UK surveillance: provision of quality assured information from combined datasets.

Surveillance information is most useful when provided within a risk framework, which is achieved by presenting results against an appropriate denominator. Often the datasets are captured separately and for different purposes, and will have inherent errors and biases that can be further confounded by the act of merging. The United Kingdom Rapid Analysis and Detection of Animal-related Risks (RADAR) system contains data from several sources and provides both data extracts for research purposes and reports for wider stakeholders. Considerable efforts are made to optimise the data in RADAR during the Extraction, Transformation and Loading (ETL) process. Despite efforts to ensure data quality, the final dataset inevitably contains some data errors and biases, most of which cannot be rectified during subsequent analysis. So, in order for users to establish the 'fitness for purpose' of data merged from more than one data source, Quality Statements are produced as defined within the overarching surveillance Quality Framework. These documents detail identified data errors and biases following ETL and report construction as well as relevant aspects of the datasets from which the data originated. This paper illustrates these issues using RADAR datasets, and describes how they can be minimised.

D2R2: an evidence-based decision support tool to aid prioritisation of animal health issues for government funding.

An evidence-based decision support tool, 'D2R2', has been developed by Defra. It contains a wide range of standardised information about exotic and endemic diseases held in 'disease profiles'. Each profile includes 40 criteria used for scoring, enabling D2R2 to provide relative priority rankings for every disease profiled. D2R2 also provides a range of reports for each disease and the functionality to explore the impact of changes in any criterion or weighting on a disease's ranking. These outputs aid the prioritisation and management of animal diseases by government. D2R2 was developed with wide stakeholder engagement and its design was guided by clear specifications. It uses the weighted scores of a limited number of criteria to generate impact and risk scores for each disease, and relies on evidence drawn from published material wherever possible and maintained up to date. It allows efficient use of expertise, as maintained disease profiles reduce the need for on call, reactive, expert input for policy development and enables rapid simultaneous access to the same information by multiple parties, for example during exotic disease outbreaks. The experience in developing D2R2 has been shared internationally to assist others with their development of disease prioritisation and categorisation systems.

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila.

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli [pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and lambda bacteriophage libraries. We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila.

Some amino acid (aa) residues within the cytolytic enterotoxin (Act) of Aeromonas hydrophila essential for biological activity were identified. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and enterotoxic activities. By deletion analysis, generation of anti-peptide Ab, and site-directed mutagenesis we showed that two regions in Act (aa 245-274 and 361-405) were very important for biological functions. As shown by competitive inhibition assays, peptide 2 (aa 245-274) blocked cytotoxic activity of Act, and aa Tyr256, Trp270 and Gly274 were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp394 and Trp396 were important for biological activities. Mutations in other regions of the toxin (e.g., Gly169, Asp170, Gly171, Trp172, Asn177,178, Asp179 and His144,209,355) also decreased biological activity. The reactivity of these mutant toxins with Ab in immunoblots was not altered. Data reported in this study suggested the role of some aa residues in biological function(s) of Act.

Enterotoxins in Aeromonas-associated gastroenteritis.

Aeromonas species produce an array of virulence factors, and the pathogenesis of Aeromonas infections is therefore complex and multifactorial. Aeromonas-associated gastroenteritis is especially a problem in young children. The potential involvement of enterotoxins in the pathogenesis of Aeromonas infections is discussed.

Aeromonas-associated diarrhea in children.

In a 27-month prospective study, Aeromonas spp. were isolated from 7.3% of children with diarrhea and from 2.2% of controls. In 32 patients with diarrhea, ranging in age from 1 to 27 months old, Aeromonas spp. were the only potential bacterial enteropathogens isolated. Principal symptoms of Aeromonas-associated diarrhea were vomiting, fever and bloody stools. Diarrhea was often self-limiting and lasted for 10 days or less in 90% of patients. No secondary spread of diarrhea among close contacts was observed and no clear-cut seasonal patterns of Aeromonas isolation were found. Aeromonas caviae was the most frequently isolated species in fecal samples of patients (24 of 29 isolates) as well as controls (5 of 7 isolates). Cholera toxin cross-reactive cytotoxic enterotoxin was produced by a vast majority of Aeromonas isolates, as compared to a non-cholera toxin cross-reactive cytotonic enterotoxin. In addition no significant correlation was observed between severity of the diarrheal disease and different Aeromonas or the quantity of enterotoxins produced. In our geographic area Aeromonas spp., and A. caviae in particular, seem to be an important and frequent cause of diarrhea in young children.

