INMCTION AND IMMUNITY, Jan. 1976, p. 92-99Copyright ©) 1976 American Society for Microbiology

Vol. 13, No. 1Printed in U.SA.

Experimental Neonatal Colibacillosis in Cows:Serological Studies

R. A. WILSON"* AND J. W. JUTILADepartment of Microbiology, Montana State University, Bozeman, Montana 59715

Received for publication 2 June 1975

Serological studies of passive immunity in experimentally induced bovinecolibacillosis was studied in a 41-cow university herd. Pregnant dams wereantigenized prepartum with two injections administered by the subcutaneousand intrammamary routes with one offour vaccine preparations (killed bacteria,live bacteria, culture supernatant, or heart infusion broth [control]). The dataindicate that 77% of the neonates born to vaccinated dams were stronglyprotected against oral challenge with Escherichia coli strain B-44. Bacterialagglutinin and passive hemagglutination titers of colostral whey directly re-flected the efficacy of the vaccines. A notable decrease in the whey titers tosomatic and capsular antigens occurred after heat treatment at 56 C for 30 min.Complicity of heat-labile immune factor(s) in protection from scouring wassuggested. The nature of the protective antigen is not clearly defined by thesestudies but there is some evidence that the K antigen may play a vital role inthis regard.

Neonatal calf diarrhea (calf scours) is anacute, often fatal infection of the young bovinepresenting a myriad of signs including diar-rhea, dehydration, malaise, prostration, andfinally death. There is evidence that Esche-richia coli can be the etiological agent of calfscours and that different strains may be in-volved (13; G. Wramby, Abstr. Vet. Bull.19:261-262, 1949). Since E. coli are common tothe intestinal tract of calves (23), it is oftendifficult to show clear etiological relationships.

Certain cellular constituents of E. coli havebeen examined for their role in promoting intes-tinal disorders including the 0 somatic anti-gen, K capsular antigens, and the entero-toxin(s) produced by some strains ofenteropath-ogenic E. coli. Although the 0 somatic anti-genic material may not be involved per se withthe pathogenicity and virulence of E. coli, theendotoxin portion of the 0 antigen may act inconcert with the virulence factors associatedwith enteropathogenic coliforms (13).

There is evidence that would implicate Kantigens in the virulence of E. coli (14). Thesestudies indicated that K antigen reduces thecomplement sensitivity of E. coli by coveringthe antigenic determinants of the 0 antigen. Ithas also been shown that certain K antigenscontribute to the tissue adherent qualities ofsome strains of E. coli. Studies by Smith andLinggood (24) indicate that the common K anti-

' Present addres: Department of Veterinary Microbiol-ogy, Syntex Research, Palo Alto, Calif. 94043.

gen (KcO) produced by certain calf and lambenteropathogenic E. coli may function in a man-ner similar to K-88 antigens in the pig (16), thatis, by facilitating adherence to the intestinalepithelium.

Agglutinin activities in sera or whey fromantigenized dams has been used in the past toassess the efficacy of E. coli vaccines (8, 12).These studies generally evaluate the effect ofroute and adjuvants upon the immune re-sponse. In a recent clinical trial, designed toevaluate the effectivenss of various E. coli vac-cines to stimulate immunity in dams and pas-sively transfer to suckling calves, a markedprotection of calves from experimentally in-duced scours was observed (19).The challengeorganisms, E. coli B-44, caused severe diarrheaonly in calves born to control dams, whereascalves born to specifically immunized damswere strongly protected. This report describesthe serological parameters of the cow/calf unitdescribed in the earlier communication (19).

MATERIALS AND METHODSTest animals. A 41-cow herd of pregnant Here-

ford dams was randomly distributed into three ex-perimental groups of nine cows each and one controlgroup of 14 cows as described earlier (19). Eachgroup was maintained in a separate outdoor pen andprovided hay once daily and water ad libitum.

