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JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1987, p. 1248-12520095-1137/87/071248-05$02.00/0Copyright © 1987, American Society for Microbiology

Atypical Campylobacters Associated with GastroenteritisWEE TEE,'* BRUCE N. ANDERSON,2 BRUCE C. ROSS,2 AND BRIAN DWYER'

Campylobacter Laboratory, Department of Clinical Pathology,1 and Virology Laboratory,2 Fairfield Hospital,Victoria 3078, Australia

Received 29 December 1986/Accepted 17 March 1987

Nine strains of Campylobacter species other than Campylobacter jejuni, Campylobacter coli, and Campylo-bacter laridis were isolated from patients with acute diarrhea. Ali nine strains showed preferred growth at 37°Cunder microaerophilic conditions. Conventional microbiological tests and DNA-DNA dot blotting were used toidentify these strains. Three of the nine Campylobacter strains hydrolyzed hippurate, reduced nitrate, producedcatalase, were resistant to cephalothin, and were shown to be highly related to C. jejuni type strains. Twostrains had negative or weak catalase activity and were hippurate negative. Three other strains hadcharacteristics similar to those of Campylobacter cinaedi. The ninth strain, isolated from a homosexual manwith antibodies to human immunodeficiency virus (human T-cell lymphotropic virus type III), showed uniquefeatures different from those of all the known campylobacters used in this study. This strain grew well at 25 and37°C and was catalase and nitrate positive, hippurate negative, and resistant to cephalothin.

Enteric pathogens of the genus Campylobacter have beenincreasingly well defined in recent years. Campylobacterjejuni, Campylobacter coli, and Campylobacter laridis arewell established etiologic agents of human gastroenteritis. Ofthese, C. jejuni is most commonly encountered. Campylo-bacter fetus is primarily a cause of systemic illness in themalnourished or immunosuppressed, although it is occasion-ally implicated as a cause of bacterial diarrhea (7).The identification and differentiation of clinical Campylo-

bacter isolates is based on a limited number of morphologi-cal and biochemical criteria, and misidentification may resultfrom atypical reactions. Alternative approaches to straindifferentiation include gas-liquid chromatographic analysisof cellular fatty acid composition (11), whole cell proteinelectrophoretic profile studies (6), and determination ofDNA base composition (16, 24).The recent emergence of new strains, such as Campylo-

bacter fennellae, Campylobacter cinaedi (5), Campylobac-ter pyloridis (14), and atypical Campylobacter strains iso-lated from children with gastroenteritis in Central and SouthAustralia (21), and the increasing appearance of reports ofunusual biochemical reactions of known campylobacters (4,7) necessitate the use of more sophisticated methods forspecies differentiation and identification.With the introduction of two temperatures, 37 and 43°C,

for routine culture of campylobacters from patients' fecalspecimens, we have isolated a number of atypical strains. Inthis study, we used DNA-DNA hybridization dot blotting todetermine whether these atypical strains belong to uniquespecies or are variants of previously described Campylobac-ter species.

MATERIALS AND METHODS

Bacterial strains. The reference strains used in this studywere C. jejuni NCTC 11351, C. coli NCTC 11366, C. laridisNCTC 11352, C. fetus NCTC 10842, "Campylobacterfaecalis" NCTC 11415, Campylobacter venerealis NCTC10352, Campylobacter sputorum subsp. mucosalis NCTC11000, Campylobacter concisus NCTC 11485, Campylobac-ter hyointestinalis NCTC 11608, C. fennellae 906, C. cinaedi

* Corresponding author.

255, N03-negative strain 093, catalase-negative or -weak-positive (CNW)-like Campylobacter sp. strain 267, and C.pyloridis 101. The reference strains (NCTC-designatedstrains) were obtained from the National Collection of TypeCultures, London, England. C. cinaedi and C. fennellaewere kindly supplied by Cynthia Fennell, Seattle, Wash.,and C. pyloridis was recovered from an antral biopsy of apatient with a duodenal ulcer. Two other strains, CNW-likeCampylobacter sp. strain 267 and N03-negative Campylo-bacter sp. strain 093, were kindly supplied by Trevor Steeleof the Institute of Medical and Veterinary Science, Adelaide,South Australia, Australia. The origin and designation ofeach of the nine atypical Campylobacter strains under inves-tigation are shown in Table 1. The nine test strains wereisolated from the feces of patients with acute diarrhealgastroenteritis admitted to this hospital. Of the nine patients,four were returned travelers from Southeast Asia, two werechildren under 3 years old, two were otherwise asympto-matic homosexual males with antibodies to human immuno-deficiency virus (human T-cell lymphotropic virus type III),and one had no apparent risk factors.