The effect of bombesin-related peptides on the phagocytic function of mouse phagocytes in vitro.

Bombesin (BBS) at doses of 0.1, 1.0, 10.0 and 100.0 nM stimulated chemiluminescence (CL) production by phagocytic cells (monocytes, macrophages and polymorphonuclear leucocytes) in mice in the presence of ZAP (opsonized zymosan particles containing luminol). These data suggest that BBS increased the phagocytic function of mouse phagocytes. BBS-related peptides, gastrin-releasing peptides (GRP)-27, GRP-14, GRP-10 and neuromedin B, also induced similar CL responses compared with BBS. The CL response elicited by BBS was depressed dramatically by various concentrations of EGTA (a Ca++ chelator), indicating that a Ca++ pathway may play a key role in the BBS-stimulated CL response.

Genetic variation in related cytolytic toxins produced by different species of Aeromonas.

Comparative analyses were performed at the immunological, biological, and genetic level to establish the degree of relatedness of a group of four cytotoxic enterotoxins and aerolysins produced by different isolates of Aeromonas: SSU [16], Ah1 [13], Ah2 [18], and Ah65 [21]. Results obtained from Western blot analysis, neutralization studies, and Southern blot analysis indicated that although the members of this group of toxins displayed structural similarities they also differed from each other at the immunological, biological and genetic level.

Stimulation of neutrophil leukocyte chemotaxis by a cloned cytolytic enterotoxin of Aeromonas hydrophila.

A cytolytic enterotoxin produced by Aeromonas hydrophila, isolate SSU, has been cloned in our laboratory. This enterotoxin lysed rabbit red blood cells, destroyed Chinese hamster ovary cells, caused fluid secretion in rat ligated ileal loops, inhibited the phagocytic function of mouse phagocytes, and was lethal to mice when injected intravenously. In this study, the effect of this cytolytic enterotoxin on the chemotaxis of human leukocytes (cell line RPMI 1788) was examined. This toxin, at concentrations from 92.5 to 370 ng/ml, significantly stimulated the chemotactic activity of human leukocytes in a dose-dependent fashion. The stimulation of leukocyte chemotaxis elicited by cytolytic enterotoxin was abolished when the toxin was neutralized, heated, or exposed to low pH values. This stimulatory effect also was inhibited by various concentrations of pertussis toxin. These results demonstrated that cytolytic enterotoxin may stimulate increased chemotaxis of human leukocytes, and suggest that human leukocytes may possess cytolytic enterotoxin receptors which may be coupled to pertussis toxin-sensitive G-protein.

Mechanism of action of a cytotonic enterotoxin produced by Aeromonas hydrophila.

In this study, we describe the mechanism of action of a cytotonic enterotoxin produced by two isolates of Aeromonas hydrophila. Isolates SSU and Ah65 are of different origin and both are capable of producing either a cytotoxic enterotoxin or aerolysin. A cytotonic enterotoxin produced by diarrheal isolate SSU, which was purified and characterized in our laboratory, elevated intracellular cAMP and PgE2 levels in cultured Chinese hamster ovary (CHO) cells. Likewise, enterotoxic activity expressed by a cytotonic enterotoxin was detected in the culture filtrate of a fish isolate (Ah65) after cytotoxic activity was neutralized with homologous aerolysin monoclonal antibodies. This cytotonic enterotoxin also elevated intracellular cAMP and PgE2 levels in CHO cells, suggesting a cholera toxin-like mechanism of action for Aeromonas cytotonic enterotoxins.