Test organism. The strain of E. coli selected forthis experiment was strain B-44 (serotype09:K:NM) furnished by H. W. Smith of Great Brit-ain.

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E. coli B-44 is nonhemolytic, prototrophic, colicinpositive, lactose positive, and demonstrates bothsmooth mucoid and rough nonmucoid morphology.This strain gives positive ligated gut loops in boththe calf and rabbit. The smooth-mucoid colony typewas used in the production of culture supernatantand live and killed vaccine, as well as for challengeof the neonatal calves.

Vaccines. The vaccines were described in a pre-vious paper (19). Briefly, B-44 was grown in brainheart infusion broth at 37 C with constant shaking.The live vaccine consisted of a 24-h culture of thetest organism which contained about 3 x 1010 viablecells/ml. The killed vaccine contained washed, for-malinized (0.2% vol/vol) B-44 organisms at a concen-tration of approximately 6 x 1010 cells/ml. The cul-ture supernatant preparation was obtained by cen-trifugation and filtration of brain heart infusionbroth culture. The concentrate of this material wasdialyzed against water at 4 C before formalin (0.20%vollvol) was added.

Vaccines were emulsified in an equal volume ofadjuvant (96% mineral oil and 4% emulsifier) foradministration. The control vaccine consisted ofbrain heart infusion broth mixed with adjuvant.

Vaccination protocol. Each cow was given 5 ml ofantigen plus adjuvant subcutaneously and 4 ml ofvaccine by the intramammary route (1 ml perquarter) twice in a 2-week interval. Antigenizationwas initiated 3 weeks before expected calving for theherd and the second dose was administered 2 weekslater.

-Neonatal challenge. The herd was under maxi-mum surveillance to ensure that newborn calveswould not have access to colostrum prior to theacquisition of the first postpartum blood specimen.At parturition, the calf was moved immediately tothe clinic and the dam was put into a chute tofacilitate collection of specimens. Calves were bledfrom the jugular vein and each calf was cultured byrectal swab. The cows were bled (20 ml) from thejugular vein and a separate sample ofcolostrum wastaken from each quarter. Thereafter, the cow/calfunit was maintained in a small pen to be certain thecalf could obtain colostrum prior to challenge. Chal-lenge organisms were administered before thecalves were 10 h old. Calves were challenged bygavage with 50 ml of an 18-h culture ofE. coli B-44.After challenge, the cow/calf unit was moved to acommon pen separate from the pregnant dams. Thecow/calf units had access to shelter and dry bedding.Time of onset, duration, and severity of diarrhea

(scored as 0, 1+, 2+, 3+, 4+) were determined foreach calf (Table 1). A zero score means no diarrheawas observed. Diarrhea of 1+ was assigned if thecalf was generally healthy and had normal rectaltemperatures (approximately 38.9 C), diarrhea, andno signs of dehydration. Diarrhea was scored as 2+if the calf had normal temperature, diarrhea, con-cavity of flanks, some dehydration indicated as par-tial recession of the eyes, and slight apathy. Thecriteria for the 3+ score was temperature higherthan 37.8 C, diarrhea, moderate dehydration indi-cated by progressive recession ofthe eyes, and weak-ness (but calf retained ability to stand). The disease

TABLsz 1. Clinical evaluation of calves after

Vaccinegroup ofdam

Control

Cs

LB

KB

challenge

Signs of neonatal calfdiarrhea:

Cow-calf Onset of Dura-no. diarrhea tion of

after diarrheachallenge (h)(h)

440-202 21 36422-209 7 40419-210 5 31443-212 7 48408-215 7 36405-221 6 27430-222 6 29427-225 11 43441-227 8 40620-229 5 32413-230 4 15446-234 4 20628-236 8 48411-240 7 33

442-201 11 8417-204 Negative 0622-207 12 48928-216 6 12630-228 9 33421-233 4 13433-235 4 72961-237 6 27410-238 17 29

