Untypable Isolates Research Articles

Comparative Analysis of Salmonella typhi by rRNA Gene Restriction and Phage Typing

Phage typing and ribotyping were used to analyze 19 isolates of Salmonella typhi from sporadic cases and two outbreaks of typhoid fever in Malaysia in 1987 and 1990. The two outbreaks were associated with phage types D1 and E1 in Penang and Alor Setar respectively, and phage types D1, E1 and A (with 3 untypeable isolates) were present among the sporadic cases. Ribotyping detected 14 ribotypes among the 19 isolates thus establishing its higher discriminative capacity compared to phage typing. The outbreak isolates were more homogeneous by ribotyping and the data also suggested that one outbreak was propagated, multi-focal in nature (Penang) while another was a point- source traceable to a single event (Alor Setar).

Molecular typing of enterohemorrhagic Escherichia coli O157:H7 isolates in Japan by using pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was applied for molecular typing of 825 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates, most of which were from 19 outbreaks and 608 sporadic cases in Japan, mainly in May to August 1996. By PFGE, the EHEC O157:H7 isolates were classified into six types (type I to V and ND [nondescript]) and UT untypeable isolates. Fifty isolates from seven outbreaks in May to June and 60 isolates from patients with sporadic cases of infection showed almost identical PFGE patterns which differed in only 1 of 22 DNA fragments. They were classified into type I. Ninety-nine isolates from 10 other outbreaks and 156 isolates from patients in the Kinki area with sporadic cases of infection obtained in the early summer of 1996 showed identical PFGE patterns, suggesting that they were derived from one huge outbreak. They were classified into type II. Type IV EHEC isolates, which had only the stx2 gene, caused another outbreak in a primary school in June. EHEC isolates of two other types, types III and V, were not related to the outbreak but were isolated in several parts of Japan. ND EHEC isolates included a variety of patterns which could not be classified into either of the types mentioned above. Twenty-five isolates could not be analyzed due to degradation of their genomic DNAs and were represented as UT. These results indicate that EHEC O157:H7 strains with various PFGE types have already spread to Japan and caused the multiple outbreaks and sporadic infections in Japan in the summer of 1996.

Hepatitis C virus replicative levels and efficiency of genotyping by specific PCR and antibody assay.

We studied factors which influence the detection of hepatitis C virus genotypes by the group-specific PCR of the sequence within the core region gene and by the newly developed genotype-specific NS4 antibody assay. Genotyping was performed on 75 hepatitis C virus carriers in Japan, where patients with hepatitis C viremia are exclusively infected with genotype 1b, 2a, and 2b. PCR failed to identify genotypes in 8 (11%) patients, whereas 12 (16%) patients, including the 8 patients mentioned above, could not be genotyped by the serological assay. Serological genotypes showed almost complete agreement with those found by the PCR except that double infection was revealed in only two of the eight patients serologically judged to be coinfected with genotypes 1 and 2. In each assay, disease activity and levels of viremia assessed by a competitive reverse transcription PCR assay were significantly lower in patients infected with untypeable isolates than in those infected with typeable ones (P < 0.01). The PCR could identify the genotypes of isolates from all 64 patients with levels of viremia of > or = 10(6) copies/ml, and the genotype-specific antibody responses were found in 60 (94%) patients. In contrast, isolates from only 3 (27%) of 11 patients with low levels of viremia (< 10(6) copies/ml) could be genotyped by the PCR (P < 0.00001), and these patients showed the genotype-specific antibody responses (P < 0.00001). These findings suggest that low levels of hepatitis C virus replication may reduce the efficiency of genotyping by serological assay as well as by PCR.

Molecular characterisation of the 3'-end of the astrovirus genome.

