 1:35 after the 2nd vaccination. Challenge exposure to virulent FVR virus resulted in a marked anamnestic immune response (mean titer of 1:207, compared with 1:12 for contact controls). In general, vaccinated cats remained alert and healthy after challenge exposure with FCV-255, whereas unvaccinated contact control cats developed definite signs of FCV disease, including central nervous system (CNS) depression (6 of 10) and dyspnea indicative of pneumonia (5 of 10). Two controls died of severe pneumonia. A mild fibrile response was detected in 28% of vaccinated cats, compared with a more severe febrile response in 78% of control cats. Some vaccinated cats developed minute lingual ulcers that did not appear to be detrimental to the health of the cat. After FVR challenge exposure, vaccinated cats were free of serious clinical signs. Five of 18 vaccinated cats had mild signs of FVR, including an occasional sneeze, low temperature, and mild serous lacrimation for 1 or 2 days. Contact controls developed definite clinical signs of FVR. The combined FVR-FCV vaccine appears to be safe and reasonably efficacious. Vaccination against FCV disease and FVR should be part of the routine feline immunization program.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses

OBJECTIVES: To measure serum polymyxin B concentration after single and repeated IV infusions in horses. ANIMALS: 5 healthy horses. PROCEDURES: In study 1, 1 mg (6,000 U) of polymyxin B/kg was given IV and blood samples were collected for 24 hours. In study 2, 1 mg of polymyxin B/kg was given IV every 8 hours for 5 treatments and blood samples were collected until 24 hours after the last dose. Polymyxin B concentration was measured as the ability to suppress nitrite production by murine macrophages stimulated with lipopolysaccharide and interferon-alpha. Urine was collected prior to the first drug infusion and 24 hours after the fifth drug infusion for determination of urinary gamma-glutamyl transferase (GGT)-to-creatinine ratios. RESULTS: In study 1, mean +/- SEM maximal serum polymyxin B concentration was 2.93 +/- 0.38 microg/mL. Polymyxin B was undetectable 18 hours after infusion. In study 2, maximal polymyxin B concentrations after the first and fifth doses were 2.98 +/- 0.81 microg/mL and 1.91 +/- 0.50 microg/mL, respectively. Mean trough concentration for all doses was 0.22 +/- 0.01 microg/mL. A significant effect of repeated administration on peak and trough serum concentration was not detected. Urine GGT-to-creatinine ratios were not affected by polymyxin B administration. CONCLUSIONS AND CLINICAL RELEVANCE: Polymyxin B given as multiple infusions to healthy horses by use of this protocol did not accumulate in the vascular compartment and appeared safe. Results support repeated IV use of 1 mg of polymyxin B/kg at 8-hour intervals as treatment for endotoxemia.     

Evaluation of the efficacy of a commercial diet in the dissolution of feline struvite bladder uroliths

Clinical signs of struvite urolithiasis resolved in an average of 19 days in cats fed either a canned or dry urine-acidifying, magnesium-restricted diet, and stones completely dissolved in 31 of 39 cats in an average of 30 days. Stones were surgically removed from five cats, and analysis revealed that these stones were not composed of struvite. The results of this study strongly suggest that the diet will dissolve naturally occurring feline struvite bladder uroliths.     

S-adenosylmethionine (SAMe) in a feline acetaminophen model of oxidative injury

S-adenosylmethionine (SAMe) is reported to have hepatoprotective and antioxidant functions. Acetaminophen (paracetamol) was used to induce oxidative damage in cats, and to then determine the effect of SAMe treatment on erythrocyte morphology, PCV, liver histopathology, thiobarbituate reacting substances (TBARS), reduced glutathione (GSH), and oxidised glutathione (GSSG).Cats receiving acetaminophen had a significant increase in methemoglobin and Heinz body production. A significant effect for the interaction of time and treatment was found for Heinz body production and changes in PCV. No significant changes were found in blood or hepatic TBARS. Blood GSH increased significantly in all cats, while the blood GSH:GSSG ratio tended to increase the most in cats given acetaminophen only. The hepatic GSH:GSSG ratio tended to increase in cats given SAMe and decrease in cats given acetaminophen, but this effect was not significant. SAMe protected erythrocytes from oxidative damage by limiting Heinz body formation and erythrocyte destruction and maybe useful in treating acetaminophen toxicity.     