Isolation and identification. All nine strains recovered frompatients' feces were isolated on 6% horse blood agar (Co-lumbia agar base) containing vancomycin (10 ,uglml), poly-myxin B (1,250 IU/ml), and trimethoprim (10 ,ug/ml). A dropof fecal suspension (1 g of feces in 10 ml of saline agitatedinto suspension with glass beads) was plated in duplicatedirectly onto selective plates. The plates were incubated at37°C (7 days) and 43°C (4 days) in 10% C02-6% 02-84% N2.All the cultures were examined daily. Oxidase-positive,gram-negative, motile, spiral or curved rods which did notgrow in air at 37°C were subjected to further characteriza-tion.

Physiological and biochemical characterization. Tests fornitrate reduction, catalase production, H2S production in thepresence and absence of 1% cysteine hydrochloride (lead-acetate strip method using brucella broth as base), growth in2.5% NaCI, growth in 1% glycine, susceptibility to naladixicacid (30-,ug disk) and cephalothin (30-,ug disk), selenitereduction, and tolerance to 0.04% triphenyltetrazolium chlo-ride were performed by methods previously described (3, 8,

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ATYPICAL CAMPYLOBACTERS AND GASTROENTERITIS

TABLE 1. Description of bacterial strains

PatientTeststrain Age Sex Occupation Overseas

Sex ~~~~~~~~~~~travelC899 ? Female Not given (friend of donor India

of C900 isolate)C900 28 yr Male Unemployed teacher IndiaC380 22 yr Female College student IndiaC033 1 yr Female ChildC790 13 mo Male ChildC948 29 yr Female Missionary worker IndonesiaC902 35 yr Male' TeacherC767 29 yr Maleb UnemployedC004 37 yr Male Geologist

a All strains were isolated from stools.b Homosexuals.

12). Hydrolysis of sodium hippurate was assessed by themethod of Hwang and Ederer (9).

Extraction and purification of Campylobacter DNA. All thestrains were subcultured onto 6% horse blood agar (Colum-bia) and incubated at 37°C microaerophilically for 6 days.Bacterial cells from 20 plates were harvested with cottonswabs and suspended in 20 ml of TE buffer (10 mM Tris, 1mM EDTA [pH 8]). The isolation and purification of theDNA was accomplished by following closely the method ofTotten et al. (23), except that DNA was precipitated withcold ethanol. The DNA pellet was dissolved in 0.42 M NaCiin TE. The purity and approximate concentration of theisolated DNA was assessed by comparing the electropho-retic patterns with those displayed by standard DNA prep-arations on agarose gels.

Preparation of DNA for hybridization. A series of eightdoubling dilutions of each DNA preparation was made in 1M ammonium acetate, and 20-,ul samples of each dilutionwere transferred to nitrocellulose filters housed in a Bio-dotapparatus (Bio-Rad Laboratories, Richmond, Calif.) usingthe conditions specified by the manufacturer. The filterswere dried in air and then baked at 80°C for 2 h beforehybridization.

Preparation of DNA probes. For each strain under study,approximately 500 ng of DNA was labeled with 10 ,uCi of[32P]dATP by nick translation (22), and unincorporated[32P]dATP was removed by column chromatography throughSephadex G75 (Pharmacia, Uppsala, Sweden). LabeledDNAs to be used as probes in hybridization reactions weredenatured by boiling for 5 min.DNA-DNA hybridization. We hybridized the DNA by the