SummaryWe have sequenced the 3′-end of the RNA genomes of 14 serotyped and 12 untyped isolates of human astrovirus. The sequences, which include all 8 serotypes, were used to predict secondary structures, postulate possible functional domains, reveal conserved regions suitable for nucleic acid amplification and perform phylogenetic analysis. The final nucleotides of the capsid protein precursor gene and the adjacent 3′-noncoding region were highly conserved and, except for 35 nucleotides with homology to a sequence in the 3′-end of a coronavirus RNA genome, unique to astrovirus family. This confirms that the 3′-end is a suitable target for universal and specific detection of astrovirus RNA. For the deduced 72 C-terminal amino acids of the capsid protein precursor, distances between the serotypes were found to vary from 0.1 substitution per site between serotypes 3 and 7 to more than one substitution per site between serotype 4 and the other serotypes. Different isolates of the same serotype were closely related, which indicates that the presently used typespecific antibodies differentiate between phylogenetically distinct groups. RNA secondary structures with minimal free energy were predicted using computer programs. Comparative sequence analysis verified the significance of certain of the predicted structural elements.

Prevalence of Salmonella organisms in swine feed

Objective To test feed and feed ingredients on swine farms for Salmonella organisms and to analyze data from these farms to determine risk factors associated with Salmonella organisms in the feed and feed ingredients. Design Epidemiologic survey and retrospective case-control study. Sample Population 30 swine farms. Procedure Samples of feed and feed ingredients and information regarding herd characteristics were collected from 30 swine farms. Samples were tested for Salmonella organisms, and data compiled from herd information forms were examined for associated risk factors between herd characteristics and isolation of Salmonella organisms. Results Salmonella organisms were isolated from 36 of 1,264 (2.8%) feed and feed ingredient samples and from 14 of 30 (46.7%) farms. Thirteen Salmonella sp serotypes and 2 untypeable isolates were cultured. Recovery of Salmonella organisms from at least 1 feed or feed ingredient on a farm was significantly associated with 6 herd characteristics (lack of bird-proofing, using farm-prepared feed for finishing-age pigs rather than purchased feed, and housing pigs in facilities other than total confinement in the growing, finishing, gestating, and breeding stages of production, respectively). Isolation of Salmonella sp was not associated with a history of salmonellosis on a farm. Clinical Implications Salmonella organisms were readily isolated from samples of feed and feed ingredients, illustrating that salmonellae are ubiquitous in a farm environment. Implementing sanitary and pestcontrol measures continues to be a prudent recommendation. Salmonella serotypes found in feed and feed ingredients have the potential to cause disease in pigs that consume the feed or, ultimately, in people that consume pork. (J Am Vet Med Assoc 1997;210: 382–385

Characterisation of Dichelobacter nodosus isolated from footrot in sheep and goats in Nepal

Cases of footrot from sheep and goats in 45 village flocks in Nepal were cultured for Dichelobacter nodosus. Six hundred and eighty-two isolates were obtained from 25 flocks, of which 527 were derived from field isolation, and 155 from a vaccination trial. Of the 527 field isolates, 477 (90.5%) were provisionally classified as serogroup E. The remainder of those classified were either serogroup B (20) or serogroup C (14). Sixteen isolates were not classifiable by slide agglutination tests with antisera to any of the nine recognised serogroups of D. nodosus. Representative serogroup E isolates were all elastase positive and produced thermostable proteases. Those of serogroup B were either elastase positive and thermostable or elastase negative and thermolabile while those of serogroup C and the untypable isolates were elastase negative and thermolabile. Initially, all D. nodosus isolates from the field were of serogroup E. To increase the likelihood of isolating other serogroups, 21 Baruwal sheep affected with footrot were treated twice 30 days apart with a vaccine based on the Nepalese variant of serogroup E. Thirty days after receiving a second dose of vaccine, 17 of 20 affected animals were free of clinical footrot compared with 5 of 17 unvaccinated animals. Whereas at the beginning of this experiment only serogroup E could be recovered, at its conclusion although serogroup E persisted in the unvaccinated animals, isolates of serogroups B and C, and one untypable isolate were obtained from vaccinates. The demonstration of two virulent strains of D. nodosus (serogroup B and E) in these investigations provided an incentive to proceed with specific vaccination as a method of footrot management in these flocks.