650-206 11 33426-208 13 48403-211 7 24450-214 Negative 0429-217 6 36649-218 5 15407-223 Negative 0937-224 Negative 0922-239 4 25

432-203 0 0618-205 7 48948-213 Born dead (cause

unknown)943-219 Negative 0921-220 13 12444-226 Negative 0404-231 Born dead (cause

unknown)439-232 Negative 0434-241 Negative 0

Scorea

1+4+4+4+4+4+4+4+4+4+4+4+3+4+

1+01+1+1+4+4+4+1+

4+4+1+01+1+001+

01+

01+0

00

a See text.

was scored 4+ when the rectal temperature was lessthan 37.8 C (often 34 to 36 C), and when the calfhaddiarrhea, severe dehydration, skin pliability,marked recession of the eyes, and was unable tostand or on occasion was prostrate.

Agglutination studies. Bacterial agglutination

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94 WILSON AND JUTILA

studies were done in accordance with methods de-scribed in Campbell et al. (5). Somatic antigensdescribed as 0 antigens were prepared from 18-hbroth culture. Cells were washed two times withsterile saline (0.85%) before boiling for 1 h. Boiledcells were then washed with sterile saline and sus-pended at a final concentration of 109 organisms/ml(nephelometric measurement). Titrations performedusing B-44 cells, boiled for 1 h, were subsequentlyshown to include anti-K antibodies indicating that 1h ofboiling does not entirely remove all of the capsu-lar material from this strain ofE. coli.

External or capsular antigens, designated as Kantigens, .were prepared from 18-h cultures of sedi-mented cells washed in the same manner as de-scribed above. Packed cells were suspended in for-malin (0.20% vol/vol) at a final concentration of ap-proximately 10' organisms/ml.

Passive hemagglutination experiments were per-formed by the method outlined in Campbell et al.(5). Endotoxin (lipopolysaccharide) was extractedfrom E. coli B-44 using the phenol water method ofWestphal and Jann (25). Sheep erythrocytes weresensitized to B-44 LPS according to the technique ofNeter et al. (20).

Blood samples. On day 1 of the experiment, justbefore the administration of antigen, a blood samplewas taken from each cow by the jugular vein. Subse-quent blood samples were obtained at calving andagain 3 months postpartum. Blood samples fromcalves were obtained at the time of birth and at 4days of age. Sera were stored at -20 C until testedand heated at 56 C for 30 min immediately beforetitrations were performed.Whey samples. Colostrum and milk samples

were treated with renin (stock solution diluted1:300) and calcium chloride in a concentration suffi-cient to create a firm clot at 42 C. Anti-O and anti-Ktiters were performed on unheated whey samplesand on whey samples that had been heated for 30min at 56 C. Passive hemagglutination titers weredetermined for heated whey only, since earlier stud-ies in this laboratory indicated an insignificant dif-ference in passive hemagglutination titers beforeand after such heat treatment.

RESULTSPassive immunization in experimental

colibacillosis: a clinical evaluation. (i) Char-acteristics of strain B-44. In the preliminarystudy of strain B-44, viable cells, filtrates, andconcentrated toxin preparations caused disten-tion of ligated intestinal segments in rabbitsand calves. Strain B-44 had both rough andsmooth colony types, and both forms causeddistention of ligated intestinal segments. Onlythe smooth form of strain B-44 was used inthese experiments. The viable cell count ofstrain B-44 grown in heart infusion broth at37 C for 24 h with vigorous aeration was approx-imately 3 x 1010 cells/ml.