method of Maniatis et al. (13). Baked nitrocellulose filterswere prehybridized at 42°C for 4 h in 50 pI of prehybridiza-tion solution (18) per cm2 before the addition of the 32p_labeled DNA probe. Hybridization was performed overnightat 42°C, and the filters were then washed twice at roomtemperature for 5 min each in double-strength SSC (l x SSCis 0.15 M NaCI plus 0.015 M sodium citrate) with 0.1%sodium dodecyl sulfate and then twice in lx SSC-0.1%sodium dodecyl sulfate for 30 min at 50°C (13). The filterswere dried in air and exposed to X-ray film between inten-sifying screens at -70°C for 1 to 2 days. The film was

developed and examined for specific hybridization of theprobe to the test samples bound to the nitrocellulose filters.Probes prepared from all strains were then examined in a

checkerboard fashion against DNA preparations from thesame organisms. We undertook a series of cross-hybrid-ization studies using these strain-specific DNAs labeled with

32 as probes of nitrocellulose filters spotted with serialdilutions of target DNA from the same 23 strains.

RESULTSFrom February 1985 to August 1986, Campylobacter

strains were isolated from fecal specimens of 157 of 1,005patients attending Fairfield Hospital for investigation ofdiarrhea. Nine strains (5.7%) showed atypical biochemicalreactions or unusual growth temperature requirements whencompared with C. jejuni (86.6%), C. coli (7.7%), or C. laridis(0%). Results of differential tests with type strains of variousCampylobacter species and the test strains are shown inTables 2 and 3. All the strains were unable to grow aerobi-cally at 37°C but grew well microaerophilically. Electronmicroscopy showed curved bacteria with a single flagellumat either one or both ends.

All except three strains grew at 37°C but not at 43°C; alltook 3 to 5 days to grow. Strains C899, C900, and C380 grewat 43°C but showed weak growth compared with that ob-tained at 37°C. These three strains were catalase positive,nitrate positive, hippurate positive, and susceptible tonaladixic acid, and they produced H2S. All were resistant tocephalothin. These strains showed biochemical reactionssimilar to those of the C. jejuni type strain, except preferredgrowth temperature.Two strains, C033 and C790, grew only at 37°C and had

the following biochemical features: negative or weak-posi-tive catalase reaction, nitrate positive reaction, cephalothinsusceptibility, naladixic acid susceptibility, and H2S-variable reaction. These strains appeared to have biochem-ical reactions similar to those shown by the CNW-likecampylobacters of Steele et al. (21).The remaining four strains, C948, C767, C004, and C902,

were oxidase and catalase positive (except C948, which wasweakly catalase positive), hippurate hydrolysis negative,and resistant to cephalothin and naladixic acid. These strainsresemble Fennell's C. cinaedi, with the exception of strainC902, which grew at 25°C as well as at 37°C.DNA-DNA hybridization of type strains using C. jejuni

NCTC 11351 as a probe demonstrated that the three hippur-ate-positive strains were highly related to C. jejuni NCTC11351 (Fig. 1). The same result was observed using each ofthese isolates as a probe, suggesting that these three strainswere very likely to be phenotypically atypical C. jejunistrains. Similarly, catalase-negative or weak-positive strainsC033 and C790 showed a high degree of DNA-DNA bindingwith the CNW-like Campylobacter sp. strain 267 of Steele etal. (21), suggesting a high level ofDNA relatedness betweenthese organisms. Strains C767, C948, and C004 were found

TABLE 2. Growth conditions of atypical Campylobacter strains

ReactionaTest Microaerophilic atmospherestrain Anaerobic Air (37'C)

25°C 37°C 43°C (37°C)

C899 - + -/W WC900 - + -/W WC380 - + -/W -C033 - + - - -C790 - + - - -C948 - + - W -C902 + + - + -C767 - + - _ _C004 - + - _ _

a W, Weak growth; -tW, no growth or weak growth.

