Vero Cell Cytotoxicity Assay Research Articles

Occurrence and Characterization of Verocytotoxigenic Escherichia coli (VTEC) Strains from Dairy Farms in Trinidad

A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P < 0.05; chi(2)). For LT production, the highest frequency was detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P < 0.05; chi(2)). Of the 61 VT-producing isolates by vero cell cytotoxicity assay tested by PCR, the VT, LT and eae genes were detected in 62.3%, 4.9% and 1.6% respectively (P < 0.05; chi(2)). Amongst the 45 E. coli isolates that were VT positive (vero cell) or VT-gene positive by PCR, 2.2%, 2.2%, 4.4% and 6.7% belonged to non-O157 strains O91, O111, O103 and O157, respectively, as determined by the Dry spot test. Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.

Immunoglobulin A Antibodies against Ricin A and B Subunits Protect Epithelial Cells from Ricin Intoxication

Epithelial cells of the respiratory and gastrointestinal tracts are extremely vulnerable to the cytotoxic effects of ricin, a Shiga-like toxin with ribosome-inactivating properties. While mucosal immunity to ricin correlates with secretory immunoglobulin A (IgA) antibody levels in vivo, the potential of IgA to protect epithelial cells from ricin in vitro has not been examined due to the unavailability of well-defined antitoxin IgA antibodies. Here we report the characterization of four monoclonal IgA antibodies (IgA MAbs) produced from the Peyer's patches and mesenteric lymph nodes of BALB/c mice immunized intragastrically with ricin toxoid. Two IgA MAbs (33G2 and 35H6) were active against ricin's lectin subunit (RTB), and two (23D7 and 25A4) reacted with the toxin's enzymatic subunit (RTA). All four IgA MAbs neutralized ricin in a Vero cell cytotoxicity assay, blocked toxin-induced interleukin-8 release by the human monocyte/macrophage cell line 28SC, and protected polarized epithelial cell monolayers from ricin-mediated protein synthesis inhibition. 33G2 and 35H6 reduced ricin binding to the luminal surfaces of human intestinal epithelial cells to undetectable levels in tissue section overlay assays, whereas 23D7 had no effect on toxin attachment. 23D7 and 25A4 did, however, reduce ricin transcytosis across MDCK II cell monolayers, possibly by interfering with intracellular toxin transport. We conclude that IgA antibodies against RTA and RTB can protect mucosal epithelial cells from ricin intoxication.

Characterization of a novel high-affinity monoclonal immunoglobulin G antibody against the ricin B subunit.

There is an urgent need for the development of a passive immunotherapy against the category B select agent ricin, a lethal ribosome-inactivating toxin composed of an enzymatic A subunit (RTA) and a single binding B subunit (RTB). To date, only one monoclonal antibody (MAb), a mouse immunoglobulin G (IgG1) against RTA called R70, has been deemed sufficiently potent in animal models to warrant further testing in humans. In this study, we have identified and characterized MAb 24B11, a murine IgG1 directed against RTB. In a Vero cell cytotoxicity assay, 24B11 was approximately two times more effective at neutralizing ricin than was R70. The equilibrium dissociation constants of 24B11 (KD = 4.2 x 10(-9) M) and R70 (KD = 3.2 x 10(-9) M) were virtually identical, suggesting that the difference in neutralization activity between the two MAbs was not due to differing affinities for the toxin. 24B11 blocked ricin attachment to galactoside receptors on primary mouse splenocytes and on the apical surfaces of human mucosal epithelial cell monolayers. Surprisingly, R70 also effectively interfered with ricin attachment to receptors on cell surfaces. Using a phage-displayed peptide library, we determined that 24B11 binds an epitope on RTB adjacent to, but not within, one of the two galactose binding domains. Finally, we demonstrate that R70 and 24B11, when combined, function synergistically to neutralize ricin in vitro, raising the possibility that these two MAbs could serve as a novel immunotherapeutic in vivo.

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx 1 and stx 2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx 1 and stx 2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx 1 gene of all the beef STEC was found to be of recently reported stx 1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC- hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion ( saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.