Enterotoxin-associated DNA sequence homology between Salmonella species and Escherichia coli.

Multiple HindIII-restriction fragments of Salmonella typhimurium and Salmonella typhi chromosomal DNA exhibited homology with the heat-labile enterotoxin (LT1) gene of Escherichia coli as determined by Southern blot analysis. A 9.4 kb HindIII restriction fragment identified in S. typhimurium and S. typhi chromosomal DNA reacted with both eltA and eltB gene probes. However, the homology of the 9.4 kb DNA fragment from these Salmonella species was greater with eltB than eltA. In addition, a synthetic oligonucleotide probe, made to a portion of the putative GM1-ganglioside binding region of cholera toxin (CT) and LT1, hybridized with the 9.4 kb DNA fragment of S. typhimurium but not with the 9.4 kb fragment found in S. typhi isolates. The hybridization of multiple restriction fragments of Salmonella DNA with eltA and eltB gene sequences further suggests duplication of the stx operon on the chromosome of these bacteria.

Insulin-like growth factors enhance phagocytosis by human neutrophils in vitro.

Polymorphonuclear neutrophils (PMNs) were isolated from human blood, and PMN phagocytosis was assessed by measuring the chemiluminescence (CL) response in the presence of ZAP (opsonized zymosin particles containing luminol). The administration of 6.5 nM of insulin-like growth factor I (IGF-I), des(1-3)-IGF-I, IGF-II or insulin to PMNs for 20 min resulted in significant increases of the CL response for all test preparations. Des(1-3)-IGF-I, a truncated IGF-I with low affinity binding to IGF binding proteins (IGFBPs), was the most potent CL stimulator. The CL production evoked by 6.5 nM of des(1-3)-IGF-I was inhibited significantly by both 0.25 and 1.0 nM of EGTA (Ca2+ chelator), or 10 microM nifedipine (Ca2+ channel inhibitor), pertussis toxin (0.05 and 1.0 micrograms/ml) or cholera toxin (5 micrograms/ml). These results suggest that IGF-I and its homologues are potent stimulators of phagocytosis and that this action is modulated by IGFBP, and may require extracellular Ca2+ and/or IGF-I receptor G-protein coupling.

Elevated cAMP in intestinal epithelial cells during experimental cholera and salmonellosis.

Cholera and salmonellosis are two diarrheal diseases in which intestinal tissue cyclic adenosine monophosphate (cAMP) concentrations are elevated. Investigations of each experimental disease were initiated to identify the specific intestinal cells containing the elevated cAMP. Epithelial cells were eluted from the mucosa of infected and control intestinal loops of adult rabbits, after which the cAMP content of the epithelial cell fractions and the lamina propria cells was extracted and assayed. The identity of the epithelial cells (in the villus tip-to-crypt cell gradient) was monitored by measuring their intracellular alkaline phosphatase activity, while scanning electron microscopy was used to visualize the effects of infection and cell elution techniques. Clearly, in both experimental cholera and salmonellosis, elevated cAMP levels were associated with crypt epithelial cells. Villus tip epithelial cells from either infection tended to contain less cAMP than those of noninfected control tissue. In Salmonella-infected loops, it was apparent that cAMP was also elevated in lamina propria cell fractions. Lamina propria cells from V. cholerae-infected intestinal loops contained only basal levels of cAMP. In vitro exposure of isolated intestinal cells from normal rabbit intestine to a cell-free lysate of Salmonella resulted in elevation of cAMP in the epithelial cells and lamina propria cells. We conclude that in experimental cholera and salmonellosis, significant elevation of the cAMP levels occurred in intestinal crypt cells, consistent with an enterotoxin-mediated mechanism. In Salmonella-infected loops, it was unclear if the increased concentration of cAMP in lamina propria cells was generated by enterotoxin released from the invasive salmonellae or by prostaglandins formed during the inflammatory response to the bacteria, or by both mechanisms.

