THE CANADIAN VETERINARY JOURNAL LA REVUE VETiRINAIRE CANADIENNE No. 8

August-aoOt 1976

Volume 17

THE EFFICACY OF A MODIFIED LIVE REO-LIKE VIRUS VACCINE AND AN E. COLI BACTERIN FOR PREVENTION OF ACUTE UNDIFFERENTIATED NEONATAL DIARRHEA OF BEEF CALVES S. D. Acres and 0. M. Radostits* by the Reo-like virus and EEC have been reported. The incidence of diarrhea and the mortality rate in calves decreased following the use of a modified live Reo-like virus (MLRV) vaccine (8, 9). An enteropathogenic strain of E. coli administered to cows in the form of either formalin killed whole cells, or live cells provided protection against the development of severe diarrhea in suckling calves challenged orally with the homologous strain (15, 16). A crude toxin preparation was also effective in preventing diarrhea (15). A formalin killed bacterin composed of six different strains of ETEC decreased the death loss due to naturally occurring diarrhea in several herds in which ETEC were isolated the previous year (13). This paper reports the results of field trials to evaluate the efficacy of a commercial MLRV-vaccinel and an experimental E. coli bacterin used separately and in combination in the prevention of diarrhea in newborn beef calves raised under commercial ranch conditions in Western Canada.

INTRODUCTION

ACUTE UNDIFFERENTIATED NEONATAL DIARRHEA of beef calves is an etiologically complex disease. Many bacteria and viruses have been incriminated as causal agents; however, considerable uncertainty exists about which ones are most important. Two infectious agents

which have been isolated from diarrheic calves in Western Canada are a Reo-like virus, now called the neonatal calf diarrhea (NCD) virus, and enteropathogenic Escherichia coli (EEC). Multiple infections of calves within the same herd with both of these agents have been demonstrated (1). The EEC possess two properties: 1) they produce enterotoxin (enterotoxigenic E. coli, ETEC) and 2) they have the ability to multiply to large numbers in the small intestine (21). The latter characteristic may be associated with the presence of K99 antigen (10, 14, 18). A Corona-like virus (25) also occurs in diarrheic calves in some areas (1, 5, 11, 25) and multiple viral infections of calves with the Reo and the Corona-like viruses have been reported (1, 5, 11). Other multiple mixed viral infections and mixed bacterial and viral infections in calves have been documented (1, 11, 26, 28). Vaccination trials against diarrhea caused *Department of Veterinary Clinical

MATERIALS

Studies,

Norden Laboratories, Lincoln, Nebraska. Dr. Acres is a fellow of the Medical Research Council of Canada.

lScourvax-Reo, Norden Laboratories, Lincoln, Nebraska. 2Samples processed by Norden Laboratories, Lincoln, Nebraska. 197

CAN. VET. JOUR., vol. 17, no. 8, August, 1976

METHODS

His-tory During the 1973 spring calving season fecal samples, rectal swabs, and nasal swabs were collected from one or more calves in 59 beef herds in Alberta and Saskatchewan. Samples were also taken from the small intestine of eight calves which had died from diarrhea in the same herds. Fecal and intestinal samples from calves in 39 of the herds were examined for the presence of Reo-like virus by the fluorescent antibody technique (FAT)2 (5, 7).

Westem College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO. This work was supported by funds from the Alberta Cattle Commission, the Alberta Agricultural Research Trust, the Saskatchewan Department of Agriculture and the National Research Council. The Escherichia coli laboratory was supported by Canada Department of Agriculture EMR Grant 7111. The modified live reo-like virus vaccine and placebo were kindly supplied by

AND

CANADIAN VETERINARY JOURNAL

Rectal and intestinal swabs from calves in 54 herds were smeared onto blood agar plates and isolated colonies of E. coli were picked from each plate and stored on trypticase-soy (TS) slants until they could be screened for the presence of ETEC by the ligated gut loop test in calves (1, 21). Reo-like virus alone was demonstrated in 13 of the 39 herds examined (33.3%), ETEC alone in 21 of 55 herds (38.1%), and both agents were found in five of 35 herds (14.3%) examined for both agents. These figures probably underestimate the herd prevalence rates of these two agents because in some herds samples were obtained from only one or two calves which had been diarrheic for several days and which had been treated with oral antibacterials. Fecal samples from all calves were also incubated for 24 hours in selenite broth and then streaked onto Hektoen-enteric agar and brilliant green agar plates for isolation of Salmonella species. None were found. Fecal and nasal samples from selected herds were also examined for the presence of the infectious bovine rhinotracheitis virus, bovine virus diarrhea virus and parainfluenza-3 virus by the methods described previously (1). None of these viruses were found. Samples collected during the 1973 calving season were not examined for the presence of Corona-like virus. Following demonstration of the above infectious agents, the vaccination trials reported here were undertaken during the 1974 calving season.

The Modified Live Reo-Like Virus Vaccine The MLRV-vaccine was supplied in boxes of 50 individual vials by the manufacturer.3 Each box contained 25 vials of vaccine and 25 vials of placebo distributed randomly and identified only by means of a number from 1 to 50. The key that identified which vials contained vaccine and which contained placebo was withheld from both the field veterinarian (SA) and the rancher. Boxes containing the vaccine and placebo were distributed to the cooperating ranches prior to the onset of calving. Preparation of Escherichia coli Bacterin Three strains of E. coli, each of which had been shown to produce enterotoxin by the ligated gut loop test and to possess the K99 antigen,4 were incorporated into a triple strain,

formalin killed bacterin. The bacterin was used in two forms; one adjuvanted with Freund's incomplete adjuvant (FICA) and one precipitated with aluminum potassium sulfate (alum). The ETEC isolated in Western Canada which can be serotyped with conventional antisera are most commonly members of serogroups 08 and 09 (1, and unpublished data). Strains S16 (08:K85:?), Ps274 (09:K(A)) and a K12 strain to which K99 antigen had been transferred were incorporated into the bacterin. It was hypothesized that K99 antibodies would prevent the attachment of K99+ ETEC to the small intestinal mucosa thereby preventing diarrhea. Strain S16 had previously been used to immunize pregnant ewes. The lambs born to these ewes were protected against diarrhea following oral challenge with the homologous strain whereas lambs from unvaccinated ewes were not protected (unpublished data). The three strains of E. coli were grown in trypticase soy broth (TSB) for six hours and then added to litmus milk and stored frozen at -70°C in 0.5% ml quantities. At the time of bacterin preparation, one litmus milk vial of each strain was inoculated separately into 20 ml of TSB and grown with constant shaking for six hours at 370C. Each 20 ml culture was then seeded separately into 5,000 ml of TSB and grown for another 18 hours with constant stirring, after which time each culture was concentrated by centrifugation to contain approximately 0.14-1.84 x 1010 viable organisms per ml. Following addition of 0.6% formalin each cell suspension was incubated overnight and then checked for sterility. Equal volumes of each of the three cell suspensions were combined to give a final suspension which contained an average of 1.13 x 1010 organisms per ml. The final suspension was combined with an equal volume of either 1) FICA or 2) sterile physiological saline (PBS) to which sufficient alum was then added to give a final solution of 2.5% aluminum potassium sulfate. This gave bacterin preparations which contained approximately 0.57 x 1010 bacterial cells per ml. Placebos were prepared by combining equal volumes of FICA or 5.0% alum with sterile PBS. The alum adjuvanted bacterin and placebo were adjusted to pH 6.8-7.0 using sterile sodium hydroxide.

