Acute Undifferentiated Necnatal Diarrhea in Beef Calves 1. Occurrence and Distribution of Infectious Agents S. D. Acres, C. J. Laing, J. R. Saunders and 0. M. Radostits*

ABSTRACT Beef calves in a 48-cow herd were studied during one calving season from birth to ten days of age to determine the presence or absence of potentially enteropathogenic bacteria, viruses, and/or chlamydia in both normal and diarrheic calves. Calves were born and raised outside in large pens unless the ambient temperature was below -10°F when calving was done inside. Fecal swabs, fecal aliquots, and nasal swabs were taken from each calf at 32, 128 ±3, and 248 ±3 hours of age and as soon after the onset of diarrhea as possible. Diarrhea was defined as that condition in which the feces contained less than 10% dry matter. Enteropathogenic Escherichia coli in feces were identified using the ligated gut loop procedure in calves and by feeding broth cultures to colostrum fed lambs seven to 16 hours old. Potentially enteropathogenic viruses were detected using a variety of methods which included tissue culture, fluorescent antibody, hemadsorption, and electron microscope techniques. Of the 40 calves studied, 32 (80%) developed diarrhea before ten days of age. Twenty-two strains of Escherichia coli which caused dilation of calf ligated intestinal loops were isolated from 11 scouring calves and from one normal calf. Nine out of ten strains of Escherichia coli which dilated ligated loops also caused diarrhea when fed to colostrum-fed lambs seven to 16 hours old. Using the fluoresof *Depa-tmcnt Radostits) and

Veterinary Clinical Studies (Acres and Department of Veterinary Microbiology (Laing and Saunder3), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Sas-

katchewan. This work was supported by funds from the Saskatchewan Horned Cattle Trust Fund, the Alberta Cattle Commission, the Alberta Agricultural Research Trust, and the National Research Council of Canada. The Escherichia coli typing was supported by Canada Department of Agriculture Extra-Mural Research Grant 7111-1972. Dr. Acres is a fellow of the Medical Research Council of Canada. Submitted May 2, 1974.

116

cent antibody technique a Reo-like virus was detected in the feces of 15 calves before, during, and after the onset of diarrhea. Four calves exCreted both loop dilating strains of E. coli and Reo-like virus in the feces before ten days of age; in all cases t-ce loop dilating E. coli were isolated from the feces prior to the demonstration of Reo-like virus. A Corona-like virus was alro demonstrated in thr.-e of the 15 calves ir.fected with Reo-like virus and a noncytopathogenic strain of bovine virus diarrhea virus was isolated from two of the 15 calves infected with Reo-like virus. A loap dilating strain of Citrobacter was isolated from one diarrheic calf. There was no consistent pattern of onset or duration of diarrhea in calves which excreted different infectious agents. Salmonella species, infectious bovine rhinotracheitis virus, parvovirus, adenoviruses, parainfluenza-3 virus, and Chlamydia species could not be demonstrated in any of the calves or their dams. No potentially enteropathogenic agents could be demonstrated in 11 of the 32 calves which scoured. These findings emphasize the complexity of the infectious aspect of the neonatal diarrhea syndrome and illustrate the difficulty in making an etiological diagnosis in field outbreaks of the calf scours complex.

RESUME Au cours de la periede de velage d'un troupeau comptant 48 vaches 'a boeuf, on a suivi de pres les veaux, jusqu'a I'age de dix jours, afin de verifier la presence ou l'absence de bacteries, de virus et/ou de chlamydia qui auraient pu s'averer ent6ropathogenes, tant chez les veaux sains que chez ceux qui manifesterent de la diarrhee. Les veaux naquirent dans des grands parcs exterieurs et ils y demeurerent; quand la temperature descendait plus bas que - 10°F, on faisait cependant veler

Can. J. comp. Med.

les vaches 'a l'interieur. On preleva des ecouvillons et dcs 62hantil!ons d2 feres, ainsi que des ecouvillans nasaux, de chacun des veaux, 'a 3_, 128 _'3 et 248 `3 heures apres leur naissance ct aussit6t que possiMe apres le debut de la d:arrhie. On considera comme diarrheiques ls feces qui contenaient l2oins que 10% de matieres seches. On identifia les souches ent6ropathagenes d'Escherichia coli en utilisant la m6thode de la ligature d'anses intestinales, chez des vearx, et en faisant ingerer des bouillons de cultures 'a des agneaux, ages de sept 'a 16 heures, qui avaient requ du colostrum. On decela les virus doues d'un pouivoir ent6ropathogine, en utilisant: les cultures tissulaires, l'immunofluorescence, l'hemadsorption et la microscopie electronique. Des 40 veaux impliques dans cette etude, 32 (89%) manifesterent de la diarrhee avant d'avoir atteint l'age de dix jours. On isola 22 souches d'E. coli qui provoquerent la dilatation d'anses intestinales ligaturees, chez 11 veaux atteints de diarrhee et chez un veau sain. Neuf sur dix de ces souches causerent de la diarrhee, lorsqu'on les fit ingerer 'a des agneaux aiges de sept 'a 16 heures et ayant regu du colostrum. L'utilisation de l'immunofluorescence permit de deceler un agent semblable a un reovirus, dans les feces de 15 veaux, avant, pendant et apres l'eruption de la diarrhee. Quatre veaux 'ages de moins de dix jours eliminerent dans leurs feces des souches d'E. coli qui provoquaient la dilatation d'anses intestinales ligaturees et un agent semblable 'a un reovirus; dans tous ces cas, l'isolement d'E. coli pr&eda la denmonstration du virus. Chez trois des 15 veaux infectes avec I'agent semblable 'a un reovirus, on demontra aussi la presence d'un agent semblable 'a un coro-navirus; on isola de deux autres veaux de ce groupe une souche non cytopathogiene du virus de Ia diarrhee vira1e bovine. On isola aussi d'un veau atteint de diarrhee, une souche de Citrobacter qui causait la dilatation d'anscs intestinales ligaturees. L'eruptien et la duree de la diarrhee des veaux qui exeretaient les differents agents infectieux ne manifesterent pas de regularite. On ne reussit pas 'a d6montrer chez les veaux, ni chez leurs meres, la presence des micro-organismes suivants: salmonelles, virus de la rhino-tracheite infectieuse bovine, parvovirus, ad6novirus, virus para-influenza 3, ou chlainydia. On, ne put isoler aucun agent doue de pouvoi_r ent6repathogene, chcz 11 des 32 veaux qui manifesterent de Ia diarrhee. Ces oiservations font ressortir Ia comnplexitd de l'aspcct iafectieux du syndronme de la diarrhee neo-natale. Elles illustrent aussi ln difficulte d'en arriver a un diagnostic etiologique

Vol. 39 - April, 1975

precis, face aux eruptions naturelles du complexe de la diarrhee du veau.

