Vol. 49, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1985, p. 949-9540099-2240/85/040949-06$02.00/0Copyright C 1985, American Society for Microbiology

Characterization of the Coliform and Enteric Bacilli in theEnvironment of Calves with Colibacillosis

PATRICIA I. PLEWS,'t MARY C. BROMEL,2t AND ITHEL A. SCHIPPER1*Department of Veterinary Science' and Department of Bacteriology,2 North Dakota State University, Fargo, North

Dakota 58105

Received 10 April 1984/Accepted 13 December 1984

In the first part of the present study the coliform and enteric bacilli in the environment of calves withcolibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli inbarnyard soils were obtained from six cattle ranches. The 0 and K serogroups of E. coli isolates obtained fromthe feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were

compared with the E. coli 0 and K serotypes found in soils. The results showed a reservoir of potentiallypathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthycalves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, totalstreptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. Inaddition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, andtheir indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC typesof coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were

compared with those isolated from unconitaminated soils. All fecal specimens were also examined for thepresence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggestthat the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil, Fromthis study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related toranch husbandary and sanitary practices.

The coliform bacteria are among those that inhabit theintestines of humans and animals. They are gram-negative,short bacilli that ferment lactose with acid and gas produc-tion in 48 h at 37°C. Members of this group include thegenera Citrobacter, Escherichia, Enterobacter, Hafnia, andKlebsiella. The coliforms can be differentiated into twogroups, the coliforms and the fecal coliforms, on the basis oftheir ability to grow at elevated temperatures. Both groupsgrow at 37°C, but only the fecal coliforms are able to grow at44.5°C. The two groups can also be differentiated by fourbiochemical tests known as the indole, methyl red, Voges-Proskauer, citrate (IMViC) series.

Escherichia coli is known to have three IMViC types:+ + - -, + - - -, and - + - -. Geldreich (7), in studies of theIMViC types found in animal feces, undisturbed soils, andpolluted soils, has designated these three IMViC types asEscherichia types, and he reported that they are predomi-nant in animal feces. The coliforms that were found to bepredominant in undisturbed soils, designated as the Entero-bacter group, have three different IMViC types: - - + +,- - + -, and - - - +. All other possible IMViC combina-tions are designated as coliforms of the intermediate type.The IMViC series and the ability of fecal coliforms to growat elevated temperatures have been used to differentiate thecoliform bacteria found in polluted surface water and waste-water (1). In addition, the association of certain IMViCtypes with animal feces, or with undisturbed soils, has beenused as an indication of the source of coliform pollution.

* Corresponding author.t Present address: Department of Biological Sciences, University

of Cincinnati, Cincinnati, OH 45221.t Present address: Great Plains Gasification Associates, Bulah,

ND 58523.

The coliform bacillus E. coli usually does not cause severedisease in normal host organisms. However, it is an oppor-tunistic pathogen that is able to cause disease when thebacilli invade extraintestinal tissue. Strains of E. coli that areable to cause disease without invasion of extraintestinaltissue are termed enteropathogenic E. coli. These strainspossess specific pathogenic properties that allow them tocolonize in animal intestines, multiply in high numbers, andinduce diarrhea or other severe forms of enteric disease.One such property is the presence of pili or fimbriae that areknown to aid in the attachment of enteropathogenic strainsto intestinal epithelial cells. These pili are proteinaceous andare classified as noncapsular K antigens. The pili of entero-pathogenic E. coli associated with diarrheal disease in pig-lets are termed K88 pili. The pili of strains associated withdiarrhea in calves and lambs are antigenically distinct fromthe K88 pili and have been designated K99 pili.The association between E. coli and enterotoxic colibacil-

losis has been well established. However, few investigationsinto the occurrence, number, and pathogenic properties ofthe E. coli strains in the environment of calves with colibacil-losis have been done. Smith and Crabb (18), employingbacteriophage typing of E. coli strains isolated from thefeces of cows and their calves, concluded that the dam didnot appear to be a frequent source of the strains responsiblefor disease and that the pen in which the calves were keptwas a more probable source. In a study of the husbandaryfactors influencing the occurrence of colibacillosis in calves,Wray and Thomlinson (19) have concluded that the use ofcalf-houses free of fecal contamination could break the cycleof infection.