Evaluation of pradofloxacin for the treatment of feline rhinitis

Forty humane society cats with suspected bacterial upper respiratory infections (URIs) were studied in order to compare amoxycillin and pradofloxacin for treatment of rhinitis and describe common pathogens. Nasal discharges were collected prior to random placement into one of three treatment groups. Cats failing to initially respond were crossed to the alternate drug. Drug toxicity was not noted. The organisms most frequently isolated or amplified pre-treatment were feline herpesvirus-1 (75%), Mycoplasma species (62.5%), Bordetella species (47.5%), Staphylococcus species (12.5%) and Streptococcus species (10.0%). No differences in clinical scores between groups over time were noted. Overall response rates for amoxycillin at 22mg/kg, q12h for seven doses (10/15 cats; 67%), pradofloxacin at 5mg/kg, q24h for seven doses (11/13 cats; 85%), and pradofloxacin at 10mg/kg, q24h for seven doses (11/12 cats; 92%) were not statistically significant. Results suggest that pradofloxacin can be a safe, efficacious therapy for some cats with suspected bacterial URI.     

Characterization of feline T and B cells

Feline peripheral-blood lymphocyte populations (n = 22) were examined for the following markers: rosette formation with guinea pig erythrocytes (GPE-T cells), rosette formation with human RBC (HRBC-T cells), rosette formation with sheep RBC, mixed rosette formation with GPE-T cells and HRBC-T cells (total T cells), erythrocyte antibody-complement rosettes, and surface immunoglobulin. An average of 28% +/- 7% (range, 16% to 39%) of the feline lymphocytes formed rosettes with GPE-T cells, and 27% +/- 7% (range, 11% to 36%), with HRBC-T cells. An average of 57% +/- 9% (range, 33% to 75%) of the lymphocytes formed mixed rosettes. The erythrocyte antibody-complement rosette-forming cells and surface immunoglobulin-bearing cells were found in peripheral blood lymphocytes (10% +/- 6% and 24% +/- 8%, respectively). The murine monoclonal antibodies OKT 11 and HuLy-m1, specific for a framework determinant of human E-rosette receptor antigens, cross-reacted with feline cell membrane molecules recognizing a bimolecular complex (45,000 to 50,000 daltons) similar to that described in persons. We investigated the distribution of these E-rosette receptor-like antigens on feline lymphocytes. By complement-mediated lymphocytotoxicity, about 30% of the feline lymphocytes expressed the antigens. When lymphocytes were treated with HuLy-m1 antibody, spontaneous rosette formation with HRBC-T cells was significantly inhibited.     

Growth and characterisation of a cell culture model of the feline blood-brain barrier

An in vitro model of the feline blood-brain barrier was developed using primary cultures of brain capillary endothelial cells derived from adult cats. They were grown in the presence of astrocytes obtained from newborn kittens. Feline endothelial cell cultures were characterised by uptake of DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) and expression of von Willebrand factor. Astrocytes were characterised based on their expression of glial fibrillary acidic protein (GFAP). Electron microscopy revealed junctional specialisation between endothelial cells. Occludin and ZO-1 expression by the endothelial cell cultures was detected by Western blot analysis. Barrier function of co-cultured endothelial cells and astrocytes was confirmed by a transendothelial electrical resistance (TEER) value of 30-35 Omegacm2 and apparent permeability coefficients (Papp) for FD-40 (FITC-dextran, 40 kDa) of 4x10(-6) cm/s and for FD-4 (4kDa) of 1.92x10(-5) cm/s. In endothelial cell monolayers grown with astrocyte-conditioned medium, the TEER value was lower (20-25 Omegacm2), and Papp of FD-40 and FD-4 was higher at 6.27x10(-6) and 3.96x10(-5) cm/s, respectively. This model should have useful applications in the examination of events occurring at the BBB early in FIV infection, and may provide knowledge applicable to HIV infection.