Survey of equine rotaviruses shows conservation of one P genotype in background of two G genotypes.

DIG-labelled ssRNA probes were prepared from variable regions of VP4 and VP7 cognate genes, and used in hybridization assays for P and G genotyping of group A cell culture-adapted equine rotaviruses and fecal samples collected from foals with and without diarrhea. The probes confirmed known P and G serotypes of sixteen cell culture-adapted strains. From one-hundred and twenty-one rotavirus-positive samples, 83 reacted when tested for their P and G genotype specific probes. From these, 71 were found to contain G3 P12 genotypes, and 11 G14 P12 genotypes. No sample reacted with H1 or L338 P and G genotype probes. This suggests that the equine rotavirus population is conservative, containing predominantly one P genotype and two G genotypes. One isolate (26/94) whose dsRNA was visualized in an agarose gel did not react with any of the equine probes, and was found to belong to G8 and P1 genotypes. This is the fourth example of a single unique equine isolate (after H1, L338, and R-22). The remaining thirty-eight untypable field isolates had no detectable dsRNA after storage for 1 to 3 years.

Intra-specific diversity within Pasteurella trehalosi based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins.

Intra-specific diversity within Pasteurella trehalosi was investigated by analysis of variation of capsular polysaccharide, and lipopolysaccharide (LPS) and outer-membrane protein (OMP) profiles. Sixty isolates of P. trehalosi, from diverse geographical locations within the UK, were examined. Capsular polysaccharide serotypes were determined by indirect haemagglutination assay; LPS and OMP profiles were compared by SDS-PAGE analysis. Capsular serotyping identified three isolates of serotype T3, 18 isolates each of serotypes T4, T10 and T15, and three untypable (UT) isolates. Analysis of LPS and OMP profiles identified six smooth LPS types and four OMP types among the 60 isolates. Forty-five (75%) of the isolates belonged to a single OMP type whereas 52 (87%) of the isolates possessed one of three LPS types. Each typing method, by itself, was not very discriminating but when the data from the three methods were combined, the 60 isolates could be separated into 14 distinct subgroups containing from one to 16 isolates as follows: serotype T3, two subgroups; serotype T4, four subgroups; serotype T10, two subgroups; serotype T15, five subgroups; UT isolates, one subgroup. Certain subgroups were associated with only one serotype whereas other subgroups were common to two or more serotypes. The subgroupings were capable of differentiating between isolates of the same serotype from the same and different geographical origins. Based on their LPS and OMP profiles, isolates of serotypes T4 and T15 were more closely related to each other than to isolates of serotype T10; serotype T4 and T15 isolates were also more heterogeneous than those of serotype T10. Certain isolates of serotype T10, recovered from a wide geographical area, were characterized by the possession of a unique capsule/LPS/OMP combination and represented a single clonal group which was responsible for a large proportion (31%) of recent disease outbreaks. Overall, a combination of capsular serotyping, and LPS and OMP typing, was found to be extremely useful for assessing diversity within P. trehalosi and should be of value for epidemiological and virulence studies.

Multiple serotypes and strains of Streptococcus suis in naturally infected swine herds.

We present a brief description of the epidemiology, clinical signs, and lesions in 21 accessions in which multiple serotypes of S. suis or multiple distinct isolates (strains) of the same capsular serotype of S. suis were identified. Isolates of the same capsular serotype that had different antibiogram profiles were considered to be distinct isolates or “strains” for the purposes of this study. Streptococcus suis isolates of the same capsular serotype and identical antibiogram profiles were considered to be identical organisms. These cases were selected to determine whether or not herds infected with multiple serotypes (or strains) of S. suis had unique features that might serve to distinguish these herds from those infected with a single serotype and strain of S. suis and to provide additional information on the development of this disease in swine. Selected cases were limited to those in which isolates were identified by capsular serotyping. Untypeable isolates of S. suis were not included in this study. Case selection criteria, data collection procedures, etc., were as previously described. 12 Of 277 accessions in which S. suis was identified, 21 accessions (7.6%) involving 37 pigs were cases in which 46 different isolates of S. suis were identified. The distribution of these isolates is shown in Tables 1 and 2. Because of the small sample size, statistical analyses were not performed. Although there was a slight increase in recovery of S. suis in the fall and winter months for all serotypes, S. suis was readily isolated throughout the year. In this study, there was a slight increase (approximately 5%) in the prevalence of serotypes 3, 7, and 8 and a decrease (approximately 10%) in the prevalence of serotype 2 from those reported previously. 12