3Scourvax-Reo, Norden Laboratories, Lincoln, Nebraska. 4Strain of E. coli kindly supplied by Dr. C. L. Gyles, University of Guelph, Guelph, Ontario.

Immunization Procedure and Collection of Samples MLRV-vaccine or placebo was administered

orally by the rancher to each calf within 24 hours of birth starting with the first calf born in the herd. The E. coli bacterin and placebo 198

NEONATAL DIARRHEA

TABLE I FOUR IMMUNIZATION COMBINATIONS USED TO EVALLTATE THE EFFICACY OF A MODIFIED LIVE REOVIRUS (MLRV) VACCINE AND AN E. coli BACTERIN AGAINST CALF DIARRHEA. TRIAL 1, HERD NOS. 1 TO 9. WESTERN CANADA 1974

E. coli bacterin to cow

MLRV-vaccine to calves Placebo to calves

Group I

4 herd

Group III 4 herd

Group II 4 herd

Groups IV 4 herd

TABLE II THE INCIDENCE RATE OF DIARRHEAa AND THE CASE FATALITY RATE ASSOCIATED WITH DIARRHEA (CFRD)b IN CALVES UNDER 30 DAYS OF AGE. HERD NOS. 1 TO 9. WESTERN CANADA 1973

Herd No. 1

Normal 18

Diarrhea

53

(75)a 2 3

4

5 6

3 75

123

99 45

167 (98) 25 (25) 82

(40) 26

(21) 10

(18) 7

8

56

430

14 (20) 125 (23)

Died 1

(1. 9)b 2

(1.2) 4

(16.0) 4

(4.9) 21

(80.8) 7

(70.0) 3

(21.4) 15

9

214

100

(12.0) 25 (25.0)

Total

1063

602

82 (13.6)

(32)

(36)

Placebo to cow

aIncidence rate of diarrhea (in brackets) = (the number of calves which developed diarrhea by 30 days of age)/(the number of calves born) X 100% bCase fatality rate associated with diarrhea (in brackets) = (the number of calves which died from diarrhea by 30 days of age)/(the number of calves which developed diarrhea by 30 days of age) X 100% were administered randomly to pregnant cows twice; the first time approximately six weeks prior to the onset of calving in the herd and a second time three weeks later. Five ml was given subcutaneously at each injection. The herd owner was unaware of which cows received bacterin or placebo. Cows and calves were identified by means of ear tags. No attempt was made to alter the existing management practices in any of the 199

herds. The decisions of when and how to treat diarrheic calves were left to the discretion of each owner. Ranchers were supplied with record cards and were asked to record the health status of each calf until 30 days of age and to collect fecal samples as soon after the onset of diarrhea as possible. Fecal samples were frozen and later examined for the presence of Reo-like virus by FAT and electron microscopy (EM) and for Corona-like virus by EM (7)5. The dry matter content (DMC) of fecal samples was determined by methods outlined previously and was used to confirm the presence of diarrhea (1). Diarrhea was considered to be present when the fecal DMC was < 10%. Two separate trials were conducted: Trial 1 - Since Reo-like virus and ETEC had occurred in several herds, Trial 1 was designed to examine the efficacy of each immunizing preparation separately and in combination. Each of eight herds were divided into four immunization groups of approximately equal size (Groups I, II, III and IV). Within each herd both the MLRV-vaccine and the E. coli bacterin in FICA were used alone and in combination according to the design shown in Table I. One other herd (Herd No. 9) was treated in a similar manner except that the cows were immunized with alum adjuvanted E. coli bacterin or placebo. The average incidence rate of, and case fatality rate associated with diarrhea (CFR-D) in Herd Nos. 1 to 9 in the previous year were 36% and 13.6% respectively (Table II). Reo-like virus had been demonstrated in the feces of calves from all nine herds and ETEC were present in Herd Nos. 3, 5 and 8 in 1973. No attempt was made to diagnose the presence of either agent prior to the onset of the trial in 1974. The MLRV-vaccine had not been used in any of these herds prior to the onset of the trial. The owner of Herd No. 2 was unwilling to have E. coli placebo administered to his cows. 5Samples processed by Norden Laboratories, Lincoln, Nebraska.

CANADIAN VETERINARY JOURNAL

Within this herd only, cows in immunization Groups III and IV were not injected. Also within Herd No. 2 the use of MLRV-placebo was discontinued during the middle of an outbreak of diarrhea and every calf was given MLRV-vaccine. Later calves were not given any treatment. Results of Trial 1 were analyzed using Friedman's Two-way analysis of variance by ranks (Friedman's X2) (4). This procedure is designed to detect any tendency for the results (expressed in percent) in one immunization group to consistently exceed or be smaller than the results in the other immunization groups. Chi square (X2) analysis (20) and one-way analysis of variance (ANOVA) (24) were also used. Trial 2 - Eight herds (Nos. 10 to 17), one of which was subdivided into two groups (for convenience called Herd Nos. 10a and 10b) participated in this trial. The incidence of diarrhea was above 27% in each of these nine herds during the previous year. Approximately one-half of the cows in Herd Nos. 10a, 11, 12 and 13 received the E. coli bacterin or placebo in FICA. One-half of the cows in Herd Nos. 10b, 14, 15, 16 and 17 received the E. coli bacterin or placebo in alum adjuvant. Reo-like virus and ETEC were present in Herd Nos. 10a, 10b and 15, and ETEC were present in Herd No. 11 during 1973. The MLRV-vaccine and placebo were delivered to Herd No. 15 and were to be used only in the event of an outbreak of diarrhea. Herd Nos. 12, 13, 14, 16 and 17 were not among the 59 herds sampled in 1973 and no attempt had been made to diagnose which infectious agents were present in these herds. Results were analyzed using the Wilcoxon Signed Rank Test (20), Chi square analysis (x2) and one-way ANOVA. Levels of probability greater than 0.05 (P > 0.05) were considered not significant (NS) while levels less *than 0.05 (P < 0.05) were considered significant (S) in both Trials 1 and 2. The incidence rate of diarrhea was defined as: the number of calves whiclh developed diarrhea bv 30 days of age x 100% the total number of calves born

