Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation

OBJECTIVE: To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. SAMPLE POPULATION: Blood samples from 6 Thoroughbreds. PROCEDURE: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. RESULTS: In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours. CONCLUSIONS: Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.     

Hematologic and plasma biochemical values of healthy hybrid tilapia (Oreochromis aureus x Oreochromis nilotica) maintained in a recirculating system.

Tilapia are cultured worldwide and are increasing in popularity among aquaculturalists in the United States; however, data regarding normal health parameters are limited. Few hematologic and plasma biochemical values of clinically normal tilapia have been reported, but these data may be key for identifying and managing disease issues in recirculating systems. Therefore, blood was collected from clinically normal hybrid tilapia (Oreochromis aureus x Oreochromis nilotica) housed in recirculating systems for the purpose of establishing normal hematologic and plasma biochemical reference ranges. Using standard clinical techniques the following hematologic values were determined: packed cell volume, plasma protein, leukocyte counts, leukocyte differentials, and thrombocyte counts. Additionally, the following plasma biochemical values were determined: albumin, total protein, globulins, albumin/globulin ratio, aspartate aminotransferase, alkaline phosphatase, glucose, uric acid, calcium, phosphorus, magnesium, sodium, potassium, chloride, urea nitrogen, and creatinine. The condition of the sample was also noted (lipemic, hemolysis, and icterus). The reference ranges reported in this study can be used in the management of cultured tilapia in recirculating systems.     

Enhanced selective extraction of hexane from hexane/soybean oil mixture using binary gas mixtures of carbon dioxide

Carbon dioxide (CO2) can effectively separate hexane from a mixture of soybean oil (SBO) and hexane with a slight coextraction of SBO. Previous research demonstrated that CO2 entrained with helium significantly reduced SBO solubility in CO2. In this study, CO2 was mixed with three gases (He, N2, or Ar) (0.5-30 vol %) to decrease SBO solubility while attempting to maintain hexane solubility. The binary gas mixtures (at 25 degrees C and 9.31 MPa) were passed through a 25 wt % hexane/SBO mixture inside a 2.5 m fractionation column. Coextracted SBO was inversely proportional to binary gas concentration, whereas residual hexane in the raffinate was proportional to binary gas concentration. The 10% binary mixture of N2 or Ar was the best compromise to obtain both low residual hexane levels (i.e., 26 ppm) and low SBO coextraction (i.e., only 40 mg). This carry-over of SBO represents a 95% reduction in SBO carry-over compared to neat CO2.     

ULTRASONOGRAPHIC CHARACTERISTICS OF LIPIDURIA IN CLINICALLY NORMAL CATS

Echoes are frequently seen in the urinary bladder of cats during abdominal ultrasound. These have been attributed to hematuria, pyuria, crystalluria, and lipid. However, sonographic findings have not been previously correlated with urinalysis. We prospectively evaluated 40 clinically normal cats via ultrasound, serum chemistry, and urinalysis. Thin layer chromatography was performed on the urine to determine the amount (mg) of lipid subfractions including diacylglycerol, triglyceride, phospholipid, free fatty acid, cholesterol, and cholesterol ester. Ninety percent (36/40) of the cats in our population had sonographic echoes suspended in the urinary bladder, with most having a subjective score of mild echoes (n = 20). None of the sonographic echoes were gravity dependent or caused distal acoustic shadowing, reverberation, or twinkle artifact. Of the cats with sonographic echoes in the urine, 66% (24/36) had no significant findings on urinalysis other than the presence of lipid. The total amount of subjective sonographic echoes was not significantly related to the total amount of fat measured on thin layer chromatography or the number of lipid droplets seen on urinalysis. An increased amount of urine diacylglycerol was significantly associated with clumping of echoes (P = 0.02) and the amount of lipid droplets seen on urinalysis (P = 0.04). An association between increased amounts of urine diacylglycerol and the amount of echoes seen on ultrasound approached significance (P = 0.05). Findings from this study support previously published theories that sonographic echoes within the urinary bladder of clinically normal cats may be due to urine lipid.     

Protection, systemic IFNgamma, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host

In the horse, conventional inactivated or subunit vaccines against equine influenza virus (EIV) induce a short-lived antibody-based immunity to infection. Alternative strategies of vaccination have been subsequently developed to mimic the long-term protection induced by natural infection with the virus. One of these approaches is the use of immune-stimulating complex (ISCOM)-based vaccines. ISCOM vaccines induce a strong antibody response and protection against influenza in horses, humans, and a mouse model. Cell-mediated immunity (CMI) has been demonstrated in humans and mice after ISCOM vaccination, but rarely investigated in the horse. The aim of this study was to evaluate EIV-specific immune responses after intra-muscular vaccination with an ISCOM-EIV vaccine (EQUIP F) containing both equine influenza H7N7 (A/eq/Newmarket/77) and H3N8 (A/eq/Borl?nge/91 and A/eq/Kentucky/98) strains. The antibody response was measured by single radial haemolysis (SRH) assay using different H3N8 EIV strains. Stimulation of type-1 immunity was evaluated with a recently developed method that measures EIV-specific IFNgamma synthesis by peripheral blood lymphocytes (PBL). The protective efficacy of this ISCOM-based vaccine against challenge infection with a recent equine influenza (H3N8; A/eq/South Africa/4/03) strain was also evaluated. Vaccinated ponies developed elevated levels of EIV-specific SRH antibody and increased percentage of EIV-specific IFNgamma(+) PBL, whereas these responses were only detected after challenge infection in unvaccinated control ponies. Vaccinates showed minimal signs of disease and did not shed virus when challenged shortly after the second immunisation. In conclusion, evidence of type-1 immunity induced by an ISCOM-based vaccine is described for the first time in horses.     

