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Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.

In a national survey in the period May 1986-December 1987, Aeromonas was isolated from 277 out of 16,857 (1.6%) samples of watery, bloody or mucous stool, from patients with diarrhoea. There was a clear seasonal pattern (less than 1% in winter, up to 3% in summer). A. caviae was isolated most frequently (49%), followed by A. sobria (35%) and A. hydrophila (15%). Some non-identifiable strains were isolated as well. Aeromonas were isolated in particular from faeces of patients aged over 70 years (predominantly A. sobria) or under 5 years (predominantly A. caviae). In 67% Aeromonas was isolated as the only possible bacterial cause of diarrhoea, but in 33% other enteropathogenic bacteria were found as well (17% Campylobacter, 14% Salmonella, 2% Shigella). In addition, all Aeromonas isolates were collected which were obtained in normal diagnostic activities in the participating laboratories, among others from blood, from pus or wound fluid, from faecal samples which did not meet the above mentioned criteria. A. caviae was the dominant species in 'other' faeces and 'various' body sites but was not isolated from blood. The results of this study do not indicate that routine examinations for Aeromonas in faeces of patients with diarrhoea are necessary.

The Widal test for detection of typhoid fever was introduced in two rural hospitals in Nigeria. Because of undetectably low O agglutination titers, 74 sera were sent overseas for further determination by means of passive haemagglutination with O 9, 12-antigen, Vi-antigen and slide agglutination with H-d antigen suspensions. This full test showed discrepancies with the normal Widal test i.e. a false negative Widal H agglutination in 6 out of 17 patients in West Nigeria and a false positive Widal H test in 28 out of 49 patients in Central Nigeria. The full test should be applicable in a small laboratory, but its value has yet to be established.

Hybridomas secreting monoclonal antibodies against the K99 antigen of Escherichia coli were produced by the fusion of spleen cells from immunized BALB/c mice with P3/X63-Ag8.653 myeloma cells. The seven hybridomas which produced the highest antibody titers in vitro, as detected by enzyme-linked immunosorbent assay (ELISA) and Perma slide agglutination test (PSAT), were chosen for antibody production in vivo. No cross reaction was observed with K88ab, F41 and P987 antigens in the ELISA. The titer of each ascitic fluid was established by the ELISA and the slide agglutination (SAT) tests. The two ascitic fluids with the highest titer in the SAT were incorporated into the set of antisera used for serotyping at our laboratory. The results were satisfactory both in terms of stability and specificity.

A mechanized microtechnique originally designed for the serotyping of E. coli was adapted to the specificity control of diagnostic Salmonella agglutinating antisera as well as for the serotyping of Salmonella. Salmonella strains sent for serotyping were inoculated into a tube with broth which was used as H antigen and, after heating also as O antigen. The agglutination reactions were carried out in clear plastic trays with U formed wells. The antigens were strained with gentian violet in order to obtain a better contrast. With only 6 monofactor O sera, 97% of 100,000 cultures received for serotyping in 1979-1982 could be O antigen typed. Eight H antisera were sufficient for the complete serotyping of 27% of these strains whereas 64% was partly typed. The technique has a high degree of versatility.

The resistance of Salmonellae to drugs has been studied in the Netherlands since 1958. In 1972, 1973, and 1974 respectively, 14241, 13086, and 22927 strains were tested for resistance to ampicillin, chloramphenicol, kanamycin and tetracycline. From 1973 all strains were also tested for resistance to trimethoprim. In the period covered, the yearly incidence of resistance to at least one of the above drugs ranged from 39.2% to 45.6% of all strains obtained from various sources (humans, animals, animal products, sewage, etc.). A new finding in the period 1972 to 1974 was that many multiply resistant strains emerged in S. typhimurium and in S. dublin isolated from calves and cattle. In 1974, 64.4% of all strains of S. typhimurium from these animals appeared to be resistant to ampicillin, tetracycline, chloramphenicol and kanamycin, and 25.5% of those of S. dublin were found to be resistant to chloramphenicol and tetracycline. Of all strains of Salmonellae examined in 1973 and 1974 respectively, 0.15% and 0.22% were resistant to trimethoprim, the main component of the twin-drug cotrimoxazol. Of the 142 strains of S. typhi isolated in 1972 to 1974 two were resistant to tetracycline only, and one was resistant to all four antibiotics. The others had a normal susceptibility pattern.

