Quantitation and confirmation of sulfamethazine residues in swine muscle and liver by LC and GC/MS

The present paper describes a liquid chromatographic (LC) method for purification of crude swine tissue extracts before gas chromatographic/mass spectrometric (GC/MS) quantitation and confirmation of sulfamethazine at low ppb levels. Fractions corresponding to sulfamethazine were collected, evaporated to dryness, N-methylated with diazomethane, concentrated, and analyzed by GC/MS. A mass spectrometer was set to selected ion monitoring (SIM) mode. Ions 233, 227, 228, and 92 m/z were detected. Ratio 227/233 m/z (sulfamethazine/internal standard, [phenyl 13C6] sulfamethazine) was used for quantitation, while ratios 228/227 and 92/227 m/z, respectively, were used for confirmation. Quantitation in spiked blank muscle tissue was tested from 100 to 1 ppb and found acceptable at all concentrations studied; coefficients of variations ranged from 4.9 to 14.4%. Similar results were obtained for liver tissue from 5 to 20 ppb; coefficients of variation ranged from 1.2 to 9.1%.     

Comparison of the pig and feline models for full thickness corneal transplantation

Purpose The goal of this study was to report on the advantages and limitations of the pig and feline models for experimental in vivo corneal transplantation. Methods Ten healthy domestic pigs and ten healthy cats were used. Full thickness penetrating keratoplasty was performed using autologous (eight cases), allogeneic (seven cases) or human xenogeneic (three cases) tissue. In two other cases, the inflammatory response to partial thickness trephination (without transplantation) was evaluated. Eyes were assessed daily before and after surgery by slit‐lamp, pachymetry, and tonometry. A transparency score ranging from 0 (opaque graft) to 4 (clear graft) was used, based on the slit‐lamp examination. Optical coherence tomography, histology, and electron microscopy were performed postmortem. Results In the pig, the mean (±SD) transparency score for the eight full thickness grafts was 0.88 ± 0.99, ranging from 0 to 3. In the feline model, the mean transparency score for the seven uncomplicated grafts was 3.93 ± 0.19, ranging from 3.5 to 4. Both negative controls without endothelium remained opaque at all time. Intraoperative tendency for iris incarceration into the wound, rapid corneal swelling, suture cheese wiring, and postoperative intraocular inflammation were the main factors jeopardizing the functional success of the corneal transplant in the pig model. Conclusion Suboptimal functional results were obtained after full thickness corneal transplantation in the pig model, while in the feline model, the same protocol yielded uneventful surgeries and clear transplants, with functional results similar to those achieved in human subjects.     

Comparison of the chemical composition of extracts from Scutellaria lateriflora using accelerated solvent extraction and supercritical fluid extraction versus standard hot water or 70% ethanol extraction

The aqueous extract of American skullcap (Scutellaria lateriflora L. (S. lateriflora), Lamiaceae) has been traditionally used by North American Indians as a nerve tonic and for its sedative and diuretic properties. Recent reports stated that flavonoids and possibly amino acids are responsible for the anxiolytic activity. As a part of our search for environmentally friendly solvents to extract the active components from medicinal plants, we used S. lateriflora in a comparison of accelerated solvent extraction (ASE) using water, and supercritical fluid extraction (SFE) using CO2 and 10% EtOH as modifier, at different temperatures. Flavonoids and amino acids were quantified by HPLC-UV and HPLC-MS, respectively. The flavonoid content was compared with conventional extraction methods (hot water extraction and 70% ethanol). The use of ASE at 85 degrees C with water as solvent gave the best results for flavonoid glycosides and amino acids, whereas SFE gave higher yields of flavonoid aglycones. However, the results obtained for total flavonoids were not significatively superior to hot water extraction or 70% aqueous EtOH extract.     

Improvements in the modified direct complement fixation test and its application in the detection of bluetongue antibodies in cattle and sheep sera.

In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors.     

Bacterial and fungal flora in healthy eyes of birds of prey.

Birds of prey are often affected with external ocular injuries that are routinely treated with antimicrobial agents used for small animals. The resident ocular bacterial and fungal flora is still unknown in birds of prey and this knowledge would be very useful in assessing the accuracy of treatments. In a study involving 65 raptors with healthy eyes, swabs were taken from both eyes to identify the resident bacterial and fungal flora. Fifty-five birds had a positive culture in one or both eyes. Both gram-positive and gram-negative organisms were isolated, with a predominance of Staphylococcus spp., which were found in 52.3% of cultures. Only two fungal species, Aspergillus spp. and Cladosporium spp. were found. The overall results of this study are similar to previous studies carried out in humans and other animals.     

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis)

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)‐affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis‐affected buffaloes. The extent of difference between normal and subclinical endometritis‐affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.     

Effect of Albizia julibrissin water extracts on low-density lipoprotein oxidization

High-value phytochemicals could be extracted from biomass prior to the current cellulosic pretreatment technologies (i.e., lime, ammonia, dilute acid, or pressurized hot water treatments) provided that the extraction is performed with a solvent that is compatible with the pretreatment. This work reports on the extraction of flavonoids from Albizia julibrissin biomass. While extracting A. julibrissin foliage with 50 degrees C water, 2.227 mg/g of hyperoside and 8.134 mg/g quercitrin were obtained, which is in the realm of what was obtained with 60% methanol. A. julibrissin foliage, flower, and whole plant extracts were tested in terms of their potential to inhibit low-density lipoprotein (LDL) oxidization. The highest inhibition was obtained with foliage water extracts, which were standardized at 2.5 microM of flavonoids. Also, the 2.5 microM foliage water extract resulted in a reduction from 43% to only 1% of the observed monocyte adherence. To have commercial application, A. julibrissin water extracts should be devoid of toxicity. The A. julibrissin foliage, flower, and whole plant water extracts were not toxic to Vero 76 cells. In summary, A. julibrissin biomass can be extracted with 50 degrees C water to yield an antioxidant stream, showing that it may be possible to couple extraction of valuable phytochemicals to the cellulosic pretreatment step.     

Equine Infectious Anemia: Sensitivity of the Agar-gel Immunodiffusion Test, and the Direct and the Indirect Complement-fixation Tests for the Detection of Antibodies in Equine Serum

The comparative values of the direct, the indirect complement-fixation and the agar-gel immunodiffusion tests were assessed for the diagnosis of equine infectious anemia. Antibodies were detected on the agar-gel immunodiffusion test as early as 18 days post-inoculation in the serums of experimentally infected horses and were readily detectable in all the subsequent bleedings. Complement-fixing antibodies, demonstrable by the direct method, were detected commencing about the same time. However, these were not long-lasting and were replaced by the non-complement-fixing antibodies demonstrable by the indirect method; although both types of antibodies could be detected in some sera at the same time. In a herd of 55 horses, 28 were positive on the agar-gel immunodiffusion test, and among these 28 horses, 24 of them reacted on either the direct or indirect complement-fixation test or both. Thirteen horses that were negative on the three tests at the first sampling, reacted on the agar-gel immunodiffusion test 43 days later. Ten of these positive animals had direct type of complement-fixing antibodies; only one had the indirect; and two of them were negative on both tests. It appeared that the AGI test was a more reliable technique than either the direct or indirect complement-fixation tests, particularly when dealing with serums which contained small amounts of antibody. The sequential appearance of the two different types of complement-fixing activity might be used to determine the evolution of the disease on a herd basis.