INTRODUCTION Calf scours is a complex disease because it has been difficult to determine the role of the many different infectious agents which have been isolated from the feces and tissues of affected calves. The early work which incriminated Escherichia coli as a primary cause of calf scours was based on the finding of a quantitative increase of the organism in the upper portion of the small intestine (7, 35). However, only recently was it shown that a limited number of strains of E. coli could act as primary enteropathogens (33). Enteropathogenic E. coli must possess two properties; they must produce enterotoxin, which can be determined using the ligated gut loop technique in the species involved (33,34), and they must proliferate to high numbers in the anterior portion of the small intestine. The ability to multiply to high numbers in the small intestine can be demonstrated by feeding the organisms to colostrum-fed calves or lambs less than 20 hours old (33). Those strains which produce enterotoxin and proliferate in the anterior small intestine usually cause diarrhea in newborn calves or lambs. In one study of epidemiologically unrelated cases of calf diarrhea only seven out of 127 calves (5.5%) were infected with strains of E. co7i which possessed the two properties noted above (33). There is no information on the occurrence of enteropathologenic strains of E. coli in beef calves in North America. Many different viruses have been isolated from the feces of diarrheic calves (2, 3, 4, 5, 6, 8, 11, 18, 20, 21, 23, 24, 28, 30, 36, 37, 32, 43) but their role in the pathogenesis of the calf scours syndrcme is not clear. Observations that diarrheic calves which are infected with obligate intracellular parasites also have increased numbers of E. coli in the upper levels of the gastrointestinal tract (30, 38) have led to the theory that infectious agents within cells lining the intestine induce a primary cnteritis which allows E. coli to attain abnorma1g.lly high numbers in the upper small initestine and contribute to the pathogeolesis of diarr hea (24, 30, 38). The objcc-tives of this study vwere: 1) to determine the frequenlcy with which certain infectious agents could be isolated from

117

newborn beef calves which were allowed to develop diarrhea spontaneously under ten days of age; and 2) to study the age distribution of the occurrence of infectious agents in the same calves.

MATERIALS AND METHODS EXPERIMENTAL ANIMALS Forty-eight mature Hereford cows which were obtained from two separate herds were randomized into four groups of 12 each approximately four months prior to the onset of calving. They were wintered and calved side by side in four separate pens which allowed an area of 250 square feet per cow. The animals in each pen were fed one of five different rations prior to calving (1). Cows were allowed to calve naturally in the pens unless the ambient temperature was below -10°F in which case they were moved into a straw-bedded shed. The calving period extended over a fivemonth interval from January 29, 1972 to May 29, 1972 during which time 40 calves were born. Further details on the management procedures and the gestation and lactation rations fed to the cows are presented in the following paper (1). SAMPLING PROCEDURE

Cows and calves were not disturbed during the first eight hours postpartum and each calf was allowed to suckle voluntarily. Beginning at eight hours after calving the cows were restrained in a cattle chute and the following samples were obtained: colostrum, serum, nasal and fecal swabs, and fecal aliquots. At 32, 80, 128±3, and 248±3 hours postpartum samples of colostrum or milk were taken from each cow by the methods outlined in the following paper (1). Serum, fecal samples, and fecal and nasal swabs were 'aken from each calf at 32 and 128 hours, and ten days of age or as soon as possible after the onset of diarrhea. Thus some calves were sampled on several successive days and samples were obtained before, during and after the occurrence of diarrhea. Fecal samples were obtained by stimulation of defecation with a gloved finger or a sterile plastic tube and collected directly from the rectum into sterile plastic cups or plastic bags.

118

Feces for virus isolation were added to Hank's balanced salt solution (BSS) which contained 1.875% bovine albumin fraction V1 and 10% antibiotic-antimycotic mixture2. Feces for Chlamydia isolation were added to storage media which consisted of tryptone broth3 containing 5.0 mg/ml of streptomycin sulfate4 and 0.5 mg/ml of kanamycin sulfate5. Feces were added to both of the storage medias to make a 20% solution of feces. Nasal swabs for virus isolation were taken by inserting a six-inch cotton-tipped swab, wetted in Hank's BSS, as far as possible into each nasal passage and then swirling it in the same solution. Samples for virus or Chlamydia isolation were frozen within two hours of collection at -60°C. Rectal swabs for bacterial isolation were taken using commercial swabs which contained their own transport media6. Samples were smeared onto blood agar and MacConkey's agar plates, and then put into selenite broth. Following incubation for 24 hours at 37°C samples were streaked on Brilliant-green agar and Hektoen-enteric agar plates for isolation of Salmonella species. Isolated colonies of E. coli picked from the blood agar plates were 0 serogrouped by routine methods. One or two colonies were picked from each blood plate and stored on trypticase-soy (TS) slants until used in the ligated gut loop procedure.

