In vitro and in vivo effects of neem tree (Azadirachta indica A. Juss) products on larvae of the sheep nose bot fly (Oestrus ovis L. Díptera: Oestridae).

Two studies were carried out in order to test the effects of neem tree extracts (Azadirachta indica A. Juss) on sheep bot fly larvae (Oestrus ovis L. Diptera: Oestridae). First, aqueous extracts from neem seeds (ASNE) at 0, 5 y 10% (w/v) concentrations were tested on larval mortality in vitro. In a second study, the effect of oral administration with neem seed meal (0, 100 y 200mg/kg) and neem leaves (1% of diet) on number of larvae found at necropsy and larval development was evaluated in experimentally O. ovis-infected sheep. Results in Experiment 1 showed a significant (P<0.05) effect of ASNE on time to L1 mortality in a dosis-dependent manner. In Experiment 2, oral administration of seeds or leaves did not affect the number of larvae found at necropsy of the sheep, but interfered with larval development and there was a tendency to reduce larval weight at the end of the infection period (55d).

Antioxidant enzymes in erythrocytes from goats seropositive to the sheep nose bot fly (Oestrus ovis L., Diptera: Oestridae) infection.

Oestrus ovis (Diptera: Oestridae) causes an important cosmopolitan parasitosis of the nasal and sinusal cavities of sheep and goats called oestrosis. Our objective was to analyze the participation of erythrocytes in the antioxidant system in goats seropositive to O. ovis infection under field conditions. Fifty female goats naturally exposed to O. ovis infection from Baja California Sur, México, were blood-sampled. Erythrocytic intracellular content was obtained from blood plasma. Oestrosis serodiagnosis was determined by ELISA. Protein, hemoglobin (Hb), superoxide dismutase (SOD), mieloperoxidase (MPO), catalase (CAT), glutathione-S-transferase (GST), and lipid peroxidation in erythrocytes were determined in both seropositive and seronegative goats. Overall seroprevalence of O. ovis infection in goats was 56%. Positive significant (P<0.05) associations were observed among systemic IgG level and protein (0.34), hemoglobin (0.43), SOD (0.32), and MPO (0.41) in erythrocytes. Protein and hemoglobin concentrations, as well as SOD and MPO activities in erythrocytes were found significantly higher (P<0.05) in seropositive than in seronegative goats. By contrast, enzymatic activities of CAT and GST and lipid peroxidation values were similar in seropositive and seronegative groups. In conclusion, there was a systemic stimulation of Reactive Oxygen Species which was efficiently scavenged by erythrocytic antioxidant enzymes in goats seropositive to O. ovis infection.

Participation of cell-surface hydrophobins for hemin binding in Helicobacter pylori.

Treatment of Helicobacter pylori cells with several chaotropic agents resulted in different degrees of inhibition in the binding of the bacteria to hemin and Congo-red dye. Polyanions also yielded a >50% inhibitory effect. Furthermore, hydrophobic interaction chromatography was used to determine the relative surface hydrophobicity of cell-associated proteins extracted with 3 mol/L urea, revealing proteins with a significant hydrophobic profile.

Sheep and goat immune responses to nose bot infestation: a review.

Oestrus ovis L. (Diptera: Oestridae) is a cosmopolitan agent of myiasis in sheep and goats. The parasitic phase begins after adult females deposit first-stage larvae (L1) into the nostrils of hosts; these larvae develop into L2 and L3 in the nasal and sinus horn cavities. Sneezing and nasal discharges are the major clinical signs in infected animals. The pathogenesis of O. ovis infection is caused by: (a) the trauma resulting from the mechanical action of spines and hooks during larval movement on mucosal membranes, and, more importantly, (b) an allergenic reaction provoked by molecules excreted/secreted by larvae, of which salivary antigens are those mainly recognized by the host's immune system. The recruitment of immune reactive cells increases gradually from the nasal to sinus cavities in infected hosts. Mast cells, eosinophils, macrophages and lymphocytes are always more numerous in infected than non-infected animals. Humoral (antibody) systemic response of immunoglobulin G (IgG) usually reaches seroconversion 2-4 weeks post-first infection and the highest levels are observed during the development of L2 and L3 larvae. Local antibody responses include specific IgG, which has been found to negatively correlate with larval survival and development. Hypersensitivity reaction, immunomodulation, immunization trials and mixed infections of O. ovis and helminths are discussed.