This paper describes the occurrence, number, and patho-genic properties of enteropathogenic E. coli in barnyard soilsamples taken at six cattle ranches. Four of these rancheswere known to have high incidences ofcolibacillosis, whereas

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950 PLEWS, BROMEL, AND SCHIPPER

the remaining two were known to have a lower incidence ofcolibacillosis. In addition, we describe the IMViC types ofthe coliform and enteric bacilli isolated from uncontami-nated soils where no cattle had been kept, from barnyardsoils contaminated with bovine feces, and from the feces ofcalves with colibacillosis diarrhea.

MATERIALS AND METHODSCollection of soil samples. Soil samples from six cooperat-

ing cattle ranches designated as herds A through F weretaken in the fall of 1979 before the 1980 calving season. Soilsamples were taken at various sites at each ranch. At eachranch soil samples were also taken at clean uncontaminatedsites where no cattle had been kept. A second set of sampleswas taken at the beginning of the 1981 calving season at aseventh ranch designated herd G. A third group of sampleswas taken in early September 1981 at the feedlots of herd G.Samples were placed in sterile Whirl-Pak polyethylene bagsand transported to North Dakota State University (NDSU)where they were stored at -20°C until tested.

Collection of fecal specimens. Fecal specimens were ob-tained from the six ranches described above during the 1980and 1981 calving seasons. Specimens were collected fromhealthy calves as well as from calves with diarrhea. In thesecond year of this study, additional fecal specimens werecollected at herd G. The samples were placed in sterileplastic containers, frozen, and then transported to NDSUand stored at 4°C until tested.

Source of control cultures. Control cultures were obtainedfrom the American Type Culture Collection, Rockville, Md.(E. coli ATCC 12795), and the NDSU Department of Veter-inary Science (E. coli 117E).

Culture media. Primary plating media for the enumerationof total aerobic heterotrophs and the enumeration of entericbacilli were plate count agar (BBL Microbiology Systems,Cockeysville, Md.), Levine eosin methylene blue agar (EMB;BBL); MacConkey agar (Difco Laboratories, Detroit,Mich.), and Hoektoen enteric agar (Hoektoen; BBL). Fecalstreptococci were counted with M-enterococcus medium(BBL) containing 3% starch. Lactose broth (BBL), withbromcresol purple solution (2 ml/liter) added as the indica-tor, lauryl sulfate broth (BBL), and E.C. medium (BBL)were used in the most probable number (MPN) determina-tion of coliforms and fecal coliforms. Simmons citrate agar(Difco), methyl red-Voges-Proskauer broth (BBL), tryptonebroth (BBL), Kleigler iron agar (KIA; BBL), lysine iron agar(BBL), and urease test medium (BBL) were used in thefurther identification of enteric isolates. Trypticase soy agar(BBL) and Trypticase soy broth (BBL) were used forsubculture and storage of isolates. Isolates of E. coli thatwere serotyped for the K99 antigen were cultivated withliquid minca medium as described by Guinee et al. (11).Enumeration of total aerobic heterotrophs. A 10-g soil

sample was blended with 90 ml of sterile distilled water athigh speed in a Waring blender for 3 min. This mixture wastreated as a 1:10 dilution and further dilutions were madewith 90- and 99-ml sterile water blanks. Dilutions wereplated in triplicate on plate count agar and distributed by thespread-plate technique (1). The plates were incubated at30°C for 24 h. Plates containing 30 to 300 colonies werecounted.