Virology - Primary fetal bovine kidney (FBK) monolayers were grown in flasks7 until confluent in Earle's balanced salt solution (BSS) containing 0.5% lactalbumin hydrolysate (LAH) and 10% heatinactivated fetal calf serum and 200 units per ml of penicillin and 200 mcg per ml of streptomycin. Monolayers were washed three times with Hank's BSS prior to inoculation. Following inoculation the FBK 'Bovine albumin fraction V. 7.5% solution in P.B.S., Grand Island Biological Company, Grand Island, New York. 2Antibiotic-Antimycotic Mixture (1OOX) - Lyophilized. Penicillin 10,000 U/ml, Fungizone 25 mcg/ml and Streptomycin 10,000 mcg/ml, Grand Island Biological Company, Grand Island, New York. 3Difco, Detroit, Michigan. 4Pfizer Company Ltd., Montreal, Quebec. 5Kantrex, Rogar/STB, London, Ontario. 6Culturette, Scientific Products Division of American Hospital Supply Corp., Evanston, Illinois. 725 cm2 Falcon Tissue culture flasks. Becton, Dickinson and Co., Mississauga, Ontario.

Can. J. comp. Med.

cells were maintained on Earle's BSS containing 0.5% LAH and 0.1% yeast extract (25) with 200 units penicillin and 20 mcg of streptomycin per ml added. Fecal samples frozen in Hank's BSS were mixed with 0.2 ml of antibiotic solution (10,000 units of penicillin and 10,000 mcg of streptomycin per ml). This mixture was allowed to stand for two hours at room temperature or overnight at 4°C, after which time 0.5 ml was inoculated on to the FBK monolayers and absorbed for one to two hours. Fresh maintenance medium was added at the time of inoculation and again at five days postinoculation. Each sample was blind-passed three times, the average passage time being 13 to 14 days. The cell cultures were examined regularly for the occurrence of cytopathic effects (CPE). At the end of each passage the supernatants containing detached cells were spun at 200 G. for 20 minutes. The cells were resuspended in a small amount of supernatant fluid and smears were made on three glass slides. Slides were examined by fluorescent antibody technique for the presence of the infectious bovine rhinotracheitis (IBR) virus and the bovine virus diarrhea (BVD) virus. Nasal samples frozen in Hank's BSS were processed in the same manner as fecal samples prior to inoculation into FBK tubes on roller drums. Growth medium was MEM Eagle's BSS containing 10% heat-inactivated calf serum, 1.0% non-essential amino acids, 100 units per ml of penicillin, and 100 mcg per ml of streptomycin. Maintenance medium, added at the time of inoculation and five days postinoculation was similar to the growth medium except that serum concentration was reduced to 4.0%. Samples were passed three times, eight to ten days per passage, and the monolayers were checked regularly for CPE. On the third passage, samples were tested for parainfluenza-3 (P13) virus by the hemadsorption procedure using guinea pig erythrocytes. Fecal samples frozen in Hank's BSS were used to make smears which were examined for the presence of Reo-like virus by the fluorescent antibody technique8. Isolation of chlamydia species was attempted according to methods published previously

(43). gConjugate kindly supplied by Dr. C. A. Mebus, Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska.

Vol. 39 - April, 1975

LIGATED GUT LOOP PROcEDURE The ligated gut loop technique was carried out following the recommendations of earlier workers (33). Fourteen Holstein calves two to seven days old were used. They were allowed to remain with their dams for a minimum of 18 hours following birth and from 18 hours up to four days of age they were transported to an isolation unit, where they were fed cow's milk twice daily at a level of 8% of body weight until 24 hours prior to surgery. A left-sided laparotomy, performed under halothane anesthesia, provided the necessary exposure of the abdominal cavity. The upper small intestine was located anterior to the left kidney and traced caudally to a point approximately 10 feet (300 cm) from the pylorus, where the first loop was tied. Intestinal loops were 10 cm long and 60 to 70 loops were made in each calf. Standard solutions for injection into test loops were prepared as follows: strains of E. coli which had been stored on TS slants were inoculated into 3.0 ml of TS broth and incubated overnight at 37°C. The next day 0.2 ml of the broth solution was pipetted on to TS plates and spread evenly. Following incubation overnight at 37°C, the growth was removed from the plates by adding 4.0 ml of PBS pH 7.2 and swirling with glass beads. The resulting solution was removed from the plates and diluted to contain 3 x 10' organisms per ml using a spectrophotometer adjusted to the density of a MacFarland No. 1 (16). Standard solutions were stored overnight at 4°C prior to injection into loops. Test loops were injected with 1 ml of standard solution and were alternated with control loops which were injected with 1.0 ml of sterile PBS only. At four or five places along the ligated section of intestine, known positive and known negative strains were inter-spaced between the test loops. Twenty-four hours following surgery, the calves were killed and the small intestine was removed and unravelled by cutting along the mesenteric border. Samples were taken from each dilated loop and known control loops to test for presence of the injected organisms. Positive loops were identified by comparing the volume of fluid present in test loops to adjacent control loops. Those strains of E. coli which dilated ligated loops were considered to be enterotoxin producing ETP strains (34).

119

ORAL INOCULATION OF LAMBS WITH E. coli

CLASSIFICATION OF DIARRHEA

Thirty-two Dorset, Finnish, or DorsetFinnish cross lambs, which included 12 pairs of twins, were fed nutrient broth cultures of E. coli when seven to 16 hours old. Lambs were allowed to remain with their ewes until about 60 minutes prior to challenge when they were placed in individual pens. The first nine lambs which were challenged were fed 200 ml of canned milk' every six hours following challenge. Lambs either developed diarrhea within 12 to 14 hours following challenge or they remained normal. The other 23 lambs were not fed 12 hours following challenge by which time they had either developed diarrhea or remained normal. Following initial adjustment of the dose those lambs which were challenged with strains of E. coli which dilated ligated loops in calves were given 5.0 ml of a 12 hour nutrient broth culture which contained an average of 3.4 x 109 total viable organisms (Table IV). Lambs challenged with strains of E. coli which did not dilate loops were given from 6 to 9 ml of nutrient broth cultures which had been incubated for variable periods of time up to 24 hours and which contained an average of total 7.6 x 109 viable organisms. Seven lambs were treated in the same manner as the challenged lambs but were not fed any E. coli. Feces from each lamb were collected by means of a plastic bag glued over the rectum. Some lambs were killed 23 to 26 hours after challenge and the small intestine was tied off in situ into several sections. When unravelled the small intestine was 22 to 25 feet long. Samples were taken from three areas of small intestine by draining the fluid contents from the lumen of a section of small intestine which was three feet long. Areas sampled were: the first three feet below the pylorus; the segment 12 to 15 feet below the pylorus; and the segment one to four feet above the ileocecal junction. Counts of viable E. coli were done by making serial dilutions of 1.0 ml of intestinal contents and plating onto MacConkey's agar plates.

