Determination of sulfamethazine and trimethoprim in liquid feed premixes by HPLC and diode array detection, with an analysis of the uncertainty of the analytical results.

Sulfamethazine (SMZ) and trimethoprim (TMP) are antibacterials used in veterinary practice. This paper describes a method for their determination in veterinary liquid feed premixes that is based on liquid chromatography with diode array detection. Gradient elution with methanol and ammonium acetate achieved excellent separation of the two analytes within 15 min without any interference from the matrix. Absorbance of the column effluent was monitored at 264 nm for SMZ and at 230 nm for TMP. Detailed analyses of the uncertainties of determinations afford estimated expanded uncertainties of, respectively, 0.2 and 0.1 w/v % for typical SMZ and TMP concentrations of 10.7 and 2.1 w/v %, respectively. At the lower end of the calibrated range of the method, the dominant source of uncertainty is the preparation of standards and the construction of the calibration line.     

Biochemical polymorphisms in Spanish Avileña-Negra Iberica cattle

SUMMARY: Thirteen biochemical blood polymorphisms were analysed in a population of 149 Spanish Avile?a-Negra Ibérica cattle. The study revealed variation at the following nine loci: HBB, CA, NP, CP, AMY1, ALB, GC, TF and PTF2. The following systems were monomorphic: CAT, DIA1, MDH1 and ME1. Using polyacrylamide-gel electrophoresis, a slow, migrating pair of bands was found in the GC protein system. This pattern is probably controlled by the GC(C) allele, described in only a few cases in cattle. Furthermore, starch-gel electrophoresis allowed the detection of a variant with intermediate mobility between the ALB(A) and the ALB(B) alleles at the albumin locus. A variant with a similar electrophoretic pattern has occasionally been reported in cattle. However, utilizing IEF under denaturing conditions, such a variant could not be differentiated from the ALB(A) allele and thus its significance is not clear. ZUSAMMENFASSUNG: Biochemischer Polymorphismus in spanischen Avile?a-Negra Iberica Rindern Insgesamt 13 biochemische Systeme wurden in einer Population von 149 spanischen Avilena-Negra-Iberika-Rindern hinsichtlich Polymorphismus analysiert. Es zeigten sich Varianten bei folgenden Loci: HBB, CA, NP, CP, AMY1, ANB, GC, TF und BTF2, w?hrend CAT, DEA, MDH1 und ME1 monomorph sind. Bei st?rke Gel-Elektrophorese wurde im Albumin-Locus eine Variante mit intermedi?rer Mobilit?t zwischen ALB(A) und ?LB(B) Allel entdeckt. Eine solche Variante wurde bisher nur sehr selten bei Rindern beobachtet. Darüber hinaus wurde bei Polyacrylamid-Gel-Elektrophorese ein langsam wanderndes Paar von B?ndern im GC-Proteinsystem gefunden. Dieses Muster ist wahrscheinlich von dem selten vorkommenden GC(C) -Allel verursacht. RESUMEN: Se analizaron trece polimorfismos bioquímicos sanguíneos en una población de 149 animales de la raza Avile?a-Negra Ibérica de ganado vacuno. El estudio reveló la existencia de variación en los nueve loci siguientes: HBB, CA, NP, CP, AMY1, ALB, GC, TF y PTF2. Fueron monomórficos los sistemas siguientes: CAT, DIA1, MDH1 y ME1. Utilizando eletroforesis en gel de poliacrilamida se encontró un par de bandas de migración lenta en el sistema de la proteína GC. Este patrón probablemente está controlado por el infrecuente alelo GC(C) , descrito en unos pocos casos en el ganado vacuno. Además, la electroforesis en gel de almidón permitió detectar en el locus de la albúmina una banda con movilidad intermedia entre los alelos ALB(A) y ALB(B) . Una variante con un patrón electroforético similar ha sido descrita en muy pocas ocasiones en el ganado vacuno. Sin embargo, la utilización de IEF en condiciones desnaturalizantes no permitió diferenciar esta variante del alelo ALB(A) y, por lo tanto, el significado de la misma no está claro.     

C-reactive protein measurement in canine saliva.

An established time-resolved immunofluorometric assay designed for measurement of C-reactive protein (CRP) in canine blood was evaluated and validated for use in canine saliva. C-reactive protein was measured in saliva specimens from 5 healthy dogs before and after the injection of casein and in 37 dogs with different disease conditions. The analytical and functional limits of detection were 0.000053 microg/ml and 0.0091 microg/ml, respectively, and intra- and interassay coefficients of variation ranged between 6.7-9.9% and 8.5-16.5%, respectively. A recovery experiment showed no significant disagreement between detected values and expected ones, and saliva CRP concentration was measured in a linear and proportional manner. A positive correlation was found between CRP levels obtained in saliva and serum samples in the experimental (R2 = 0.76) and clinical studies (R2 = 0.70). The assay was able to detect significant differences between salivary CRP levels in healthy dogs and dogs with inflammatory processes. These results suggest that saliva can be used for CRP measurement in dogs. The use of saliva presents the advantage of an easier and less stressful sampling method for the animals, which might be performed outside of hospital environments.     

Validation of a human immunoturbidimetric assay to measure canine albumin in urine and cerebrospinal fluid.

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0-225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.     

Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.     

Comparison of Crinipellis perniciosa isolates from Brazil by ERIC repetitive element sequence-based PCR genomic fingerprinting

Fifty isolates of Crinipellis perniciosa originating from Theobroma cacao , Heteropterys acutifolia and Solanum lycocarpum , from six states within Brazil, were characterized through ERIC-PCR, representing the first application of this method for molecular characterization within C. perniciosa . Phenetic analysis of banding patterns revealed a separation of isolates on the basis of host of origin, with T. cacao -derived isolates showing only a 0·2 similarity level to a cluster comprising the isolates from H. acutifolia and S. lycocarpum . Considerable intraspecific variability was observed within C. perniciosa isolates from T. cacao , with distinct groups observed correlating with geographical origin. Given that a number of isolates from T. cacao from the Amazon region grouped with isolates from Bahia state, this work discusses the possibility that current C. perniciosa populations pathogenic on T. cacao in Bahia originated from the Amazon region, rather than from alternative host plants.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

Use of two doses of cloprostenol in different intervals for estrus synchronization in hair sheep under tropical conditions

Tropical Animal Health and Production - This study evaluated the effect of two doses of prostaglandin at different intervals on reproductive parameters of crossbred ewes. In Experiment 1, 30 ewes...