(ii) Response of calves to oral challengewith live E. coli. A calf scour index was de-

rived by determining the mean diarrhea scorefor each experimental group from the data pre-sented in Table 1. The control calves had ascour index of 3.7, which is markedly higherthan the indexes calculated for the calves in thevaccine groups.The calf scour index indicates that the calves

in the killed vaccine (KB) group had the lowestindex, 0.3, followed by 1.3 and 1.9 for the calvesin the live bacteria (LB) and culture superna-tant (CS) groups, respectively (the index repre-sents an average of the degree of scours basedon the clinical evaluation of calves in eachgroup). The onset of diarrhea (Table 1) variedbetween 4 and 21 h after the calves were chal-lenged and was essentially the same for boththe control and vaccine groups. The durationvaried from 12 to 72 h, at which time the calveseither had expired or were removed from theexperiment.

Clinically, all calves gaining a score of 4+diarrhea had profuse watery diarrhea whichappeared several hours before the initial onsetof the accompanying signs of dehydration(sunken eyes). As dehydration progressed apa-thy and anorexia were noted, and then the calfwas unable to stand, body temperature de-creased, and if untreated, the calf became pros-trate and died. Bacteremia was not detecteduntil the calves were moribund. At necropsy,gross pathological changes were not seen.

(iii) Bacteriological evaluation of fecalspecimens. Fecal swabs collected from 19 of 39calves at time of challenge did not producegrowth of E. coli on Tergitol-7 agar withtriphenyltetrazolium chloride. In fecal swabscollected from the remaining calves at chal-lenge a few coliforms of the rough form wereisolated. Only two calves were excreting signif-icant numbers of coliform organisms exhibitingsmooth colony at the time of challenge.

After calves were challenged with strain B-44, the population ofE. coli increased, with themajority being smooth and mucoid and havingmorphological properties identical to those ofstrain B-44. Smooth organisms resemblingstrain B-44 were initially isolated from fecalswabs of every calf between 5 and 24 h afterchallenge. The appearance of the various col-ony types remained constant for 4 days of fecalswab collections. Usually, rough colony typesemerged and became approximately equal innumber to the smooth, mucoid form by day 4 ofcollection.

Serological studies. (i) 0 agglutinins ofthe dam. The titration of the preimmune serafrom all dams yielded undetectable or negativeO agglutinin titers. The titration of cow serum2 weeks after a single antigenization revealed

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SEROLOGICAL STUDIES ON COW COLIBACILLOSIS 95

TABLE 2. Cow serum titers 2 weeks after a single antigenization and at parturition

Anti-Ob Anti-Kb PHAbExperimen-tal groupa 2 weeks post- At parturi- 2 weeks post- At parturi- 2 weeks postin- At parturi-

injection tionc injection tionc jection tionc

C <10d <10d <10d <lod 930d 1,274dCS <10 21 <10 <10 2,416 5,440LB 20 63 10 14 1,719 6,948KB 80 110 10 14 2,321 6,656

a C, Unvaccinated dams.b Anti-O, Reciprocal of the mean titer of agglutinins to E. coli B-44 boiled cells; Anti-K, formalinized E.

coli B-44 cells; PHA (passive hemagglutination), E. coli B-44 LPS-sensitized sheep erythrocytes.Eight to 66 days from the time of the second antigenization.

d Serum heated at 56 C for 30 min.

TABLE 3. Summary of statistical analysisa of results

Data analyzed Significance between groups"

Anti-O (2 weeks pi) C CS LB KBAnti-O (at parturition) C CS LB KBAnti-K (2 weeks pi) C CS LB KBAnti-K (at parturition) C CS LB KBPHA (at 2 weeks) C CS LB KBPHA (at parturition) C CS LB KB

a Test of comparison among means by leastsquare difference. PHA, passive hemagglutination;pi, postinfection.

n Means not underlined by the same line are differ-ent from each other (P < 0.05).

that neither dams in the control group nor inthe culture supernatant group had detectable 0agglutinins in their serum (Table 2). Dams vac-cinated with LB had a mean titer of 20 com-pared to a mean titer of 80 in the sera of thedams antigenized with KB. These values arenot significantly (P > 0.05) different (Table 3).The mean titer in serum from the KB group,however, is significantly higher than the con-trol group.