Dry matter content (DMC) of feces was determined by drying approximately 10 gm of feces to a constant weight. Diarrhea was considered present when the feces contained less than 10% DMC. Feces with a DMC of 10 to 14% were considered abnormal but not diarrheic, and above 14% DMC was considered normal. Bouts of diarrhea separated by three or more days on which the feces were normal were considered to be separate occurrences of diarrhea. Diarrheic calves were allowed to progress as long as possible and treatment was begun only if they appeared to be in danger of dying. Treatment consisted of fluids and electrolytes10 and chloramphenicol1' administered intravenously. A complete pathological examination was done on each calf that died.

9Carnation Co. Ltd., Wetaskiwin, Alberta.

120

RESULTS INCIDENCE OF DIARRHEA Thirty-two of the 40 calves (80%) developed diarrhea before ten days of age with the peak incidence occurring at one day of age (Fig. 1). The morbidity in each of the four pens was: 1) 10/11, 2) 7/10, 3) 7/9, and 4) 8/10. No calves developed diarrhea on days three or ten. The onset of diarrhea in affected calves was sudden and dehydration and weakness developed in a few hours. The disease was indistinguishable from acute diarrhea seen commonly in nursing beef calves under ten days of age in Western Canada. The dry matter content of the feces was usually below 4%; the lowest value recorded was 2.1%. Three calves died from the effects of the diarrhea and a fourth was killed in extretiis after it developed gangrene of the limbs from cold exposure. LIGATED GUT LoOP TECHNIQUE

One hundred and seventy strains of E. coli were examined for their ability to dilate ligated loops. One hundred and sixtytwo of these strains were isolated from 10hI,otcnie solution of sodium bicarbonzte and sodium ch!oride whith contained 78 mEq Lt. HCO:,, 78 mEq/1. Cl, and 156 imEq/l Na, Wcstezn College of Veterinary Medicine Phan-nmacy. PRogar-Mycine 100, Rogar/STB, London, Ontario.

Can. J. comp. Med.

of the 23 ETP strains (95.7%) were recovered from calves in which the dry matter content was less than 14% at the time the samples were taken, while only one ETP IS, !9 strain (4 %) was recovered from calves a passing normal feces at the time of samp6 ling. The three calves in which the fecal DMC was between 10 and 14% and which I excreted loop dilating E. coli had all scoured m and excreted loop dilating E. coli on pre5 5 3 5 7 5 Io * 32*1. 2 vious days (Table II). These findings inAGE AT ONSET OF COURS IDAIS) dicate a strong relationship between the Fig. 1. Number of calves which started to scour from presence of ETP E. coli in the feces and one to ten days of age. the occurrence of diarrhea. None of the eight strains which had been isolated from cow feces dilated ligated loops. The 23 potentially enteropathogenic calves between 32 hours and ten days of age (Table I) and eight strains were re- strains of bacteria were recovered from 13 covered from the feces of cows eight hours different calves (Table II). Five calves following calving. Twenty-two strains of excreted loop dilating strains of E. coli on E. coli and one strain which belonged to the more than one day and in three of these Citrobacter species caused dilation of calves loop dilating strains which belonged ligated loops (Table II) and are classified to the same 0 serogroup were isolated over as enterotoxin producing (ETP) strains a period of several days. Loop dilating and as potential enteropathogens. The 23 strains which belonged to serogroup 08 bacterial isolates which dilated ligated loops occurred in calves in all four pens, but it represent 14% of the strains which were was also apparent that within each pen examined from all calves. Thirty percent different calves excreted ETP strains of (19/64) of the strains examined from calves E. coli which belonged to at least two difin which the fecal DMC was than 10 % ferent serogroups. Within the herd and dilated loops while less than one percent within the pens there was a tendency for (1/74) of the strains isolated from calves several calves born in consecutive order in which the fecal DMC was above 14 % to excrete loop dilating E. coli (Table II). Loop dilating strains of E. coli were caused loop dilation (Table I). Twenty-two #A

.j

ii_ I

TABLE I. Dry Matter Content (DMC) of Fecal Samples Examined for the Presence of Loop Dilating Escherichia coli and for Reo-like virus Reo-like Virus

E. coli Strains Tested in

.......

72

Strains Positive 1

10 - 14%.....

26

3

11.5

11

< 10%

64

l9d

29.70

51

162

23d

14.2

91

DMC of Feces

at Sampling > 14%

Total

in

Ligated Loops

Percent Positive 1.4c

Samples Examined by FATv 29

Samples or Suspect Positive

Positiveb 7 (4,3) 4

(2,2) 11

(5,7) 22

(11,11)

Percent Positive or

Suspect

Positive 24.1

27.5 21.6

24.2

aFluorescent antibody technique bFigures in brackets represent the number of positive samples, and the number of suspect positive samples eSignificantly different at the .005 % level dIncludes one strain which belonged to Citrobacter species and one strain of E. coli isolated from the ileum of a calf which died of diarrhea. For convenience of discussion the Citrobacter stain is included with the E. coli

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April, 1975

121

TABLE II. Enterotoxin Producing Strains of Escherichia coli as Defined by the Ligated Loop Test

Penl...

Pen 2

Pen 3

Pen 4

...................

...................

...................