LOW-FIELD MAGNETIC RESONANCE IMAGING OF EARLY SUBCHONDRAL CYST-LIKE LESIONS IN INDUCED CRANIAL CRUCIATE LIGAMENT DEFICIENT DOGS

Six healthy adult male mongrel dogs underwent cranial cruciate ligament transection in the left stifle. Survey radiography of both stifles and low-field (0.064 T) MRI of the left stifle were performed preoperatively and at 2, 6, and 12 weeks postoperatively. Focal changes in signal intensity were seen with MRI in the subchondral bone of the medial tibial condyle at 2 and 6 weeks postoperatively. At 12 weeks postoperative, a cyst-like lesion was detected using MRI in the subchondral bone of the medial tibial condyle in 4 of 6 dogs and a less defined lesion at this site in the remaining 2 dogs. The cyst-like lesion was spherical in shape and showed typical characteristics of fluid with low signal intensity on T1-weighted images, high signal intensity on T2-weighted images and high signal intensity on inversion recovery images. The lesion was seen in the subchondral bone of the caudal medial and/or middle region of the tibial plateau slightly cranial to the insertion of the caudal cruciate ligament. No subchondral cysts were seen in the tibia on radiographs. Histopathologically, the tibia was characterized by a loose myxomatous phase of early subchondral cyst formation.     

Weaning requirements of larval mulloway,Argyrosomus japonicus

Mulloway (Argyrosomus japonicus) is an emerging aquaculture species in Australia, but there is a need to improve the production technology and lower costs, including those associated with larval rearing and live feeds. Three experiments were conducted to determine appropriate weaning strategies from live feeds, rotifers (Brachionus plicatilis) and Artemia, to cheaper formulated pellet diets. Experiment 1 examined the effects of feeding Artemia at different levels [0%, 50% or 100% ration of Artemia fed from 18 days after hatching (dah); based on current hatchery protocols] and a pellet diet from two larval ages (14 or 23 dah). In addition, rotifers were supplied to larvae in all treatments for the duration of the experiment (14–29 dah), at which time all larvae were successfully weaned onto the pellet diet. No significant (P>0.05) differences existed between the growth of fish fed a 50% and 100% ration of Artemia; however, fish fed a 0% ration of Artemia had significantly (P<0.05) reduced growth. The time of pellet introduction had no significant (P>0.05) effects on the growth of larvae. Experiments 2 and 3 were designed to determine the size [total length (TL), mm] at which mulloway larvae selected Artemia equally or in preference to rotifers, and pellet (400 μm) equally or in preference to Artemia respectively. Each day, larvae were transferred from a holding tank to experimental vessels and provided with rotifers (2 mL^?1), Artemia (2 mL^?1) or a combination of rotifers (1 mL^?1) and Artemia (1 mL^?1) (Experiment 2), and Artemia (2 mL^?1), a pellet diet or a combination of Artemia (1 mL^?1) and a pellet diet that was broadcast every 15 min (Experiment 3). After 1 h, a sub‐sample of larvae was randomly selected from each replicate vessel (n=5) and the gut contents were examined under a light microscope. Mulloway larvae began selecting Artemia equally to rotifers at 5.2 ± 0.5 mm TL and selected pellets equally to Artemia at 10.6 ± 1.8 mm TL. Our results have led to the establishment of weaning protocols for larval mulloway, which optimize larval growth while reducing feed cost by minimizing the amount of Artemia used during production.     

Are composts from shredded leafy branches of fast-growing forest species suitable as nursery growing media in arid regions?

The morpho-physiological quality of seedlings is negatively affected by the wide scale use of forest soils as substrates in developing countries. With the objective of finding long-term sustainable supply of growing media, compost was produced from shredded branches of three fast growing species (Acacia cyanophylla (AA), Acacia cyclops (AS) and Eucalyptus gomphocephala (EG). The composting process covered three different periods over the course of a year. Pile temperatures were monitored daily and the composts were routinely sampled and analyzed for 19 chemical variables. Although composting is feasible year-round in arid climates, compost produced in the humid cool conditions of autumn, winter and early spring reaches the maturation phase more quickly than compost produced under hot, dry summer conditions. It also requires less turning and water. The evolution of the composting process and quality of the final product can be assessed using three chemical variables (C/N, pH, EC). Seed germination rates in the three types of compost were similar to that in a peat:vermiculite substrate and vigorous high quality seedlings were produced in the two acacia composts. However, compost-grown seedlings had significantly smaller shoots and root systems than those produced in peat substrate. Principal components analyses showed that the quality of a compost-based substrate is reproducible and that its final chemical composition can be predicted from its raw organic materials. The EG composts had higher pH than the acacia composts, whereas the AA and EG composts were higher in mineral salts than the AS.