Anti-enteropathogenic Escherichia coli immunoglobulin Y isolated from eggs laid by immunised Leghorn chickens

IgY, the egg yolk immunoglobulin, equivalent to the IgG from mammals, has been used in veterinary practice for passive immunisation against bacterial or viral infectious diseases. Enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhoea in Brazil and other developing countries. Our aims were to isolate immunoglobulin IgY from egg yolk laid by EPEC -immunised Leghorn chickens and to study its reactivity to the antigens from this pathogen, including some virulence factors. Leghorn chickens were immunised with a bacterial suspension intramuscularly (three hens) or intravenously (three hens) or with PBS (two hens). Eggs were collected over a period of 17 weeks. IgY isolation procedures were carried out by salt precipitation (ammonium sulphate, in solid form) followed by centrifugations and dialysis. Final preparations were submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS - PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All immunised animals developed good levels of antibodies reactive to whole bacteria or lipopolysaccharide (LPS), in contrast to the control ones. Immunoblottings allowed the recognition of several antigenic fractions of bacterial antigens, some of which had a molecular weight compatible with bacterial virulence factors, confirming the efficacy of the immunisation and the adequacy of the method.     

Hemangiosarcoma of the third eyelid in a cat

A case of hemangiosarcoma of the third eyelid in a 15-year-old Domestic Short-haired cat is reported. A small red mass protruding from the anterior surface of the third eyelid in the left eye was present. Only mild bilateral conjunctivitis and nuclear sclerosis were revealed on initial ophthalmic examination. After anti-inflammatory treatment, surgery and cryotherapy were performed. On histopathologic examination the mass was located just beneath the conjunctival epithelium, suggesting that the conjunctiva was the tissue of origin. The tumor showed high cellularity and was composed of pleomorphic spindle and polygonal cells arranged in interlacing bundles and solid sheets. The presence of slit-like spaces between tumor cells containing erythrocytes demonstrated the vascular differentiation. Mitotic activity was moderate, but atypical mitoses were detected. The surgical area healed uneventfully and after 7 months there was no recurrence. To the authors' knowledge, this is the first report of conjunctival hemangiosarcoma of the third eyelid in a cat.     

Accuracy of serum beta-hydroxybutyrate measurements for the diagnosis of diabetic ketoacidosis in 116 dogs

The aim of this study was to evaluate the accuracy of serum beta-hydroxybutyrate (beta-OHB) measurements for the diagnosis of diabetic ketoacidosis (DKA) in dogs. One hundred sixteen diabetic dogs were prospectively enrolled in the study: 18 insulin-treated (IT) diabetic dogs that had a positive urine ketone test and 88 untreated, newly diagnosed diabetic dogs. Venous blood gas tensions and pH, serum glucose and urea nitrogen (SUN), and electrolyte (Na+, Cl-, and K+) and urine acetoacetate (AA) concentrations were measured concurrently with serum beta-OHB concentrations. On the basis of laboratory findings, the patients were assigned to I of 3 groups: diabetic ketoacidosis (n = 43); diabetic ketosis (DK, n = 41); and nonketotic diabetes (NDK, n = 31). Serum beta-OHB concentrations differed significantly (P < .001) among the study groups. Although marked differences in beta-OHB concentrations were found, a considerable overlap exists between the distributions of dogs with DK and those with DKA. The overall accuracy of beta-OHB determination as a diagnostic test for DKA, determined by the area under the receiver operating characteristic (ROC) curve, was 0.92. In the 1.9- to 4.8-mmol/L range, serum beta-OHB determination sensitivity varied from 100 to 35.7%, whereas specificity varied from 39 to 100%. The cutoff value of 3.8 mmol/L showed the best equilibrium between specificity (95%), sensitivity (72%), and likelihood ratio (14.8). We concluded that the quantitative measurement of serum beta-OHB may be a potential tool for diagnosing and monitoring ketosis and ketoacidosis in diabetic dogs.     

Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.     

Canine echinococcosis: an alternative for surveillance epidemiology

The essential activities for programmes of cystic echinococcosis control are the census of all dogs from the program and identification of parasitised animals. Currently, in South America evaluations and epidemiological surveillance are based on the administration of arecoline hydrobromide. This method has the disadvantage of increasing environmental pollution and risk for operators and owners of treated dogs. A genus-specific ELISA capture method has been employed for recently issued faeces and the confirmation of positive examination was performed by dog autopsies. Our work presents an alternative method based on collection of dry field-dispersed faeces, followed by serological diagnosis by Copro-ELISA and confirmation by Copro-Western blot. If Copro-ELISA were used to define positive samples of dry faeces, the Copro-Western blot assay would provide 70% sensitivity and 100% specificity. Global efficiency of the system using dry faeces would reach 76%, allowing epidemiological surveillance to be oriented to analysis of surface units instead of dog as measurement unit.     