Detection and characterization of Shiga toxin-producing Escherichia coli from seagulls.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from a seagull in Japan were examined. A total of 50 faecal samples was collected on a harbour bank in Hokkaido, Japan, in July 1998. Two different STEC strains, whose serotypes were O136:H16 and O153:H-, were isolated from the same individual by PCR screening; both of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing active Stx2 and Stx1, respectively. They harboured large plasmids, but did not carry the haemolysin or eaeA genes of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, pulsed-field gel electrophoresis analysis (PFGE), and the stx genes sequences, the isolates were different. Phylogenic analysis of the deduced Stx amino acid sequences demonstrated that the Stx toxins of seagull-origin STEC were closely associated with those of the human-origin, but not those of other animal-origin STEC. In addition, Stx2phi-K7 phage purified from O136 STEC resembled Stx2phi-II from human-origin O157:H7, and was able to convert non-toxigenic E. coli to STEC. These results suggest that birds may be one of the important carriers in terms of the distribution of STEC.

Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.

Detection and Genetical Characterization of Shiga Toxin‐Producing Escherichia coli from Wild Deer

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90-kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR-based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2-producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.

Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.

Detection and long-term existence of Shiga toxin (Stx)-producing Escherichia coli in sheep.

The isolation and characterization of Shiga-like toxin (Stx)-producing Escherichia coli (STEC) from sheep are described. The distribution of stx genes in E. coli isolates was detected by PCR. When brain heart infusion (BHI) broth and novobiocin supplemented m-EC broth (N-mEC) were used as enrichment culture for the isolation of STEC, N-mEC, compared to BHI, showed clearly lower efficiency. Finally, 5 STEC isolates from 4 sheep were isolated and characterized by biochemical and genetical analysis. All of them were confirmed by ELISA and Vero cell cytotoxicity assay for the production of Stx. Moreover, some strains carried hemolysin and eaeA genes and harbored large plasmids. Based on their plasmid profiles, antibiotic patterns and PCR-based DNA fingerprinting analysis using random amplified polymorphic DNA (RAPD), all isolates were different from each other. Three of the isolates were identified to belong to serogroups O2, O153 and O165, respectively, and the STEC strains belonging to these serogroups had been isolated from STEC outbreaks in humans. Four months after the first isolation in July 1997, STEC from sheep #1 was isolated again. A new isolate, HI-11, was identified as STEC O2:Hnt. Simultaneously, 2 STEC, which were genetically and phenotypically different from each other, were isolated from the same sheep at intervals of 4 months. These results demonstrate that sheep may be an important animal for studying human STEC infections, and that further epidemiological surveys on STEC are necessary.

Neurotoxicity of intrathecal Shiga toxin 2 and protection by intrathecal injection of anti-Shiga toxin 2 antiserum in rabbits

The initial brain lesions in rabbits given intravenous Shiga toxin 2 (Stx2) were noted at 24 h in an area around the third ventricle (Fujiiet al.,Infect Immun1996, 64: 5053–60). This result implied that Stx2 is present in the cerebrospinal fluid (CSF) despite the fact that the toxin was administered intravenously. We measured Stx2 activity in CSF by using a Vero cell cytotoxicity assay at various times after an intravenous injection of Stx2. Stx2 was detected from 2 h after the injection, and its concentration in CSF remained at a high level for a further 6 h. Fifty percent lethal doses (LD50) of Stx2 were measured in rabbits after intravenous and intrathecal Stx2 injections; The LD50after an intrathecal injection of Stx2 was 0.36 μg/kg, which was 9.2-fold lower than that of an intravenous injection of Stx2 (3.4 μg/kg). Magnetic resonance images obtained after an intrathecal Stx2 injection (5 μg/kg) were compared with those obtained after an intravenous Stx2 injection (5 μg/kg). At 48 h, the cerebellar lesions had spread from the area in contact with the CSF on a T2-weighted image, which suggests that the intrathecal Stx2 may invade the cerebellum directly. We then examined whether anti-Stx2 antiserum injected intrathecally protects rabbits against brain damage. Eighty percent of the rabbits infected with Stx2 at 5 μg/kg died within 8 days from brain damage. Rabbit anti-Stx2 sera (with titres of ×16 and ×64 by the Ouchterlony precipitation method) were administered into the CSF space through the cisterna magna. All the rabbits (n=10) survived when they were given an intrathecal injection of rabbit anti-Stx2 antiserum 2 h before the intravenous injection of Stx2. Our results suggest that a leakage of Stx2 into the CSF from the choroid plexus causes brain damage, and that an intrathecal injection of anti-Stx2 antiserum could be a therapy for acute encephalopathy caused by Stx2-producingEscherichia coli.