The diarrhea-specific neonatal mortality rate (NMR-D) was defined as: the number of calves which died from diarrhea by 30 days of age x 100% the total number of calves born

the case fatality rate associated with diarrhea (CFR-D) was defined as: the number of calves which died from diarrhea by 30 days of age the number of calves which developed diarrhea by 30 days of age

x

200

RESULTS Trial 1 - The Effect of Immunization on the Incidence of Diarrhea

The incidence of diarrhea and the CFR-D for calves in each of the four immunization groups are shown in Table III. The incidence of diarrhea was high (>35%) in Herd Nos. 1, 2 and 3. Herd Nos. 4, 5 and 6 experienced an intermediate incidence (10-12%) and Herd Nos. 7 and 8 a low incidence ( 0.05, NS) among the incidences of diarrhea in calves which received the four immunization combinations. Within each individual herd there were no significant differences (Chi square procedure) among the incidences of diarrhea in calves in the four immunization groups. Further analysis of the data in Table III by the Chi square procedure is shown in Tables IVa, b and c. When the total number of calves in each of the four immunization groups were considered (Table IVa), the incidence of diarrhea in the calves in Group III (E. coli placebo to cow MLRV-vaccine to calf) appeared to be significantly lower than in the other three groups of calves (X2 = 8.34, P < 0.05, S). However, closer inspection of Table III indicated that this apparent significance resulted from the fact that there were only 13 calves in Herd No. 2 which fell into immunization Group III, whereas there were 47, 45 and 39 calves in Herd No. 2 which fell into immunization Groups, I, II and IV, respectively. Since all of the calves in Herd No. 2 developed diarrhea, the low number of calves in immunization Group III from this ranch resulted in the erroneous indication that there was a significant difference among incidences of diarrhea on all ranches. Elimination of Herd No. 2 and recalculation of the Chi-square value on the remaining seven herds indicated that there were no significant differences in the frequency of diarrhea calves among any of the four immunization groups (X2 = 1.20, P < 0.05, NS, Table IVa). The total number of calves in Table III was divided into two groups based on the immunization history of their dams only (E. coli bacterin vs. placebo, Table IVb). The apparently higher (X2 = 6.47, P < 0.01, S) frequency of

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0.05, NS.

to

calf

Placebo

to

calf

Total 284 682 966

Diarrhea 159 (30.6) 125 (27.9) No diarrhea 323 359 518 Total 448 aIncidence of diarrhea. X2 on Herd Nos. 1 to 8 = 0.90, P > 0.05, NS. X2 after elimination of Herd No. 2 = 0.04, P > 0.05, NS.

diarrheic calves in the group born to cows P > 0.05, NS, Table V). Similarly, there were which received the E. coli bacterin was again no significant differences between the NMR-D due to the low number of calves in Herd No. in calves when the immunization history of the 2 which fell into immunization Group III. dams only (E. coli bacterin vs. placebo) was Analysis of the data after removal of Herd considered, or when the immunization history No. 2 indicated that there were no significant of the calves only (MLRV-vaccine vs. placebo) differences (X2 = 0.40, P > 0.05, NS) in the was considered (calculations not shown). frequency of calves which became diarrheic There were no significant differences among based on the immunization history of the cows the CFR-D for calves in each of the four only. immunization groups on a herd basis (FriedWhen the immunization history of the man's x2 = 2.85, P > 0.05, NS). However, calf only was considered (MLRV-vaccine vs. on the basis of the total number of calves in placebo) there were no differences in the fre- all eight herds the CFR-D was significantly quency with which calves became diarrheic higher for calves in immunization Group III (Table IVc). (placebo to cow - MLRV-vaccine to calf) (X2 = 10.35, P < 0.005, S; Table VI). There were Trial 1 - The Effect of Immunization on the no significant differences between the CFR-D when the immunization history of the dams Diarrhea-Specific Neonatal Mortality Rate (NMR-D) and the Case Fatality Rate only (E. coli bacterin vs. placebo) or of the Associated with Diarrhea (CFR-D) calves only (MLRV-vaccine vs. placebo) was Analysis of the data in Table III indicated considered (calculations not shown). that there were no differences among the NMR-D for calves in each of the four immun- Trial I - Other Epidemiological Observations ization groups either on a herd basis (Fried- Used to Evaluate the Efficacy of the man's x2 = 3.15, P > 0.05, NS; calculations Immunization Combinations not shown) or on the basis of the total numFour other epidemiological observations ber of calves in the eight herds (X2 = 6.95, which were used to evaluate the efficacy of the 202

NEONATAL DIARRHEA

TABLE V THE DIARRHEA SPECIFIC NEONATAL MORTALITY RATE (NMR-D)a IN CALVES CLASSIFIED ACCORDING TO IMMUNIZATION COMBINATION. TRIAL 1, HERD Nos. 1 to 8 E. coli bacterin to cow (FICA) No. of calves which

MLRV-vaccine to calf

Placebo to calf

Placebo to cow (FICA)

MLRV-vaccine to calf

Died 5 (2.1)a 10 (3.9) 11 (5.3) Did not die 237 248 195 Total born 242 258 206 "The diarrhea-specific neonatal mortality rate (in brackets) = the number of calves which died from diarrhea by 30 days of age X 100% the total number of calves born

Placebo to calf 4 (1.5) 256 260

Total 30 (3.1) 936

966

x2 = 6.95, P > 0.05, NS. TABLE VI THE CASE FATALITY RATE ASSOCIATED WITH DIARRHEAa (CFR-D) IN CALVES CLASSIFIED ACCORDING TO IMMUNIZATION COMBINATION. TRIAL 1, HERDS Nos. 1 TO 8

E. coli bacterin to cow (FICA) No. of calves with

MLRV-vaccine to calf

Placebo to calf

Diarrhea-died

Placebo to cow (FICA)

MLRV-vaccine to calf

5 (6.3) 10 (11.6) 11 (23.9) Diarrhea-did not die 74 76 35 Total 79 86 46 aThe case fatality rate associated with diarrhea (shown in brackets) = the number of calves which died from diarrhea by 30 days of age X 100% the total number of calves which developed diarrhea by 30 days x2 (calves in all 4 immunization groups) = 12.28, P < 0.01, S. x2 (immunization groups I, II, IV vs III) = 10.35, P < 0.005, S.

Placebo to calf 4 (5.5)

69 73

Total

30 (10.6) 254 284

immunization combinations are shown in listed above for either of these two series of Table VII. Analysis of the data from Herd calves. Nos. 1 to 5 only using Friedman's two-way Within Herd No. 9 the ratio of diarrheic to ANOVA by ranks indicated that there were nondiarrheic calves and the incidence of diarno significant differences in: 1) the average rhea in immunization Groups I to IV respecage of calves at the onset of diarrhea, 2) the tively were: 1/11 (8.3%), 14/130 (9.7%), average duration of diarrhea or 3) the average 12/106 (10.2%) and 0/12 (0%). There were duration of treatment for diarrhea among no significant differences. Only one calf died calves in any of the four immuization groups. in this herd. One-way ANOVA indicated that within Herd No. 3 the calves born to vaccinated cows were Trial 1 - The Average Age of Calves at the older at the onset of diarrhea (mean 7.02 Time of Administration of MLRV-vaccine days) than calves born to control cows (mean or Placebo 3.75 days) (P < 0.001, S). Recurrent bouts The average ages of calves in Herd Nos. 1 of diarrhea in the same calf was not a major to 7 at the time of administration of the problem within any of the eight herds (Table MLRV-vaccine or placebo were within the VII). During the outbreak of diarrhea in recommended 24 hour time limit (Table Herd No. 2 the use of MLRV placebo was VIII). Calves in Herd No. 2 were older than stopped and a series of 11 calves born in suc- calves in the other herds at the time of imcession were given the MLRV vaccine. Fol- munization; six calves in this herd were older lowing this the next 21 calves born were not than 24 hours when immunized. The average given vaccine or placebo. There were no age of calves when the MLRV-vaccine or noticeable changes in the three observations placebo was administered did not differ be203

CANADIAN VETERINARY JOURNAL

TABLE VII FOUR EPIDEMIOLOGICAL OBSERVATIONS USED TO EVALUATE THE EFFICACY OF THE FOUR IMMUNIZATION COMBINATIONS. TRIAL 1, HERDS NoS. 1 TO 8 E. coli bacterin to cow

Observations Average age of calvesat onset of diarrhea (days)

Herd No.