Porcine and bovine strains of E. coli suspected to be enterotoxigenic, were tested for heat-labile enterotoxin (LT) production by means of a test measuring the stimulation of the synthesis of 3151 cyclic adenosin monophosphate (cAMP test) and rounding mouse adrenal tumor Y1 cells (Y1 test), and for heat-stable enterotoxin (ST) production by means of the baby mouse test (BM test). The test results were compared with those of the ligated gut test (LG test) using pig and calf intestine. One hundred and eleven strains of human origin were tested in the cAMP test, the Y1 test and the BM test. All strains were tested for the presence of the attachment factors K88, K99, P987 and CF10407. Seventy-four of 117 bovine strains produced only ST and all 74 ST-producing strains possessed K99 antigen. None of the 43 nonenterotoxigenic strains possessed K99. The presence of K99 antigen is therefore a reliable indicator of enterotoxigenicity in bovine strains. Porcine strains produced LT, ST or LT + ST. The K88 antigen was found in 52 out of 101 enterotoxigenic strains, K99 and P987 in 7 and 6 strains respectively and the latter 13 strains produced only ST. P987 was only detected after subculturing in liquid medium to enhance pilus formation. Eleven out of 111 strains of human origin stimulated cAMP synthesis but failed to round Y1 cells, whereas 4 known LT producers were positive in both tests. Attachment factors were found only in 2 of the latter 4 strains.

Strains of Escherichia coli isolated from faecal specimens of ten infants receiving breast milk, six receiving a cow-milk preparation with iron supplement (5 mg/l) and six the preparation without iron supplement (less than 0.5 mg/l), were serotyped and examined for their haemagglutinating activity. The Escherichia coli flora of breast-fed and bottle-fed infants consisted of one resident strain, accompanied by one or more transient strains. Changes in the serotype of the Escherichia coli flora and in the frequency of occurrence of strains associated with urinary tract infections were more often seen in bottle-fed than in breast-fed infants. In breast-fed and bottle-fed infants without iron supplement most strains of Escherichia coli were non-haemagglutinating, while most strains in infants bottle-fed with iron supplement showed mannose-resistant haemagglutination. It is concluded that human milk favours the establishment of a stable non-pathogenic Escherichia coli flora and that a low iron content in standard cow-milk preparation favours colonization with non-adherent strains of Escherichia coli.

The K99 antigen of Escherichia coli can be detected more readily in cultures grown on Minca medium at 37 degrees C for 6 to 8 h or grown on Minca plus 1% Iso VitaleX for 20 to 24 h.

E. coli strains were isolated from the urine of patients with acute cystitis in general practice and from the faeces of a comparable reference group of healthy individuals. These strains were serotyped and tested for virulence in an experimental mouse model. Of 30 cystitis-strains 18 were virulent, and of 30 faeces-strains 15 were virulent. It is concluded that the cystitis-strains were not more often virulent than the faeces-strains. O antigens commonly found among urinary E. coli isolates were present in 60% of the cystitis-strains and in 37% of the faeces-strains. K antigens commonly found in urinary E. coli strains were present in 33% of the cystitis-strains and in 12% of the faeces-strains. Neither the presence of common urinary O-antigens, nor the presence of common urinary K antigens could be associated with virulence of the isolated strains. However, it is suggested that certain O and K antigens (O2,O6, K23) may be associated with virulence for the urinary tract.

A device for mechanical application of either drops of phage suspensions or antibiotic-containing tablets was developed. The device consists of a Transport Apparatus which transports petri dishes from one side of the apparatus to the other in such a way that the plate and the lid of a petri dish become entirely separated at a particular stage. Either a Phage Applicating System or an Antibiotic Tablet Dispenser can be connected to the apparatus. In this way a maximum of 27 phages of 7 antibiotic tablets per plate can be applied pneumatically.