Total aerobic heterotrophs in the fecal specimens wereenumerated by a similar method with modifications adaptedto specimen size and consistency. Whenever possible, 1.0 gof fecal material was used. If specimens contained less than1.0 g, 10 ml of sterile saline (0.85% NaCI) was added to the

entire sample, and the sample was blended with a Vortexmixer to give an even suspension. One milliliter of thissuspension was then used. A total of 1 g or 1.0 ml ofspecimen was blended with 99 ml of sterile distilled water athigh speed for 3 min in a Waring blender. Serial dilutionswere made and plated in triplicate on plate count agar by thespread-plate method (1). Plates were incubated and countedas described above.Enumeration of enteric bacilli. Serial dilutions of soil

samples and fecal specimens prepared as described abovewere plated in triplicate by the spread-plate method (1) onEMB, Hoektoen, and MacConkey agar. Plates were incu-bated at 37°C for 24 and 48 h. The lactose-fermenting andnon-lactose-fermenting colonies were differentiated andcounted.Enumeration of fecal streptococci. Fecal streptococci in

soil samples and fecal specimens were counted by inoculat-ing M-enterococcus medium with 1.0 ml of an appropriatelydiluted sample. A pour-plate method was employed (1).After solidification, the plates were incubated at 35°C for 48h. Plates were counted and flooded with iodine and in-spected for starch hydrolysis. All samples were plated intriplicate.Enumeration of total coliform bacteria. Total coliforms

were determined by the multiple tube fermentation method(1) by using a five-tube test. Lactose broth and lauryl sulfatebroth were inoculated with 1.0 ml of diluted sample. Alltubes were incubated at 37°C for 24 and 48 h. The MPN wasdetermined from tables published in previously establishedstandards (1). Broths with gas in the Durham tube and achange in the indicator to yellow were considered positive.A loopful from positive tubes was streaked on EMB andMacConkey agars. The presence of typical coliform colonieswas considered positive for total coliforms.Enumeration of fecal coliform bacteria. The presence of

fecal coliform bacteria was determined by inoculating E.C.medium with a 0.01-ml calibrated metal loop from eachpositive lactose and lauryl sulfate broth tube. Tubes wereincubated in a 44.5°C water bath for 24 h. A tube wasconsidered positive if growth and gas production occurred.A loopful from positive tubes was streaked on EMB andMacConkey agars. The MPN was determined as describedabove.

Identification of enteric bacilli. Representative colonies ofenteric bacilli were selected from EMB, Hoektoen, andMacConkey plates, and the colonies were transferred toKIA tube medium. These isolates were identified to thegenus or species level by biochemical and serological meth-ods (4).

Identification of coliform isolates. Bacterial colonies with atypical E. coli morphology and appearance on primaryplating medium were counted; representative colonies wereselected and transferred to KIA tube medium. In addition,selective plate media were streaked with inocula from posi-tive lactose broth cultures, and typical coliform colonieswere transferred to KIA medium. These primary isolateswere tested for the ability to grow at 44.5°C in E.C. medium.Isolates capable of growth at elevated temperatures and anyatypical isolates that grew poorly or produced very little orno gas in E.C. medium were biochemically characterized bytheir IMViC reactions. The identity of atypical strains givinga + + - - IMViC series was confirmed with API 20E strips(Analytab Products, Plainview, N.Y.).Typing sera. The following poly OK typing sera were

used: (i) E. coli OK poly A containing antibodies to antigens026:K60, 055:K59, 0111:K58, and 0127:K68 (Difco), and

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(ii) E. coli OK poly B containing antibodies to antigens086a:K61, 0119:K69, 0124:K72, 0125:K70, 0126:K71, and0128:K67 (Difco).

All isolates showing agglutination with poly A and poly Bantisera were then serotyped with the individual sera. The Ktyping sera used were purchased from the Department ofVeterinary Science, Pennsylvania State University, Univer-sity Park, Pa. Additional K typing sera used were the gifts ofH. W. Moon of the Animal Health Center, Ames, Iowa, andSteven Clegg of the University of Iowa, Iowa City, Iowa.

Slide agglutination. Isolates of E. coli were subcultured in

Trypticase soy broth before serotyping for 0 group antigensand the K88 antigen. Strains tested for K99 antigen were firstsubcultured in Trypticase soy broth with vigorous aerationat 37°C. Logarithmic-phase cells were then transferred toliquid minca medium and grown with vigorous aeration at37°C for 8 to 10 h. The cells were sedimented by centrifuga-tion, and the supernatant was drawn off. The pellet was

dispersed with a Pasteur pipette, and the suspended cellswere typed with K99 antisera.