1 2 3 4 5 6 7 8 Average 1 duration 2 of 3 diarrhea 4 (days) 5 6 7 8 Average 1 duration 2 of 3 treatment 4 for 5 diarrhea 6 (days) 7 8 Proportion 1 of 2 calves 3 in which 4 diarrhea 5 occurred 6 more than 7 once 8 aNo cases of diarrhea occurred. bData not recorded by rancher.

Placebo to cow

MLRV-vaccine to calf

Placebo to calf

MLRV-vaccine to calf

Placebo to calf

11.4 6.7 6.0 8.0 14+ 14+ 2.5 N.C. 1.7

12.8 4.0 7.6 10.3 6.5 8.0 11.0 N.C. 1.7 N.R. 2.6 2.3 3.8 6.0 N.R. N.C. 2.4 N.R. 2.8 1.5

10.3 6.2 3.1 8.0 6.0 N.C. N.C. N.C. 1.4 N.R. 1.6 3.0 5.0 N.C. N.C. N.C. 2.3 N.R. 1.5 2.4 4.0 N.C. N.C. N.C. 4/15 N.R. 1/9 0/5 0/4 N.C. N.C. N.C.

13.0 4.8 3.4 12.0 N.C. 7.0 N.C. 14+ 1.9 N.R. 2.1 3.3 N.C. 2.3 N.C. 3.0 2.7 N.R. 2.1 2.4 N.C. 3.0 N.C. 3.0 1/17 N.R. 1/7 0/5 N.C. 0/3 N.C. 0/2

N.R.b 3.0 1.5 4.7

2.0 1.0 N.C. 2.6 N.R. 3.4 1.3 4.7 1.0 1.0 N.C. 2/14 N.R. 1/8 0/4 0/3 0/1 0/2 N.C.

4.5 N.R. N.R. N.C. 3/12 N.R. 1/16 0/4 0/6 0/2 0/1 N.C.

tween calves which remained normal and those which became diarrheic in any of the herds.

Trial 1 -The Occurrence of Reo- and Corona-like Viruses in Fecal Samples Thirty-six fecal samples from calves in Herd Nos. 1 to 5 were examined by FAT and EM (Table IX)6. Reo-like virus only was found in Herd No. 1, Corona-like virus only in Herd No. 4, and both viruses in Herd Nos. 2 and 3. Reo-like virus was not found in two samples from calves in Herd No. 5. Reo-like virus was found in 14 of 36 (39%) calves examined; eight by FAT, seven by EM, and one by both FAT and EM. Five of the 14 (36%) calves which were positive for Reolike virus plus five additional calves (total 10) 6Samples processed by Norden Laboratories, Lincoln, Nebraska. 204

were positive for Corona-like virus. Reo-like virus particles were seen by EM in the feces of one calf which was not diarrheic (Calf No. 1). The fecal DMC of this calf at the time of sampling was 13.4%. Reo-like virus was demonstrated in five of 12 (42%) calves examined in Herd Nos. 1 and 3 which had received the MLRV-vaccine, and in nine of 24 calves which were not given MLRV-vaccine. However, there were no significant differences in the proportion of calves which were positive for Reo- or Corona-like virus between those which received MLRVvaccine or placebo (Table X). In Corona-like virus infected herds (Herd Nos. 2, 3 and 4) the CFR-D was significantly higher (X2 =10.28, P < 0.025, S) in calves in immunization Group III (E. coli placebo to cow - MLRV-vaccine to calf) than in calves in the other three immunization groups. There was no significant difference in the CFR-D among calves in the

NEONATAL DIARRIHEA

TABLE VIII THE RANGE AND AVERAGE AGE (HOURS) OF CALVES AT THE TIME OF ADMlNISTRATION OF THE MLRV-VACCINE OR PLACEBO. TRIAL 1, HERD Nos. 1 TO 7a Immunization Groupb II III IV Normal Diarrhea Normal Diarrhea Normal Diarrhea Normal Diarrhea 1 1-4 2-160 1-6 1-4 1-4 1-3 1-4 1-5 5d 2 2 2 2 2 2 3 2 N.C.e 1-28 N.C. 4-26 N.C. 3-25 N.C. 3-25 15 17 13 15 3 2-10 2-8 1-8 2-8 2-8 2-8 1-8 4-14 5 5 4 4 4 5 5 7 4 1-20 1-3 1-14 1-10 1-32 2-20 1-6 1-2 5 2 4 4 7 7 3 2 5 L-15 3-5 1-20 1-12 1-22 1-4 i-14 N.C. 5 5 6 6 5 2 7 6 2-14 0 3-19 4-7 N.C. 2-20 4j-20 6-9 8 4 10 6 11 10 8 7 1-30 2-3 j-18 0 j-6 j-24 N.C. N.C. 8 3 5 4 2 4 aThis data not recorded for Herd No. 8. bI = E. coli bacterin to cows-MLRV-vaccine to calves, II = E. coli bacterin to cows-Placebo to calves, III = Placebo to cows-MLRV-vaccine to calves, IV = Placebo to cows-Placebo to calves. oRange. dAverage to nearest hour. eNo calves in this group. Herd No.

I

four immunization groups from Herd Nos. 1, 5, 6, 7 and 8.

were no significant differences betwen immunized and control groups in the following parameters: 1) the average age of calves at the onset of diarrhea, 2) the average duration in days of diarrhea, 3) the average duration in days of treatment for diarrhea and 4) the proportion of calves in which diarrhea reoccurred. Reo-like virus was demonstrated by FAT in nine of 17 (53%) fecal samples and Corona-like virus was detected by EM in two of five (40%) fecal samples from calves in Herd Nos. lOa- and 10b. One of the above calves, which became diarrheic at six days of age, was positive for both viruses. No samples were available from Herd No. 14. The MLRV-vaccine and placebo which had been delivered to Herd No. 15 were not used.