The hereditary characters of organisms are bound up in the DNA of their genomes. Genetic information is expressed by a complicated mechanism. The process by which this occurs and the control of the degree of expression in bacteria and a number of the other lower organisms are known only in broad outline. There usually is no exchange of DNA between organisms of unrelated species in nature. "Foreign" DNA can be linked to bacterial plasmid DNA and multiplied in the laboratory as a recombinant DNA molecule in bacteria using the recently discovered so-called restriction enzymes. Expression of bacterial DNA can thus be accomplished in a different, unrelated bacterium. In principle, this means that the natural barriers between unrealated species of organisms may be passed by. Human and animal DNA can also be multiplied in bacteria by this method. Previous recombinant DNA studies showed that the mode of expression of genetic information in the higher organisms differs markedly from that in bacteria. The initially sanguine expectations regarding the practical use of recombinant DNA research, for instance in the production of biologically important substances by bacteria, will therefore possibly not be realized at short notice. On the other hand this technique has added considerably to the knowledge of expression of genes within a short space of time. This is particularly true of the mechanisms of pathogenicity of E. coli, the bacterium which plays such an important role in veterinary medicine. The recombinant DNA technique is briefly reviewed in the present paper. The uses are broadly outlined, and one of these, which could be of importance in veterinary medicine, is described in greater detail. International discussions of potential hazards, and therefore also of the admissibility of this type of experiment, are griefly summarized.

The genetic and molecular properties of the plasmids in Salmonella typhimurium phase type 201 isolated are described. Such strains are resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, kanamycin, and several other antimicrobial drugs, and are highly pathogenic for calves. These strains have been encountered with increasing frequency since 1972 in West Germany and The Netherlands. We show that isolates of this phage type constitute a very homogeneous group with regard to their extrachromosomal elements. These bacteria carry three small plasmids: pRQ3, a 4.2-megadalton (Md) colicinogenic plasmid; pRQ4, 3.4-Md plasmid that interferes with the propagation of phages; and pRQ5, a 3.2-Md cryptic plasmid. Tetracycline resistance resides on a conjugative 120-MD plasmid pRQ1, belonging to the incompatibility class H2. Other antibiotic resistance determinants are encoded by a nonconjugative 108-Md plasmid pRQ2. Transfer of multiple-antibiotic resistance to appropriate recipient strains was associated with the appearance of a 230-Md plasmid, pRQ6. It appears that pRQ6 is a stable cointegrate of pRQ1 and pRQ2. This cointegrate plasmid was transferable with the same efficiency as pRQ1. Other conjugative plasmids could mobilize pRQ2, but stable cointegrates were not detected in the transconjugants. Phase type 201 strains carry a prophage, and we show that phage pattern 201 reflects the interference with propagation of typing phages effected by this prophage and plasmid pRQ4 in strains of phage type 201.

Escherichia coli K88ab and K88ad antigens were isolated from K88 antigen-producing E. coli K12 transconjugants and purified by means of repeated column chromatography. Injection of about 1 to 2 mg of the purified antigens into rabbits yielded specific K88ab and K88ad antisera. Attempts to obtain specific K88ac antiserum by means of this procedure failed.

We modified the rabbit model for enteric infection by Vibrio cholerae developed by Spira et al. and designated the RITARD (for removable intestinal tie-adult rabbit diarrhea) model (20). Our modification DISC comprises a permanent ligation of the cecum (C) to prevent resorption of the fluid secreted by the small intestine, a temporary ligation of the small intestine (S) to enable the bacteria to colonize, and duodenal inoculation (DI) of the challenge material. The main difference between RITARD and DISC is that in the latter model the challenge material is injected into the duodenum approximately 10 cm distal to the stomach instead of into the jejunum. Four out of 5 V. cholerae strains tested, including 2 serotypes and 2 biotypes, were able to elicit a massive and usually fatal cholera-like diarrhea. The virulence depended strongly on the culturing conditions. One strain, C5, caused fatal diarrhea in a dose of about 1000 organisms, even if the temporary ligation was omitted (DIC model). Other modifications were the DIS and the DI model in which the permanent ligature of the cecum or both ligatures were omitted. Duodenal inoculation of organisms in a dose of 100 X the minimum infective dose (MID) in the DIS or DI model did not cause any disease symptom. However, such inoculations were found to cause protection against subsequent challenges with 100 X MID of homologous and heterologous organisms up to 52 weeks after duodenal inoculation. Subcutaneous injection with classical, whole cell cholera vaccine gave only partial protection of short duration. This model might contribute to the understanding of the pathogenesis of cholera as well as to the improvement of efficacy testing of cholera vaccines.

