Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

Composition and structure of helminth communities in two populations of Pipistrellus pipistrellus (Chiroptera: Vespertilionidae) from Spain.

The community composition and structure of helminths of Pipistrellus pipistrellus (Schreber, 1774) from two widely separated Spanish localities, El Saler (n = 42) and the San Pedro pothole (n = 34), were determined and compared. Five species of trematodes, Plagiorchis (Plagiorchis) sp., Lecithodendrium (Lecithodendrium) linstowi Dollfus, 1931, Prosthodendrium (Prosthodendrium) sp., Pycnoporus heteroporus (Dujardin, 1845) and Parabascus semisquamosus (Braun, 1900), and one species of cestode, Hymenolepis pipistrelli López-Neyra, 1941, were found. The two bat populations harboured the same helminth species and showed the same trematode dominance, but the most important differences between the two helminth community structures were attributable to L. (L.) linstowi and H. pipistrelli. The mean species richness in the two localities was not significantly different. The mean number of helminth species per infected bat, mean infracommunity abundance and mean infracommunity diversity showed significant differences between both localities. The number of helminths per bat in both populations displayed an aggregated distribution. Results indicate that the different characteristics of the P. pipistrellus foraging area in both localities are important in determining the composition and structure of helminth communities in this bat species. This is the first study of a Palaearctic bat helminth community.     

Effects of dietary level of ruminally protected choline on performance and carcass characteristics of finishing beef steers and on growth and serum metabolites in lambs

Ruminally protected choline (RPC) was evaluated in two experiments. In Exp. 1, beef steers (n = 160; average initial BW = 350.9 kg) were fed a 90% concentrate diet with either 0, .25, .5, or 1.0% RPC (DM basis) for 112 to 140 d. Feeding .25% RPC increased ADG 11.6% compared with 0% RPC, but responses diminished with increasing RPC level (cubic response, P < .10). Daily DMI was not affected by RPC level, but feed:gain was improved 6.8% with .25% RPC compared with 0% RPC, and responses diminished with increasing RPC level (cubic response, P < .10). Carcass yield grade increased linearly (P < .10) as RPC level increased, but marbling score was lower for all three RPC-containing diets than for the 0% RPC diet (quadratic response, P < .05). In Exp. 2, 20 Suffolk lambs (initial BW = 29.8 kg) were fed an 80% concentrate diet for 56 d with the same RPC levels as in Exp. 1. Serum triglycerides (TG) and cholesterol (CLSTRL) were measured in weekly blood samples, and intensive blood samples were collected on d 28 and 56 to evaluate serum insulin (INS), GH, and NEFA. For the 56-d feeding period, ADG responded quadratically (P < .10) to RPC level, but DMI and feed:gain were not affected. Serum INS and NEFA concentrations increased linearly (P < .05) and serum GH responded cubically (P < .05) to RPC level on d 28, but no differences were noted on d 56. Serum TG concentrations in weekly samples increased linearly (P < .10) with RPC level, but, averaged over all weeks, serum CLSTRL concentrations did not differ (P > .10) among treatments. Quantities of carcass mesenteric (P < .05) and kidney fat (P < .10) increased linearly, but longissimus muscle and liver fat contents did not differ (P > .10) among RPC levels. Supplementing RPC in high-concentrate diets improved performance, but results were not consistent among RPC levels or between cattle and sheep. Potential effects of RPC might be mediated through alterations in fat metabolism and(or) metabolic hormones related to fat metabolism.     

Morphologic, Molecular, and Pathogenic Characterization of Diaporthe phaseolorum Variability in the Core Soybean-Producing Area of Argentina

ABSTRACT Isolates of the Diaporthe/Phomopsis (D/P) complex were collected in the main soybean producing area of Argentina during the 1996-97, 1997-98, and 1998-99 growing seasons. Twenty-three morphologic characters related to type of colonies, stroma, pycnidia and conidia, presence of perithecia, and asci length were studied by principal component analysis (PCA). Genomic DNA were analyzed by the random amplified polymorphic DNA (RAPD) technique. From both studies, 18 isolates were identified as D/P complex and grouped in four major taxa: (i) Diaporthe phaseolorum var. meridionalis, (ii) D. phaseolorum var. caulivora, (iii) D. phaseolorum var. sojae, and (iv) Phomopsis longicolla. In addition to distinguishing interspecific and intraspecific variability, molecular markers allowed the detection of differences among isolates within the same variety. Pathogenicity was assayed in the greenhouse, by the toothpick method, inoculating the D/P isolates to soybean genotypes carrying different resistance genes (Rdc1, Rdc2, Rdc3, and Rdc4) against soybean stem canker (SSC). Pathogenic analysis distinguished two main groups: (i) the SSC-producing isolates, including D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora, and (ii) the non-SSC-producing isolates, including D. phaseolorum var. sojae and P. longicolla. Cultivar RA-702 (susceptible control) was compatible with both D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates; meanwhile, Tracy-M (Rdc1 and Rdc 2 genes) was incompatible with D. phaseolorum var. meridionalis but compatible with D. phaseolorum var. caulivora isolates. The fact that Rdc1 and Rdc2 together (as in Tracy-M) confer an almost immune reaction to all assayed isolates of D. phaseolorum var. meridionalis but were ineffective against the D. phaseolorum var. caulivora isolates evaluated suggests that the virulence or avirulence genes in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora are different. Moreover, physiological races of D. phaseolorum var. meridionalis were detected by using differential soybean genotypes carrying distinct single Rdc genes. As far as we know, this is the first report on the existence of physiological races of D. phaseolorum var. meridionalis in South America. Selective pressure due to deployment of resistant host cultivars may have changed the frequency of the virulence or avirulence genes within the population of D. phaseolorum var. meridionalis. On the whole, our results show that pathogenic variability of D. phaseolorum in the core soybean-producing area of Argentina is higher than previously recognized.     