Assays of sera obtained at the time ofparturi-tion detected antibody activity in the sera of theCS-treated dams but the titers (21) were verylow. A slight increase in titer was seen in thesera of dams in the KB group (110), whereas athreefold increase (63) was observed in the seraof dams antigenized twice with LB.

Investigations conducted a year after thesetitrations were performed indicated that theprocedure used to prepare the 0 somatic anti-gens was not rigorous enough to eliminate all ofthe capsular material from the bacterial cells.Therefore, the 0 agglutination titers reportedmay have included a minor contribution byanti-K antibodies present in the sera and fail toexpress the exact titer. Additionally, K antigen

TABLE 4. Agglutinin titers of heated and unheatedcolostral whey from immune and nonimmune dams

Experi- Anti-O"' Anti-Kbmental U-Un- Heated haegroup heated Heated heated

C 19 10 10 10 2,720CS 77 27 14 10 12,581LB 134 38 39 16 21,809KB 276 190 69 54 37,050

a.b.d See Table 2.

TABLE 5. Summary ofstatistical analysisa of resultsin Table 4

Data analyzed Significance between groupsb

Anti-O (unheated) C CS LB KBAnti-O (heated)c C CS LB KBAnti-K (unheated) C CS LB KBAnti-K (heated) C CS LB KBPHAd (heated) C CS LB KB

a.b See Table 3.c Heated at 56 C for 30 min.dSee Table 2, footnote b.

present on the cells may have obscured 0 deter-minants.

Colostral whey from immune and nonim-mune dams was titrated before and after heat-ing at 56 C for 30 min. A very low titer of anti-Oactivity was detected in the unheated wheyfrom the control dams, whereas mean titers of276, 134, and 77 were measured in unheatedwhey from dams antigenized twice with KB,LB, and CS, respectively (Tables 4 and 5). Afterheating, the titers dropped 32% in the KBgroup, 52% in the LB group, and 65% in the CSgroup. All of the titers, however, were stillsignificantly high-er than the mean whey titersof the nonimmunized dams. Agglutinating ac-

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TABLE 6. Calf serum titers at birth and at 4 days old

Experimental Anti-Ob Anti-Kb PHAb

groUpa At birth 4 days old At birth 4 days old At birth 4 days old

C <10C <10C <10o <loc <40c 24cCS <10 56 12 60 40 214LB <10 90 <10 240 <10 1,920KB <10 97 <10 121 <10 1,496

a,b.c See Table 2.

tivity was not detectable in whey from milkobtained 3 months postpartum in dams givenCS or bacterins.

(ii) 0 agglutinins of the calf. The 0 agglu-tinin titers were estimated on serum harvestedfrom calves before colostral feeding and 4 dayspost-parturition. The results of these titrationsare found in Table 6. The data show that few ofthe calves had antibody activity (mean = 10).Less than three calves in each of the antigen-ized groups demonstrated titers at birth andnone of the titers exceeded 40.The data show a significant (Tables 6 and 7)

increase in the antibody titers in the sera of 4-day-old calves born to KB-, LB-, and CS-vacci-nated dams as contrasted to the precolostralserum. None of the control calves acquired de-tectable serum antibody against the test orga-nism by 4 days of age.

(iii) K agglutinins of the dam. Cow serumobtained before antigenization and again 2weeks later failed to agglutinate a formalinizedpreparation of E. coli B-44. At parturition, noantibody activity was detected in the serum ofdams in the control nor CS groups. A very weakhumoral response, to the capsular antigens,was detected only in the sera of dams that hadbeen immunized twice with KB or LB.

Titrations were performed on whey beforeand after heating at 56 C for 30 min. Bothheated and unheated whey from control damswere devoid of antibody activity against thecapsular antigens. An insignificant titer (mean= 14) was measured in the unheated whey fromdams in the CS group. The antibody contribut-ing to this activity was inactivated by heat. Themean titers of the KB and the LB groups of 69and 39, respectively, are significantly (P < 0.01)different (Table 3) and are also significantlyhigher than the control titers.