RESULTS

All soil and fecal samples in the present study were

analyzed for total aerobic heterotrophic bacteria, total strep-tococci, fecal streptococci, total coliforms, and fecal coli-forms. Additionally, E. coli plate counts were done on allfecal samples. All counts represent the logarithm of viablecount per gram of soil or gram of feces. The mean value andthe standard deviation are given followed by the number ofpositive samples.The seven soil samples taken from areas where no cattle

had been kept contained only aerobic heterotrophic bacte-ria, (8.24 + 0.78, 7) and total coliforms (3.87 ± 0.92, 7).However, streptococci and fecal coliforms we're not found inuncontaminated soil samples. In addition, these organismswere not found in uncontaminated soil samples taken atherds E, F, and G (data not shown). Twenty-two soilsamples taken in feedlots, barns, and pastures contained allthe bacterial types expected, including both fecal strepto-cocci and fecal coliforms. Their values were as follows: totalaerobic heterotrophic bacteria, 8.59 ± 0.69, 22; total strepto-cocci, 4.77 ± 1.14, 21; fecal streptococci, 4.17 ± 1.46, 6;total coliforms, 4.02 ± 1.57, 22; and fecal coliforms, 3.75 ±

1.52, 17. In contrast to the results obtained from the ranchesdescribed above, eight soil samples from herds E and F didnot contain fecal streptococci or fecal coliforms. However,similar counts were obtained for the following: total aerobicheterotrophic bacteria, 8.79 ± 1.07, 8; total streptococci,4.42 1.26, 4; and total coliforms, 4.70 + 1.70, 5.

Bacterial numbers in the feces of 51 calves with diarrheawere as follows: total aerobic heterotrophic bacteria, 9.39 ±

0.99, 45; fecal streptococci, 6.95 ± 1.13, 12; total coliforms,7.89 1.75, 50; fecal coliforms, 9.97 ± 1.60, 38; and E. coli,8.44 1.09, 38. Bacterial numbers in the feces of six healthycalves were as follows: total aerobic heterotrophic bacteria,9.96 ± 0.60, 6; total streptococci, 8.42 + 1.98, 5; fecalstreptococci, 9.99, 1; total coliforms, 8.81 2.25, 6; fecalcoliforms, 8.64 ± 2.51, 6; and E. coli, 8.86 1.80, 6.A comparison of the number of total aerobic heterotrophic

bacteria in the feces of healthy calves with the numbers oftotal aerobic heterotrophic bacteria in the feces of diseasedcalves by the F test for sample variance indicated that therewas no detectable significant difference in the varianceobserved (P > 0.05). Similarly, the F test showed that therewas no detectable significant difference in the variance in the

number of total streptococci or the number of E. coli in thefeces of these two groups of animals.

Parr (15) has reported the decline and eventual disappear-ance of E. coli from fecal samples stored in an ice box.Evidence of a similar decline in viable E. coli numbers wasseen in the present study. The data presented in Table 1show the decline in total coliform, fecal coliform, andfermentative enteric bacilli in nine fecal specimens withincreasing storage time. The number of E. coli found indiarrheal specimens in the present study was lower thanexpected. The possibility of the loss of viable count becauseof prolonged storage is a reasonable explanation for thelower counts.

Starch-hydrolyzing Streptococcus species were detectedin the feces of one of six healthy calves and in the feces of 12of 51 diseased calves. The streptococci found were not of theenterococcal group but were group D streptococci, as deter-mined by the method of Facklam (6). Because these strainswere of bovine origin and rapidly hydrolyzed starch (12),they were designated as fecal streptococci. This speciesoccurs in fresh feces (12) but is known to perish rapidlyoutside of the animal body. It is likely that this organism waslost in the majority of the fecal specimens during storagebefore analysis could be performed.