Trial 2 - The Effect of Immunization of the Dam with E. coli Bacterin on the Incidence of Diarrhea in Calves The incidence of diarrhea and the CFR-D for calves born to cows immunized with the E. coli bacterin or placebo adjuvanted with either FICA (Herd Nos. 10a, 11, 12 and 13) or with alum (Herd Nos. lOb, 14, 15,-16 and 17) are shown in Table XI. The incidence of diarrhea in immunized and control groups of calves was significantly lower than the herd incidence during the preceding year in all except Herd Nos. 10a, 10b and 14 (calculations not shown). Analysis of the data from all nine herds using the Wilcoxon Signed Rank Test indiDIscUSSION cated that there was no difference in the incidence of diarrhea or the CFR-D between imNeither the MLRV-vaccine nor the E. coli munized and control groups on a herd basis. bacterin used alone or in combination effecThe incidence of diarrhea was significantly tively reduced the incidence or severity of lower in the immunized as compared to the diarrhea. The MLRV-vaccine did not reduce control group within Herd No. 14 (x2 = 13.9, the severity of diarrhea in three herds (Herd P < 0.005, S) but not within Herd Nos. 10a Nos. 2, 3 and 4) in which Corona-like virus and 10b. Neither the NMR-D nor the CFR-D was present during the trial. Within these differed between immunized and control three herds the CFR-D was significantly groups in these three herds. higher in calves given the MLRV-vaccine One-way ANOVA on the data from Herd only (immunization Group III) than in the Nos. 10a, 10b and 14 indicated that there other three immunization groups. 205

CANADIAN VETERINARY JOURNAL

RESULTS

OF

TABLE IX FECAL EXAMINATION FOR REO AND CORONA-LIKE VIRUSES IN 36 CALVES IN FOUR IMMUNIZATION GROUPS. TRIAL 1, HERD NoS. 1 TO 5 Result of fecal examination for

Herd No.

Calf No.

Immunization Combinationa

1

1 2 3 4 5 6

I II III IV IV IV

2

7 8 9 10 11 12 13 14 15 16 17 18 19

Reo-like virus FATb EM c

_d

+e

+

NE9

NE

-

NE NE NE

NE NE NE

1/6

1/2

0/2

NE

NE + NE NE + +

I II II II II II II II II II IV IV IV

+

NE

20 21 22 23 24 25 26 27 28 29 30

II II II II III III III IV

31 32 33 34

II II III IV

35 36

I

-

III

-

+ +

+

5

± NE + 3/8

NE + + NE + 7/8

NE NE + NE NE NE

NE NE + NE NE NE

+

+

4/11 4

-

+ NE NE

3/13 3

Corona-like virus EM c

0/4

0/2

NE

NE + NE 3/4 NE NE

NE NE NE 2/4

NE 0/1 NE NE 0/0

NE 1/1 NE NE 0/0

Age at Onset of Diarrhea

ND'

4 7 30 12 5

5 4 NRh

5 6 7 6 6 6 7 4 4 6 7 5

14± 5 7 6 2 2 4 5 2

9 7 9 9

14+

NR

Total Positive Results 8/36 7/15 10/15 aI = E. coli bacterin to cows-MLRV-vaccine to calves, II E. colibacterin to cows-placebo to calves, III = Placebo to cows-MLRV-vaccine to calves, IV Placebo to cows-placebo to calves. bFluorescent antibody technique. cElectron microscopy. Diagnostic procedure performed by Norden Laboratories, Lincoln, Nebraska. =

=

dNegative. epositive. fWas not

diarrheic when sampled at 12 days of age. Fecal DMC gNot examined by this procedure. hData not recorded.

206

=

13.4%.

NEONATAL DIARRHEA

TABLE X THE PROPORTION OF FECAL SAMPLES POSITIVE FOR REO OR CORONA-LIKE VIRUS CLASSIFIED BY IMMUNIZATION HISTORY OF THE CALVES ONLY. TRIAL 1, HERD NoS. 1 TO 5 Reo virusa

Calves immunized with

MLRV-vaccine (GroupsI +III) Placebo (Groups II + IV) Total

Corona Virus Pos. Neg.

Pos.

Neg.

Total Examined

5

7

12

3

3

6

9 14

15 22

24 36

7 10

2 5

9 15

= 0.06, P > 0.05, NS x2 aIncludes results of fluorescentxI antibody and electron microscopy procedures.

=

Total Examined

1.25, P > 0.05, NS

TABLE XI THE INCIDENCE OF DIARRHEA AND THE CFR-D IN CALVES UNDER 30 DAYS OF AGE CLASSIFIED ACCORDING TO THE IMMUNIZATION STATUS OF THEIR DAMS. TRIAL 2, HERD Nos. 10 TO 17

Immunization given to dam E. coli bacterin (FICA)a

Herd No.

Normal

10a

32

Diarrhea 13

(28.9)b 11

59

2

(3.3) 12

23

0

(0) 13

Subtotal

115 229

5

Placebo (FICA),

Died

Normal

3

32

(23.1)0 0

(0) 0

(0) 0

(4.2)

(0)

20

3

(8.0)

(15.0)

E. coli bacterin (alum adjuvant) Normal 10b

24

Diarrhea 12

(33.3) 14

109

13

(10.7) 15 16

17

Subtotal

168 80 48 429

4

(2.3) 2 (2.4) 0

Died 2

(16.7) 0

23

109 223

31 93

0

74

(0)

31

2 (6.5)

aFreund's lete adjuvant. blncidence incomp of diarrrhea. cCFR-D.

207

1

(23.8)

(10.0)

0 (0) 0 (0) 7

0 (0) 0 (0) 0

(6.0)

(0)

17 (7.1)

1 (5.9)

14

(31.1) 167

(0) 0

10

Died

Placebc (alum adjuvant) Normal Diarrhea Died

(0) 0 (0)

(0) (6.7)

59

Diarrhea

39

(29.5)

22

387

2

3

(21.4) 1

(2.6) 0

(1.2)

(0)

5 (6.3) 0 (0) 60

0 (0) 0 (0) 4

(13.4)

(6.6)

CANADIAN VETERINARY JOURNAL

These results are different from published reports which claimed that the incidence of diarrhea and the morality rate decreased in Reo-like virus infected herds following the use of an oral MLRV-vaccine (8, 9). Our observation that the MLRV-vaccine did not reduce the incidence of diarrhea in Coronalike virus infected herds agrees with the observations made previously (8, 9). However, the results of the present trial differ from one previous report which suggested that the use of a MLRV-vaccine reduced the severity of diarrhea in Corona-like virus infected herds (8). There are several obvious differences between the experimental design of this trial and the two previous reports which may explain the different results obtained. This trial was conducted on a double blind basis: neither the rancher nor the field veterinarian knew which calves received vaccine or placebo. The order in which vaccine and placebo were administered was randomized before the trial was started so that vaccinated and control calves within each herd were exposed to the same environmental and management conditions. The health status of all calves from birth to 30 days of age was recorded while the trial was in progress. Neither of the two earlier trials was conducted on a blind or even a randomized basis. Vaccine, but not placebo, was left with the rancher and was administered by him. Vaccination was started at the beginning of calving or at some unspecified time during the calving season, and control calves were either those born during the same year before vaccination was started, or those born during the previous year (8, 9). There are several potential sources of bias which may have influenced the results of these trials: 1) The health status of vaccinated calves was recorded during the trial whereas that of the control calves was estimated retrospectively. Thus it is possible that information on vaccinated calves was more accurate than on control calves, in which case data for the two groups are not comparable (6). 2) Ranchers may have expected vaccinated calves to be healthier than calves which were not vaccinated in the previous or the same year and this may have influenced the observations which they reorded (17). 3) The incidence of diarrhea fluctuates within and between calving seasons, therefore it is difficult to compare the effect of a vaccine used one year with no vaccine in the previous year. The experimental design of the earlier work was based on the hypothesis that if control 208

