VTechWorks Repository :: Browsing by Author "Furr, Martin O."

Browsing by Author "Furr, Martin O."

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Aerosol delivery of Rhodoccocus equi IgG to the lungs of ponies
Foley, Alicia Kate (Virginia Tech, 2013-08-09)
The objective of this study was to determine if R. equi IgG purified from commmercially available hyperimmune R. equi plasma and delivered to the lungs of adult ponies would cause a local inflammatory response and if increases in total and R. equi specific IgG occurred post administration. IgG was purified and concentrated from plasma via protein G affinity chromatography. A cross over study was performed. Eight healthy adult ponies were randomly assigned to two groups of four; each pony acted as its own control. Either the IgG product or 0.9% Saline was delivered via a vibrating mesh nebulizer during the first treatment phase. During the second treatment phase ponies recieved the oppostie treatment. A 4 week washout period was allowed between phases. Bronchoalveolar fluid was recovered using a low volume endoscopic technique prior to aerosolization (time 0), and at 1 hr, 6 hrs, and 24 hours post administration. The BAL fluid total IgG concentration and R. equi specific IgG titers were determined via ELISA and cytologic analysis was performed. No clinically significant local inflammatory response was identified in response to IgG treatment. While total IgG concentrations were increased at T1 compared to T0, no significant effects of time were found (P=0.19). However, overall significantly higher concentrations of total IgG were found after administration of saline when compared to IgG administration (P=0.023). While the R. equi specific titer increased at T1 after IgG administration, no significant difference was identified between treatment or time (P=0.261). Overall the individual response to IgG was variable. It is possible that the protein rich IgG acted as a relatively hypertonic solution and caused fluid influx from the pulmonary parenchyma after treatment thereby diluting the total IgG present when compared to saline administration. This conclusion cannot be verified as BAL dilution correction was not performed. However, it is unknown what titer or level of increased IgG is nessecary to assist with prevention of disease. Future research should focus on the effect of R. equi specific IgG on pulmonary cells to determine if administration of local R. equi specific IgG would alter intrapulmonary immune responses to R. equi.
Cardiovascular and Hematological Effects of Hetastarch and Hypertonic Saline Solutions During Experimental Endotoxemia in Horses
Pantaleon, Lucas Guillermo (Virginia Tech, 2005-06-30)
Justification: Endotoxemia and sepsis are major causes of mortality in horses, resulting in significant economic losses for the equine industry. Objective: To determine the effects of the combination of Hypertonic Saline Solution and Hetastarch in endotoxemic horses. Animals: Eighteen horses divided into three groups of six. Procedure: All horses received a total dose of intravenous E. coli endotoxin infused at 50 ug/kg; divided into a bolus infusion of 20 ug/kg followed by 30 ug/kg given over 30 minutes. After induction of endotoxic shock; group I (control) received a bolus (15 ml/kg) of isotonic solution, group II (isotonic solution) received a bolus (60 ml/kg) of balanced polyionic crystalloid solution and group III (Hypertonic saline plus Hetastarch) received a bolus of 5 ml/kg of hypertonic saline, followed by a bolus of 10 ml/kg of Hetastarch. Hemodynamic and hematological parameters were measure at different time points. Results: Hemodynamic, biochemical and hematological differences were observed among the three groups. Conclusions and Relevance: the use of large volume crystalloid fluid resuscitation causes volume overload, exerting deleterious effects on the cardiovascular and pulmonary systems. The use of small volume resuscitation (HSS-HES) showed a trend towards better cardiovascular and pulmonary function, without the deleterious effects of volume overload. Abnormalities with regard to coagulation were not seen for the time period of the experimental protocol and the dose regimen used for HSS-HES. Small volume resuscitation in critically ill horses shows promise for its beneficial effects in cardiovascular and pulmonary functions.