Strain 687A 688C

Calf Number, 8-2-1 8-2-1

760C 760D 899B 939B 988A 1054 1194D 1272Dd 1732D 762A 782D 1163D 759C 759D 780D 845A 990A 1048B 1065Ce 1045 1164B

9-3-1 9-3-1 14-5-1 14-5-1 14-5-1 18-3-2 21-4-2

Age (days)

0 serogroup 8 8

5 6 2 2 1 3 7 1

untyped

1

Citrobactero

15 8

ll9b ll9b ll9b

25-5-2

1

8

32-8-2 10-5-3 10-5-3

5 2 4

107c 15 8

22-8-3 11-2-4 11-2-4 11-2-4 11-2-4

1

untyped

i 1 2 5

untyped 8 untyped

16-4-4 16-4-4 16-4-4

1 3 5

17-5-4 23-6-4

1

8

8 8 8

untyped untyped

1

-The first number is the birth order of the calf within the herd, the second number is the birth order of the calf within the pen, and the third number is the number of the pen bInconsistent reaction - see text for details -Kindly typed by Dr. I. COrskov, International Escherichia Center (W..H.O.). Statens Seruminstitut, Copenhagen, Denmark dThis calf did not scour .Recovered from ileum at necropsy

oStom

of

* Strais of

E.coi tested

in

toops

Ecoii positiv* in loops

40.

'IAIs30. i 20

IR FL

32Hrs. 2

3

X

5

nLLft¶n

5

r

AGE OF CALVES FROM WHICH E. ci WERE

.5

9

10

ISOIATEO CAYSI

Fig. 2. Number of strains of E;. coli positive by the ligated loop procedure compared to the total number of strains tested.

122

most frequently isolated from scouring calves at 32 hours of age (Fig. 2). Eight of the 11 (73%) calves which started to scour when one day old excreted loop dilating strains of E. coli or Citrobacter when sampled at 32 hours of age. Twentytwo of the 23 (91%) potentially enteropathogenic E. coli or Citrobacter isolated were recovered from calves under six days of age and no loop dilating strains of E. coli were detected in calves older than seven days even though a significant number of strains were tested from calves eight, nine and ten days of age. In all calves but one the initial isolation of loop dilating strains of E. coli from the feces correspond closely with the onset of diarrhea (Fig. 5). Strains 899B, 939B, and 988A, all from calf 14-5-1 gave inconsistent results serologically and in the ligated loop procedure.

Can. J.

comp.

Med.

These three strains were tested for their ability to dilate ligated loops 13 times in six different calves. Seven of the 13 loops which were injected with one or the other of these strains dilated; the other six loops did not dilate. Organisms which belonged to O group 119 could not be consistently recovered from the loops. All other strains listed in Table II consistently caused dilation of ligated loops with the exception of strains 1045 and 1054, which were tested in only one calf. Eleven 0 serogroups of E. coli have been reported to include strains which are potential enteropathogens for calves based on the ligated gut loop technique (Table III). Strain 1732D which belongs to 0 serogroup 107 represents an 0 serogroup not previously reported to include loop dilating strains. Four other 0 serogroups have been listed as possibly containing

bovine enteropathogens (15). Isolation of two strains which belong to 0 serogroup 15 (760C, 762A) which dilated ligated loops confirm that members of this serogroup can produce enterotoxin and may be enteropathogenic. No conclusion can be reached about members of serogroup 01119.

ORAL INOCULATION OF LAMBS WITH E. coli Of the 23 lambs fed strains of E. coli which had been shown to dilate ligated calf loops, 18 developed severe diarrhea, two developed transient diarrhea, and three did not show any signs of diarrhea (Table IV). The Citrobacter strain also caused diarrhea when fed to one lamb. Strains B44 and B117, previously reported as being enteropathogenic for calves and lambs (33) were included as positive controls. Strain 3422 which was isolated from a calf not in

TABLE III. Summary of Bovine Enterotoxigenic Escherichia coli as Defined by the Ligated Loop Technique No. of Strains 2 3

1 1 2 2 2

1 1 3 2

3 2

10 7 5 2 1 32 6 4 2 1 3 6

Antigenic Structure

09:K9:H*

0101 :K :T-I

09:K30 08:K *H 0101 :K- :H9 08:K- :H2 Untyped 08, 09, 026, 0101

015O, 017a, 0115O, 0119 089:K.:NM 09:K.:NM Untyped 022 :K-H1 0101 :K(A) :H* 026 :K60(B) :H* 08 0101 0141 011 017, 026, 065, 0124 untyped 08:K(85) :H27b 08:K- :H* 015:K :H0107:K :H.b 0119 ? Untyped

Donor Country of Origin Canada (1), Belgium (1) Smith & Halls (33) Canada (1), Belgium (1) 1967 Holland (1) Britain U.S.A. El Nageh (12) France 1970

not stated

Gyles (17) 1972

U.S.A.

Tennant et al (40) 1972

U.S.A. France

Myers et al (27) 1973 Corboz & Becker (9) 1973

France

Shoenaers & Kaeckenbeeck (31) 1973

Canada

W.C.V.M. 1973

*O groups listed as possibly containing enteropathogenic strains bKindly typed by Dr. I. Orskov, International Escherichia Centre (W.H.O.) Statens Seruminstitut, Copenhagen, Denmark

Vol. 39

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123

TABLE IV. Results of Oral Challenge of Suckling Newborn Lambs with Escherichia coli

Lamb No. lAb 2AO 3 4Ac 5Ac 6A 7Ab 8A 9A 1OAc 11A 12 l3Ae 14 15 16 17 18 19 20 21 10B 22Ac 1B 23 24 7B 8B 25 26 27 6B

Total No. Organisms Age at 0 Group (x 109) Strain C:hallenge (hrs) Lambs challenged with loop dilating strains of E. coli 8 3.0 760D 10 8 0.5 760D 7-8 1.5 8 760D 8 8 N.D.d 760D 12 8 N.D. 760D 16 8 760D N.D. 9 8 1.5 990A 7 9 22.0 8 B44e 8 1.5 1272D 8-9 8 N.D. 1272D 7-13 8 N.D. 7 1065C 15 0.1 760C 5 15 8 760C N.D. 8 759C 13 12 8 1.8 759D 9 107 1732D N.D. 12-13 8 1048B 2.5 12 Cf 1194D N.D. 10 B44 9 1.6 12 8 11 B117e 2.0 8 89 B117 5.1 3422 101 3.0 8-9 3422 101 7-8 0.5

Lambs challenged with non-loop dilating strains of E. coli 8 1896B 10 3.0 8 1896B 2.9 10-12 8 1896B 10 4.8 8 2.4 7 500C 9 8 2021C 34.0 2021C 9 6 3.5 0119 1072D 10-11 2.8 0119 1072D 7 N.D. 1049 9 015 N.D.