calves developed diarrhea due to the Reolike virus they would contaminate the calving grounds with the virus and allow infection and disease to occur in vaccinated calves before protection developed (8). It was also thought that vaccinated calves may shed the vaccine virus and indirectly vaccinate contact calves thereby making meaningful comparison between the two groups difficult. No evidence was presented to substantiate these views. However, if these considerations are valid it suggests that the double blind technique, while reliable for evaluating drugs in individual patients, may not be reliable for the evaluation of vaccines in herds of animals where vaccinates and nonvaccinates mingle. There are at least two possible explanations for the results observed in the trials reported here: 1) The MLRV-vaccine and the E. coli bacterin were ineffective in preventing diarrhea caused by their respective agents under the conditions of the trial. 2) Enteropathogens other than Reo-like virus and ETEC were present. The assumptions made in the experimental design of Trial 1 were that: 1) the use of the MLRV-vaccine would reduce the incidence or severity of diarrhea in Reo-like virus infected herds and 2) there would be some additional effect from the use of E. coli bacterin in at least Herd Nos. 3, 5 and 8. The use of both immunization procedures together should have eliminated any tendency for Reolike virus or ETEC to obscure the effect of either the MLRV-vaccine or the E. coli bacterin used separately. Both assumptions are invalid in the presence of any other enteropathogens. The efficacy of the MLRV-vaccine against Reo-like virus cannot be evaluated in Herd Nos. 2, 3 and 4 because Corona-like virus was present in these herds. Similarly, the efficacy of the E. coli bacterin against ETEC cannot be evaluated in Herd No. 3. The possibilitv cannot be eliminated that Corona-like virus, or some other enteropathogen, was also present before or during the trial in Herd Nos. 1, 5, 6, 7, 8 and 9. Also, it was assumed that because ETEC were present in Herd Nos. 3, 5, 8, 10, 11 and 15 in 1973, that they would be present in these herds in 1974. This was not confirmed. The MLRV-vaccine did not reduce the incidence of diarrhea in herds infected with Corona-like virus (Herd Nos. 2, 3 and 4). Within these three herds the CFR-D was significantly higher in calves given the MLRVvaccine only (immunization Group III) than in the other three immunization groups. This

NEONATAL DIARRHEA

observation suggests that use of the MLRVvaccine alone may be contraindicated in herds infected with Corona-like virus. Explanation of the increased CFR-D in vaccinated calves would require knowledge about the immunological mechanism of action of the MLRVvaccine and about the factors which influence the temporal, spatial and numerical relationships between the Reo and Corona-like viruses within an individual calf and within a herd; such knowledge is not currently available. The efficacy of the MLRV-vaccine will probably vary among herds even in the absence of enteropathogens other than the Reolike virus. In the present trial the average age of calves at the time of vaccination was well within the recommended 24 hour age limit. However, exposure to virulent organisms probably occurs immediately after birth and may precede vaccination if the environment is heavily contaminated or if the population density is high. It has also been suggested that the protective effect of vaccines can be overwhelmed if vaccinated calves are exposed to diarrheic calves (8, 27). In our study Reolike virus was found in five vaccinated calves (Calf Nos. 1, 20, 21, 22, 29, Table IX) and in nine unvaccinated calves (Calf Nos. 2, 8, 9, 13, 15, 17, 19, 23, 26, Table IX). Since it is not possible to distinguish the vaccine virus from the field strain the significance of these observations cannot be evaluated. The MLRV-vaccine should be administered to conventionally raised calves under controlled conditions in order to determine the following: 1) the time following vaccination at which resistance to oral challenge develops, 2) the challenge dose of virulent virus necessary to overcome resistance and 3) the degree of shedding of the vaccine strain of the virus to contact calves. Germ free, colostrum deprived calves which were given a MLRVvaccine when six to seven hours old were resistant to challenge 48 to 72 hours later (9). However, the development of resistance in the intestines of calves which are contaminated with normal microflora may follow a different pattern than in germ free calves. It is also possible that a high colostral antibody titer could neutralize the vaccine strain of the virus in the intestines of suckling calves. Three strains of E. coli which produced enterotoxin (ENT+) and which possessed the K99 antigen (K99 +) were included in the bacterin used in this study because all EEC which have been examined are ENT+ (1, 10, 12, 21) and most are K99+ (10, 14, 18, 23). The enterotoxin produced by most calf EEC is similar to the heat stable (ST) form of 209

enterotoxin produced by porcine EEC (3, 10, 22, 23) which is not neutralized by antiserum (23). Therefore it is unlikely that immunization with an ENT+ calf strain would stimulate the production of enterotoxin neutralizing antibody. The K99 antigen may function in calf EEC to facilitate adhesion of the bacteria to intestinal epithelium in a manner similar (18, 23) but not identical to the K88 antigen (29) possessed by some porcine K88+ EEC (19). It was hypothesized that antibodies against K99 antigen would be secreted into the dam's colostrum and be ingested by the calf where they would prevent attachment of K99+ EEC to the small intestinal epithelium thereby preventing diarrhea. This method is effective against K88+ EEC in pigs (19) and may explain the reduced incidence of diarrhea seen in calves born to vaccinated cows in Herd No. 14 and the delayed onset of diarrhea in calves in Herd No. 3. The etiology of calf diarrhea is complex; many different bacteria and viruses may be the infectious cause while environmental, immunological and nutritional causes often appear to play a role. Because of this complexity it is unrealistic to hope to develop a vaccine which will prevent the disease complex on a routine basis. This is unlike many other diseases which are caused by a single agent and for which vaccines are highly effective. The presence of multiple enteropathogens is common in diarrheic calves and the extent of this multiplicity has been underestimated or ignored. In this and earlier studies (8, 9) Corona-like virus has been found in from 20 to 46% of herds infected with Reo-like virus, Reo-like virus and ETEC have been found in 14% of herds and infections with other multiple mixed bacterial and viral enteropathogens have been reported (1, 11, 26, 28). Newer and more reliable diagnostic techniques combined with epidemiological field investigation of herd outbreaks of neonatal diarrhea will undoubtedly reveal the presence of a greater frequency of mixed infections and generate some vital information about the noninfectious aspects of the disease. In this study the MLRV-vaccine and the E. coli bacterin used alone or together were ineffective in herds infected with Reo-like virus and ETEC. A recently introduced ReoCorona virus vaccine did not reduce the incidence of diarrhea in six of 21 (29%) or the mortality rate in seven of 21 (33%) herds infected with one or both viruses7 (27). 7Norden Laboratories. Reo-Corona viral calf diarrhea vaccine modified live viruses bovine cell line origin (oral). Field trial report. 1975.

