Anne M. Field

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes.

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.

The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775

A possible colonization factor, E8775, has previously been described for enterotoxigenic Escherichia coli strains of serogroups O25, O115 and O167. Re-examination of these strains by immunodiffusion has revealed that the antigenic nature of this factor, renamed putative colonization factor (PCF) 8775, is more complex than was first thought. All the strains of serogroup O25 tested possessed two antigenic components, termed CS4 and CS6, and gave mannose-resistant haemagglutination (MRHA) of human and bovine erythrocytes. Spontaneous variants possessing CS6 only did not give MRHA. Strains of serogroups O115 and O167 had the antigenic components CS5 and CS6, and gave MRHA of human, bovine and guinea-pig erythrocytes. Using immune electron microscopy, the components CS4 and CS5 were identified as fimbriae. No fimbriae were associated with CS6.

Blood donor screening for parvovirus B19.

A programme of blood donor screening for parvovirus B19 was conducted from January to May 1990. The main aim of the study was to identify a B19 positive donation that could be used as a source of viral antigen for diagnostic serology. Out of 24,000 donors tested one was positive for B19 antigen by counter current immunoelectrophoresis and over 100 ml of undiluted B19 containing material was obtained. However, much of the positive donation was incorporated in a plasma pool of 28 donations. An acid dissociation technique was used to recover B19 antigen from immune complexes formed in the plasma pool.

Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166.

Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.

In vitro propagation of parvovirus B19 in primary foetal liver culture.

The culture of parvovirus B19 in foetal liver tissue has been described recently. We have established the technique in our laboratory and studied parameters affecting the yield of B19 virus. Replication of the virus was detected by radioimmunoassay for B19 antigen and dot blot hybridization assay of B19 DNA, and the virus was localized by immunofluorescence and thin section electron microscopy. B19 DNA and antigen production became detectable at day 2 and reached a maximum at day 5. Virus particles were seen mainly in cell nuclei, but some cytoplasmic membranes were lined with virus particles. The amount of virus produced depended on the age of the foetus and the cell culture and the concentration of erythropoietin and interleukin 3 in the culture medium.

Comparison of Vero-cytotoxin-encoding Phages from Escherichia coli of Human and Bovine Origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.

Electron microscopic reporting of gastrointestianal viruses in the United Kingdom, 1985–1987

We examined some epidemiological features of the viruses associated with gastrointestinal illness, using national data reported by electron microscopists in the United Kingdom. During the 3 years analyzed (1985–1987), a total of 1,993 positive detections of astroviruses, caliciviruses, coronaviruses, and small round structured viruses (SRSVs) were reported. In 1 year of this period, 8,210 rotaviruses were reported. More than 90% of the astroviruses and caliciviruses were detected in children under 5 years of age, while coronaviruses and SRSVs were detected in adults as well as children. Detections of astroviruses increased in the winter and were infrequent during the summer, a seasonal pattern similar to that observed for rotaviruses. There was some variability between reporting regions in rates of detection of fecal viruses. We have attempted to identify the reasons for this. We make suggestions for improving the detection of human fecal viruses, and we recognize the need for continued surveillance of these agents.

Expression of plasmids coding for colonization factor antigen II (CFA/II) and enterotoxin production in Escherichia coli

Two plasmids transferred from enterotoxigenic Escherichia coli (ETEC) of serotype O6. H16 and biotypes A and C coded for mannose-resistant haemagglutination (MRHA) and production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both plasmids were nonautotransferring being mobilized most efficiently by the R plasmid R100-1. They were similar in their genetic properties being incompatible with each other and plasmids of the Inc group FI. The wild-type strains produced the colonization factor antigen II (CFA/II) which was made up of different coli surface antigens (CS). The biotype A strains produced CS1 and CS3 while the biotype C strains produced CS2 and CS3. These three antigens have the ability to cause MRHA. When plasmids coding for MRHA were transferred to K12 strains, the degree of haemagglutination was markedly reduced and only CS3 was produced. When both plasmids were transferred back into biotype A strains, good MRHA was restored and the strains produced CS1 and CS3. In a biotype C strain CS2 and CS3 were formed. The production of the antigens was compared by enzyme-linked immunosorbent assay (ELISA). The strains were also examined by electron microscopy where it was found that CS1 and CS2 were fimbrial antigens while CS3 was not.

Diagnostic Virology Using Electron Microscopic Techniques

Publisher Summary This chapter illustrates the development of the use of electron microscopy in viral diagnosis. The field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are also included where relevant. It is reported that the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section immune electron microscopy (IEM). The application of negative stain IEM has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. Viral antigens can also be detected in thin sections of infected cells by IEM with suitably labeled specific antibodies. Confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is also frequently useful.

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6

Summary: Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than 0 6 and produced colonization factor antigen 11, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransfer-ring plasmid, NTP165, from a strain of E. coli 0168.H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTPl65 depended on the recipient strain : a biotype A strain of serotype 0 6. H 16 expressed CS1 and CS3; a biotype C strain of serotype 0 6. H 16 expressed CS2 and CS3; strain K 12 and strain El9446 of serotype 0139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype 0139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lostthe plasmid coding for CS antigens produced both CSl and CS3 after the introduction of NTP165.Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.