Results 4

+ +

4 -

+ + + + + + + + + + + + + + + + -

= transient diarrhea; - = no diarrhea a + = diarrhea; bOther twin challenged with a strain of E. coli which belonged to the same 0 group but which did not dilate loops Other twin present but not challenged dNot done eObtained from W. J. Sojka, Central Veterinary Laboratory, Weybridge, Surrey

;Citrobacter

this study was also included because it had been used as a positive control in the

ligated loop procedure.

Ten of the 23 strains of E. coli which dilated ligated loops (Table I) were fed to lambs and nine of the ten strains caused diarrhea (Table IV). Five strains of E. coli which failed to dilate ligated loops were fed to nine lambs; none of these strains caused diarrhea. In three of these cases the lamb challenged was one member of a pair of twins, the other member of which was challenged with a strain of E. coli which belonged to the same 0 serogroup but which did dilate ligated loops (lamb nos. 1A - 1B, 7A - 7B, 8A - 8B, Table IV). In

124

all three cases only the lambs which received the strains of E. coli which dilated ligated loops developed scours. Those strains which dilated ligated calf intestinal loops and which also caused diarrhea when fed to lambs are considered primary entero-

pathogens.

Six of the seven lambs which were not fed any E. coli did not show any signs of diarrhea. One lamb did develop persistent scours and it was later found to be infected with the loop dilating strain used to challenge its twin (lamb no. 13A) which had also developed diarrhea. The number of E. coli present in the small intestinal contents of those lambs

Can. J. comp. Med.

o Fd gehs O

mbw flor Roe-t

wires

* positive * suspect positive

,Oa

25

IL

20

,1

I

3 .'ft

I.EI S

L I

AGE CF CALVS OAS)

*

9

I

Fig. 3. Number of fecal samples pooitive or suspect positive for Reo-like virus compared to the total number of fecal samples examined.

which developed diarrhea was significantly higher than in those lambs which, did not develop diarrhea (Table V). Lamb no. 3 which was challenged with a strain of E. coli which dilated ligated loops did not develop diarrhea. The number of E. coli present in the small intestinal contents of this lamb was much lower than the number present in lamb no. IA which was challenged with the same strain but which did develop severe diarrhea.

DEMONSTRATION OF REO- AND CORONA-LIKE VIRUSES

Initially 21 frozen fecal samples were sent to Nebraska to demonstrate the presence of Reo- and Corona-like viral agents."2 Six of 21 samples were positive or suspect positive for the presence of Reo-like virus (24) using the fluorescent antibody technique, and three of four samples examined by electron microscopy were positive for the Corona-like agent (35). After obtaining these results, 91 fecal samples which were frozen in Hank's BSS and which had been collected from 37 calves were examined for the presence of the Reo-like virus by the fluorescent antibody technique. Sixteen of the 91 samples were the same as those sent to Nebraska. The results obtained following blind processing and reading of the slides were the same as those obtained in Nebraska with one exception. Of the 91 fecal samples examined, 11 were positive for the presence of Reo-like virus and 11 were suspect positive (Table I and '?Samples kindly processed by Dr. C. A. Mebus, Department of Veterinary Science. University of Nebraska, Lincoln.

Vol. 39

April, 1975

Fig. 3). Fifteen of these 22 positive or suspect positive results (68.1%) were from calves in which the DMC of the feces was less than 14% at the time the samples were collected. Reo-llke virus was de'-ected in six calves which were not scouring when the virus first appeared in the feces (Table VI). Five of these calves subsequently developed scours but one (calf no. 25-5-2) remained normal throughout the study period. The longest interval between the appearance of the virus in the feces and the onset of diarrhea, except for calf no. 25-5-2, was six days (calf no. 27-7-1). In seven calves the Reo-like virus was detected following the onset of diarrhea and in one of these calves (no. 14-5-1) the virus was demonstrated seven days after the onset of diarrhea. In three calves it was possible to demonstrate the virus before and after the onset of diarrhea. Twenty of the 22 (91%) positive or suspect positive readings for Reo-like virus were in animals five to ten days of age (Fig. 3). The two suspect positive readings in calves younger than five days of age were from animals which were not scouring at the time of sampling (Figs. 3 and 5). Six of the calves which excreted Reo-like virus also excreted potentially enteropathogenic E. coli or Citrobacter before ten days of age (Table VI). Coronalike virus was demonstrated in the feces of three calves, two of which were six days old and one which was seven days old. All three of these calves were also positive for Reo-like virus and all were scouring at the time of sampling (Table VI).

