Specificity of the carbon immunoassay (CIA) test for the diagnosis of Toxoplasma infections.

A rapid and low cost procedure, the carbon immunoassay (CIA) test, was evaluated for the diagnosis of Toxoplasma gondii infections. Using a closely related parasite (Besnoitia jellisoni) as antigen, and homologous or heterologous immune sera, it was demonstrated by light and electron microscopy that CIA is a very reliable and specific test. As it is neither expensive nor time-consuming, it can be recommended for general and routine laboratory use.     

Molecular cloning,purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.     

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

Sperm Morphology in the Domestic Cat, and its Relation with Fertility: A Retrospective Study

Knowledge about normal ranges in semen quality and the association between sperm morphology and fertility in felids is limited. The aims of this retrospective study were to (1) define a normal spermiogram in cats; (2) evaluate possible effects of season, age and breed on sperm morphology; and (3) evaluate the relationship between sperm morphology and fertility. Semen samples collected by electroejaculation from 52 cats were evaluated for sperm morphology. The cats constituted two groups: a general population of cats (n = 48) and cats examined because of poor breeding records (n = 4). The general population was divided into household (n = 20), pedigree (n = 19) and colony cats (n = 9) and into three age classes, <12 months, 12-59 months and >or=60 months. The median percentage of normal spermatozoa in the general population was 44.0% (range 1.0-91.0%). Criteria were tentatively set for what was considered a normal spermiogram. The mean percentage of normal spermatozoa was higher during February to July than during August to January (p < 0.05). Pedigree cats had a lower mean percentage of normal spermatozoa than did household cats (p < 0.05). Age had no effect on the percentage of normal spermatozoa but was positively correlated with the percentage of proximal droplets. Of the cats with <40% normal spermatozoa (n = 19), all those with known breeding records (n = 11) had produced litters. The four cats examined because of poor breeding results had higher percentages of different sperm abnormalities than tentatively stipulated for the normal spermiogram. In two of these cats both sperm morphology and fertility changed over time.     

Quantification of airborne spores of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l⁻¹, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

Management of inappetant sheep during export by sea

In the first of 2 experiments, a simulated voyage was conducted to examine the effects of various treatments on bodyweight change and feeding frequency of inappetant sheep at the end of lot-feeding (non-feeders). The treatments, applied during simulated shipping, were: normal quantities of feed and length of troughs; extra trough length; extra feed. Adult Merino wethers (n = 108) were used in each treatment. A voyage to the Middle East was then conducted to establish whether shipboard mortality could be reduced by separating non-feeders (n = 305) from feeders (n = 5,620) late in the feedlot hase and housing the groups separately aboard ship. A control group of non-feeders (n = 215) mixed with feeders (n = 5,732) was used for comparison. Bars (marker bars), containing a coloured dye, were attached to feed troughs to mark sheep that fed. Most non-feeders (82%) began eating when placed in shipping pens in both studies. However, there was no significant difference in percentage of sheep that fed between non-feeders given extra trough length or extra feed compared with non-feeders given standard management at any stage of simulated shipping. There was no significant difference in mean bodyweights between treatment groups on days 1, 8 and 15 of simulated shipping. Differences in bodyweight on d 22 were probably associated with different levels of gut fill. Death rates were not significantly different in separated and control groups (1.1%, 0.9%, P = 0.6) in the voyage of 14 d to the Middle East.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.