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TABLE 3. Biochemical characteristics of Campylobacter speciesReactiona

Strain Nitrate Hippurate H2S Growth 0.04% 2.5% Selenite Naladixic CephalothinCatalase Oxidase reduction hydrolysis witehib with TTCC NaCI reduction acid (30.kug (30-etg disk)

cysteHnew glycine disk)

C899 + + + + + + + - + S RC900 + + + + + + + - + S RC380 + + + + + + + - + S RC033 -/W + + - + - - - + S SC790 -/W + + - - - - - - S SC948 -/W + + - + - - - + R RC902 + + + - - - - - + R RC767 + + + - - + + - + R RC004 + + + - - + R R

a S, Susceptible; R, resistant; -/W, weak reaction or no reaction.b Lead-acetate strip.C Triphenyltetrazolium chloride.

to react strongly to a C. cinaedi probe and moderately to a C.fennellae probe, as shown in Table 4. However, the probemade from strain C902 was uniquely specific, showing noDNA-DNA hybridization with any of the type strains andreference strains, including the two strains provided byFennell. Probes made from each individual type strain actedspecifically. In all cases, the genetic grouping agreed withthe phenotypic characteristics of the group, the probe react-ing only with biochemically or physiologically similarstrains.

DISCUSSIONSince King (10) associated thermophilic vibrios with diar-

rhea in humans and with the later development and estab-lishment of selective culture techniques by Skirrow andBenjamin (19, 20) and Butzler et al. (1, 2), culture methodshave been progressively refined to provide optimal recoveryof thermophilic C. jejuni from fecal specimens. However,recent reports by Fennell et al. (5), Steele et al. (21), and

Megraud and Bonnet (15) have shown that the widely usedmethod of microaerophilic incubation at 43°C for 3 to 4 dayswith selective media may preclude isolation of other cam-pylobacters. Indirect support of these observations has beenmade by Paisley et al. (17), who found that in diarrheal stoolsexamined directly by dark-field microscopy for organismswith morphology and motility typical of Campylobacterspecies, C. jejuni was not recovered by culture in 38% ofcases. In this study, we incubated cultures at 37°C for up to7 days, as well as at 43°C for 4 days, using Skirrow medium,which is cephalothin free.The problems and difficulties in differentiating Campylo-

bacter species using the limited biochemical tests availablein most laboratories are well-known. The presence of hip-purate-negative strains of C. jejuni has added more confu-sion to the identification of Campylobacter species. Thethree hippurate-positive strains we describe here are, webelieve, atypical strains of C. jejuni. They conform to theexpected biochemical and physiological profile of C. jejuni,

1 2 3 4 5 6 7 8 3 10 11 12 13 14 15 1 5 17 18 1 )

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FIG. 1. DNA-DNA hybridization dot blot showing differentiation of Campylobacter strains. Doubling dilutions ofDNA prepared from teststrains were placed onto nitrocellulose filters and hybridized with 32P-labeled probe DNA from C. jejuni NCTC 11351. Lanes: 1, C. jejuniNCTC 11351; 2, C. coli NCTC 11366; 3, C. laridis NCTC 11352; 4, C. fetus NCTC 10842; 5, "C. faecalis" NCTC 11415; 6, C. jejuni (patient);7, C. venerealis NCTC 10352; 8, C. sputorum subsp. mucosalis NCTC 11000; 9, C. concisus NCTC 11485; 10, CNW-like Campylobacter sp.strain 267; 11, N03-negative Campylobacter sp. strain 093; 12, strain C380 (patient); 13, C. pyloridis (patient); 14, C. hyointestinalis NCTC11608; 15, C. fennellae 906; 16, C. cinaedi 255; 17, strain C033 (patient); 18, strain C899 (patient); 19, strain C900 (patient).

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ATYPICAL CAMPYLOBACTERS AND GASTROENTERITIS

TABLE 4. DNA relatedness of atypcial campylobacters to representative Campylobacter type and reference strainsusing probes made from all strains

DNA relatednessa of test strain:Reference or type strain

C899 C900 C380 C033 C790 C948 C902 C767 C004

C. jejuni NCTC 11351 + + +C. coli NCTC 11366 - - - - - - - - -C. laridis NCTC 11352C. fetus NCTC 10842 - - - - - - - -

"C. faecalis" NCTC 11415 - - - - - - - -

C. venerealis NCTC 10352 - - - - - - - -

C. sputorum subsp. mucosalis - - - - - - - -

NCTC 11000C. concisus NCTC 11485 - - - - - - - -

C. hyointestinalis NCTC 11608 - - - - - - - -

C. cinaedi 255 - - - - - + - + +C. fennellae 906 - - - - - M - M MC. pyloridis NCTC 101 - - - - -

CNW-like Campylobacter sp. - - - + + - - - -strain 267

N03-negative Campylobacter + + +strain 093a +, Strong positive hybridization; - no detectable hybridization; M, moderately strong hybridization.