Efficacy of hyperimmunized plasma in the treatment of horses with acute diarrhea
Atherton, Rachel Paget (Virginia Tech, 2007-05-24)
The aim of this study was to evaluate the use of a hyperimmunized plasma containing high concentration of antibodies against Clostridium difficile, Clostridium perfringens and Salmonella sp in a referral population of equine colitis cases. A prospective, blinded clinical trial was undertaken. Horses were enrolled if they were over 1 year old, duration of diarrhea at presentation was less than 72 hours, they had not received equine plasma within the last 3 months and the serum total protein was greater than 4mg/dl. Horses were randomized to receive hyperimmunized plasma, control plasma (collected from non-immunized horses) or no plasma therapy. Clinical parameters were recorded and a fecal score (2 -14) assigned (every 6 hours) based upon diarrhea frequency, volume and consistency, for a total of 72 hours. A score less than 5 was considered normal. Fecal consistency was observed until resolution, discharge or death. Complete blood counts and biochemical profiles were collected at admission, 24 and 72 hours and at admission, 24 hours and 48 hours respectively. Forty two horses were enrolled and 38 horses completed the study. At study admission clinical and clinicopathological parameters, other than fecal frequency score were comparable between the groups. Fecal frequency score was significantly different between the treatment groups (p=0.003). The mean duration of diarrhea was 40.7±9.8 hours (mean ±SEM), 119.2±56.1 hours and 72.0±24.5 hours for the hyperimmunized plasma, normal plasma and control groups respectively. This data confirms the hyperimmunized plasma used in this study decreased the time to resolution of diarrhea.
Equine Protozoal Myeloencephalitis. Preliminary Investigation of Protozoan-Host interactions in the horse
Goehring, Lutz Steffen (Virginia Tech, 1998-08-14)
Equine Protozoal Myeloencephalitis is the most frequently diagnosed neurologic disorder of horses in the united states, which is caused by the protozoan organism Sarcocystis neurona. The disease has a profound impact on the American Horse Industry. This impact includes prolonged and expensive treatment without a guaranteed return to a previous level of use for the individual horse. Poor respponse to and prolonged duration of treatment may suggest an immune mediated impariement of host response. There is limited information about the direct interaction between the pathogen and the host. In two in vitro experiments we investigated a) whether the presence of the protozoan organism can influence mitogen-stimulated peripheral blood mononuclear cells (PBMCs), suggesting a direct influence of the protozoan organism on cells of the immune system, and b) if cerebrospinal fluid (CSF) from horses with EPM has an effect on mitogen-stimulated PBMCs, suggesting that the microenvironment of the site of infection influences the course of disease. Experiment 1: Mitogen simulated PBMCs from EPM affected and control horses were co-cultured with fragments of freeze thawed bovine turbinate cells that were infected with S. neurona merozoites. Compared to controls PBMCs co-cultured with S. neurona fragments were the only cells that showed a decreased proliferation (p<0.05). A difference between EPM affected and control horses could not be detected (p>0.05). These results may imply that the persistence of S. neurona infection in the horses CNS is, in part, due to a pathogen-derived mechanism that attentuates the hosts immune response. Experiment 2: Mitogen stimulated PBMCs from a horse affected with EPM and a control were co-cultured n the presence of CSF from EPM affected and uninfected controls. Prior to co-culture the CSF was fractionated by a filtration process over two microfilter units. An identical volume of NaCl (0.9%) served as a control for the volume of CSF that was added. The proliferation assay revealed a deviation of the response depending on cell donor and CSF fraction used. The effect was independant of the protein concentration of the CSF fraction, and a decrease in lymphocyte proliferation was not caused by increased cellular death. This suggests the presence of subsets within the CSF which have a stimulatory of suppressive influence on the cells in culture. The effect was cell donor dependant which implies a difference in lymphocyte subsets between the two horses that were used.
Evaluation of clinical methods of pulmonary gas exchange assessment in the standing horse
Davis, Michael S. (Virginia Tech, 1995-07-05)
There are limited methods of assessing pulmonary function in horses at rest. In this study, we developed clinical techniques to measure gas exchange efficiency in horses. These techniques were then used to evaluate horses with varying degrees of lower respiratory disease. Three groups of horses (Group 1: asymptomatic, n=6; Group 2: symptomatic only with rebreathing, n=11; Group 3: symptomatic at rest, n=9) were selected based on physical exam, transtracheal aspirate, and thoracic radiographs. Blood samples were obtained from the transverse facial artery and jugular vein. Maximal end-tidal CO₂ tension (E_TCO₂) was measured by an infrared capnograph through a facemask. Alveolar O, tension, alveolar dead space fraction (V_DB/V_T), and physiologic shunt fraction (Q_S/Q_T) were calculated using standard formulas. Horses with both mild and severe signs of lower respiratory disease had significant (p<0.05) differences in gas exchange indices at rest compared to asymptomatic horses. Albuterol was administered to seven of the Group 2 horses from a metered-dose inhaler through an equine facemask at a dose of 90 μg per 100 kg. Blood samples and tidal gas samples were obtained 15 minutes post-treatment, and Q_S/Q_T and (V_DB/V_T) were calculated. Albuterol caused significant (p<0.05) hypoxemia 15 minutes following inhaled administration. This was accompanied by a significant increase in Q_S/Q_T, suggesting that the hypoxemia was due to increases in which the ratios of ventilation to perfusion were decreased.