Since 1974, tetracycline resistance in salmonellae of human and porcine origin has decreased nation-wide in the Netherlands. This decrease had coincided with the ban on incorporation of tetracycline in animal feeds for growth-promotion.

Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad. We propose to include this antigen into the international E. coli typing scheme. Ultrasonic extracts of field strains of E. coli possessing the K88ab, K88ac, or K88ad antigen and their E. coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C. By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis. When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic. The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E. coli K-12. Anodic and cathodic K88ac antigens could not be distinguished serologically. The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow. We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture. It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes.

Escherichia coli strains isolated from pigs suspected to have succumbed to E. coli enterotoxicosis and not belonging to the commonly incriminated (classical) serotypes (O8:K87:K88, O45:K88, O138:K81:K88, O141:K85:K88, O147:K89:K88, O149:K91;K88, and O157:K88) were tested for enterotoxigenicity in the ligated gut test (LGT) using pig intestine. Of 202 strains tested, 54 strains belonging to 13 different O groups were positive in the LGT. Four of these strains had K88 antigen and one possessed K99 antigen. The majority of the strains was not agglutinated by any of the standard OK antisera. Four new K antigens ("K200", "K442", "K2346" and "K2347") were provisionally designated. K200 was found in pig enterotoxigenic strains belonging to O group 8 and carrying flagellar antigen H31 and in non-enterotoxigenic non-motile strains of O group 8, as well as in O group 20 strains isolated from calves succumbing to E. coli septicemia in two countries. The provisional antigen K2346 was encountered in 18 enterotoxigenic strains with various O antigens from two countries. It is proposed to include these two K antigens into the international E. coli antigens scheme. Attempts to demonstrate a common antigen in the nonclassical enterotoxigenic strains lacking K88 and K99 antigens failed.

Approximately 20,000 strains of Salmonella were screened annually for resistance to tetracycline, chloramphenicol, kanamycin and ampicillin since 1959, and also to trimethoprim since 1978. Tetracycline-resistant strains increased in human subjects and pigs from 1961. After the ban on incorporation of tetracycline in animal feeds for nutritive purposes in 1974, the proportion of tetracycline-resistant strains in pigs and human subjects decreased. In veal calves, the number of strains of S. typhimurium and S. dublin resistant to multiple drugs increased from 1972. Strains resistant to multiple antibiotics in man were mainly isolated from adoptive children from Indonesia. No further spread of these strains was observed. So far, strains similar to those in calves resistant to multiple drugs were only incidentally isolated from human patients.

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as "medium vesicles" and "medium lipopolysaccharides", were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

Previous studies have shown that cholera, as well as protective immunity against infection with Vibrio cholerae, can be induced in the rabbit. This protection is long-lasting (up to 30 months) and is characterized on challenge by rapid, symptom-free disappearance of V. cholerae from the intestine; we therefore believe this to be vibriocidal protection. In this study, we analysed the humoral and secretory immune response against various subcellular V. cholerae components in vibriocidally protected, non-vibriocidally protected, and unprotected animals. Only vibriocidal protection was found to be associated with high levels of biliary IgA directed against lipopolysaccharide O antigen. We did not find such a correlation between either type of protection and response in serum. Therefore, anti-lipopolysaccharide antibodies are essential in protection against experimental infection with V. cholerae.