DEMONSTRATION OF BOVINE VIRUS DIARRHEA VIRUS Non-cytopathogenic BVD virus was demonstrated in two calves, one five and one ten days of age, by the fluorescent antibody technique after the third tissue culture passage. Both calves were also positive for Reo-like virus at the same time (Table V). The ten day old calf was diarrheic and the five day old calf became diarrheic three days following demonstration of the viruses in the feces (Fig. 5). DETECTION OF OTHER INFECTIOus AGENTS No Salmonellae were isolated from more than 200 rectal swabs. One hundred and fifty fecal samples tested in tissue culture were negative for the presence of IBR virus and no CPE was found to suggest the pre-

125

TABLE V. The Distribution of Escherichia coli in the Small Intestine of Lambs 23 to 26 Hours Following Oral Challenge

Log1o Viable Count of E. coli per Lamb Number 1A 15 17

Challenge Strain

760D 759D 1048B

0 Group 8 8 8

ml or Small Intestinal Tract Contents Level, 1 2 3 7.70 9.20 9.28 9.46 9.04 10.03 7.00 9.08 9.64 8.05 9.11 9.65

Loops Reaction Diarrheab + + + + +

Mean 3 23 25

760'D 1896D 2021C

8 8 9

+ -

-

Mean a + dilated loops; - did not dilate loops b + feces < 10% DMC: - feces > 14% DMC e 1 = 1-3 feet distal to pylorus; 2 = 12-15 feet distal to pylorus; 3 Mean viable counts are significantly different (.05%) at all levels

sence of parvovirus (5). No Chlamydia were isolated from 127 fecal samples from both cows and calves; 107 nasal swabs from cows and calves were negative for IBR, PI3 and adenoviruses (23). AGE AND FREQUENCY DISTRIBUTION OF INFECTIOUS AGENTS

5.14 5.08 4.84

6.46 5.00 5.31

4.00

4.84

5.31

1-4 feet proximal to ileocecal junction.

pathogen started to scour when one day old but virus i could not be demonstrated until three, fivie and seven days of age in these calves (1 Fig. 5). Calves in this group scoured aIn average of 5.2 days before ten days of aage. Calf nos. 20-7-3, 28-8-1 and 33-9-1 we:,re treated with intravenous fluids and chlor:amphenical starting at two, eight and eightt days of age respectively. Four c:Ialves excreted both ETP E. coli and Reo-lilike virus before ten days of age. In all fouir calves the E. coli were isolated prior to tlhe demonstration of Reo-like virus in the fec es (Fig. 5). The same results were found in tthe calf which excreted the Citrobacter strrain and Reo-like virus. The presence of b)oth an ETP strain of E. coli and

One or more types of potentially enteropathogenic microorganisms was demonstrated in 22 of the 40 calves (55.0%) before ten days of age. Potential enteropathogens could be demonstrated in 21 of 32 calves which scoured (65.6%) (Table VII). Sixteen of the 21 scouring calves (76%) excreted either ETP E. coli only, Reo-like virus only, or both ETP E. coli and Reo-like virus. Loop dilating E. coli were demonstrated in scouring calves one to seven days old and Reo-like virus was detected in scouring calves five to ten days old (Fig. 4). Six of the seven calves which excreted ETP E. coli as the only detectable enteropathogen began to scour when one or two days of age (Fig. 5). Calves in this group scoured an average of 3.4 days before ten days of age. Calf nos. 18-3-2 and 17-5-4 B'* were treated with intravenous fluids and i chloramphenicol starting at one and six is days of age respectively. Calf no. 18-3-2 z -I died at 12 days of age following a persistent diarrhea. Three of the calves which excreted ReoT like virus as the only detectable entero- ETP 4E.coli

126

=

3.83 4.33 3.83

o Cd wich iC Wd * Ciw"Nhi secreted loop Xu11

* Cdw whio

exereed Rea-til,

ICoi virus

L iJ number of diarrheic calves which excreted and/or Reo-like virus.

Can. J. comp. Med.

TABLE VI. Age at Demonstration of Virus and Age at Onset of Diarrhea in those Calves which Excreted Reo-like Virus

Pen 1

Pen 2

Pen3

Pen 4

Age at Onset of Fluorescence' Diarrhea (Days) 1 SP

Calf Number 14-5-ld

Age at Time of Sampling (Days) 8

Feces at Time of Samplingb S

27-7-1

1

N

SP

7

28-8-1

3 10

S S

SP SF

1

33-9-1 12-2-2

7 5 7

S A A

SP P P

1 6

21-4-2d

7 10

S

P Spe

1,7g

S

25-5-2d

5 10

N A

P SP

30-7-2

5 7

N S

32-8-2d 19-6-3 20-7-3 40-9-3 16-4-4d

10 5

S N

SP SPf P

5

10 5

23-6-4d

5 6 10

7

pe

5 8

S

P

1

S A N S N

P SP P

2,79 1

Pf

1,60

P

1,6 SPI &The first number is the birth order of the calf within the herd; the second number is the birth order of the is the pen number calf within the pen, the third number bS = scours, A = abnormal, N = normal *SP = suspect positive, P = positive dCalves which excreted ETP E. coli or Citrobacter before ten days of age eCalves also positive for BVD virus at this time fCalves also positive for Corona-like virus at this time gDiarrhea stopped for three days or longs and reoccurred on the second day listed

35-9-4

6

Reo-like virus on the same day was demonstrated in only one calf (no. 16-4-4). This calf developed acute, severe diarrhea (DMC 4.5%) at one day of age which lasted 48 hours. On days three, four and five the calf did not scour but it was too weak and dehydrated to nurse. No treatment was given and death occurred on day five. Loop dilating strains of E. coli were isolated from rectal swabs taken from this calf at one and three days of age and from the ileum at necropsy. Fecal samples were negative for Reo-like virus on day one and positive on day five just prior to death. Calves which excreted both loop dilating E. coli and Reo-

Vol. 39 - April, 1975

S

like virus scoured an average of 3.4 days before ten days of age. Of the nine strains of bacteria shown to be primary enteropathogens (Table IV), four were recovered from calves which excreted loop dilating E. coli only, and five were recovered from calves which excreted both loop dilating E. coli and Reo-like virus. In those calves in which the fecal DMC was less than 10% there was no significant difference between the fecal DMC of those calves which excreted loop dilating E. coli (mean DMC = 5.3%) and the fecal DMC of those calves which excreted Reo-like virus (mean DMC = 6.7%). 127

I0

Er.-I lEer

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r=:

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.