Heating the wheys from the KB group de-creased the antibody activity by 22% while de-creasing the activity in the LB groups by 51%.None of the milk specimens tested had detecta-ble agglutinating antibody 90 to 120 days aftercalving.

(iv) K agglutinins of the calf. Except for the

TABLE 7. Summary of statistical analysis a of resultsin Table 5

Data analyzed Significance between groupsbDataanalyzed ~ ~ ~ ( < 0.05)

Anti-O (at birth) C CS LB KBAnti-O (4 days old) C CS LB KBAnti-K (at birth) C CS LB KBAnti-K (4 days old) C CS LB KBPHA (at birth) C CS LB KBPHAC (4 days old) C CS LB KB

a,b See Table 3.c See Table 2, footnote b.

low titer of 12 in the CS vaccine group, serumspecimens obtained from precolostral calveslacked agglutinating activity to the K antigensofE. coli B-44 (Table 6). However, considerableantibody activity was apparently acquired fromcolostrum by the calves in the CS, LB, and KBgroups. Thus, calves in the KB group had amean titer of 240 compared to titers of 12 and 60in sera of calves in the LB and CS groups,respectively. The difference observed amonggroups was not statistically significant (P >0.05) (Table 7).

(v) Passive hemagglutination activity ofthe dam. Antibody activity against the LPSmoiety of B-44 was not measured in cow serumprior to antigenization. Two weeks after a sin-gle antigenization and again at parturition thecows were tested for serum passive hemaggluti-nation activity. The results are given in Table4.

After a single antigenization, the serum pas-sive hemagglutination activities detected in thevaccine groups were not significantly different(Table 5). However, the titers were signifi-cantly different at parturition. The serum titersshowed a threefold increase in all of the vaccinegroups, whereas titers in the control group didnot show an appreciable change.The highest colostral whey titer detected in

the KB group had a mean titer of 37,000 ascornapred to a titer of 12,500 in the CS groupand 21,000 in the LB group (Table 4). The differ-

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ence among these groups is not signiflcant atthe 0.05 level (Table 5). However, the resultsare significantly different between the vaccinegroups and the controls. Contrasting the meanwhey titers to the serum titers in the vaccinegroups indicated that two to six times moreanti-LPS activity was present in the whey ofimmune cows.The relative difference between the vaccine

groups can be seen in the result of the 0 anti-body titers measured in unheated whey (Table4).

DISCUSSIONThese studies, designated to evaluate the effi-

ciency of three different vaccine preparations,indicated that subcutaneous and intramam-mary antigenization of preparturient dams af-forded significant protection to the neonatalcalves. Seventy-seven percent of the calvesborn to vaccinated dams were protected againstan oral challenge with E. coli B-44. On thecontrary, only two of 14 calves born to dams inthe control group failed to reach a 4+ scourcondition after oral challenge with the test orga-nism.A semiquantitative index, derived from the

mean diarrhea score for each group of calves,showed that the KB vaccine was superior to theother three preparations. Next in the order ofefficiency were the LB and CS vaccine prepara-tion. Interestingly, the CS preparation appar-ently contained enough somatic and capsularmaterial to elicit a response sufficient to reactwith the challenge organisms and protect theneonates.

It was noted early in the experiments thatchallenging with strain B-44 after 20 h of agewas clearly less detrimental to the calf than anoral challenge administered within the first 10h of life. The only calf in the control group thathad less than 3+ diarrhea had been challengedbeyond 12 h of age; hence, a relationship be-tween age and susceptibility was suggested.