Influence of grazing dormant native range or winter wheat pasture on subsequent finishing cattle performance, carcass characteristics, and ruminal metabolism

A winter grazing/feedlot performance experiment repeated over 2 yr (Exp. 1) and a metabolism experiment (Exp. 2) were conducted to evaluate effects of grazing dormant native range or irrigated winter wheat pasture on subsequent intake, feedlot performance, carcass characteristics, total-tract digestion of nutrients, and ruminal digesta kinetics in beef cattle. In Exp. 1, 30 (yr 1) or 67 (yr 2) English crossbred steers that had previously grazed native range (n = 38) or winter wheat (n = 59) for approximately 180 d were allotted randomly within previous treatment to feedlot pens (yr 1 native range = three pens [seven steers/pen], winter wheat = two pens [eight steers/pen]; yr 2 native range = three pens [eight steers/pen], winter wheat = four pens [10 or 11 steers/pen]). As expected, winter wheat steers had greater (P < 0.01) ADG while grazing than did native range steers. In contrast, feedlot ADG and gain efficiency were greater (P < 0.02) for native range steers than for winter wheat steers. Hot carcass weight, longissimus muscle area, and marbling score were greater (P < 0.01) for winter wheat steers than for native range steers. In contrast, 12th-rib fat depth (P < 0.64) and yield grade (P < 0.77) did not differ among treatments. In Exp. 2, eight ruminally cannulated steers that had previously grazed winter wheat (n = 4; initial BW = 407 +/- 12 kg) or native range (n = 4; initial BW = 293 +/- 23 kg) were used to determine intake, digesta kinetics, and total-tract digestion while being adapted to a 90% concentrate diet. The adaptation and diets used in Exp. 2 were consistent with those used in Exp. 1 and consisted of 70, 75, 80, and 85% concentrate diets, each fed for 5 d. As was similar for intact steers, restricted growth of cannulated native range steers during the winter grazing phase resulted in greater (P < 0.001) DMI (% of BW) and ADG (P < 0.04) compared with winter wheat steers. In addition, ruminal fill (P < 0.01) and total-tract OM digestibility (P < 0.02) were greater for native range than for winter wheat steers across the adaptation period. Greater digestibility by native range steers early in the finishing period might account for some of the compensatory gain response. Although greater performance was achieved by native range steers in the feedlot, grazing winter wheat before finishing resulted in fewer days on feed, increased hot carcass weight, and improved carcass merit.     

Foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.

Résumé

Le virus de la fièvre aphteuse est un aphtovirus de la famille des Picornaviridae et l'agent de la maladie animale la plus importante sur le plan économique. En tant que picornavirus typique, le virus de la fièvre aphteuse est nu, sous forme d'icosaèdre et son génome comprend un acide ribonucléique monobrin avec environ 8500 nucléotides et une polarité positive. L'acide ribonucléique de ce virus est infectieux et il se réplique par l'intermédiaire d'un brin d'ARN moins, complémentaire. La réplication de l'acide nucléique de ce virus conduit à des erreurs, de telle sorte que les populations virales comprennent un ensemble de mutants (quasi espèce) plutôt qu'une séquence génomique bien définie. Par suite, le virus de la fièvre aphteuse est génétiquement et antigéniquement varié. Ceci entraîne des difficultés importantes pour le diagnostic, la prévention et la maîtrise de la fièvre aphteuse. Une connaissance plus approfondie de la complexité et de l'évolution de la population de ce virus a conduit à des besoins pour une nouvelle génération de vaccines aphteux. Ceci est lié au débat actuel sur le choix d'une politique de vaccination ou de non-vaccination dans la lutte contre la fièvre aphteuse.     

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.)

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.     

Levamisole is a potent enhancer of gilthead seabream natural cytotoxic activity

Gilthead seabream (Sparus aurata L.) head-kidney (HK) leucocytes were incubated with 10(3) to 10(-4) ng levamisole/ml for 4, 24 or 48 h and then assayed for their natural cytotoxic activity against xenogeneic tumor cells. This activity was slightly increased after 24 h of incubation. In a second experiment, fish specimens were fed 0, 75, 150 or 300 mg levamisole/kg diet for 10 consecutive days. The fish were then fed a commercial non-supplemented diet and sampled 0, 1, 2, 3, 4 and 6 weeks post-administration of levamisole. The cytotoxic activity was found increased along the experiment and remained greatly enhanced at the end. In conclusion, levamisole enhanced seabream natural cytotoxic cell activity both in vitro and in vivo and had a great and lasting action when administered by feeding.