Purification and partial characterization of a cytotonic enterotoxin produced by Aeromonas hydrophila.

This report describes the purification and partial characterization of a cytotonic enterotoxin produced by a human diarrheal isolate (SSU) of Aeromonas hydrophila. The extracellular enterotoxin was purified by (NH4)2SO4 precipitation, hydrophobic column chromatography, and chromatofocusing. The highly purified enterotoxin exhibited a molecular mass of 44 kDa and an isoelectric point in the range of 4.3 - 5.5 as determined by chromatofocusing. Western blot analysis using Aeromonas anti-enterotoxin revealed a single band at 44 kDa; however, cholera antitoxin failed to detect the enterotoxin antigen. This non-cholera toxin cross-reactive (non-CTC) enterotoxin was biologically active in vivo as determined by rabbit ligated ileal loop and rabbit skin vascular permeability assays. Biological activity also was in vitro by this toxin as measured by the elongation of Chinese hamster ovary (CHO) cells. The enterotoxic activity associated with this molecule was neutralized completely by homologous antibodies but not by cholera antitoxin. The purified toxin preparation was free of hemolytic and cytotoxic activities as determined by its inability to lyse rabbit red blood cells or damage CHO cells, respectively. Furthermore, this toxin induced the elevation of cAMP in CHO cells suggesting thereby that the mechanism of action of Aeromonas non-CTC enterotoxin may be similar to heat-labile enterotoxins of Escherichia coli and Vibrio cholerae.

Effect of Aeromonas hydrophila enterotoxins on function of mouse phagocytes.

Two enterotoxins produced by Aeromonas hydrophila isolate SSU have been characterized in this laboratory. One is a cholera toxin cross-reactive cytolytic enterotoxin (CTC toxin) and the other is a non-cholera toxin cross-reactive cytotonic enterotoxin (non-CTC toxin). The two enterotoxins are capable of causing fluid accumulation in animal models; however, only the CTC toxin is lethal to mice and expresses hemolytic as well as cytotoxic activities. In this study, we have investigated the effects of these two toxins on mouse phagocytes. Four hours after intraperitoneal injection of a sublethal dose (460 micrograms/kg of body weight) of CTC toxin, the chemiluminescence (CL) response of phagocytes in mouse blood was depressed significantly when compared with that observed for controls (intraperitoneal injection of only Hanks' balance salt solution, non-CTC toxin or before treatment with CTC toxin). When fresh whole blood was incubated with various concentrations (2.3, 11.5, 23, 230, 2300 ng/ml) of CTC toxin for 1.5 hr at 37 degrees C, the CL response of blood phagocytes was reduced strikingly in a dose-dependent fashion; however, non-CTC toxin did not inhibit the CL response. The inhibitory effect induced by CTC toxin of the phagocytic function not only was abolished completely, but phagocytosis was enhanced in the presence of interferon-gamma (IFN-gamma). In addition, IFN-gamma alone induced the largest enhancement of the CL response in mouse phagocytes. These results demonstrated that CTC toxin inhibits the phagocytic ability of phagocytes either in vivo or in vitro and that IFN-gamma pretreatment can overcome this toxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-linked immunosorbent assay for detection of type A streptococcal exotoxin: kinetics and regulation during growth of Streptococcus pyogenes.

We describe the detection and quantitation of type A streptococcal exotoxin (erythrogenic toxin, streptococcal pyrogenic exotoxin) by an enzyme-linked immunosorbent assay. This sensitive and specific technique detected microgram amounts of type A exotoxin and was useful for studying the kinetics and regulation of type A exotoxin production during the growth of Streptococcus pyogenes NY5. Maximum production of type A exotoxin was observed during the mid-log phase of growth, similar to the production of other streptococcal extracellular products. When S. pyogenes NY5 was grown at 42 degrees C, decreases in both growth and type A exotoxin production were observed. The results obtained when we studied the influence of nutrient additives and metal ions on the production of type A exotoxin led to the conclusion that none of these factors significantly affected type A exotoxin synthesis and that regulation was constitutive.