Serological characterisation and antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pigs in Spain

Seventy-one isolates of Actinobacillus pleuropneumoniae isolated from the lungs of pigs in outbreaks of pleuropneumonia in Spain were serotyped by indirect haemagglutination. Serotype 4 (42.2 per cent), serotype 7 (22.5 per cent) and serotype 2 (12.8 per cent) were predominant, whereas serotypes 1, 3, 6, 8, 9, 12 and untypable isolates were present only in small numbers. Serotypes 1, 2, 4 and 7 originated mainly from cases of acute pleuropneumonia, whereas serotypes 3, 6, 8, 9 and 12 were associated with chronically infected herds. The susceptibility of the isolates to 20 antimicrobial agents was determined by agar disc diffusion. Most were susceptible to cefuroxime, cefaclor, cefazolin, kanamycin, tobramycin, gentamicin, oxolinic acid, ciprofloxacin, enoxacin, thiamphenicol, colistin and trimethoprim/sulphamethoxazole. Marked resistance was found with amoxicillin, ticarcillin, oxytetracycline, doxycycline and metronidazole. Rifampicin, fosfomycin and tiamulin were the agents most effective against the isolates tested.

Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization.

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.

Antibiotic susceptibility of 206 Haemophilus influenzae isolates collected from children in central Italy.

Susceptibility of 206 H. influenzae isolates was evaluated by disk diffusion method for 11 antimicrobial agents. No isolates were found to be resistant to third-generation cephalosporins, amoxicillin+clavulanic acid, gentamicin and ciprofloxacin. Four untypable isolates (1.9%) were beta-lactamase producing ampicillin-resistant; one of these was also resistant to chloramphenicol. The rate of resistance against rifampin was 0.5 percent.

Capsular Polysaccharide Serotypes of Coagulase-Positive Staphylococci Associated with Tenosynovitis, Osteomyelitis, and Other Invasive Infections in Chickens and Turkeys: Evidence for New Capsular Types

To study the role of capsular polysaccharides in staphylococcal infections in chickens and turkeys, we surveyed 101 coagulase-positive isolates obtained from investigators in geographically diverse locales in the United States and Canada. Fifty-six isolates were obtained from chickens; 51 (91%) were type 5, and 5 (9%) were type 8. Forty isolates were obtained from turkeys; 13 (33%) were type 5, 15 (38%) were type 8, and 12 (29%) were untypable. Five type 5 isolates were obtained from poultry of uncertain species. Two untypable turkey isolates were studied further. Rabbit serum obtained after immunization with formalin-killed staphylococci was type-specific as assessed by a tube agglutination test and promoted opsonophagocytosis of the isolate that had been used for immunization. Chromatography of carboxy-reduced capsular polysaccharide from one of the isolates suggested that mannoseaminouronic acid was present in the unreduced polysaccharide. The spectrum obtained by 13C-nuclear magnetic resonance suggested the presence of N-acetyl sugars such as mannoseaminouronic acid and 6-deoxy sugars such as N-acetyl fucosamine. The structure of the polysaccharide differed from that of the type 5 and type 8 capsular polysaccharides. Thus, poultry isolates can be classified into at least four serotypes on the basis of their capsular polysaccharides. This observation provides a new focus for investigation into the epidemiology and virulence determinants of this important pathogen in chickens and turkeys.

