Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA

Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs.     

Epidemiology of Fasciola hepatica infection in the Paraíba River Valley, S?o Paulo, Brasil

The study revealed that although Lymnaea columella and Stenophysa marmorata were found in the same habitat, only L. columella harbored intramolluscan stages of F. hepatica. The population density of L. columella cyclically decreased between September and February, and increased from March to September. The drought that occurred between June and November 1981 dramatically reduced the number of snails collected. Larger snails were collected in March-July and November-December, while the smaller snails were collected in January-February and August-October, suggesting that there may be two generations of L. columella per year. The highest prevalence levels of infection in L. columella were: first year, June 8.82%, September 9.09% and October 10.52%; second year, March 6.25% and April 6.89%; third year, July 7.69%, August 10.25%, April 17.91% and May 13.91%. Results obtained with tracer sheep showed that the largest numbers of metacercariae were on pasture between June and October, and between March and April, but the infection was present during most of the year.     

A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera pavonina L. seeds and its effect on Callosobruchus maculatus

The cowpea weevil Callosobruchus maculatus is one of the major pests of Vigna unguiculata cowpea. Digestion in the cowpea weevil is facilitated by high levels of cysteine and aspartic acid proteinases. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (ApTI) isolated from Adenanthera pavonina seeds showed activity against papain. The inhibition of papain by ApTI was of the noncompetitive type, with a K(i) of 1 microM. ApTI was highly effective against digestive proteinases from C. maculatus, Acanthoscelides obtectus (bean weevil), and Zabrotes subfasciatus (Mexican bean weevil) and was moderately active against midgut proteinases from the boll weevil Anthonomus grandis and the mealworm Tenebrio molitor. In C. maculates fed an artificial diet containing 0.25% and 0.5% ApTI (w/w), the latter concentration caused 50% mortality and reduced larval weight gain by approximately 40%. The action of ApTI on C. maculatus larvae may involve the inhibition of ApTI-sensitive cysteine proteinases and binding to chitin components of the peritrophic membrane (or equivalent structures) in the weevil midgut.     

A family 11 carbohydrate-binding module (CBM) improves the efficacy of a recombinant cellulase used to supplement barley-based diets for broilers at lower dosage rates

1. Exogenous microbial beta-1,3-1,4-glucanases and hemicellulases contribute to improving the nutritive value of cereals rich in soluble non-starch polysaccharides for poultry. 2. In general, plant cell wall hydrolases display a modular structure comprising a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Based on primary structure similarity, CBMs have been classified in 50 different families. CBMs anchor cellulases and hemicellulases into their target substrates, therefore eliciting efficient hydrolysis of recalcitrant polysaccharides. 3. A study was undertaken to investigate the effects of a family 11 beta-glucan-binding domain in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum that were used to supplement a barley-based diet at lower dosage rates. 4. The results showed that birds fed on diets supplemented with the recombinant CtLic26A-Cel5E modular derivative containing the family 11 CBM or the commercial enzyme mixture Rovabio Excel AP tended to display improved performance when compared to birds fed diets not supplemented with exogenous enzymes. 5. It is suggested that at lower than previously reported enzyme dosage (10 U/kg vs 30 U/kg of basal diet), the beta-glucan-binding domain also elicits the function of the recombinant CtLic26A-Cel5E derivatives. 6. Finally, the data suggest that exogenous enzymes added to barley-based diets act primarily in the proximal section of the gastrointestinal tract.     

Role of a family 11 carbohydrate-binding module in the function of a recombinant cellulase used to supplement a barley-based diet for broiler chickens

1. Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs have been classified into 52 different families, based on primary structure similarity. These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate eliciting efficient hydrolysis of the target polysaccharides. 2. A study was undertaken to investigate the importance of a family 11 CBM, displaying high affinities for barley beta-glucans, in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum used to supplement a barley-based diet for broiler chicken. 3. The results showed that birds fed on diets containing the recombinant CtLic26A-Cel5E modular derivatives or the commercial enzyme mixture Rovabio Excel AP displayed improved performance when compared with birds fed on diets not supplemented with exogenous enzymes. 4. It is suggested that the enzyme dosage used in this study (30 U/kg of basal diet), was probably too high for the efficacy of the family 11 CBM to be noticed. It remains to be established if the targeting effect resulting from the incorporation of CBMs in plant cell wall hydrolases may be effective at lower exogenous enzyme dosages.     

Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis

Biofilm formation where bacterial cells adhere to a surface and surround themselves in a polysaccharide matrix is thought to be an important factor in disease initiation and persistence for many bacterial species. We have examined biofilm formation by Mycoplasma mycoides subsp. mycoides small colony using a simple model without an air/liquid interface and have found that adherent Mmm SC was more resistant to many stresses, including heat, osmotic shock and oxidative stress. Biofilms of Mmm SC also exhibited remarkable persistence and were able to survive for up to 20 weeks in stationary phase. Significant variation was seen between Mmm SC strains in their ability to form a biofilm and the morphology of the biofilm produced with some strains unable to produce microcolonies. Proteomic analysis found that a number of proteins linked to adherence were over-expressed in biofilms compared with planktonic cells.     

Corpus Luteum–Endometrium–Embryo Interactions in the Dairy Cow: Underlying Mechanisms and Clinical Relevance

Conception rates of dairy cows are currently declining at an estimated 1% every year. Approximately, 35% of embryos fail to prevent luteolysis during the first three weeks of gestation. Interactions between the corpus luteum, endometrium and embryo are critical to the successful establishment of pregnancy and inadequacies will result in the mortality of the embryo. For example, as little as a one day delay in the post-ovulatory rise of progesterone has serious consequences for embryo development and survival. Recently, we found that LH support, degree of vascularization and luteal cell steroidogenic capacity were not the major factors responsible for this luteal inadequacy, but are nevertheless essential for luteal development and function. Progesterone acting on its receptor in the endometrium stimulates the production of endometrial secretions on which the free-living embryo is dependent. However, their exact composition and effects of inadequate progesterone remains to be determined. The embryo is recognized through its secretion of interferon tau (IFNT), which suppresses luteolytic pulses of prostaglandin F_2α. In the cow, it is most likely that IFNT inhibits oxytocin receptor up-regulation directly and does not require the prior inhibition of oestrogen receptor α (ESR1). Unravelling the precise luteal-endometrium and embryo interactions is essential for us to understand pregnancy establishment and development of strategies to reverse the declining fertility of dairy cows.