Simultaneous Flow Cytometric Analysis of Phagocytosis and Oxidative Burst Activity in Equine Leukocytes

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.     

Evaluation of relationships between weather patterns and prevalence of sorghum ergot in the Texas panhandle

ABSTRACT Sorghum ergot caused by Claviceps africana was observed for the first time in the United States in Southern Texas in 1997. That year there was a widespread ergot epidemic in hybrid seed production fields in the Texas Panhandle. However, occurrence of the disease has been sparse during the past 3 years, easing fears that the hybrid seed industry in the region might be endangered. To determine whether climatic factors were associated with observed variations in prevalence of ergot, weather data (temperature, precipitation, and relative humidity) were collected from seven weather stations in the Texas Panhandle. Sorghum ergot prevalence data for the period 1997 to 2000 were collected from records of seed companies in the Panhandle and related to weather variables. Results showed that, in the southern section of the Panhandle, maximum temperature and precipitation between 1 and 15 August were associated (r(2) = 0.98, P = 0.001 and r(2) = 0.81, P = 0.0193, respectively) with variations in the prevalence of ergot during the 4-year period. In the northern section, only maximum temperature during 16 to 31 July was significantly associated (r(2) = 0.91, P = 0.0111) with disease prevalence. Over all, 1997 was wetter and cooler, during the 1 to 15 August period, than each of the subsequent 3 years. In addition to creating humid conditions for ergot development, precipitation was associated with suppression of maximum temperature, enhancing ergot-favorable temperature conditions. Examination of historic weather data for the region showed that there were many instances in the past where temperature depression was associated with a rise in cumulative precipitation, creating ergot-favorable conditions similar to those in 1997. Cross-spectral analysis was used to determine whether such association is periodic. Weather data from five of the seven locations in the region showed peaks of significant coherency ( alpha< 0.05) at 2 to 4 years and 7 to 10 years or greater, indicating the existence of a periodic cycle in the temperature-precipitation association. The results of the investigation suggested that association of precipitation with temperature depression is a primary factor in development of ergot in the Texas Panhandle, and such association has a periodic cycle.     

Flow cytometry: clinical applications in equine medicine

The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell surface proteins. Other approaches have been used to elucidate changes in cell function and DNA content. This review is intended to provide the reader with the fundamental uses of flow cytometry. Examples of clinical applications in equine patients include immune-mediated hemolytic anemia, immune-mediated thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia.     

Pythiosis with bone lesions in a pregnant mare

A 9-year-old pregnant mare was referred for evaluation of a nonhealing wound of 8 weeks' duration on the lateral aspect of the left forelimb. A soft tissue mass encircled the proximal two thirds of the metacarpus; radiography revealed a moderate periosteal reaction affecting metacarpal bone i.v. Histologic and immunohistochemical examinations revealed eosinophilic granulomatous inflammation and Pythium sp in the soft tissues. The mare was treated for 12 days with antimicrobials, medicated wound dressings, debridement, and i.v. administration of sodium iodide; radiography revealed progression of the bone lesions. The mare was treated by regional arterial perfusion with miconazole and excision of affected soft tissues and the distal two thirds of metacarpal bone i.v. The mare recovered without complications and gave birth to a healthy foal. Regional perfusion of antifungal agents provides high concentrations in soft and osseous tissues and permits use of low dosages of agents administered by other routes, which reduces cost, adverse effects, and teratogenic effects.     

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9–36.7%, but was eliminated by the use of the F(ab)₂ fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs) test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.     

Pacemaker implantation in a ferret (Mustela putorius furo) with third-degree atrioventricular block.

A 7.5-year-old castrated male ferret (Mustela putorius furo) was diagnosed with third-degree atrioventricular (AV) block. A monopolar epicardial pacemaker system was implanted, resulting in a regular, paced cardiac rhythm with third-degree AV block at 140 beats per minute. Over the next 2 months, the ferret developed anorexia, interstitial pneumonia, intermittent diarrhea, and hind-limb weakness and had a slow and progressive recovery. The ferret developed clinical signs of congestive heart failure 4 months after the surgery, resulting in its death 3 weeks later. Necropsy results attributed the death to cardiac failure due to extensive myocardial mineralization. To the authors' knowledge this is the first published report of surgical report of surgical pacemaker implantation in a ferret.     

T-shaped malformation of the ventral colon in a Thoroughbred filly with colic

A 4-month-old Thoroughbred filly presented for abdominal pain was diagnosed with a T-shaped malformation of the ventral colon at exploratory laparotomy. Following resection and anastomosis of the large colon, no further episodes of abdominal pain occurred during a 12-month follow-up. Acute dehiscence of the linea alba occurred as a complication of the initial laparotomy, but was successfully managed following additional surgical repair. T-shaped malformation of the ventral colon has not previously been reported and is considered a congenital malformation of mesocolon formation.     

Failure of passive transfer in foals: incidence and outcome on four studs in New South Wales

Objective To determine the regional incidence and effectiveness of treatment of failure of passive transfer (FPT) in foals. Design A study of disease incidence. Animals Eighty-eight foals and 57 mares from four studs in the practice area of the Rural Veterinary Centre were tested. Procedure Foals were tested for their serum IgG and total serum protein (TSP) concentration within the first 72 hours of life. Colostrum was collected from mares and specific gravity determined. FPT and partial failure of passive transfer (PFPT) of immunoglobulins was diagnosed when serum IgG concentrations were < 4 g/L and 4 to 8 g/L respectively. Owners of foals diagnosed with FPT were offered treatment with 1 to 2 L plasma (TSP > 70 g/L); 9 (64%) of the affected foals were treated. Results Fourteen foals (16%) had FPT whereas 15 (17%) had PFPT. There were significant differences between the mean TSP concentration in foals with FPT (42.6 ± 4.2 g/L), PFPT (48.1 ± 3.9 g/L) and those acquiring adequate passive immunity (58.9 ± 5.5 g/L) (P < 0.01). Sixteen (29%) mares had pre-suck colostral specific gravity < 1.060 and 12 (71%) foals raised by these mares had FPT or PFPT. The incidence of severe disease (categorised by a sepsis score > 11, positive culture of bacteria from blood or disease requiring hospitalisation) in all foals in the first 2 months of life was 10%. However, none of the nine foals with FPT that received plasma experienced severe disease. In contrast, foals with PFPT had an increased susceptibility to severe disease (P < 0.001) when compared with normal foals. Conclusion Treatment of foals with FPT may reduce the subsequent incidence of severe disease. Pre-suck colostral specific gravity and foal TSP may be used to predict the likelihood of FPT and PFPT. Even though the number of foals studied is small the results highlight the importance of optimal management practices in reducing the incidence of FPT and disease associated with this process.