CANADIAN VETERINARY JOURNAL

These results suggest that other methods, in addition to the use of selected vaccines, must be used to prevent diarrhea. The principles of control of acute diarrhea in newborn calves include: 1) removal of the calf from the contaminated environment immediately after birth, 2) removal of the source of infection from the environment in which the calf is born, 3) increasing the nonspecific and specific immunity of the newborn calf (2) and 4) reducing environmental stress. Applying all these principles in beef herds means: 1) providing adequate nutrition for the pregnant female, 2) calving heifers and cows separately, 3) the planning, preparation and use of calving grounds separate from the wintering grounds in order to minimize contamination at the time of birth, 4) the provision of constant surveillance and assistance at calving time to ensure that calves are born with a minimum of stress and traumatic injury or are not weak because of difficult parturition, 5) avoiding overcrowding in the calving area, 6) ensuring that each newborn calf obtains a liberal quantity of colostrum immediately after birth even if it means forced feeding (2 kg of colostrum for a 35 kg calf within five hours after birth), 7) the immediate removal of diarrheic calves from the calving yards and main herd into a hospital pen for immediate treatment and 8) minimizing environmental stress factors if possible. SUMMARY A modified live Reo-like virus (MLRV) vaccine and a formalin killed bacterin which contained three strains of enteropathogenic E. coli were evaluated for efficacy against neonatal diarrhea in beef calves. Nine Reo-like virus infected herds, three of which were also infected with enterotoxigenic E. coli participated in Trial 1. Each herd was divided into four immunization groups of approximately equal size and the MLRV-vaccine and the E. coli bacterin were administered as follows: Group 1, E. coli bacterin to cows - MLRVvaccine to calves; Group II, E. coli bacterin to cows - placebo to calves; Group III, placebo to cows - MLRV-vaccine to calves; Group IV, placebo to cows - placebo to calves. The E. coli bacterin in Freund's incomplete 210

adjuvant was administered to cows twice before calving; the MLRV-vaccine was given orally to calves within 24 hours of birth. With the exception of one herd there were no significant differences among the four immunization groups in the incidence, or the severity of diarrhea as evaluated by: 1) the average age of calves at the onset of diarrhea, 2) the average duration of diarrhea, 3) the average duration of treatment for diarrhea, 4) the reoccurrence of diarrhea in the same calves and 5) the diarrhea - specific neonatal mortality rate. Corona-like virus was found in three herds and may be one of the reasons why the immunization procedures were ineffective in these herds. The case fatality rate associated with diarrhea was significantly higher in calves in Corona-like virus infected herds which were given the MLRV-vaccine only (Group III) than in calves in the other three immunization groups. Nine herds, all of which had a problem with diarrhea the previous year, participated in Trial 2. The E. coli bacterin in Freund's incomplete adjuvant or in alum adjuvant was administered to one-half of the cows in each herd and placebo was given to cows in the other half. Diarrhea was a problem in only three of the nine herds during the trial. The incidence of diarrhea was significantly lower in the immunized as compared to the control group in one of the three herds. Reo and Corona-like viruses were found in calves in the two herds in which the E. coli bacterin did not prevent diarrhea. Possible reasons for the lack of efficacy of the MLRV-vaccine and the E. coli bacterin are discussed and management procedures for the prevention of neonatal diarrhea are sug-

gested. RESUME Les experiences decrites dans cet article visaient a evaluer l'efficacite d'un vaccin attenue antireovirus et d'une bacterine formolee, contenant trois souches d'E. coli enteropathogenes, comme moyens de prevenir la diarrhee neonatale chez des veaux de races de boucherie. La premiere experience portait sur neuf troupeaux ou sevissait l'infection par le virus reo; trois de ces troupeaux etaient aussi aux prises avec des E. coli enterotoxinogenes. On divisa chacun des troupeaux en quatre groupes d'immunisation comptant a peu pres le meme nombre de sujets et on proc6da de la fagon suivante: dans le premier groupe, on administra la bacterine aux vaches et le vaccin aux veaux; dans le deuxieme groupe, on administra aussi la bacterine aux vaches, mais les veaux ne re9urent

NEONATAL DIARRHEA

qu'un placebo; dans le troisieme groupe, on donna un placebo aux vaches et on vaccina les veaux; dans le quatrieme groupe, vaches et veaux ne re9urent qu'un placebo. On injecta la bacterine E. coli, dans l'adjuvant incomplet de Freund, aux vaches, 'a deux reprises avant le velage; les veaux recurent le vaccin, par le voie buccale, en dedans de 24 heures apres leur naissance. Sauf dans un troupeau, on n'observa pas de difference appreciable entre les quatre groupes d'immunisation, quant 'a l'incidence ou a la gravite de la diarrhee, comme le revelerent: 1) la moyenne d'age des veaux lors de l'eclosion de la diarrhee, 2) la duree moyenne de la diarrhee, 3) la duree moyenne de la therapie anti-diarrheique, 4) la recurrence de la diarrh6e chez les memes veaux et 5) le taux de mortalite attribuable 'a la diarrhee neo-natale. La presence du virus corona dans trois troupeaux constituerait probablement l'une des raisons expliquant 1'echec de la vaccination des veaux de ces troupeaux. Le taux de mortalite attribuable 'a la diarrhee neo-natale s'avera sensiblement plus eleve chez les veaux des troupeaux infectes par le virus corona et qui ne recurent que le vaccin anti-reo, comparativement aux veaux des trois autres groupes d'immunisation. Le deuxieme experience portait sur neuf troupeaux qui avaient tous connu des troubles de diarrhee neo-natale, I'annee precedente. On injecta la bacterine E. coli, dans I'adjuvant incomplet de Freund ou dans l'adjuvant "a I'alun, 'a la moitie des vaches de chacun des troupeaux, tandis que I'autre moitie ne regut qu'un placebo. Au cours de cette experience, la diarrhee s'avera un probleme dans seulement trois des neuf troupeaux. Elle accusa une incidence sensiblement moins elevee chez le groupe vaccine que chez celui qui ne l'etait pas, dans l'un des trois troupeaux; on recouvra les virus reo et corona des veaux des deux troupeaux oiu la bacterine n'avait pu prevenir la diarrhee. Les auteurs commentent les raisons plausibles du manque d'efficacite du vaccin attenue anti-reo et de la bacterine E. coli; ils suggerent aussi des procedes de regie susceptibles d'aider a prevenir la diarrhee neonatale. ACKNOWLEDGMENTS

This work was undertaken with the technical assistance of L. Churchill, V. Lacroix and F. Shearer. The help and cooperation of Dr. Roy Berg and his staff at the University of Alberta Kinsella Ranch is greatly appreciated. We also express our thanks to the manv practicing veterinarians and ranchers who cooperated in this studv. The advice and support of Drs. G. Ward, J. R. 211

Saunders, B. Rouse and C. H. Bigland are also gratefully acknowledged.

1.

2. 3. 4. 5.

6.

7.

8.