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.

Pheno-genotyping of verotoxin 2 (VT2)-producing Escherichia coli causing haemorrhagic colitis and haemolytic uraemic syndrome by direct analysis of patients' stools.

The subtype of verotoxin 2 (VT2) found in 22 VT2-positive stool samples from severely diseased Italian and German children with haemorrhagic colitis or haemolytic uraemic syndrome, or both, and that produced by the corresponding VT-producing Escherichia coli (VTEC) strains isolated from the stools were studied by cytotoxicity seroneutralisation assays and by polymerase chain reaction (PCR) amplification of the VT2 B-subunit gene, followed by restriction fragment length polymorphism (RFLP) analysis. The free faecal toxin was serotyped as the classical VT2 in 21 stool samples, and as the VT2 variant VT2c in one. For all but one of the VTEC isolates, the toxin phenotype was consistent with the type of VT produced in vivo and found in the corresponding stool samples. Genotyping was in agreement with phenotyping for those strains harbouring a single type of VT2 gene. Three O157:H7 isolates carrying both VT2 and VT2c genes had the VT2 phenotype, instead of the expected VT2c phenotype. Direct PCR analysis of stools detected VT genes in only 11 of 20 VT-positive stool samples suggesting that the Vero cell cytotoxicity assay is more sensitive in diagnosing VTEC infection. Immunological and genetic subtyping of VT2 performed directly on stool samples from patients with haemolytic uraemic syndrome could be a useful complementary approach to understanding the role of the different types of VT in this syndrome.

Application of monoclonal antibody-based sandwich ELISAs to detect verotoxins in cattle faeces

Sandwich elisas, which use monoclonal antibodies for both the capture and the completion stages, were developed for the detection of verotoxins I and II (non-variant). The ELISAs were used to investigate the incidence of verotoxins in 304 samples of bovine faeces collected from cases of enteritis, and 113 samples collected from normal animals. For most samples, the tests were carried out directly on faeces, and on colony sweeps which were taken from cultures grown from the faeces. These colonies were either grown directly from faeces (mixed colony sweep) or from a coliform culture purified from this (purified colony sweep). All the positive ELISA reactions were investigated by Vero cell cytotoxicity assays for the confirmation of verotoxins. The largest numbers of ELISA-positive reactions which were confirmed by cytotoxicity assays were obtained with mixed colony sweeps. Verotoxin activity was confirmed in 74 (24 per cent) of the samples from cases of enteritis and from 35 (31 per cent) of the normal faeces samples. The occurrence of ELISA reactions unconfirmed by cytotoxicity assays and high cytotoxicity levels with low ELISA readings indicated the presence of non-toxic verotoxin epitopes and the presence of toxic verotoxin variants in the samples.

In vitro assays for detection of diphtheria toxin

Subunit-specific antipeptide antibodies have been used to develop a dot blot immunoassay for detection of diphtheria toxin (DT) produced by toxigenic strains of Corynebacterium diphtheriae. A detection limit of 0.1–1.0 ng DT/ml was obtained. DT is cytotoxic to cultured Vero cells and cell viability if conveniently assessed using a tetrazolium dye, MTT. This has been used as the basis of a cytotoxicity assay for DT, which has a detection limit of 20 pg/ml of biologically active toxin. Sixty isolates of C. diphtheriae were assayed for toxigenicity. There was complete concordance between the Vero cell cytotoxicity assay and guinea pig intradermal assay in vivo, demonstrating the utility of the cytotoxicity assay.