In addition to the bacteriological examination of fecalspecimcens, the specimens were examined for the presenceof rotavirus by four methods: agar-gel immunodiffusion(E. R. Gion, M.S. thesis, NDSU, Fargo, 1980), fluorescentantibody tissue culture (Gion, M.S. thesis), counterimmuno-electrophoresis (5), and enzyme-linked immunosorbent as-say (5). Rotavirus was detected in two of the healthy animalsand in eight of the diseased calves. To determine whetherthe presence of rotavirus had any effect on the numbers oftotal aerobic heterotrophic bacteria, total streptococci, andtotal coliforms, a multivariate analysis of variance wasperformed by a statistical analysis system Manova proce-dure (10). The presence of rotavirus was found to have nosignificant effect on the number of total heterotrophic bac-teria, total streptococci, or total coliforms (P > 0.5), nor wasthere evidence of an overall effect by rotavirus when thethree groups of organisms were considered together by theManova procedure.

Table 2 shows the identification, the IMViC type, and thefrequency of occurrence of the enteric and coliform bacilliisolated from uncontaminated soils, barnyard soil previouslycontaminated with bovine feces, and the feces of calves withcolibacillosis.

Isolates of E. coli were tested for the presence of 100-type antigens (026, 055, 086a, 0111, 0119, 0124, 0125,

TABLE 1. Effect of the length of storage time of fecal samples onbacterial counts

Log of counts (MPN) of: Log of plateFecal counts of Days ofsample Total Fecal fermentative storage

coliforms coliforms enteric bacilli

32N2 10.1 10.1 10.0 172N6 9.6 9.6 9.8 3431N2 9.7 9.7 9.6 483N3 8.2 8.2 7.6 4828N2 7.4 7.4 7.4 547N1 8.9 7.5 9.5 839N1 7.7 7.7 7.6 83501 4.5 0.0 0.0 97832 3.8 0.0 0.0 97

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TABLE 2. Coliforms and enteric bacilli identified in uncontaminated control soil, barnyard soil, and calf feces and IMViC typesFrequency of occurrence (%) in the following samples:

Organism Type no. IMViC type Control soil Barnyard soil Calf feces(n = 50) (n = 50) (n = 60)

Escherichia typesCitrobacter species 1 + + - - 0 16 0Enterobacter agglomerans 1 + + - - 0 2 0Escherichia coli 1 + + - - 0 14 29Klebsiella oxytoca 1 + + - - 0 0 2Citrobacter freundii 4 - + - - 19 10 13Enterobacter agglomerans 4 - + - - 4 2 6Escherichia coli 4 - + - - 0 0 2Hafnia als'ei 4 -+-- 8 4 0Klebsiella ozaenae 4 - + - - 0 6 9Escherichia coli 10 + - - - 0 0 2

Enterobacter typesEnterobacter spp. 2 - - + + 13 0 2Enterobacter cloacae 2 - - + + 0 10 0Klebsiella pneumoniae 2 - -+ + 0 2 2Enterobacter spp. 14 - - + - 2 0 0Hafnia alvei 14 --+- 0 0 2

Intermediate typesCitrobacter spp. 5 - + - + 0 4 0Citrobacter freundii 5 - + - + 0 0 6Hafnia alvei 5 -+-+ 21 0 0Klebsiella ozaenae 5 -+-+ 21 0 0Klebsiella pneumoniae 5 - + - + 0 0 4Yersina enterocolytica 5 - + - + 0 0 2Citrobacter spp. 6 + + - + 0 8 2Citrobacter diversus 6 + + - + 0 8 0Enterobacter cloacae 7 - +++ 0 2 0Klebsiella oxytoca 7 - + + + 0 0 2Klebsiella pneuinoniae 7 - +++ 0 0 9Serratia spp. 7 -+++ 0 4 0Klebsiella oxytoca 8 + + + + 0 0 2Enterobacter cloacae 9 + - + + 0 2 0Klebsiella oxytoca 9 + - + + 13 6 0Hafnia alvei 12 -++- 0 0 4Yersina pseudotuberculosis 13 ---- 0 0 2

0126, and 0128) and for two K-type antigens (K88 and K99)as well. Isolates from two soil samples and three fecalspecimens obtained from herd A were found to be serologi-cally identical. These strains agglutinated in all but one ofthe 0-typing sera (0126). These strains were K88- andK99+. Similarly, four soil isolates and two fecal isolatesfrom herd C were found to be identical. The strains werepositive for all 0-type antigens except 0124, 0126, and0128. As with the isolates from herd A, these strains wereK88- and K99+.