IgG antibody response against salivary gland antigens from Oestrus ovis L. larvae (Diptera: Oestridae) in experimentally and naturally infected goats.

The aims of this study were to analyze the systemic IgG responses against third-instar salivary gland (L3SG) antigens by ELISA in Oestrus ovis experimentally infected kids (EIK) and in naturally exposed adult goats (NEG). Firstly, kids (n=4 per group) were assigned to receive intranasally 0, 12, 24, 36, and 48 first-instars in experimental infections. Blood samples were taken from EIK at Days 0, 14, 42 and 67 post-infection. At necropsy (Day 67), larval number and developmental instars were recorded. In an epidemiological study, blood serum samples were collected from 448 grazing NEG (n=20 flocks) in Baja California Sur, Mexico. Results showed that larval establishment rate was similar in EIK groups. Systemic IgG response reached the threshold after Day 42, but humoral response was not statistically different among EIK groups receiving experimental infections. In NEG, all surveyed flocks (100%) showed specific systemic IgG antibodies to L3SG antigens and the overall goat oestrosis prevalence was 59.2%. In conclusion, larval L3SG antigens were effective in detection of specific systemic IgG antibodies against O. ovis infected kids and goats by ELISA.

Specific IgG antibody responses in Oestrus ovis L. (Diptera: Oestridae) infected sheep: associations with intensity of infection and larval development.

Larvae of Oestrus ovis (Diptera: Oestridae) are ubiquitous parasites of nasal and sinusal cavities of sheep and goats. According to the chronobiology of O. ovis infections in Sardinia and the seasonal pattern of the IgG response, the optimal period to investigate the relationships between O. ovis larval populations and intensity of local and systemic IgG antibody responses was mid-July in the summer season. Sarda x Lacaune ewes (n=186), divided into three ram-families were used in the study. Systemic and local IgG responses were measured by ELISA tests using second stage larval crude extracts (L2CE) and L2 (L2SGC) and L3 (L3SGC) salivary gland contents as coating antigens. The number of larval instars, larval length of L1, L2 and L3 larvae, and larval weight of L2 and L3 larvae were individually recorded after ewe necropsy. Negative correlations among larval establishment and/or larval development on the one hand and intensity of local or systemic IgG responses on the other hand were found in two out of three studied ram-families.

Proteolytic activity in salivary gland products of sheep bot fly (Oestrus ovis) larvae.

This study identified and characterized hydrolytic enzymes in salivary gland products of Oestrus ovis larvae. Third instars were collected from the heads of slaughtered goats. Salivary glands were extracted, their products obtained by centrifugation and the enzymatic profile determined. Optimum pH, temperature of maximum proteolytic activity, thermal stability, and resistance of salivary gland products were determined on collagen and subclasses of proteases were identified using protease inhibitors. Zymograms were used to determine the molecular weight of proteases. Antigenic protein bands were revealed by immunoblotting using sera obtained from experimentally infested goats. Seven positive enzymatic activities were detected in salivary gland products: acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase (C4), esterase lipase (C8), leucine arylamidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Optimum pH for proteolytic activity was 8.0; proteolytic activity increased with temperature (10-50 degrees C) then drastically decreased at 60 degrees C. Proteases in O. ovis salivary gland products belong to the serine subclass. In Zymograms, bands of proteolytic activity were detected in the 20-63 kDa range; the immunoblot showed three antigenic bands, one of them related to a protease band (63 kDa). Serine proteases in O. ovis salivary gland products are most likely involved in larval nutrition and host immuno-modulation.

Sexual comparisons in immune ability, survival and parasite intensity in two damselfly species.

Recent evolutionary studies have suggested that females have a more robust immune system than males. Using two damselfly species (Hetaerina americana and Argia tezpi), we tested if females produced higher immune responses (as phenoloxidase and hydrolytic enzymes), had a higher survival (using a nylon implant inserted in the abdomen and measuring survival after 24h) and fewer parasites (gregarines and water mites) than males. We also tested whether immune differences should emerge in different body areas (thorax vs. abdomen) within each sex with the prediction that only females will differ with the abdomen having a higher immune response than their thorax since the former area, for ecological and physiological reasons, may be a target zone for increased immune investment. Animals were adults of approximately the same age. In both species, females were more immunocompetent than males, but only in H. americana females were immune responses greater in the abdomen than in the thorax. However, there were no differences in survival and parasite intensity or the probability of being parasitised between the sexes in either of the two species. Thus, this study lends partial support to the principle that females are better at defending than males despite the null difference in parasitism and survival.