Evaluation of Pgg-Glucan, a Novel Immunomodulator, in in Vitro and Ex Vivo Models of Equine Endotoxemia
Sykes, Benjamin William (Virginia Tech, 2003-06-16)
Justification - Endotoxemia is an important contributor to mortality and loss of use in the horse and results in significant losses to the equine industry on an annual basis. Objective - To determine the effect of PGG-Glucan on the cytokine response to endotoxin in the horse. Animals - Part 1; 6 adult horses. Part 2; 12 adult horses. Procedure - Part 1; Whole blood was collected, aliquoted, and incubated in vitro in four groups; saline control, endotoxin (LPS) (100 ng/ml), PGG-Glucan (0.1, 1.0, 10 and 100 Î¼g/ml) and LPS (100 ng/ml) plus PGG-Glucan (0.1, 1.0, 10 and 100 Î¼gg/ml). Supernatants were collected at 0, 6 and 12 hours and assayed for tumor necrosis factor Â£\ (TNFÂ£\) activity. Part 2; Horses received either PGG-Glucan (1 mg/kg) or an equal volume of isotonic saline (0.9% NaCl) IV over 15 minutes. Twenty four hours later blood was collected and mononuclear cells isolated for cell culture. Cells were treated with LPS (100 ng/ml) and RNA extractions were performed at 0, 6, 12, 24 and 48 hours. Relative mRNA expression of TNFÎ±, interleukin-1Î² (IL-1Î²),, interleukin-10 (IL-10) and interferon-Î³ (IFN-Î³) was determined by reverse transcription and real time polymerase chain reaction. Results - Using an in vitro endotoxin challenge method PGG-Glucan altered the production of TNFÎ± in a dose-dependent manner. PGG-Glucan had no effect upon the ex vivo cytokine mRNA expression of TNFÎ±, IL-1Î², IL-10 or IFN-Î³. Conclusions and Relevance - Although mild changes were observed in TNFÎ± production in vitro, it is not likely that PGG-Glucan will have a significant effect upon clinical endotoxemia.
Evaluation of the toxic effects of eltenac (4-((2,6-dichlorophenyl) amino)-3-thiophene acetic acid), a nonsteriodal anti-inflammatory drug, in horses
Goodrich, Laurie Ruth (Virginia Tech, 1996-05-15)
A controlled study was performed to determine the potential toxic effects of the new nonsteroidal anti-inflammatory drug, eltenac (4-[(2,6-dichlorophenyl) amino]-3-thiopheneacetic acid), in horses. Four treatment groups were formed, each composed of 6 horses. The drug was injected intravenously, once daily, at a dose rate of 0.5 mg/kg, 1.5mg/kg or 2.5 mg/kg for 15 days. A control group was injected with sterile saline solution. Parameters assessed were hay and water consumption, daily clinical observations (evaluation of attitude, mentation, pulse and respiratory rate, fecal consistency, skin condition, and color and hydration of mucous membranes), physical examinations, complete hemogram, coagulation profiles, serum biochemical profiles, urinalysis, gastroscopic examinations and gross post-mortem and histopathological examinations on all organ systems. All examiners were blinded to group assignment and dosage levels until the completion of the study. A few glandular gastric ulcers, mild in severity, developed in seven animals during the treatment period. This occurred more often in horses treated with high doses of eltenac (P=.02). A dose dependent change of WBC count and neutrophil count were noted. Total protein, albumin and globulin levels had dose dependent decreases. One horse in the high dose group (2.5mg/kg) developed ventral edema as well as hypoproteinemia. N one of the horses in any of the dosage groups exhibited depression or anorexia. Gross post-mortem and histologic examination did not reveal any signs of drug related gastrointestinal, renal or hepatic abnormalities. Minimal toxic effects of eltenac given intravenously were greatest in horses treated with 2.5 mg/kg of the compound for 15 days.