It is known that newborn calves absorb pro-teins more efficiently during the first 18 to 20 hof life (10), and further, that strains of entericorganisms begin to establish in the intestinaltract at 18 to 24 h of age (23). It would followthen that transfer of bacterial enterotoxins aswell as immunoglobulins would be influencedby the time related physiological mechanismsin the neonatal intestine. In addition, it is possi-ble that endotoxin and enterotoxin substancesare being released from dead and metabolicallyactive bacteria, respectively, during the timeperiod when the small intestine is exquisitelysensitive and vulnerable to molecular cytotoxicreactions. These factors quite likely contribute

to field cases of neonatal diarrhea (scours) aswell.The time of onset and duration of the clinical

syndrome caused by strain B-44 (profuse diar-rhea, severe dehydration, apathy, decrease inbody temperature and morbidity, death) andthe absence of bacteremia before the moribundstate was reached are typical of field cases ofthe disease. Bywater (4) reported that physiolog-ical changes caused by strain B-44 enterotoxinincluded a net increase in secretion of fluid,sodium, bicarbonate, and chloride from theblood into the small intestine of the calf. Simi-lar physiological changes in calves have beenassociated with naturally occurring neonataldiarrhea (7, 11, 18).

Results of the serological studies indicatethat, 2 weeks after a single antigenization, asignificant agglutinin response to 0 somaticand K antigen was not detectable in serum ofdams. At parturition (8 to 66 days after thesecond antigenization), only the anti-O serumtiters were significantly different from titers ofthe control dams. Anti-K activity still was notdetected in the sera of the controls or the vac-cine groups at parturition. These data wouldagree with the findings of others, that is, the 0-agglutinating titers in sera of vaccinated damsare generally higher than are the titers de-tected against capsular antigens of the vaccinestrain (12).The present studies would indicate that the

KB, LB, and CS vaccines, in that order, wereeffective in eliciting responses to the 0-somaticantigens. No such ordering of vaccine efficacywas possible using anti-K titers. Therefore,since both anti-O and anti-K antibody activityare highly likely to be involved in passive im-munity, an ordering of the effectiveness of KB,LB, and CS vaccines from the results of serumactivity of the dam is questionable.

Since the importance of colostrum for thesurvival of the calf has been established (1, 9,15), an evaluation of the three vaccines for pro-duction of protective factors in colostrum wouldbe an appropriate indication of vaccine effi-ciency. Since colostrum-derived antibody hadbeen shown by many investigators to be a criti-cal factor in passive immunity in neonatalcalves (2, 3, 26), it would follow that an effectivevaccine should elicit protective antibodies thatwill predominate in the dam's colostrum.The somatic antigens remaining after heat

treatment (boiling for 1 h) were reacted againstboth heated (56 C, 30 min) and unheated wheyobtained from individual quarters of the udderof each cow. In all groups, the titers detected inthe unheated whey were distinctly higher thancorresponding titers in the heated specimens.

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The greatest response, measured as agglutinat-ing activity, was noted in whey from damsantigenized with KB. The agglutinating titerin the LB group was one-half the titer detectedin the KB group. Surprisingly, the mean agglu-tinin response made by dams in the CS group

was, again, one-halfthe titer detected in the LBgroup. The low agglutinin titers of the controldams were most likely due to low avidity, cross-

reactive antibodies directed against the com-

mon antigen carried by all Enterobacteriaceae.One of the most interesting findings in the

agglutination studies centered about the effectof heat upon the whey titers. For example, LBand KB anti-O titers were not significantlydifferent (P > 0.05) in unheated whey but differsignificantly in heated specimens. CS titerswere not different (P > 0.05) from control titersafter the whey samples were heated (whey sam-

ples were heated prior to titration).The effect of heat upon whey agglutinins

(anti-O) was seen in the results of the anti-Ktitrations as well. The anti-K titers in colostralwhey were analyzed by the least square differ-ence statistical test. Titers ofheated whey fromdams antigenized with CS were not signifi-cantly higher than the titers in the controlgroup. In addition, the mean anti-K titer in theLB group was significantly (P < 0.05) lowerthan the mean titer in the KB group. However,both the KB and LB groups had mean titerssignificantly higher (P < 0.05) than the mean