DISCUSSIONThe absence of streptococci and fecal coliforms in the

uncontaminated soil samples from all six cattle ranchestaken at sites where no cattle had been kept demonstratesthat these organisms are not normal soil inhabitants. Theirpresence in barnyard soils subject to fecal contaminationindicates that bovine feces are the source of these organ-isms. This is in agreement with the findings of Geldreich (7).The repeated isolation of K99-positive enteropathogenic

E. coli from barnyard soils and the recovery of serologicallyidentical enteropathogenic E. coli from soil samples col-lected in the fall and from the feces of calves with colibacil-losis born at the same locations indicate a reservoir ofenteropathogenic E. coli in barnyard soil contaminated with

bovine feces. Furthermore, the results of this study suggestthat the occurrence and survival of fecal coliforms in barn-yard soils is related to ranch husbandary and sanitarypractices. The absence of fecal coliforms and fecal strepto-cocci at herds E and F should be noted. These two rancheswere known to have had an incidence of neonatal calfdiarrhea that was significantly lower than that at herds Athrough D where fecal coliforms and fecal streptococci wereconsistently isolated from barnyard soils. Husbandary prac-tices at these two ranches included the clearing of manurefrom the lots and the barnyards where calves were born andreared. This was done as soon as spring weather conditionspermitted the moving of cattle to pasture locations.

After the cattle were removed, these sites were allowed tostand empty until the fall. The lower incidence of diarrhea incalves born at these two ranches provides circumstantialevidence that calves may acquire disease-causing entero-pathogenic E. coli strains from contaminated barnyard soil.The pattern of coliform survival in soils observed in this

study was similar to previously reported survival patterns (2,3, 13). Although E. coli is known to die off rapidly in soils,the survival of enteric bacteria in soil is increased, andregrowth is possible when sufficient organic matter andmoisture are present (9). Removal of the manure from thebarnyards at herds E and F eliminated moisture-retaining

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organic material. It appears that this practice is sufficient toeliminate fecal coliforms from contaminated soil.

Although the ecology of coliforms and fecal coliforms wasthe primary concern of this study, the occurrence of anotherbacterial species used as an indicator of fecal pollution wasstudied. Streptococci were found in slightly higher numbersthan the coliforms in contaminated soil at herds A throughD. A similar pattern has been reported in studies of thesurvival of indicator bacteria in aquatic environments (8,14), where enterococci have been found to survive longerthan coliforms. These findings suggest that the survivalpatterns of streptococci in contaminated soils are similar tothose in polluted surface waters.The results of the present study are in agreement with

those of Smith (16, 17) who did not observe any difference inthe principal bacterial flora of healthy and diseased animals.He reported an increased number of E. coli in a group ofseverely ill animals. However, in the present study, in whichthe severity of disease was not determined, the number of E.coli in diarrhea specimens was lower than expected. Thepossibility of a loss of viable count because of prolongedstorage has been discussed previously. In addition, had theseverely ill animals been treated as a separate group, ahigher count of E. coli might have been observed. Thenumber of E. coli in feces of both healthy and moderately illcalves do not appear to be significantly different, thussuggesting that E. coli do not need to be present in highnumbers to cause mild or moderate disease.

In studies by Geldreich (7) on the occurrence of differentIMViC types of coliform bacteria in soil and animal feces, 12IMViC types were reported. In the present study twoadditional types are reported, 13 (----) and 14 (--+-)(Table 2).