with the exception of preferred growth at 37°C and at aslower rate of growth (3 to 5 days as opposed to 2 days).They are of interest because all three strains were importedfrom India. We have yet to isolate these atypical strains fromAustralians who have not traveled abroad.The two catalase-negative strains are morphologically and

biochemically similar to CNW-like strains recovered fromremote aboriginal communities in Central and South Austra-lia as described by Steele and colleagues (21). DNA-DNAhybridization showed that they are highly related to thereference strain CNW-like Campylobacter sp. strain 267.We have not found any nitrate-negative campylobacters inour patient population.Three of the nine strains had biochemical reactions and

high DNA-DNA relatedness similar to those of the type 2Campylobacter-like organisms isolated from homosexualmen with proctitis in Seattle, Wash. This organism has beenofficially designated C. cinaedi. The remaining Campylobac-ter-like organism, C902, had phenotypic characteristics sim-ilar to those of the others, but DNA-DNA hybridizationdifferentiated it from all the other campylobacters.The clinical significance of these campylobacters is not

clear, although all of these patients presented with gastro-enteritis. Conventional enteric bacterial pathogens (salmo-nellae, shigellae, Aeromonas sp., Plesiomonas sp., vibrios,Clostridium difficile) and fecal parasites, including Giardiasp., Entamoeba sp., Cryptosporidium sp., and Blastocystishominis, as well as rotavirus, were sought but not found.Information about the serological response and more de-ta-led epidemiological data are required to clearly establishthe pathogenic role of these atypical Campylobacter strains.Our data show that C. jejuni strains are biochemically and

physiologically variable, especially in relation to preferredgrowth temperature and cephalothin susceptibility. The def-inition of C. jejuni must therefore be expanded to includethese variabilities. DNA-DNA hybridization, as used in thisstudy, is relatively time-consuming but is undoubtedly anextremely useful tool to differentiate organisms to the spe-cies level when conventional methods fail. Phenotypic char-acteristics reflect the expression of genes from only a smallportion ofDNA of an organism. DNA-DNA hybridization isa measure of shared nucleotide sequences throughout the

bacterial genome. This method bas not been sufficientlystandardized for routine use in taxonomic studies; however,it will demonstrate whether the test strain is a new strain oris related to existing known strains by its degree of related-ness. We plan to streamline our procedure to enable a morerapid determination of the relatedness of new isolates toknown Campylobacter strains and to fractionate the probeDNAs to identify pools ofDNA which more readily discrim-inate between related and unrelated strains.

ACKNOWLEDGMENTS

We thank Patrick Edwards for technical assistance.We also thank the Special Purposes Fund, Fairfield Hospital, for

financial assistance to purchase items of equipment used in thisstudy.

LITERATURE CITED

1. Butzler, J. P., P. Dekeyser, M. Detrain, and F. Dehaen. 1973.Related vibrio in stools. J. Pediatr. 82:493-495.

2. Butzler, J. P., and M. B. Skirrow. 1979. Campylobacterenteritis. Clin. Gastroenterol. 8:737-765.

3. Cook, G. T. 1950. A plate test for nitrate reduction. J. Clin.Pathol. 3:359-362.

4. Edmonds, P., C. M. Patton, T. J. Barrett, G. K. Morris, A. G.Steigerwalt, and D. J. Brenner. 1985. Biochemical and geneticcharacteristics of atypical Campylobacter fetus subsp. fetusstrains isolated from humans in the United States. J. Clin.Microbiol. 21:936-940.

5. Fennell, C. L., P. A. Totten, T. C. Quinn, D. L. Patton, K. K.Holmes, and W. E. Stamm. 1984. Characterization of Campylo-bacter-like organisms isolated from homosexual men. J. Infect.Dis. 149:58-66.

6. Ferguson, D. A., Jr., and D. W. Lambe, Jr. 1984. Differentiationof Campylobacter species by protein bapding patterns in poly-acrylamide slab gels. J. Clin. Microbiol. 20:453-460.