Glucocorticoid Receptor Density and Binding Affinity in Horses with Systemic Inflammatory Response Syndrome
Hoffman, Crystal Joyce (Virginia Tech, 2014-06-03)
There were three objectives of this study. The first was to determine if commercially available fluorochromes could be used to determine the glucocorticoid receptor (GR) density and binding affinity (BA) in equine peripheral blood mononuclear cells. The second was to determine if there was a correlation between elevated plasma cortisol and GR density or binding affinity in healthy adult horses. The third objective was to evaluate the HPA axis in adult horses presenting with systemic inflammatory response syndrome (SIRS), and to determine where any alterations in HPA axis function occur in these patients compared to healthy adults. For the first part of the study, peripheral venous blood was collected from 3 healthy research horses on 3 days. Peripheral blood mononuclear cells were isolated using Ficoll gradient centrifugation. Phycoerythrin (PE)-CD44 was then used to extracellularly label leukocytes, and then an intracellular GR antibody was used to determine a baseline measurement of GR density and fluorescein isothiocyanate (FITC)-dexamethasone was used to determine binding affinity via flow cytometric analysis. Comparison of control samples to those for CD44, GR density, and GR binding affinity showed a statistically significant difference for all samples (P<0.0001, P<0.0001, and P<0.0001 respectively). This showed that the CD44, GR antibody, and FITC-dexamethasone could successfully be used to analyze equine peripheral blood mononuclear cells for GR activity. For the second part of the study, an ACTH stimulation test was performed on 8 healthy horses in order to induce an increase in endogenous cortisol production. Plasma cortisol levels, GR density, and GR binding affinity were measured at baseline, 4, 8, and 24 hours after treatment. Median basal cortisol concentration was 4.9, range 3.2-6.1 μg/dl. This initially increased following ACTH stimulation to 5.6, range 4.8-7.4 μg/dl, then showed a significant decrease by 8 hours post ACTH administration to 1.4, range 1.1-2.7 μg/dl (P=0.0221). No correlation was observed between plasma cortisol concentration in healthy horses and GR density or binding affinity (r=-0.145, P=0.428 and r=0.046, P=0.802, respectively). For the third phase of the study, horses (N=10) with systemic inflammatory response syndrome (SIRS) were compared to healthy, age and sex matched controls (N=10) presenting for lameness evaluation or ophthalmologic examination. Blood was collected from SIRS cases and controls on presentation to the Equine Medical Center. A CBC, serum biochemistry, and serum ACTH and cortisol measurements were performed. GR density and binding affinity were also determined. Nonsurvivors had a significantly decreased GR binding affinity (P=0.008) and demonstrated a trend towards an increase in the ACTH:cortisol ratio. ROC analysis was performed for serum ACTH and cortisol concentrations, the ACTH:cortisol ratio, GR density and GR binding affinity, and triglycerides to determine cut-off values associated with nonsurvival. These were then used to analyze this population using Fischer's exact test to determine the odds ratio (OR) associated with nonsurvival for each variable. This revealed that a serum triglyceride concentration greater than 28.5 mg/dl was associated with nonsurvival (OR=117, 95% CI, 1.94-7060). The other variables were not found to be significantly associated with nonsurvival, although a Delta BA% of less than 35.79% was found to be closely associated with nonsurvival (OR=30.33, 95% CI, 0.96-960.5). Additionally, a significant negative correlation was detected between the plasma ACTH concentration and Delta BA% (r=-0.685, P=0.029) and the ACTH:cortisol ratio and the Delta BA% (r=-0.697, P=0.025). This study showed that nonsurviving horses with SIRS had a significantly decreased GR binding affinity compared to survivors, and a tendency toward an increase in their ACTH:cortisol ratios. This confirms that HPA axis dysfunction occurs in adult horses with SIRS as tissue resistance to glucocorticoids, and potentially relative adrenal insufficiency as well. These results suggest that there are horses with SIRS that might benefit from "physiologic" doses of synthetic glucocorticoids to complement their relative adrenal insufficiency in addition to their poor tissue sensitivity. Further research should focus on methods to more rapidly determine which horses might benefit from treatment with glucocorticoids on presentation, as well as to more accurately determine prognosis for survival.