The phage type and antibiogram of 5199 S. typhi murium strains from 13 countries and of 4949 strains from The Netherlands was determined. For a number of countries it could be established that types predominating in pigs or calves predominate also in humans in that country. However, the predominant phage types were not the same in the various countries. Type 505 predominated in The Netherlands and, to a lesser degree, also in Belgium and the German Federal Republic. Type 131 predominated in Poland and was also frequently found in the German Democratic Republic. In the German Democratic Republic, type 450 was the most frequently found type. Phage patterns not accepted as a type and occurring one country only, were found to predominate in Italy, Hungary, Israel and Sweden. The majority of antibiotic-resistant strains belonged to a limited number of phage types only.

Escherichia coli strains isolated 1985-1988 in Spain from patients with diarrhoea were examined; 1170 strains were isolated from 582 sporadic cases of diarrhoea in children, and seven strains were associated with seven outbreaks of diarrhoea. Strains positive for STa enterotoxin production in the infant mouse test were also assayed for production of LT enterotoxin on Vero cells and by a coagglutination test. Thirty-one strains were STa positive: 28 were isolated from 16 (2.7%) sporadic cases of diarrhoea and three were responsible for outbreaks. The majority of STa+LT- strains from both outbreaks and sporadic cases were serotype O153:H45 and expressed the CFA/I colonization factor antigen. Enterotoxigenic STa+LT- strains of serotype O27:H7 and STa+LT+ CFA/II+ strains of serotype O6:K15:H16 were also isolated frequently from sporadic cases.

The common antigen of calf enterotoxigenic strains of Escherichia coli, recently established as the K99 antigen, was studied by means of the slide agglutination test and immunoelectrophoresis. Specific antisera were obtained by absorption of crude antisera with ultrasonicates of autologous cells grown at 18C or by injection into rabbits of the purifies K99 antigen obtained by preparative electrophoresis. The K99 antigen was usually undetectable in calf enterotoxigenic E. coli cultures with capsular K antigens of the A variety grown at 37C on commercially available nutrient agar plates designed for the isolation of Enterobacteriaceae, but was rapidly detectable when grown on a buffered semi-synthetic medium at pH 7.5 (Minca medium). An alternative procedure for the isolation and identification of calf entertosigenic E. coli strains from feces, using the Minca medium, is proposed. K99 was found in 70 of 74 strains of E. coli, the enterotixigenicity of which was established in the ligated gut test in calves. None of the 20 cultures negative in the ligated gut test possessed K99 antigen. The K99 antigen is therefore probably a useful diagnostic tool for the identification of calf enterotixigenic E. coli strains, taking into account that K99 and enterotoxigenicity are controlled by different plasmids.

Cholera disease can be induced in the rabbit by duodenal inoculation (DI) of Vibrio cholerae organisms after ligation of the cecum (C) (DIC model). When ligation of the cecum is omitted, no disease symptoms develop. In contrast, the animals are primed which becomes apparent as vibriocidal protection upon challenge with V. cholerae in the DIC model. This protection coincides with high anti-O antigen IgA levels in the bile. The O antigen was shown to be the protective antigen and it must be presented by live organisms. A non-enterotoxigenic mutant of V. cholerae induced protective immunity in the rabbit but was reported to cause mild diarrhea in human volunteers. Looking for alternatives, we applied cholera toxin, known as a mucosal adjuvant, together with killed V. cholerae cells to rabbits. Unfortunately, the minimum adjuvant dose was equal to the minimum toxic dose. A Salmonella typhimurium strain expressing also the V. cholerae O antigen induced systemic rather than local immunity which was not protective. Several Escherichia coli strains were able to elicit a local immune response, but the animal to animal differences were considerable. Therefore, V. cholerae itself was thought to be the most appropriate carrier organism. Some non-enterotoxigenic and auxotrophic mutants of V. cholerae were able to prime and did not show any undesired side-effects in the DIC model. Therefore, further attenuation of non-toxigenic V. cholerae strains by means of stable deletions in nutritional genes seems to be the most promising way to obtain acceptable vaccine candidates.

Eleven Salmonella typhi isolates were recovered from patients with typhoid in Indonesia. These motile isolates had neither H:d nor H:j antigen but had a new H antigen, designated Z66 in this paper. After passage through semi-solid agar medium with anti-Z66 serum (in U-tube), 7 strains were agglutinable by H:d antiserum. No strain was able to express the three H factors. In case of typhoid fever caused by such strains, conventional Widal test may not detect anti-H:d agglutinins in patients' sera.