7. Harvey, S. M., and J. R. Greenwood. 1983. Probable Campylo-bacter fetus subsp. fetus gastroenteritis. J. Clin. Microbiol.18:1278-1279.

8. Hébert, G. A., D. G. Hollis, R. E. Weaver, M. A. Lambert, M. J.Blaser, and C. W. Moss. 1982. 30 years of campylobacters:biochemical characteristics and a biotyping proposal for Cam-pylobacterjejuni. J. Clin. Microbiol. 15:1065-1073.

9. Hwang, M.-N., and G. M. Ederer. 1975. Rapid hippurate hy-

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drolysis method for presumptive identification of group Bstreptococci. J. Clin. Microbiol. 1:114-115.

10. King, E. O. 1957. Human infections with Vibrio fetus and arelated Vibrio. J. Infect. Dis. 101:119-128.

11. Leaper, S., and R. J. Owen. 1981. Identification of catalaseproducing campylobacter species based on biochemical charac-teristics and on cellular fatty acid composition. Curr. Microbiol.6:31-35.

12. Leuchtfeld, N. W., and W.-L. L. Wang. 1982. Hippurate hydrol-ysis by and triphenyltetrazolium tolerance of Campylobacterfetus. J. Clin. Microbiol. 15:137-140.

13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

14. Marshall, B. J., and J. R. Warren. 1984. Unidentified curvedbacilli in the stomach of patients with gastritis and pepticulceration. Lancet i:1311-1314.

15. Megraud, F., and F. Bonnet. 1986. Unusual campylobacters inhuman faeces. J. Infect. 12:275-276.

16. Owen, R. J., and S. Leaper. 1981. Base composition, size andnucleotide sequence similarities of genome deoxyribonucleicacids from species of the genus Campylobacter. FEMS Micro-biol. Lett. 12:395-400.

17. Paisley, J. W., S. Mirrett, B. A. Lauer, M. Roe, and L. B. Reller.1982. Dark-field microscopy of human feces for presumptivediagnosis of Campylobacterfetus subsp. jejuni enteritis. J. Clin.

Microbiol. 15:61-63.18. Rigby, P. W. J., M. Diekmann, C. Rhoades, and P. Berg. 1977.

Labelling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

19. Skirrow, M. B. 1972. Campylobacter enteritis: a "new" disease.Br. Med. J. 2:9-11.

20. Skirrow, M. B., and L. Benjamin. 1980. "1001" campylobac-ters: cultural characteristics of intestinal campylobacters fromman and animals. J. Hyg. 85:427-442.

21. Steele, T. W., N. Sangster, and J. A. Lanser. 1985. DNArelatedness and biochemical features of Campylobacter spp.isolated in Central and South Australia. J. Clin. Microbiol.22:71-74.

22. Thomas, P. S. 1980. Hybridization of denatured RNA and smallDNA fragments transferred to nitrocellulose. Proc. Natl. Acad.Sci. USA 77:5201-5205.

23. Totten, P. A., C. L. Fennell, F. C. Tenover, J. M. Wezenberg,P. L. Perine, W. E. Stamm, and K. K. Holmes. 1985. Campylo-bacter cinaedi (sp. nov.) and Campylobacter fennellae (sp.nov.): two new Campylobacter species associated with entericdisease in homosexual men. J. Infect. Dis. 151:131-139.

24. Ursing, J., M. Walder, and K. Sandstedt. 1983. Base composi-tion and sequence homology of deoxyribonucleic acid ofthermotolerant campylobacters from human and animalsources. Curr. Microbiol. 8:307-310.

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ERRATUM

Atypical Campylobacters Associated with GastroenteritisWEE TEE, BRUCE N. ANDERSON, BRUCE C. ROSS, AND BRIAN DWYER

Campylobacter Laboratory, Department of Clinical Pathology, and Virology Laboratory, Fairfield Hospital, Victoria 3078, Australia

Volume 25, no. 7, p. 1248, column 1, paragraph 5, lines 4 and 5: "NCTC 10352" should read "NCTC 10354."Page 1250, Fig. 1 legend, fine 4: "NCTC 10352" should read "NCTC 10354."

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