Glucose and insulin dynamics associated with continuous infusion of dextrose or dextrose and insulin in healthy and endotoxin-exposed horses
Han, Janet (Virginia Tech, 2008-06-20)
The objective of the study was to investigate and characterize the effects of a continuous rate infusion of dextrose or dextrose and insulin on glucose and insulin dynamics in both healthy and endotoxin-exposed horses. Administration of a low dose of endotoxin has been used in horses to mimic the clinicopathologic changes seen in endotoxemia, including the development of an inflammatory response. Our hypothesis was that a continuous rate infusion of insulin at a rate of 0.07 IU/kg/hr would prevent the development of hyperglycemia induced by administration of dextrose in both healthy and endotoxin-exposed horses. Nine healthy adult horses were used in the study. In Phase 1 of the experiment, horses received a saline infusion or a dextrose infusion in a balanced crossover design. In Phase 2 of the experiment, horses received a dextrose and insulin infusion, both prior to and after receiving a low dose of endotoxin (no LPS group and LPS group respectively) in a balanced crossover design. Blood samples were collected at regular intervals throughout both phases for measurement of plasma glucose and insulin concentrations. Infusion of dextrose alone resulted in hyperglycemia for nearly the entire study period. Insulin concentration was also increased in comparison to the saline infusion. When comparing the dextrose treatment group to the combined dextrose and insulin treatment group (no LPS group), the insulin levels were significantly greater over time in the latter group and resulted in maintenance of euglycemia. When comparing the no LPS group to the LPS group, both the glucose and insulin concentrations were higher in the LPS group but euglycemia was still achieved. These results serve to validate the dose of insulin used in this study (0.07 IU/kg/hr) in regards to effective prevention of hyperglycemia when administered concurrently with a dextrose infusion. Hyperglycemia was prevented in both healthy and endotoxin-exposed horses. In addition, the dose of insulin used was demonstrated to be safe, as hypoglycemia did not occur in any of the horses.
Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro
Frellstedt, Linda (Virginia Tech, 2010-06-23)
Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of "endotoxin tolerance" (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET. Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model.
The influence of equine bone marrow derived stem cells on the response of cultured peripheral blood mononuclear cells to endotoxin
MacDonald, Elizabeth Steward (Virginia Tech, 2015-10-05)
Endotoxemia is a major cause of morbidity and mortality in horses. The presence of large amounts of circulating endotoxin inititates a number of cell signaling pathways leading to a systemic inflammatory response. Activation of these pathways causes the release of a number of pro- and anti-inflammatory mediators. An overwhelming release of these mediators leads to the development of clinical signs associated with endotoxemia. Treatment options are limited mostly to supportive care at this time. Mesenchymal stem cells (MSCs) have been shown to have anti-inflamamtory and immune modulatory effects that may have some benefit for the treatment of horses with endotoxemia. To evaluate the effect of equine MSCs on the response to endotoxin challenge, the study was performed on two different stem cell lines with peripheral blood mononuclear cells (PBMCs) used as controls. After stimulation with endotoxin, secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon gamma (IFN-γ) were determined by ELISA. The immunogenic properties of MSCs were assessed with a one-way mixed lymphocyte reaction. In addition, the ability of MSCs to alter production of cytokines from stimulated PBMCs was assessed. TNF-α was not produced by MSCs when compared to PBMCs (p = < 0.001). There was no significant difference between MSCs and PBMCs in the production of IL-6. IL-10 production was significantly different (p = <0.001) at 6 and 12 hours with MSCs producing more than PBMCs in one stem cell line only. MSCs did not stimulate proliferation of PBMCs. Co-incubation of MSCs with PBMCs decreased the production of TNF-α in both stem cell lines although it was not statistically significant (p = 0.4 and 0.9) at either time point. IL-6 secretion was suppressed at twelve hours with co-incubation. IL-10 production was increased with co-incubation in one stem cell line. MSCs secrete soluble factors that can alter PBMC cytokine production and they do not appear to be immunostimulatory. These findings have potential implication for treatment of equine inflammatory conditions.