Studies on thermophilic cellulolytic fungi.

Three thermophilic cellulolytic fungi, Chaetomium thermophile var. coprophile, Sporotrichum thermophile, and Thermoascus aurantiacus were studied to determine the conditions for a high rate of cellulose degradation. The range of temperature over which good growth occurred was determined first in a temperature gradient incubator; the optimum temperature was then established in shake flask cultures. T. aurantiacus had the highest optimum growth temperature range (46 to 51 C), whereas S. thermophile had the broadest range over which good growth occurred (36 to 43 C). Optimum temperatures for the three organisms, T. aurantiacus, S. Thermophile, and C. thermophile were 48, 40, and 40 C, respectively. It was found that the addition of an organic carbon and nitrogen source to a cellulose mineral solution medium markedly increased the rate of cellulose degradation. The surfactant, Tween 80, which has been reported to be of value in the production and recovery of the enzyme, cellulase, was shown to be detrimental to the degradation of cellulose in culture. In the medium used, S. thermophile gave the highest rate of substrate utilization; 56% of the cellulose was hydrolyzed in 72 h. The average degree of polymerization of cellulose decreased from 745 to 575.

Stimulation of specific Aeromonas antibody secretion in rat intestine by gamma-aminobutyric acid.

This study was undertaken to assess a possible role for gamma-aminobutyric acid (GABA) in the regulation of intestinal secretion of IgA and IgG antibodies. Rats were immunized with culture supernatant of Aeromonas hydrophila isolate SSU. This culture supernatant contains a number of toxins that may be considered virulence factors. After 24 days of immunization, rats were anesthetized and a 10-cm intestinal segment was intubated and ligated at both ends in situ. The intestinal loop was perfused with phosphate-buffered saline (PBS). The effluents were collected for measurement of IgA and IgG by the ELISA. When compared with the effect of intravenous administration of normal saline in the control group, intravenous injection of GABA (30 mg/kg) resulted in a significant increase of IgA and IgG secretion in the experimental group. These stimulatory effects of GABA on secretion of IgA and IgG were abolished by bicuculline, a GABA-receptor antagonist, and by atropine, indicating that the GABA-stimulated secretion of IgA and IgG was mediated via the GABA receptors and cholinergic muscarinic receptors. These results suggest that GABA may participate in the nervous regulation of intestinal secretion of IgA and IgG antibodies in the rat.

Bombesin: an activator of specific Aeromonas antibody secretion in rat intestine.

The effect of bombesin (BBS) in modulating the secretion of specific Aeromonas antibodies in rat intestine was determined. Rats were immunized with the culture supernatant of Aeromonas hydrophila, isolate SSU. This culture supernatant contained a number of toxins that may be considered virulence factors. After 24 days of immunization, rats were anesthetized and a 10-cm intestinal segment was perfused with phosphate-buffered saline. The effluents were collected for measurement of IgA and IgG by the enzyme-linked immunosorbent assay. When compared with the effect of intravenous administration of normal saline in the control group, intravenous injection of BBS (20 micrograms/kg) in the experimental group caused a significant increase in rat intestinal IgA and IgG in perfusates. The stimulatory effects of BBS on the presence of IgA and IgG were depressed partially by proglumide, a receptor antagonist of cholecystokinin (CCK) and gastrin. Treatment with pentagastrin (250 micrograms/kg) accelerated intestinal secretion of IgA, but failed to stimulate intestinal IgG secretion. In addition, intravenous injection of CCK-8 (120 ng/kg) evoked the intestinal secretion of either IgA or IgG. These findings demonstrated that BBS, gastrin, and CCK can stimulate antibody secretion in rat intestine and the stimulatory effect of BBS may be mediated partially via release of CCK and gastrin. These results suggest that neuropeptides such as BBS and gastrointestinal hormones, eg, CCK and gastrin, may participate in the regulation of intestinal secretion of IgA and IgG antibodies, respectively, in rats.