Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.     

Genetic and environmental parameters for mature weight in Angus cattle

Genetic and environmental variances and covariances and associated genetic parameters were estimated for weaning weight, asymptotic mature weight, and repeated mature weights. Data consisted of a set of weight measurements of 3,044 Angus cows born between 1976 and 1990. Mature weight was predicted by individually fitting Brody growth curves (asymptotic weight) and by using weights repeatedly measured after 4 yr of age. Variance and covariance components for mature weight were estimated by REML from a single-trait animal model with asymptotic weight, a two-trait animal model with asymptotic and weaning weight, and a two-trait animal model with repeated weights and weaning weight. Weaning and cow contemporary groups were defined as fixed effects. Random effects for weaning weight included direct genetic, maternal genetic, and permanent environmental effects; and for mature weight, direct genetic and repeated measurements (if in the model). Heritability estimates for weaning weight were similar for both two-trait models (.53 and .59). Estimates of heritability for mature weight were .44, .52, and .53 for the single-trait model with asymptotic weight, two-trait model with asymptotic weight, and two-trait model with repeated measures weights, respectively. The estimate of the genetic correlation between mature and weaning weight was higher for the repeated measures model (.85 vs. .63). A lower heritability estimate for mature weight from the single-trait model was likely due to postweaning culling. Therefore, a genetic evaluation of mature weight from field data should include a trait recorded earlier in life that is less subjected to selective data reporting.     

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.     

Sire marbling score expected progeny difference and weaning weight maternal expected progeny difference associations with age at first calving and calving interval in Angus beef cattle.

Field records from the American Angus Association were used to study the associations of sire marbling score EPD and sire weaning weight maternal (milk) EPD with age at first calving (AFC) and calving interval (CI). Cows were selected based on the accuracy of their sire's milk (> or =.7) or marbling (> or =.6) EPD. The data were screened using biological constraints, and regression models were used to identify records that were greater than 5 SD from the mean. The AFC was modeled for both milk and marbling data sets to account for effects of year, sire EPD, and their interaction. The CI was subdivided into first, second, and mature calving interval traits and modeled to account for state, year, calf sex, calf birth weight (BW), calf weaning weight (WW), sire EPD, and interactions of EPD with year and state. Derivative-free REML was used to estimate heritability and genetic correlations for AFC and CI. Sire milk EPD and marbling EPD were predictors of AFC (P < .001); however, pooled estimates were unreliable because of state x EPD interactions (P < .001). Increases in sire milk EPD resulted in reductions in AFC; however, there was no consistent pattern to effects of marbling EPD increases. Models accounted for < 8% of variation in AFC. Sire milk EPD was not a predictor of first, second, or mature CI (P > .1). Sire marbling score EPD was not a predictor of second, or mature CI (P > .1); however, it was associated (P = .059) with first CI, although regression estimates varied across states and prevented pooling. The BW, sex, and WW were predictors of CI (P < .001). Increases in BW resulted in longer mature CI, and mature CI decreased as WW increased. The AFC was heritable (.22), and CI traits had heritabilities ranging from .01 to .03. The AFC was genetically correlated with first CI (-.6) and mature CI (-.93). Genetic correlations between CI traits were uninterpretable because of low additive genetic variances. In conclusion, sire marbling score and milk EPD do not seem to be reliable predictors of AFC or CI. The BW and WW have significant but small effects on AFC and CI. Selection for AFC is possible, but earlier calving heifers may have longer calving intervals.     

Serotypes and electrophoretic protein profiles of Pasteurella haemolytica isolated from pneumonic ovine lungs.

Serotypes and SDS-PAGE protein profiles of P. haemolytica isolated from pneumonic ovine lungs were investigated. Of 268 P. haemolytica isolates, 232 (86.6%) were serotypable. A total of 12 serotypes were recognized in 20 different geographic origins of central Turkey. The most common serotype was A2, followed by A7, A1 and T4. Serotypes A13, A14, A16 and T15 could not be detected. In SDS-PAGE, marked differences between major bands of biotype A and T strains were found. In numerical analysis of protein profiles, biotype A and T strains were separated at 58% similarity level. Biotype A isolates produced a cluster at 80% similarity level, and biotype T isolates at 92% similarity level. No single cut off level was able to discriminate between each serotype studied and isolates could not be clustered on the basis of their geographic origins.     