Investigation into the Presence of Helicobacter in the Equine Stomach by Urease Testing and Polymerase Chain Reaction and Further Investigation into the Application of the 13C-Urea Blood Test to the Horse
Hepburn, Richard James (Virginia Tech, 2004-06-14)
Equine gastric glandular mucosal ulceration can have a prevalence of 58%, yet its etiology is poorly understood. In man Helicobacter pylori is the most common cause of gastritis and peptic ulcer disease. Helicobacter is uniquely able to colonize the stomach, via the action of cytoplasmic urease. Different Helicobacter species have been isolated from many mammals but none has yet been cultured from the horse. Three tests used to identify human Helicobacter infection were applied to the horse. Test 1: PCR amplification of Helicobacter specific DNA, n=12. Test 2: the Pyloritek™ rapid urease test (RUT), n=15. Test 3: the 13C-urea blood test, n=8. Gastroscopy and antral biopsy was performed in all horses. All horses demonstrated the presence of Helicobacter specific gene material by PCR. Biopsy specimens from 7/15 horses were urease positive by RUT. Significant 13C enrichment of the body CO2 pool was found in all horses after intragastric administration 13C-urea (p<0.05). As Helicobacter is currently the only known gastric urease positive microorganism, the demonstration of this activity in horses positive by PCR strongly supports the presence of an equine gastric Helicobacter species. Variations of 13C-urea blood test were further examined and a single protocol was found to be most applicable. As the horse is a hind gut fermenter, the effect of cecal urease on the test was examined by laparoscopic intracecal administration of 13C-urea. Significant cecal urease activity was demonstrated however the timing of peak 13C enrichment may limit any effect on the gastric test to 90 minutes onwards.
Local Administration of Botulinum Toxin Type-B in the External Anal Sphincter of Horses Produces Transient Reduction of Peak Anal Pressure
Adam-Castrillo, David (Virginia Tech, 2003-06-27)
Toxins produced by the Gram-positive bacteria Clostridium botulinum cause transient chemodenervation of mammalian muscle. The toxin binds to specific proteins within cholinergic presynaptic nerve terminals which regulate the release of acetylcholine in the synaptic space resulting is loss of muscle activation and function. Local injections with botulinum toxins are currently used in humans for the treatment of disorders that benefit from prolonged neuromuscular blockade such as strabismus, blepharospasm, focal dystonias, spasticity, tremors, and anal fissures. Injections with botulinum toxin type A into the internal or external anal sphincter cause relaxation of the anal canal and allow healing of chronic anal fissures. Perineal lacerations in mares, which occur during foaling often dehisce after surgical repair due to the high pressure across the incision resulting from accumulation of feces in the rectum. We hypothesized local injections of Clostridium botulinum type B toxin into the external anal sphincter could cause a decrease in anal pressures, thus reducing the incidence of dehiscence if used before surgical repair of perineal laceration in mares. The purpose of this project was to determine the effects of BTB injection in the external anal sphincter in normal horses. Our hypothesis was that local injection of BTB would result in transient reduction of anal tone without causing clinical side effects. Peak and resting anal sphincter pressures of horses were measured with a custom made rectal probe connected to a pressure transducer. Pressures were measured before treatment and after injection with Clostridium botulinum type B toxin (BTB) or saline. Dose titration with 500, 1000, 1500 and 2500 units of BTB was completed. The horses' physical changes, behavior, and anal pressure were recorded. Injection of 1000 units of BTB produced significant reduction in peak anal pressure from days 2 to 84 when compared to control animals (P<0.05). Maximal effect of the toxin was observed within the first 15 days after injections followed by a slow return to baseline over 168 days. Injection in the anal sphincter with 2500 units of BTB in one horse produced signs of depression, generalized weakness, and dysphagia for 14 days. Clinical side effects were not observed in horses after injections with 500, 1000, or 1500 units of BTB. In summary, local injections of botulinum toxin type-B in the external anal sphincter of horses caused transient relaxation of the anus and reduction of peak anal pressures. Systemic side effects were observed in one horse, which suggested a narrow dosage range to avoid toxicity. Further research to test the effects of botulinum toxin in clinical cases is needed to determine the full potential of this treatment modality.
Methods to Detect Apoptosis in Equine Peripheral Blood Neutrophils from Normal Healthy Adult Horses
Wereszka, Marta (Virginia Tech, 2007-06-15)
Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils. To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils. Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells. In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS. In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05. The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05. All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide. Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours. Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001). Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC. The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%). These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies.