titers in control whey. These data indicate thatthe CS was not as effective in stimulating anti-K agglutinins as KB or LB. Moreover, the effi-cacy of the KB and LB vaccine preparations,reflected by anti-O and anti-K titers, was appro-priately reflected in the scouring index as well.The effect ofthe factor(s) upon the agglutinat-

ing activity in whey is apparent; the effect invivo is unknown. Whether or not the heat-labile factor(s) represents an antibody to com-

mon enterobacterial antigen was not deter-mined.The strong relationship between agglutina-

tion titers of unheated whey and vaccine effec-tiveness suggests the complicity of a heat-labileimmune factor(s) in protection from scouring.The passive hemagglutination titers in se-

rum and whey of vaccinated cows, 2 weeks post-antigenization and at parturition, were signifi-cantly higher than titers observed in the sera

and whey of control cows. None of the mean

titers in any of the vaccine groups, however,were significantly different. The relative differ-ence seen, however, is similar to differencesnoted in the data for anti-O titers ofboth heatedand unheated whey. Furthermore, the related-ness of mean passive hemagglutination and

anti-O titers in whey specimens correlate wellwith the clinical findings.

Since endotoxin has not been shown to be acritical effector in E. coli enterotoxemias, it isdoubtful that the passive hemagglutination an-tibody detected in passive hemagglutination as-says functions in the protection against E. coli.However, immunological or physiological activi-ties contributing to the death of enteric orga-nisms coupled with a breakdown of the integ-rity of the intestinal mucus membrane (pro-duced by enterotoxin) may require the interac-tion of endotoxin neutralizing antibodies. It isapparent from these studies that anti-LPS ag-glutinating antibody is present in significantamounts in the serum and whey of the vacci-nated dams, and like the anti-O agglutinins,deserve consideration as significant factors inscouring.

Since absorption of colostral antibodies is lim-ited to the first 24 to 36 h of life (9), it was notsurprising that at birth these calves did notexhibit agglutinating activity against eitherthe 0 or K antigens. Of particular interest wasthe passive hemagglutination activity detectedin precolostral calf serum in each of thegroups, including the control calves. In uteroresponse to an LPS moiety could account for theantibody activity since immunocompetence hasbeen demonstrated in another ungulate, theovine (22, 21), and more recently in the fetalcalf (6).In contrast to the control calves, the calves in

the vaccine groups showed an increase in se-rum agglutinin activity by day 4. The mean 0titers were not significantly different betweenimmune groups, whereas the 0 titers in colos-trum were significantly different. Perhaps themost significant observation in these studiesrelated to the marked increase in the anti-Kactivity in sera of calves born to all vaccinateddams. The response was most pronounced inthe KB and LB groups and stands in contrast tothe lack of anti-K activity in the dams' sera.These results strongly suggest then that aneffective response to 0 somatic and K antigensof E. coli was produced in dams and that pas-sive transfer of immunity to calves via colos-trum was manifest as a marked diminution ofthe symptoms of and death from scouring. Thenature of the protective antigen is not clearlydefined by these studies but there is some evi-dence that the K antigen may play a vital rolein this regard.

ACKNOWLEDGMENTSWe acknowledge the assistance of the Veterinary Re-

search Laboratory (MSU) personnel and F. Newman, J.Catlin and C. Myers for their involvement in the clinicalinvestigations.

INFECT. IMMUN.

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SEROLOGICAL STUDIES ON COW COLIBACILLOSIS 99

This study was supported by Allied Professional HealthGrant 5TO1-AI-00131-09 and United States Public HealthService Grant AI-06552-09 from the National Institute ofAllergy and Infectious Diseases.

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VOL. 13, 1976

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