Diagnosis, therapy and endocrinologic parameters of persistent follicles in mares in comparison with preovulatory follicles]

During the 1997 breeding season persistent follicles were diagnosed in 17 mares. In 16 of these mares a total of 17 follicles were transabdominally punctured and the steroids oestradiol, progesterone and testosterone were measured in the follicular fluid and in blood serum. In ten mares serving as a control group preovulatory follicles were punctured. The follicular fluid of the persistent follicles revealed a very high variability of the steroid concentrations. Depending on the steroid ratio within the follicles, eight follicles were rated as being intact, three follicles were undergoing atresia and five follicles were luteinized. Because of the high oestradiol levels of the follicular fluid within the control group, all of these follicles were considered to be intact. In both groups, no correlation of the steroid concentration between serum and follicular fluid was detectable. This fact argues against a passive diffusion of the steroids through the follicular wall. By puncturing the persistent follicles it was possible to bring the affected mares back into a physiological oestrus cycle within a normal dioestrus period.     

Immunohistochemical study of the inflammatory infiltrate associated with feline cutaneous squamous cell carcinomas and precancerous lesions (actinic keratosis).

The distribution of T lymphocytes (CD3+), B lymphocytes (CD79+), immunoglobulin-containing plasma cells (IgG, IgM and IgA), macrophages (Mac387+) and MHC Class II antigen was analysed in the inflammatory infiltrate associated with cutaneous squamous cell carcinomas (SCC) from 23 cats. Peri-tumoural skin (12 cases) and precancerous lesions of actinic keratosis (nine cases) were also evaluated for the expression of MHC Class II. The results revealed that an abundant inflammatory infiltrate was associated with the majority of SCC. This infiltrate was composed mainly of CD3+ T lymphocytes, B cells (CD79+) and IgG-bearing plasma cells, and the intensity of infiltration increased with the degree of invasiveness of the tumour. The number of CD3+ T cells and CD79+ cells was significantly increased in well-differentiated SCC compared with moderately differentiated tumours, whereas the number of IgM+, IgA+ plasma cells and Mac387+ macrophages was low or moderate and did not change significantly with histologic grade or invasiveness. MHC Class II antigen was expressed by infiltrating lymphocytes and macrophages, and by fibroblasts. A variable number of neoplastic cells (10% to 80%) in 10 SCC, and keratinocytes of basal layers in seven of nine cases of actinic keratosis also expressed MHC Class II, whereas keratinocytes of normal skin were always negative for this antigen. These results suggest that CD3+ T lymphocytes, CD79+ B cells and IgG-bearing plasma cells may participate in down-regulation of tumour growth, since these cell types were particularly numerous in well-differentiated and mildly invasive SCC, as well as in actinic keratosis. The expression of MHC Class II by neoplastic cells could enhance this local anti-tumour immune response.     

Identification of stable normalization genes for quantitative real-time PCP, in porcine articular cartilage

Background: Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results: Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein-zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions: BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.     

Bacterial cholangiohepatitis in a dog

A 9-year-old female Yorkshire terrier was presented for vomiting and diarrhea. Blood chemistry tests revealed hepatic dysfunction, cholestasis, and inflammation. Liver ultrasonography and liver biopsy were consistent with cholangiohepatitis. Fine-needle aspiration of the gallbladder revealed the presence of bacteria later identified as Clostridium spp. The cholangiohepatitis was successfully treated.     

First genetic characterization of equine adenovirus type 1 (EAdV-1) in Turkey

Equine adenovirus type 1 (EAdV-1) is a cause of repiratory tract infection in equids. In present study for the first time in Turkey, the prevalence of EAdV-1 in nasal swab samples obtained from horses showing respiratory symptoms was investigated by polymerase chain reaction (PCR), and molecular characterization of the hexon gene detected in the Turkish (TR) strain was performed. Overall, the prevalence of EAdV-1 was found low (1.4%) as indicated by a positive PCR reaction from the nasal swab extracts tested. Phylogenetic analysis based on the partial sequences of the hexon gene of a TR-EAdV-1 strain with those of previously isolated AdVs from different mammals and an EAdV-1 M1 strain showed that the EAdV-1 strains were placed into a unique cluster. Although the TR-EAdV-1 strain was closely related to CAV-1, CAV-2 and bat adenovirus reference strains, larger-scale studies are necessary to better understand the molecular epidemiology and population structure of EAdV-1 in Turkey.