Purification and quantitative description of Rhodococcus equi IgG designed for aerosol nebulization to foals
Beebe, Dale (Virginia Tech, 2011-06-29)
The objective of this study was to purify IgG from commercially available hyperimmune Rhodococcus equi plasma and to assess the delivery of IgG as an aerosol to the equine lung. IgG was purified from plasma, and the IgG concentration of both the plasma and the purified IgG was determined by ELISA. The purified IgG was aerosolized using a vibrating mesh nebulizer and aerosol characterization was performed using cascade impaction. The purified IgG was nebulized to six healthy adult horses in order to assess the efficacy of pulmonary delivery and safety of administration. Bronchoalveolar fluid was retrieved endoscopically using a low volume technique prior to aerosolization (time 0) and at 0.5, 4 and 24 hours post aerosolization. The BAL fluid IgG concentration was determined and cytologic analysis was performed. The IgG concentrations of the plasma and purified IgG were 2,175 mg/dL and 1,145 mg/dL, respectively. The MMAD of the purified IgG aerosol was 4.7 microns. The mean BAL fluid IgG concentration increased 61% from 19.33 µg/dL at time 0 to 31.5 µg/dL at 0.5 hours, but this increase was not significant (P=0.603). No significant change was observed in inflammatory cell numbers over time or at any time point during the study. This study demonstrated that IgG antibodies were purified at a concentration acceptable for nebulization, and that the nebulization unit generated aerosol particles from the IgG solution of appropriate size for pulmonary delivery. Nebulization of purified IgG to adult horses was well tolerated and caused no local or systemic adverse effects.
The Role of Neutrophil Apoptosis in Horses with Acute Abdominal Disease
Krista, Kathryn Morton (Virginia Tech, 2012-04-26)
Neutrophils, the chief phagocytic cells in most mammals, are critical in the inflammatory response. Regulation of neutrophil activity occurs through several mechanisms, including apoptosis. Dysfunction of neutrophil apoptosis has been implicated as a cause of organ damage in hyper-inflammatory conditions in human patients. This pilot study investigated apoptosis in circulating neutrophils from horses with surgical lesions in the large and small intestine. We hypothesized that delayed neutrophil apoptosis occurs in peripheral blood of horses undergoing surgery with acute abdominal disease, compared with elective orthopedic cases. Adult horses undergoing surgery for acute abdominal disease (N=10) and elective orthopedic surgery (control) (N=10) were studied. Peripheral blood was collected preoperatively and postoperatively. Neutrophils were isolated using Percoll gradient. Cells undergoing apoptosis were determined by flow cytometry using a commercially available staining kit (Annexin V-PE Apoptosis Detection Kit I, BD Pharmingen™). The Mann-Whitney U test was used to detect significant differences in neutrophil apoptosis between the two groups as well as between lesion types in the abdominal surgery group. Correlations between neutrophils in apoptosis and postoperative parameters were detected using Spearman's rank correlation coefficient. No significant differences in percentages of apoptotic neutrophils between groups were found; however, a significantly lower percentage of neutrophil apoptosis was present in horses with strangulating intestinal lesions versus nonstrangulating lesions. Current investigations about neutrophil apoptosis in human medicine may result in therapeutic intervention to prevent organ damage in hyper-inflammatory states. Understanding the role of neutrophil apoptosis in equine acute abdominal disease may guide the use of new treatments as they become available.
The Role of the CD14 molecule in equine endotoxemia
Guedes Alves da Silva, Adriana (Virginia Tech, 2012-06-14)
Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases.
