Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate.     

Nutritional Enhancement of Biocontrol Activity of Candida sake (CPA-1) against Penicillium expansum on Apples and Pears

Pome fruits are poor in nitrogenous compounds and the addition of nitrogen can improve colonisation of the fruits by antagonists. Twenty-two nitrogenous compounds were evaluated for their effect on Candida sake (CPA-1) growth in vitro. Ten compounds that induced greater growth were applied with the antagonist to wounded fruits to evaluate their effect on enhancing control of Penicillium expansum. Calcium chloride and 2-deoxy-D-glucose were also tested. L-serine and L-aspartic acid enhanced biocontrol by C. sake against P. expansum on apples. On apples and pears, ammonium molybdate, calcium chloride and 2-deoxy-D-glucose improved the capacity of the antagonist to control P. expansum. The addition of ammonium molybdate at 1 mM allowed C. sake to be used on apples and pears at a lower concentration without diminishing control. Similar results were observed with the addition of calcium chloride to the antagonist. 2-deoxy-D-glucose at 6 and 18 mM enhanced biocontrol on pears by over 81%, but on apples the improvement of biocontrol was observed only at 6 mM. In cold storage, the combination of ammonium molybdate and C. sake completely eliminated the incidence of blue mould on pears, and reduced its severity and incidence by more than 80% on apples.     

Histomorphometry and quantification of nucleolar organizer regions in bovine thyroid containing methylthiouracil residues

In order to study the morphology and morphometry and to characterize and quantify the nucleolar organizer regions (NORs) of bovine thyroids containing methylthiouracil (MTU) residues, five animals were orally treated with a suspension of MTU (5 g/animal/day) for 20 days (group A). This treatment protocol was interrupted 5 days before the the animals were slaughtered. Six animals receiving placebos composed group B. A third group (group C) was composed of normal thyroids obtained from a slaughterhouse. All glands were previously assessed for detection of antithyroid residues by chromatography, and only those glands from MTU-treated animals were positive. Follicles of glands from group A showed wide variation in size and shape. There was a predominance of small follicles covered by multiple layers of columnar cells, sometimes forming papillary projections into the lumen, characterizing severe interfollicular and intrafollicular adenomatosis. Many follicles had vacuolated cells with nuclei showing karyolysis or pyknosis and reduced amounts of a low-density and very excavated colloid. They also showed higher follicular epithelia and larger proportions of their structural components when compared with glands of groups B and C. In the thyroids from group A, the argyrophilic nucleolar organizer region-associated proteins (AgNORs) were greater in number, with small ones scattered all over the nucleus. Although the size of AgNORs in thyroids from groups B and C was variable, these AgNORs were fewer and larger than were those in glands from group A. In conclusion, the MTU induces proliferation and regressive changes in follicular cells, and the AgNOR technique is efficient to distinguish different degrees of thyroid hyperplasia.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.     

Antibody response to a fragment of the protein G of VHS rhabdovirus in immunised trout

A fragment (called frg#11, amino acids, aa 56-110) of the protein G (pG) of viral haemorrhagic septicaemia virus (VHSV) was designed after previous results showed it to be recognised by approximately 40% of the trout immunised to VHSV [Dis. Aquat. Organ. 34 (1999) 167]. frg#11 was then cloned, expressed, purified and used to study the production of antibodies to its epitopes in trout immunised to VHSV. Anti-frg#11 trout antibodies could be detected in serum from individual trout surviving VHSV exposure, immunised by injection with purified VHSV or DNA-immunised with its pG gene whereas it was not detected in non-infected and non-immunised trout. The trout serum antibodies which reacted more strongly by ELISA using solid-phase frg#11 (continuous or linear epitopes on the sequence of the pG) had the lowest VHSV-neutralising activity (epitopes which are pG conformation-dependent). Because antibodies recognising continuous as well as conformation-dependent epitopes of the pG seem to be involved in protective trout immunological responses to VHSV, the estimation of anti-frg#11 antibodies could help to the dissection of the complex trout antibody response to VHSV infections. In addition, these preliminary results suggest that the determination of anti-frg#11 antibodies might also be used to complement in vitro viral neutralising assays which seem to be restricted to pG conformation-dependent epitopes.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

Improved Control of Postharvest Decay of Pears by the Combination of Candida sake (CPA-1) and Ammonium Molybdate

ABSTRACT The potential enhancement of Candida sake (CPA-1) by ammonium molybdate to control blue and gray mold caused by Penicillium expansum and Botrytis cinerea, respectively, on Blanquilla pears was investigated. In laboratory trials, improved control of blue and gray molds was obtained with the application of ammonium molybdate (1, 5, 10, and 15 mM) alone or in combination with C. sake at 2 x 10(6) or 2 x 10(7) CFU ml(-1) on Blanquilla pears stored at 20 degrees C. In semicommercial trials at 1 degrees C for 5 months, the efficacy of C. sake at 2 x 10(6) CFU ml(-1) on reducing P. expansum and B. cinerea decay was enhanced more than 88% with the addition of ammonium molybdate 5 mM in the 1999-2000 season. In two seasons, the performance C. sake at 2 x 10(6) CFU ml(-1) plus ammonium molybdate was similar to or greater than that of C. sake at 2 x 10(7) CFU ml(-1). Similar control of blue mold was obtained on pears stored under low oxygen conditions. The preharvest application of ammonium molybdate did not reduce postharvest blue mold decay. The population of C. sake on pear wounds significantly decreased in the presence of ammonium molybdate 1 and 5 mM at 20 and 1 degrees C.     

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.     

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.