Serratia marcescens O antisera prepared with boiled bacterial suspensions gave relatively low titers when tested with boiled broth cultures in tube or tray agglutination tests, but extremely high titers when tested in the haemagglutination test with conserved sheep erythrocytes coated with alkali-treated heat extract. However, an antiserum prepared with boiled O group 6 bacteria, did not react at all. A proper O6 antiserum could only be prepared if the O6 cells were not heated at 100 degrees C. The antigenic relationships between standard strains of S. marcescens were investigated by means of haemagglutination tests. The antigenic relationships already described in the literature were confirmed. Additional reciprocal relationships were observed, some of which could be explained by 8 antigenic factors which are designated I-VIII. O antisera could be made monospecific by absorption. It was confirmed that standard strain O6H3 has two antigenic determinants which should be written as O6ab, that standard strain O6H8 has O6bc and that O14 has O6b. Standard strain O7 was found to possess O6a in addition to the specific determinant O7, whereas the antigen O6c turned out to be identical with O8. The standard strain O14 has, in addition to O6b only the factor hitherto described as Co12, 13, 14 and no specific O14 antigen. We propose to redesignate Co12, 13, 14 as O14 and to write the formula of O12 and O13 strains possessing this antigen as O12, 14 and O13, 14 respectively. O antigen O23 should then be written as O14, 23. For similar reasons we propose to redesignate Co2, 3 as O25. The O antigen O20 was found to be related to the antigens O16ab, O16ac and O16acd in such a way that it should be written O16ae. For similar reasons O22 should be written as O10ac. When serotyping Dutch isolates of Serratia marcescens, hitherto undescribed antigenic combinations such as O2, 16ab; 03, 6a; O3, 21 and O4, 6b were encountered. For H antigen typing we found the slide agglutination with cultures grown on semisolid medium the most appropriate.

A scanning electron microscopic study was carried out to compare the in vivo pathogenicity of two strains of Vibrio cholerae in an adult rabbit ligated-gut test model. V. cholerae C5 (serotype Ogawa, biotype El Tor), a motile strain possessing hemagglutinating activity in vitro, and C21 (serotype Ogawa, classical biotype), a nonmotile strain possessing no hemagglutinating activity, were tested. Tissue samples from small intestinal loops were examined 3, 6, 9, and 12 h postinoculation. Contradictory to most published data, neither hemagglutinating activity nor motility appeared to be essential prerequisites for the pathogenesis of cholera in the experimental animal model used: nonmotile hemagglutinin-negative strain C21 adhered to and colonized the small intestine at least to the same extent as did motile hemagglutinin-positive strain C5. Maximum colonization was seen at 9 h postinoculation for both strains. C5 and C21 vibrios caused comparable damage to the villi of the small intestine. The villous epithelium showed only mild changes during the first 9 h postinoculation. However, after 12 h the epithelium was seriously damaged concomitant with a decrease in the number of vibrios. Many villi showed partial or total denudation, owing to repelled epithelium, leaving a bare basal lamina with only some to moderate numbers of vibrios attached. Since similar changes were induced by pure cholera enterotoxin, these changes were likely the result of excessive fluid accumulation. From this study it is concluded that, at least in the animal model used, factors other than hemagglutinating activity and motility may also play a role in the association of V. cholerae with the small intestinal surface.

Antisera were raised with heat-killed vaccines prepared from 25 Aeromonas hydrophila strains, 4 A. sobria strains and one A. caviae strain. Twenty-seven of these 30 antisera gave high titers when tested in the microtiter tray agglutination (MTA) test with their homologous antigen heated at 100 degrees C for 30 min. Three O antisera gave low titers in the MTA test but reacted to high titers in the haemagglutination (HA) test with conserved sheep red blood cells coated with alkali-treated heat extract. All 27 antisera showing high titers in the MTA test, showed even higher titers in the HA test. Therefore, the HA technique was employed to type 306 strains isolated from surface and drinking water, from food samples and from faeces of human patients with diarrhea. Of 155 A. hydrophila strains 21 (14%) could not be typed. For A. sobria and A. caviae, the percentage of untypable strains was 46% and 68% respectively. Many A. sobria and A. caviae strains reacted to titer in A. hydrophila O antisera. A limited number of strains reacted to titer in more than one specific O antiserum. Immunoelectrophoretic studies indicated that the O antigen is often represented by more than one precipitation line and that true K antigens occur in Aeromonas.