Serum calcitonin gene-related peptide concentrations in the horse and their relationship to the Systemic Inflammatory response
Mitchell, Emma (Virginia Tech, 2006-06-28)
Systemic inflammation is a leading cause of mortality and morbidity in both human and equine intensive care patients. This systemic inflammatory response may be due to insult from bacterial, viral, fungal or parasitic invasion or from trauma or hypoxemia. Local and systemic release of a wide variety of endogenous pro-inflammatory mediators results in activation of the innate immune system in order to resolve the insult. In sepsis this initial appropriate host response becomes amplified and deregulated leading to refractory hypotension and multiple organ dysfunction. The exact incidence of sepsis (SIRS due to bacterial infection) has not been reported in the equine literature (Roy 2004). Since early recognition and treatment of sepsis are associated with improved outcome the search for markers to accurately predict presence of sepsis and likelihood of survival continues. The serum concentration of both procalcitonin and its related molecule CGRP have been documented to increase in humans with SIRS, yet no literature exists as to the production or role of CGRP in equine patients with SIRS. This study showed that equine CGRP was produced in detectable quantities by healthy adult horses and neonatal foals less than two weeks of age using a rat Ã¡-CGRP ELISA. The low percentage recovery of CGRP from samples and the high lower limit of detection for the assay prevented establishment of a normal concentration range of CGRP in healthy horses. In both adult horses and foals with documented SIRS, CGRP concentrations were significantly increased at time of presentation to the hospital (p<0.0002, p<0.003 respectively). A trend towards increased serum CGRP concentration was present in anaesethized horses exposed to endotoxin, but this was not statistically significant (p< 0.067).
Serum concentrations of lidocaine and its metabolites after prolonged infusion in healthy horses
Dickey, Emma Jane (Virginia Tech, 2009-06-11)
Lidocaine continuous-rate infusions (CRI) are the most commonly used prokinetic in equine practice for the treatment of post-operative ileus and are also increasingly being used in pain management, such as in cases of severe laminitis, and are often used for prolonged durations. To date only limited time/concentration relationships of lidocaine administered as a short term (24hours) CRI to horses are reported. This study examined the time/concentration profile of lidocaine and its active metabolites (GX, MEGX) during a 96 hour lidocaine infusion in eight mature healthy horses. Serum lidocaine concentrations reached steady state by three hours and did not accumulate thereafter. The serum concentration of lidocaine was above the target therapeutic concentration (980ng/ml) only at 6 and 48 hours. The serum lidocaine concentration did not reach the range described as potentially causing toxicity (>1850ng/ml). The MEGX metabolite did not accumulate over time, while the GX metabolite accumulated significantly up to 48 hours and then remained constant. The serum concentrations of lidocaine, MEGX and GX were below the limit of detection within 24 hours of discontinuation of the infusion. None of the horses developed any signs of lidocaine toxicity during the study. It was concluded that the metabolism of lidocaine was not significantly impaired by prolonged infusion, contrasting with studies in dogs and humans. No adverse effects were observed in this study, which with the lack of lidocaine accumulation suggests that prolonged infusions are safe. However the accumulation of GX, a potentially toxic active metabolite, is cause for concern.
Time and Concentration Relationships of Gentamicin in Serum and Bronchial Lavage Fluid of Horses Administered Gentamicin Intravenously and by Aerosol
McKenzie, Harold Cantrell III (Virginia Tech, 1999-01-28)
This study was performed to compare the delivery of the antimicrobial gentamicin to the respiratory tract of adult horses following aerosol and intravenous administration. Nine adult horses were used in a crossover design. Aerosol administration of gentamicin was performed using a close fitting facemask and an ultrasonic nebulizer. Intravenous gentamicin was administered via a jugular venous catheter. Samples of pulmonary epithelial lining fluid were collected by bronchial lavage performed at 0.5, 4, 8 and 24 hours after gentamicin administration. All samples were analyzed for gentamicin concentration, and cytologic examination was performed on aliquots of bronchial lavage fluid from times 0.5, 8 and 24 hours. Comparisons were made using the Wilcoxon signed-rank test. The bronchial lavage fluid gentamicin concentration after aerosol administration was significantly greater (p<0.05) than after intravenous administration at 0.5, 4, and 8 hours. The bronchial lavage fluid total nucleated cell count increased significantly (p<0.05) from 0.5 to 24 hours following both routes of gentamicin administration, with the increase observed following aerosol administration being significantly greater (p<0.05) than that observed following intravenous administration. A significant increase in neutrophil count was detected between bronchial lavage fluid samples taken at 0.5 hours and 24 hours, regardless of route of gentamicin administration. We conclude that aerosol administration of gentamicin to the equine respiratory tract achieves bronchial lavage fluid gentamicin levels that are significantly higher than levels obtained following intravenous administration for at least the first 8 hours after administration, while inciting a mild inflammatory response.

Browsing by Author "Furr, Martin O."

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