Identification to the Genospecies Level of Aeromonas Strains Isolated from Raw Meat and Carp, and Evaluation of Their Biological Activities

During the period from January to October 1992, a total of 214 samples (100 raw pork, 94 raw chicken and 20 carp) obtained in Ibaraki prefecture were examined for the presence of Aeromonas. It was found that 42 (42%) of 100 raw pork samples, 30 (32%) of 94 raw chicken samples and 13 (65%) of 20 carp were contaminated by some Aeromonas. The population of Aeromonas in contaminated samples was mostly below 102cfu/g. Attempts to identify the isolates by the biochemical method (phenospecies) revealed that 26 (19%) of 137 isolates belonged to Aeromonas hydrophila groups, 5 (2.3%)to A. caviae, 59 (27.5%) to A. sobria groups, and 4 (1.9%) to A. jandaei; the rest 10 (4.7%) were untypable.On the basis of genetic classification by DNA-DNA hybridization in microdilution wells, 13 of 26 A. hydrophila group isolates were reidentified as A. hydrophila. Eight of 87 A. sobria group isolates were identified as A. sobria and 74 as A. veronii biotype sobria. All of 7 A. jandaei isolates hybridized with the type strain. One each of the 10 untypable isolates hybridized with A. sobria and A. veronii biotype sobria.All of the isolates were investigated for hemolytic activity to rabbit and sheep erythrocytes, and cytotoxicity to HeLa 229 cells. Many strains showed both activities in A. hydrophila groups, A. veronii biotype sobria and A. jandaei, suggesting that these activities were associated. In A. caviae and A. sobria, many strains lacked these activities.

Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis

Polyvalent coagglutination reagents (PRs) have been evaluated for the serotyping of Streptococcus suis. Monovalent antisera produced against 28 S. suis reference strains have been grouped to obtain five different pools. A total of 249 field isolates previously identified and belonging to different serotypes were tested with PRs prepared by two different procedures: (i) monovalent coagglutination reagents were individually prepared and mixed in equal proportions, and (ii) antisera were mixed in equal proportions before the addition of the Staphylococcus aureus suspension. Only antisera tested by a tube agglutination test with 2-mercaptoethanol and presenting titers of 1:32 or higher were used. Results obtained with PRs prepared by both procedures were similar, and there was a very good correlation between the capsular type of the isolate and the reaction obtained with PRs. Thus, from a practical viewpoint, it is suggested that PRs be prepared by the first procedure. To isolates, were tested in parallel with both the PRs and the monovalent coagglutination reagents over a 1-year period. Ninety-nine percent of the typeable and all of the untypeable isolates were correctly identified. Serotyping with PRs is suggested to be a very useful and reliable screening procedure, particularly when a large number of S. suis isolates have to be serotyped. In addition, the choice of antisera to be included in a given pool is facultative and should be oriented to the needs of a region or a country.

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.

Characterization of six new capsular types (23 through 28) of Streptococcus suis

Six new capsular types of Streptococcus suis (types 23 to 28) are described. All reference strains were isolated from diseased pigs and were morphologically and biochemically similar to previously described capsular types 1 to 22. Clear and specific reactions were obtained for each of the new capsular types with three different typing techniques; no cross-reactions were detected among them or with other S. suis capsular types. Their capsular material presented similar ultrastructural characteristics, as shown by electron microscopy, and fimbriae similar to those described for other capsular types of S. suis were observed. When untypeable field isolates were tested with antisera raised against the six new capsular types, capsular type 23 appeared to be the most prevalent, representing more than 50% of all these isolates. Most isolates were recovered from cases of pneumonia, septicemia, and meningitis. Presumptive biochemical identification described for S. suis capsular types 1 to 22 may also be used for capsular types 23 to 28.

