The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus.

AIMS: The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. METHODS AND RESULTS: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. CONCLUSIONS: The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.

Simple and direct detection of Aeromonas hydrophila infection in the goldfish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies.

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross-reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross-reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1-8, AS1-6, AC1-5, Aeromonas veronii and Aeromonas jandaei) without cross-reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 105 to 107 CFU mL⁻¹. However, the sensitivity of detection was increased to 10²-10³ CFU mL⁻¹ after inoculation of the sample in tryptic soy broth for 3-6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests.

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW.

AIMS: The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae. METHODS AND RESULTS: A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65 degrees C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non-cholerae Vibrio isolates and 37 non-Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2.2 x 10(3) CFU ml(-1) or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2.2 x 10(4) CFU g(-1) or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction. CONCLUSION: The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection. SIGNIFICANT AND IMPACT OF THE STUDY: The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae. This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus.

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.

Three more novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii.

In addition to five FMRFamide-like peptides (FLPs) previously isolated from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii (16), three more new FLPs (Mar-FLP6-8) were identified from minor immunoreactive fractions of 5,000 eyestalk extracted in methanol/acetic acid/water: DGGRNFLRFamide, GYGDRNFLRFamide and VSHNNFLRFamide. These three peptides share 5-6 common residues at the C-terminus with Mar-FLP1,2 and 3. This evidence reveals that the structural diversity and complexity of the FLP family in M. rosenbergii are similar to that found in other invertebrate species.

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon.

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.

Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Heptapeptide (YANAVQV-NH2 = T-) and octapeptide (YANAVQTV-NH2 = T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T- antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37-39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T- antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24 +/- 5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34 +/- 4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T- antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.

Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.

Heterogeneity of cholecystokinin/gastrin-like immunoreactivity in the nervous system of the nematode Ascaris suum.

A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8.

Neuronal localization of serotonin in the nematode Ascaris suum.

We have used immunocytochemical techniques to investigate the distribution of serotonin-like immunoreactivity in the nematode Ascaris suum. Antisera raised against serotonin (5-hydroxytryptamine, 5-HT) conjugated to bovine serum albumin (BSA) labelled a pair of neurons in the pharynx of both sexes and five cells in the ventral cord of the male tail. The labelling was blocked by 5-HT or by 5-HT conjugated to BSA. The 5-HT-immunoreactive cells in the pharynx resemble neurosecretory cells and are probably homologous to the neurosecretory motor neurons (NSM) in Caenorhabditis elegans; the cells in the male tail appear to be motor neurons that are homologous to CP neurons in C. elegans. Other cells that stain with 5-HT antisera have been observed in C. elegans but are not seen in Ascaris.

Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum.

By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extracts, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans.

Motor behavior and motor nervous system function in the nematode Ascaris suum.

Ascaris suum has a nervous system that is very simple both numerically and morphologically. It comprises only 298 neurons almost all of which are extremely simple in shape. Extensive anatomical descriptions of the morphology of neurons and of their synaptic connections, together with the study, by using intracellular recording techniques, of their physiological properties, have led to a prediction of how the motor nervous system controls behavior. Subsequent discovery of endogenous neuropeptides that have potent activity on subsets of the motor neurons suggests that the description of the motor circuitry is more complex than is apparent from its anatomy.

A versatile dot-ELISA method with femtomole sensitivity for detecting small peptides.

Several protocols for conjugating peptides in situ to a protein carrier on paper, nitrocellulose, or nylon membranes were explored for their usefulness in dot-ELISA detection of the peptides. The most sensitive method in which peptide diluted in bovine serum albumin is applied to nitrocellulose, then fixed with glutaraldehyde, can detect several peptides, ranging from 4 to 38 amino acids in length, at the level of 2-10 fmol. Both immunohistochemical grade antisera and monoclonal antibodies have been used successfully. The method may be a useful alternative to radioimmunoassay since there is no requirement for radiolabelled peptide, or (for quantitation) for known quantities of unlabelled peptide. The method has been used to monitor, semiquantitatively, the fractionation of FMRFamide-like or CCK-like peptides from the nematode Ascaris, and to detect peptide-like immunoreactivities in tissue extracts.

Neuropeptide diversity in Ascaris: an immunocytochemical study.

An immunocytochemical method was used for localization of various peptide-like substances in the Ascaris nervous system. Out of 45 antipeptide antisera, 12 demonstrated immunoreactivity in different subsets of neurons; these 12 antisera were raised against luteinizing hormone-releasing hormone (LHRH), Aplysia peptide L11 (L11), Aplysia peptide 12B (12B), small cardioactive peptide B (SCPB), neuropeptide Y (NPY), FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), alpha-melanocyte stimulating hormone (alpha MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF), and vasoactive intestinal peptide (VIP). Several peptide-like substances were colocalized to the same neuron. Our results suggest that Ascaris, like other organisms, contains multiple peptidergic systems.

Generation of monoclonal antibodies against a nematode peptide extract: another approach for identifying unknown neuropeptides.

Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.

Effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: a light and electron microscopic study.

Using a ligation method, rat rectal epithelium was exposed to 2% sodium salicylate, and light and electron microscopic methods were used to assay for: 1) permeability of the epithelium to a marker dye, trypan blue, and 2) damage expressed in terms of disruption of the epithelial surface. Rectal mucosa was exposed to salicylate at pH 4.8, 7.0, and 9.0, and the effects of pretreatment with phlorizin were also studied. Results indicated that 2% sodium salicylate does very little damage to rectal epithelial cells at pH 7.0 while enhancing their permeability to trypan blue, an effect that is reversed upon washing out the sodium salicylate. The major cellular change induced by salicylate was a reduction in the length or distribution of glycocalyx filaments on microvilli of epithelial cells. It was also noted that pretreatment with phlorizin counteracted some of the effects of salicylate treatment.