Isolates belonging to three genera other than Escherichiaproduced a classical E. coli IMViC type 1 (Table 2). A totalof 10 coliforms isolated that gave an IMViC type 1 notbelonging to the genus Escherichia were identified. Further-more, the Enterobacter agglomerans identified was culturedfrom E.C. broth incubated at 44.5°C. The Durham tubecontained gas, but no E. coli was found when a loopful ofthis broth culture was streaked onto EMB medium. Thesefindings underscore the limitations of the use of the IMViCtest as a sole criterion in the identification of fecal coliforms.They also emphasize the importance of the completed testfor fecal coliforms by the MPN method.No coliform bacteria producing the classical E. coli IMViC

type 1 were detected in uncontaminated soils. However,three genera producing the Escherichia IMViC type 4 wereisolated. However, this IMViC type was reported by Geld-reich (7) to constitute 3.3% of the coliforms present inundisturbed soils. The intermediate type S was isolatedfrequently from uncontaminated soils in this study. This is inagreement with Geldreich's conclusion that this is a predom-inant type in undisturbed soils (7).According to Geldreich (7), the IMViC type 9 constituted

2.9% of the coliforms isolated from undisturbed soils, butwas not found in polluted soils, and constituted less than1.0% of coliforms found in livestock feces. In the presentstudy, this type was found in uncontaminated and barnyardsoils but was not found in the feces of calves with colibacil-losis. These findings suggest that the IMViC type 9 is a soiltype.

Geldreich reported three predominant IMViC types inlivestock feces: 1, 3, and 6. They represented 99% of thecoliforms isolated from livestock feces. However, in thepresent study, two of these types, 1 and 6, constituted only

33% of the coliforms isolated from calf feces. Furthermore,type 3 was not found in any of the fecal samples. The data ofGeldreich (7) for livestock feces were obtained from theexamination of 31 fecal samples: 10 from sheep, 11 fromcows, and 10 from hogs. There is no information in thisreference about the age or the state of health of theseanimals; however, the fecal specimens examinated in thisstudy were obtained from newborn calves and this mayaccount for the noted discrepancy.

Unexpectedly, in the present study, IMViC types 2, 4, 5,6, 7, and 8 constituted 61% of the coliforms isolated fromcalf feces. These six types have been reported by Geldreich(7) to occur at the highest frequency in undisturbed soils. Itis interesting that these IMViC types were not those com-monly associated with livestock feces, but rather they areassociated with undisturbed and polluted soils. This suggeststhat the predominant IMViC types found in the feces ofcalves with colibacillosis originate in the soil. If newborncalves acquire enteropathogenic E. coli from contaminatedsoils, as the 0- and K-type serotyping of soil and fecalcoliform isolates in the present study suggests, it seemsreasonable that the coliforms that are naturally present insoils also would be present in their feces.

ACKNOWLEDGMENTSPartial funding for this work was provided by the North Dakota

Beef Commission, 107 S. 5th St., Bismarck, N.D.We thank Robert B. Carlson for assistance in the statistical

analysis of the data presented here.

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2. Bell, R. G., and J. B. Bole. 1978. Elimination of fecal coliformsfrom soil irrigated with municipal sewage lagoon effluent. J.Environ. Qual. 7:193-196.

3. Edmonds, R. L. 1976. Survival of coliform bacteria in sewagesludge applied to a forest clearcut and potential movement togroundwater. Appl. Environ. Microbiol. 32:538-546.

4. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacteriaceae, 3rd ed., Burgess Publishing Co., Minneap-olis, Minn.

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6. Facklam, R. R. 1980. Streptococci and aerococci, p. 88-110. InE. H. Lennette, A. Balows, W. J. Hausler, and J. P. Truant(ed.), Manual of Clinical Microbiology, 3rd ed. American Soci-ety for Microbiology, Washington, D.C.

7. Geldreich, E. E. 1966. Sanitary significance of fecal coliforms inthe environment. Water Pollution Control Research Series,Publication no. WP-20-3. Cincinnati Water Research Labora-tory, Cincinnati, Ohio.

8. Geldreich, E. E., H. F. Clark, and C. B. Huff. 1964. A study ofpollution indicators in a waste stabilization pond. J. WaterPollut. Cont. Fed. 36:1372-1479.

9. Gerba, C. P., C. Wallis, and J. L. Meknick. 1975. Fate ofwastewater bacteria and viruses in soil. ASCE J. Irrig. DrainageDiv. 101:157-174.

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