Effectiveness of Cyanothece spp. and Cyanospira capsulata exocellular polysaccharides as antiadhesive agents for blocking attachment of Helicobacter pylori to human gastric cells.

The effect of cyanobacterial polysaccharides (from Cyanothece spp. and Cyanospira capsulata) on the binding of Helicobacter pylori to gastric epithelial cells was evaluated. The antiadhesive action on Kato III and HeLa S3 human gastric cell lines was established.

Superoxide dismutase as modulator of immune function in American white shrimp (Litopenaeus vannamei).

The immunomodulatory action of superoxide dismutase (SOD) and its possible use as an indicator of immune responses in American white shrimp (Litopenaeus vannamei) were studied. Juvenile shrimp were immersed in aerated beta-glucan and sulfated polysaccharide solutions for 6 h. SOD activity in haemocytes and muscle was quantified to evaluate whether beta-glucan and sulfated polysaccharide induce immunostimulatory activity. Haemocytes and muscle showed similar increased levels of SOD activity (1.5- and 1.4-fold that of control, respectively). Total haemocyte count decreased within the first 24 h after challenge with immunostimulants, but total haemocyte count and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulfated polysaccharide is sufficient to generate an increase in the antioxidant activity of L. vannamei SOD.

Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to beta-glucan and sulphated polysaccharide.

Juvenile American white shrimp (Litopenaeus vannamei) were immersed in aerated beta-glucan and sulphated polysaccharide solutions for 1, 3 and 6 h. Superoxide anion and SOD activity in haemocytes and muscle were investigated to evaluate whether beta-glucan and sulphated polysaccharide induce any immunostimulatory activity. Haemocytes and muscle showed different levels of superoxide anion generation and SOD activity (2.0 and 14 times that of control, respectively) when shrimp were immersed for 6 h in aerated sea water containing beta-glucan and sulphated polysaccharide. Total haemocyte count (THC) decreased within the first 24 h after challenge with immunostimulants, but THC and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulphated polysaccharide is capable of generating an increase in the respiratory burst of L. vannamei haemocytes.

Mucosal immune response of spotted sand bass Paralabrax maculatofasciatus (Steindachner, 1868) orally immunised with an extracellular lectin of Aeromonas veronii.

To assess the immunogenic and immunoprotective role of the extracellular lectin from Aeromonas veronii (MCBP), which has affinity for mucosal constituents such as mucin, lactoferrin, immunoglobulins and collagen, spotted sand bass (Paralabrax maculatofasciatus) were orally immunised either with soluble MCBP, adjuvant-conjugated MCBP or immobilised MCBP on latex microspheres. The results suggest that the MCBP is capable of eliciting protective immunity against A. veronii infections when administered orally. The highest mucosal immune response was elicited in fish immunised with MCBP covalently linked to cholera toxin B subunit (CTB) or to Escherichia coli heat-labile toxin (hLT). MCBP-CTB was found to elicit immunoprotection against a challenge with live Aeromonas cells with a relative percent survival of almost 70% and without the expression of the severe histopathological alterations induced by A. veronii.

Differences in the susceptibility of American white shrimp larval substages (Litopenaeus vannamei) to four vibrio species.

The rapid expansion of commercial culture of penaeid shrimp is threatened by Vibrio diseases affecting survival and growth. These opportunistic microorganisms are considered part of the normal ecosystem of penaeid shrimp and cause diseases only under conditions that favor them over the host. Shrimp larvae show different susceptibility to these pathogenic agents. In the present work, we report on a comparative study of the susceptibility of all American white shrimp (Litopenaeus vannamei) larval substages to four potentially pathogenic Vibrio species (V. harveyi, V. parahaemolyticus, V. alginolyticus, and V. penaeicida). Strains of these bacterial species were used to infect nauplii, protozoea I-III, mysis I-III, and postlarvae 1 by immersion challenge at 10(3), 10(5), or 10(7) cfu mL(-1) for 30 min. V. alginolyticus infection had no significant effect on survival rate, compared to control, in all shrimp larvae and at all doses tested. Shrimp larvae infected with V. alginolyticus showed a high survival rate compared to other Vibrio species at the three dose levels. V. penaeicida produced a significant mortality effect (P < 0.01) in all shrimp substages and only in postlarvae 1 at low infection dose (10(3) cfu mL(-1)). V. harveyi and V. parahaemolyticus induced significant mortality rates (P < 0.01) only at high doses in shrimp larvae. In summary, shrimp larvae demonstrated an age susceptibility that depends on the Vibrio species and dose level.