Isolation and Characterization of Streptococcrcs Suis Capsular Types 9-22

The incidence and biochemical patterns of Streptococcus suis capsular types 9-22 are presented. Of 148 untypeable (with types 1-8 antisera) isolates of S. suis recovered from diseased pigs, 10% were not capsulated. Of the remaining 134 isolates, only 53% belonged to capsular types 9-22; capsular types 22 and 9 were the most prevalent, representing 19% and 13%, respectively. Capsular type 15 (de Moor's group T Streptococcus) is reported here for the first time in North America since it was described in 1963 in Europe. Of 188 untypeable isolates recovered from clinically healthy pigs, 25% were noncapsulated. Of the remaining 141 isolates, 90% belonged to the new capsular types, and 87% were identified as 1 of 4 types: 17, 18, 19, and 21. Capsular types 12 and 20 were not detected among the Canadian isolates. Almost half of strains were arginine dihydrolase-negative, and 45% fermented mannitol, which is seldom a positive test with capsular types 1-8. Although some strains were negative with salicin or trehalose, none were negative for both sugars. Only 54% of isolates tested with 1 rapid multitest system were correctly identified as S. suis. A tentative biochemical profile that might be used with a microplate identification system is also presented. Biochemical identification using the conventional system instead of the rapid multitest system is preferable.

Proposed revisions to the serological typing system forTreponema hyodysenteriae

Antisera were prepared in rabbits against seven well-characterized strains of Treponema hyodysenteriae of known serotype, and reacted in agarose gel double immunodiffusion tests (AGDP) with lipopolysaccharide (LPS) extracted from 18 Western Australian isolates of the organism. Eight isolates were provisionally typed by this method, but sera raised against one 'typed' and two 'untypable' local isolates reacted in an unexpected fashion with LPS from other local and type strains. Serum raised against the 'typed' local isolate reached with LPS from other previously untyped local isolates: this indicated the presence of more than one major LPS antigen amongst certain local isolates, and was confirmed by cross-absorption of sera. Sera raised against apparently untypable local isolates reacted with LPS from certain type organisms, thus suggesting the presence of complex antigenic relationships between LPS antigens. The serotyping system for T. hyodysenteriae which was proposed by Baum & Joens (1979) uses unabsorbed antisera and is made unworkable by these observations. Instead we propose placing organisms which share common LPS antigens into serogroups A to E, members of which are defined by their reactivity with unabsorbed sera raised against a type organism for the group. We suggest strains B78, WA1, B169, A1 and WA6 respectively as being the most suitable type organisms for the five serogroups identified so far. Isolates possessing additional unique LPS antigens can be regarded as serotypes within the serogroup. However the serotype of an isolate can only be established if antiserum is prepared against it, and this serum continues to react homologously after cross-absorption with bacteria from other serotypes within the serogroup.

In vitro and in vivo Characterization of Avian Escherichia coli. I. Serotypes, Metabolic Activity, and Antibiotic Sensitivity

Over a 2 1/2-year period (January 1981 to June 1983), 177 Escherichia coli isolates were obtained from 145 field-reared broiler flocks in the Delmarva peninsula, and 20 were obtained from clinically normal day-old hatchery chickens representing an additional 17 flocks in Delmarva. Ninety-one isolates obtained from the field-reared birds between 2 and 8 weeks of age were associated with complicated air-sac disease. Serotyping efforts demonstrated a predominance of O2, O35, and O78 serogroups and a large number of untypable isolates. More than 50% of the isolates in each of the three dominant serogroups were collected from broilers with colibacillosis, but they were never detected in the yolk-sac samples of clinically normal day-old hatchery chickens. In vitro biochemical characterization of the E. coli isolates revealed variable rates of carbohydrate fermentation and amino acid decarboxylation. No common characteristics appeared to be shared by the predominant serogroups isolated from clinically affected birds, although several serogroup-specific reactions were noted. The majority of the isolates were sensitive to chloramphenicol, gentamicin, nalidixic acid, ormethoprim-sulfadimethoxine, spectinomycin, neomycin, and ampicillin. About half of the isolates were sensitive to nitrofurantoin and the sulfa compounds. Less than 25% of the isolates were sensitive to streptomycin, erythromycin, tetracyclines, novobiocin, penicillin, bacitracin, and lincomycin.