Identifying the location of olive fruit abscission

For high quality oil, table olive characteristics, and reduction of alternate bearing tendencies, it is often desirable to harvest olive fruits prior to full physiological maturity, that is, before the natural separation zone has differentiated. Olive fruit abscission can occur at different positions, mainly fruit-pedicel, pedicel rachis and peduncle-branch, apparently varying according to cultivar, fruit weight, and fruit maturation. Precisely identifying the location of separation is critical for studying the differentiation of the abscission zone or zones and testing the effectiveness of harvest procedures, including the mode of action of different fruit-loosening chemicals. We determined the olive fruit abscission zone position under natural conditions, throughout the complete fruit maturation period, for two cultivars differing in fruit size. The major separation zone for the cultivars studied was between the fruit and pedicel. For cv. Picual, abscission in zones other that fruit-pedicel occurred in less than 15% of the fruit at all dates throughout the maturation period, whereas in cv. Hojiblanca, the percentages of fruits detached at peduncle-shoot (27%) and pedicel-rachis (19%) were initially substantial, and then decreased progressively during fruit maturation. Our data clearly indicate both varietal and temporal differences which should be taken into account not only when testing fruit-loosening compounds but in extrapolating test results to different varieties or dates. Fruit weight did not appear affect the abscission location. The method we used, of counting the different detached units, is simple and effective for determining the position of fruit abscission.     

In vitro effects of malathion and O,O,S-trimethyl phosphorothioate on cytotoxic T-lymphocyte responses

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in technical formulations of malathion, has been shown to be associated with a high incidence of pneumonia in rats and to be highly immunosuppressive in mice. Based on these findings, an in vitro model was established to study the effect of this and other organophosphorus compounds on murine cytotoxic T-lymphocyte (CTL) responses. The organophosphorus compounds were tested for their ability to block in vitro generation of CTL responses to alloantigen and/or the expression of these cytotoxic responses. Responses were generated in C57Bl/6 (H-2^b) spleen cells to mitomycin C-blocked P815 (H-2^d) tumor cells. The cytotoxicity of the cultured splenocytes to P815 target was measured using a 4-hr chromium release assay. These data demonstrated that malathion was able to block the ability of splenocytes to sensitize to P815 at concentrations as low as 25 μg/ml, but was not able to block the expression of cytotoxicity by mature killer T cells. The same was true for OOS which had been activated by preincubation with rat liver postmitochondrial supernatant (PMS). Activated OOS blocked the generation of CTL responses at concentrations as low as 75 μg/ml while having no effect on mature cytotoxic cells. In fact, both malathion and activated OOS were no longer able to suppress CTL responses if treatment was performed as early as 24 hr after exposure to antigen. Additionally, it was demonstrated that when malathion was preincubated with PMS it was no longer suppressive and that OOS without activation failed to suppress CTL responses.     

Arbuscular mycorrhizal colonization of vascular plants from the Yungas forests, Argentina

In Argentina, the Yungas forests are among the ecosystems most affected by human activity, with loss of biodiversity. To assess the mycorrhizal status in these ecosystems, the roots of 41 native plant species belonging to 25 families were collected throughout the year from two sites of the Yungas forests. Roots were washed and stained to study the presence of arbuscular mycorrhizas (AM). Morphological types of arbuscular mycorrhizas (Arum and Paris-type) and colonization patterns were identified and photographed. All plants presented AM colonization. The AM colonization patterns varied from single intracellular aseptate hyphae, coils, appresoria, to vesicles and/or arbuscules. Among the species studied, the Paris-type colonization showed to be dominant. Results confirmed that AM hosts are predominant in the Yungas of South American forests.     

Biflavonoids from Podocalyx loranthoides

Analysis of the ethyl acetate extract of the aerials parts of Podocalyx loranthoides led to the isolation of I 7, II 4'-dimethylamentoflavone (1) and II 4'-methylamentoflavone (2). Compound (1) gave a moderate effect against Leishmania mexicana promastigotes.