Production and secretion of collagen-binding proteins from Aeromonas veronii.

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.

Anti-adhesive activity of sulphated exopolysaccharides of microalgae on attachment of red sore disease-associated bacteria and helicobacter pylori to tissue culture cells.

Because of the affinity of certain bacterial species for sulphated glycoconjugates exposed on the epithelial cells of susceptible hosts, we hypothesized that sulphated exopolysaccharides of microalgae can be used in anti-adhesive therapies against bacterial infections, both in cold- and warm-blooded animals. In this study we found that adhesion of the human pathogen Helicobacter pylori to the HeLa S3 cell line, and adhesion of the fish pathogens Vibrio campbellii, V. ordalii, Streptococcus saprophyticus, and Aeromonas veronii to spotted sand bass primary tissue culture cells, can be effectively blocked with the various sulphated exopolysaccharides used.

Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture.

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

Analysis of the interaction of Aeromonas caviae, A. hydrophila and A. sobria with mucins.

Aeromonas species are known to be involved in human gastrointestinal diseases. These organisms colonize the gastrointestinal tract. Aeromonas hydrophila, A. caviae, and A. sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date. Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A. caviae, A. hydrophila, and A. sobria strains. Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure. The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase. Binding of the various horseradish peroxidase-labeled mucins by A. caviae, A. hydrophila, and A. sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources. The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A. hydrophila than for A. caviae and A. sobria. Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins. The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A. caviae strain A4812 (95 and 44 kDa); A. hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A. sobria strain 48739 (95 and 43 kDa). Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media. The culture conditions greatly influence the expression of A. hydrophila mucin-binding activity.

Cell-surface lectins of Azospirillum spp.

Cell-surface lectins were screened in seven strains of Azospirillum brasilense and A. lipoferum. The presence of lectins was determined by particle agglutination assays employing latex beads coated with neoglycoproteins and by Western blot with neoglycoproteins labeled with horseradish peroxidase as a probe. Seven strains were agglutinated with the assayed sugar residues. The highest agglutination was with fucose and glucose and to a lesser extent with mannose residues. Cell-wall proteins extracted from two Azospirillum spp. strains exhibit lectin-like activities. We believe that lectins are present in the cell-wall of Azospirillum spp.

A collagen-binding protein produced by Aeromonas hydrophila.

The gastrointestinal pathogen Aeromonas hydrophila strain A186 produces a collagen-binding protein (CNBP) which is found extracellularly and loosely associated with the cell surface. The cell-associated CNBP was purified by sequential ammonium sulphate precipitation, size-exclusion chromatography and ion-exchange chromatography, or by sequential ammonium sulphate precipitation and affinity chromatography with collagen-Sepharose. The purified CNBP was homogeneous in SDS-PAGE, and had a mol. wt of c. 98 kDa. Cyanogen bromide cleavage of the CNBP destroyed collagen-binding activity; however, enzymic digestion with Staphylococcus aureus V8 protease generated > 10 polypeptide fragments, from which a 30-kDa polypeptide contained the strongest collagen-binding activity. Binding of collagen by the CNBP was restricted to the alpha1 (I) chain of the collagen molecule and binding seemed to involve both the carbohydrate moieties and certain peptide sequences on the collagen. Collagen-saccharides generated by alkaline hydrolysis inhibited collagen binding by A. hydrophila. Also, glycosidase digestion and chemical alteration of the carbohydrate residues of collagen reduced its ability to be bound by the CNBP. Collagen-homologous synthetic peptides inhibited binding of 125I-collagen by the bacteria.

Helicobacter pylori interacts with heparin and heparin-dependent growth factors.

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 x 10(-12)M), and also heparan sulfate and heparin with higher affinity (Kd 9 x 10(-9)M) than FGFs bind to heparin (10(-8) - 10(-9)M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanisms. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.