Samonella typhimurium bacteriophage type 505 is the most frequently encountered phage type in the Netherlands and its neighboring countries. Phage type 505 was analyzed with regard o the interference with propagation of the typing phages by the prophages and plasmids, present in the type strain S. typhimurium 505...

The virulence of faecal and urinary Escherichia coli strains was studied in relation to serotype, haemolysin production and haemagglutination pattern. By means of an experimental mouse model E. coli strains can be divided into avirulent (I), mouse nephropathogenic (II), and generally virulent (III) strains. Virulent group II and group III strains were more often haemolytic and haemagglutinating than avirulent group I strains. Presence of K antigen could not be associated with virulence. Discriminant analysis for qualitative variables revealed that no combination of the investigated properties contributed more to a strain's virulence level than did one single property. It is concluded that other virulence factors, apart from haemolysin production in group II strains and haemagglutinins in group III strains, must be involved in the determination of a strain's virulence level. All O2, O6 and O18 ac strains tested were virulent, and by far the most O75 strains were avirulent, whereas other O groups were more variable with regard to virulence. Pyelonephritis strains were more often mannose-resistance haemagglutinating than faecal and other urinary isolates, indicating that mannose-resistant adhesins may be important in the pathogenesis of pyelonephritis.

From 1975-1980, about 130 000 Salmonella strains isolated from various sources were tested for resistance to ampicillin, chloramphenicol, kanamycin, tetracycline and trimethoprim. Following the ban on incorporation of tetracycline in animal feeds for nutritive purposes, tetracycline resistance in S. typhimurium and S. panama strains of porcine origin dropped from about 90% in 1974 for both species, to about 34% and 1%, respectively, 1980. The incidence of resistance in human strains concurrently decreased from about 80% in 1974 to 25% and 1%, respectively, in 1980. The build-up of multiple resistance in bovine S. dublin and S. typhimurium strains, already started in 1973-74, has continued. Recently, phage type 193 S. typhimurium strains have become predominant and they are invariable resistant to ampicillin, chloramphenicol, tetracycline, kanamycin, neomycin, streptomycin, sulphonamide and trimethoprim. Up to now, type 193 strains were hardly encountered in human patients, but the number of human isolates is slowly increasing. A fairly large number of multiply resistant strains belonging to S. oranienburg, S. schwarzengrund, S. typhimurium and, recently, S. krefeld have been isolated from adoptive children from the Far East.

E. coli strains were isolated from urine specimens of hospitalised patients with acute pyelonephritis, acute cystitis or asymptomatic bacteriuria (ABU), and tested for virulence in an experimental mouse model. Of 12 pyelonephritis-strains 11 were shown to be virulent and 1 avirulent; of 12 cystitis-strains 4 were virulent and 8 avirulent; of 12 ABU-strains 5 were virulent and 7 avirulent. It is concluded that, while no difference in virulence was found between cystitis- and ABU-strains, pyelonephritis-strains were more often virulent than cystitis- and ABU-strains. No associations could be shown between virulence of the isolated strains and the presence of antibody-coated bacteria in the urine. Common urinary O types were not more often virulent than other O types. No relationship was seen between virulence and the presence of K antigen or the presence of particular K types.