REFERENCES ACRES, S. D., C. J. LAING, J. R. SAUNDERS and 0. M. RADOSTITS. Acute undifferentiated neonatal diarrhea in beef calves I. Occurrence and distribution of infectious agents. Can. J. comp. Med. 39: 116-132. 1975. BLOOD, D. C. and J. A. HENDERSON. Veterinary Medicine, 4th Edition. p. 42. London: Bailliere Tindall. 1974. BYWATER, R. J. Dialysis and ultrafiltration of heat-stabile enterotoxin from Esclierichia coli. J. med. Microbiol. 5: 337-343. 1972. DANIEL, W. W. Biostatistics: A Foundation For Analysis in the Health Sciences. pp. 351355. New York: John Wiley and Sons. 1974. LABORATORY METHODS FOR DETECTINCG CALF DIARRHEA (SCOURS) VIRUSES. Norden Laboratories, Lincoln, Nebraska. 1973. NIACMAHON, B. and T. F. PUGH. Epidemiology Principles and Methods. pp. 257-258. Boston: Little, Brown, and Co. 1970. MEBUS, C. A., N. R. UNDERDAHL, NM. R. RHODES and M. J. TWIEHAUS. Calf diarrhea (scours) reproduced with a virus from a field otutbreak. Univ. of Nebraska agric. Exp. Stn Res. Bull. 223: 1-16. 1969. MEBUS, C. A., R. G. WHITE, E. L. STAIR, M. B. RHODES and M. J. TWIEHAUS. Neonatal calf diarrhea: Resuilts of a field trial us-

ing a Reo-like virus vaccine. Vet. Med. :mall Anim. Clin. 67: 173-174, 177-178. 1972. 9. MEBUS, C. A., R. G. WHITE, E. P. BASS and M. J. TWIEHAUS. Immunity to neonatal calf diarrhea viruLs. J. Am. vet. med. Ass. 163: 880-883. 1973. 10. MOON, H. W., S. C. WHIPP and S. NI. SKARTVEDT. Enterotoxigenic Eschierichia coli associated with diarrheal disease of calves. An.

J. vet. Res. 1976. In Press. 11. MORIN, M. Study on the etiology of neonatal calf diarrhea in the province of Quebec. Interim report. Can. vet. J. 16: p. xiv, February 1975. 12. MYERS, L. L. Characterization of E.scericihia coli obtained from newborn calves with diarrhea. Infection & Immunity 11: 493-496. 1975. 13. MYERS, L. L. Vaccination of cows with an Escherichia coli bacterin for the prevention of naturally occurring diarrheal disease in their calves. Am. J. vet. Res. 1976. In Press. 14. MIYERS, L. L. and P. A. M. GUINEE. OCcurrence and characteristics of enterotoxigenic Escherichia coli isolated from calves with diarrhea. Infection & Immunity 1976. In Press. 15. MYERS, L. L., F. S. NEWMAN, R. A. WILSON and J. E. CATLIN. Passive immunization of calves against experimentally induced enteric colibaccillosis by vaccination of dams. Am. J. vet. Res. 34: 29-33. 1973. 16. NEWAlAN, F. S., L. L. MYERS, B. D. FIRE-

CANADIAN VETERINARY JOURNAL

HAMMER and J. E. CATLIN. Prevention of experimentally induced enteric colibacillosis in newborn calves. Infection & Immunity 8: 540-543. 1973. 17. NEWMAN, F. S., L. L. MYERS, B. D. FIREHAMMER and J. E. CATLIN. Licensing and use of the calf scours vaccine - Part II. An analysis of Scourvax-Reo. Proc. 77th a. Meet. U.S. Anim. Hlth Ass. 59-64. 1973.

18. 0RSKOV, I., F. 0RSKOV, H. W. SMITH and W. J. SoJKA. The establishment of K99, a thermolabile, transmissible Escherichia coli K antigen, previously called "Kco", possessed by calf and lamb enteropathogenic strains. Acta path. microbiol. scand. 83: 31-36. 1975. 19. RuTTER, J. M. and G. W. JONES. Protection against enteric disease caused by Escherichia coli - a model for vaccination with a virulence determinant. Nature, Lond. 242: 531-532. 1973. 20. REMINGTON, R. D. and A. M. SCHORK. Statistics with Applications to the Biological and Health Sciences. New Jersey: Prentice-Hall. 1970. 21. SMITH, H. W. and S. HALLS. Observations by the ligated intestinal segment and oral inoculation methods of Escherichia coli infections in pigs, calves, lambs, and rabbits. J. Path. Bact. 93: 499-529. 1967. 22. SMITH, H. W. and C. L. GYLES. The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Escherichia coli of porcine origin. J. med. Microbiol. 3: 387-410. 1970. 23. SMITH, H. W. and M. A. LINGGOOD. Further observations in Escherichia coli entero-

toxins with particular regard to those produced by atypical piglet strains and by calf and lamb strains: the transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains. J. med. Microbiol. 5: 243-250. 1972. 24. SNEDECOR, G. W. and W. G. CocHRAN. Statistical Methods. Sixth Edition. Ames: Iowa State University Press. 1973. 25. STAIR, E. L., M. B. RHODES, R. G. WHITE

26.

27.

28.

29.

and C. A. MEBUS. Neonatal calf diarrhea: purification and electron microscopy of a Corona virus-like agent. Am. J. vet. Res. 33: 1147-1156. 1972. STORz, J. Multiple mixed viral and chlamydial infections of new-born calves with enteric disease. Presented at the Symposium on Enteric Infections of the Neonatal Calf, 110th Annual AVMA meeting. Philadelphia, July 16-19. 1973. THURBER, E. T., E. P. BAss and W. H. BECKENHAUER. Field trial evaluation of a Reo-Coronavirus calf diarrhea vaccine. J. Am. med. vet. Ass. 1976. In Press. WALDHALM, D. G., R. F. HALL, W. A. MEINERSHAGEN, E. STAUBER and F. W. FRANK. Combined effect of neonatal calf diarrhea virus and Providencia stuartii on suckling beef calves. Am. J. vet. Res. 35: 515-516. 1974. WILSON, M. R. and A. W. HOHMANN. Immunity to Escherichia coli in pigs: adhesion of enteropathogenic Escherichia coli to isolated epithelial cells. Infection & Immunity 10: 776-782. 1974.

ABSTRACT Studies on the flora, populations and sensitivity pattern of micro-organisms in bovine semen. N. A. Fish, 0. Onoviran and R. B. Truscott (Vet. Micro. and Immun., Ont. Vet. Coll., Guelph, Ontario). This study involved sampling semen obtained from groups of bulls in artificial insemination centres. Bacterial isolates from semen consisted primarily of Bacillus, Micrococcus, Pseudomonas and Proteus species. Other isolations included Enterobacter, Escherichia, Candida, Corynebacterium and Serratia. Swab samples obtained from artificial insemination equipment revealed organisms similar to above and fungi. Bacterial numbers on the equipment and in semen samples ranged from 104-105 per ml, and yeast and mould count 5 x 101. The various identified bacterial genera isolated were subjected to antibiotic sensitivity tests. The majority of above or212

ganisms were resistant to penicillin and streptomycin.

Microbiological examinations were conducted on unprocessed and fresh semen and penial swabs for Mycoplasma species. In the examination of 132 penial swabs, 87 (65.9%) and 46 (34.9%) were positive for large colony and T-strains respectively. Corresponding figures for these organisms in the unprocessed semen samples were 32 (22.9%) and 34 (24.3%), in the processed semen 1 (2.4%) and 6 (14.3%). An evaluation of antibiotics for Mycoplasma species indicates that a combination of lincomycin and spectinomycin were lethal to large colony mycoplasmas but were only inhibitory to the T-strains at the same concentrations. Research Rostrum of the Twenty-seventh Cana-

dian Veterinary Medical Association Annual Convention, Halifax, Nova Scotia 1975.