The DIC model (Duodenal Inoculation with ligation of the Cecum in rabbits) was employed to study experimentally induced cholera and the related protective immunity. Duodenal inoculation (DI) without ligation of the cecum with live V. cholerae organisms did not cause any disease symptom but induced protection against subsequent challenges with homologous and heterologous organisms for up to 24 months. After 30 months this protective immunity began to decrease. A similar protective immunity could be induced by administration of the A- B+ derivative CVD101 of V. cholerae strain 395. This type of experiment can only be done successfully with conventional, healthy rabbits held under low stress conditions. A so-called specific pathogen-free rabbit breed was found to be entirely unsuitable. Duodenal inoculation with heat- or merthiolate-inactivated V. cholerae for a prolonged period of time by means of an intestinal osmotic minipump did not induce protection. Injection of heat-inactivated V. cholerae material into the Peyer's patches sometimes led to protection, suggesting that a thermostable antigen, possibly lipopolysaccharide, is one of the major protective antigens. Duodenal administration of a combination of inactivated V. cholerae serotypes Ogawa and Inaba cells and 1 mg B subunit of the V. cholerae enterotoxin by up to three inoculations protected only 3 out of 12 rabbits against challenge. The results obtained on the rabbit model are discussed in relation to the efficacy of this vaccine in human volunteers and in a recent field test.

The intracellular localization of enterotoxin in Escherichia coli AP1, a strain of porcine origin which produces high levels of heat-labile, but no heat-stable enterotoxin, has been examined. The cytoplasmic and outer membranes of this strain both contained enterotoxin activity, while the membranes isolated from a serologically related non-enterotoxigenic strain (E. coli AP2) also of porcine origin, did not show enterotoxin activity. The periplasmic fraction isolated from the enterotoxigenic strain contained considerable enterotoxin activity, but this activity was associated with outer membrane fragments present in the periplasmic fraction. Thus, of the total cellular enterotoxin activity, about 55%, 15% and 30% were present in the outer membrane, cytoplasmic membrane and the cell cytoplasm, respectively. The specific activity of enterotoxin was 20 units per mg protein in the cytoplasm and 90 and 150 units per mg protein in the cytoplasmic and outer membranes, respectively.

Of 1,004 isolates of Escherichia coli obtained during the spring of 1975 in seven different states from calves with diarrhea, 124 isolates were enterotoxigenic based upon ability to cause distention of the calf ligated intestinal segment. Isolates of enterotoxigenic E. coli (ETEC) were obtained from calves in six of the seven states. ETEC were detected in calves in 118 of 355 herds in Montana during the 1974 and 1975 spring beef calving seasons. The occurrence and serotypes of ETEC isolated from calves in states outside Montana were similar to ETEC isolated from calves in Montana. One hundred and fourteen of the 124 isolates of ETEC were placed in one of six different groups upon agglutination in OK antiserum. Serotyping of 35 of the 124 isolates of ETEC indicated the following serotype for isolates in each group: group 1, O9:K35; group 2, O101:K30; group 3, O8:K85; group 4, O20:K?; group 5, O8:K25; and group 6, O101:K28. Determination of the presence of K99 antigen indicated that 28 of 35 isolates of ETEC had K99 antigen, whereas the antigen was not detected in any of the 10 isolates of non-ETEC studied.

Cholera disease can be induced in the rabbit by ligation of the cecum (C) followed by duodenal inoculation (DI) of virulent Vibrio cholerae organisms (DIC model). When the cecum is not ligated, DI does not induce disease. In contrast, the animals are primed which becomes apparent upon challenge with live V. cholerae in the DIC model. Such animals are vibriocidally protected. This protection is characterized by absence of disease symptoms, rapid disappearance of V. cholerae from the feces and presence of high levels of anti-lipopolysaccharide Immunoglobulin A in the bile. The present study shows that primed rabbits can also be boosted by duodenal administration of killed, smooth V. cholerae cells. On the other hand, killed cells cannot prime. The minimal lethal dose of a rough derivative of a smooth strain C5, designated R5 and lacking the O antigen part of the LPS, was 100,000 times higher than that of its parent strain C5, in the DIC model. Rabbits which had been duodenally immunized with strain R5 and were subsequently challenged with the smooth strain C5, all developed diarrhea and two out of eight died. This result supports an earlier observation that the specific O antigen part of the V. cholerae LPS is an essential prerequisite for the induction of protective immunity in the rabbit.