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-

-

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irS

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-

FECESC 10 % 0.M C FECES 10-14% 0DMC. FECES> 14 SD C

_

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St re SC

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which scoured and which did not excrete any potential enteropathogens began to scour when they were six, seven, or eight days old. These calves were nursing cows whose lacteal secretions contained higher levels of total solids and fat in milk and a lower level of ash in total milk solids from 72 to 144 hours prior to the onset of scours (i.e. at the 80 hour sampling) than cows whose calves did not become diarrheic on days six, seven, or eight. The significance of these findings are discussed in the following paper (1). Three calves died as a direct result of scouring. Calf no. 16-4-4 died at five days of age and ETP E. coli and Reo-like virus were detected prior to death as described above. Calf no. 18-3-2 died at 12 days of age. An ETP strain of E. colti was isolated from this calf at one day of age when scouring started. Calf no. 33-9-1 started to scour at one day of age and died when 47 days old after repeated attacks of diarrhea. Reo-like virus on day seven was the only enteropathogen recovered from this calf.

I7

TABLE VII. Distribution of Potentially Enteropathogenic Agents in Scouring Calves

a

of enterodemonstration of 5. Times potential pathogens in all calves with diarrhea from birth to ten days of agee.

Fig.

Three calves

were

positive for both Reoon the same day.

Corona-like viruses

and

In all three calves the time of detection of

the viruses coincided with the onset of diar-

creted

a

the

to

(no. 23-6-4)

One of these calves

rhea.

loop

dilating

strain

of

E.

coli

ex-

prior

appearance of either virus in the

feces. Bovine virus diarrhea

strated in

two

other

ten

the

calves,

no.

-virus five

demon-

was

days old and

days old. Both calves or suspect

were

positive for preReo-like virus at these times. Calf

either positive sence of

one

19-6-3 did not start to

third day after the feces. Calf loop dilating

until the

scour

of

21-4-2

Citrobacter

also excreted

the

strain

one

when

the

32

calves

which

did

group scoured an average

of

2.3

days

were ten days old and tended at the onset of diarrhea than

before to

be

calves older in whidch potential enteropathogens

were

detected

128

Scouring Calves 7 6 3 2 1 1

viruses

Citrobacter + Reo-like virus + BVD virus

1

21

Total infected calves No

I1

infectious agents demonstrated

32

Total

'Only four of 40 calves examined for

presence

of

Corona-like virus

(Fig. 5).

Seven

of the

DISCUSSION

scoured

not excrete any demonstrable (34.4%) enteropathogenic organisms. Calves in this

they

Number of

detection of the viruses in no.

day old.

Eleven

Infectious Agent(s) Demonstrated in Feces Loop dilating E. coli only Reo-like virus only E. coli + Reo-iike virus Reo- + Corona-like virusess Reo-like virus + BVD virus E. coli + Reo- Corona-like'

11

calves

One or more potentially enteropathogenic bacteria or viruses were isolated from 66% of the calves which scoured during this epizootic. This rate of detection of enteropathogens compares favourably with studies on human populations where bacteria and viruses can be isolated from an average of

Can. J.

comp.

Med.

35% of infants with gastroenteritis (10). Fourteen percent of 162 strains of E. coli which were isolated from all of the calves dilated ligated gut loops and 30 % of the strains isolated from calves with diarrheic feces dilated loops. There was a significant correlation between the prasence of loop dilating E. coli in the feces and the occurrence of diarrhea. Eighteen of 22 (82%) loop dilating strains of E. coli were recovered from calves in which the fecal DMC was under 10% while only one of 22 (4.5%) loop dilating strains of E. coli was recovered from calves with a fecal DMC over 14%. Strains of E. coli which dilated ligated loops were isolated from 12 of 32 (37%) calves which scoured which is a higher incidence of loop dilating strains of E. coli than has been found in other studies (9, 12, 31, 33). Strains of E. coli which dilated ligated loops could be isolated from feces and intestinal tract contents of only seven of 127 (5.5%) epizootiologically unrelated cases of scours in calves under ten days of age from several countries (33). The treatment status of the calves and the time after the onset of diarrhea at which the samples were collected was not known. In France three separate studies have reported the incidence of loop dilating strains of E. coli in calves as 6.1% (12), 10% (31), and 14% (9). The higher incidence of loop dilating strains of E. coli found in this study may have been due to the fact that calves were sampled prior to treatment and on several different days following the onset of diarrhea which would ensure a greater chance of isolating enteropathogens if they were present for short periods of time. Also the strains of E. coli examined by the ligated loop technique in the other four studies reported in the literature were not recovered from calves within one herd or during an epidemic of diarrhea. The strains of E. coli examined in this study were all isolated from a group of calves raised under confined conditions during an epidemic situation. There was ample opportunity for any of the enteropathogenic agents which were present to spread from one calf to another. This is supported by the fact that six of the loop dilating strains of E. coli which were isolated were serotype

08:K(85):H27 (Table III). Members of serogroup 08 were the most frequently isolated loop dilating strains of E. coli but no one serogroup predominated in the diarrheic calves. Although some diar-

Vol. 39

-

April, 1975

rheic calves excreted members of the same O serogroup for several days, other loop dilating strains of E. coli which belonged to different serogroups were isolated from the same pen. Similar variations in the E. coli population of diarrheic calves have been reported using phage typing (32). No enteropathogenie E. coli were isolated from calves older than seven days. Others have isolated five loop dilating strains of E. coli from calves all of which were less than three days old (40). Enterotoxemic colibacillosis has been reported in calves up to 30 days of age (15). Eighteen of 23 (78%) lambs which were fed strains of E. coli or Citrobacter which dilated ligated loops developed diarrhea while none of nine lambs which were fed strains of E. coli which did not dilate loops developed diarrhea. The number of E. coli present in the small intestinal contents of these lambs which developed diarrhea was significantly higher than in those lambs which did not develop diarrhea. These results agree with those published previously (33). One lamb which was challenged with an enteropathogenic strain of E. coli and which did not develop diarrhea had a very low number of E. coli in the small intestinal tract contents. The presence of low numbers of enteropathogenic E. coli in the small intestine of calves and lambs without resulting diarrhea has been reported (27, 33). The isolation of an ETP strain of E. coli from the feces is not sufficient evidence to incriminate that strain as the cause of diarrhea without demonstrating that it is present in high numbers in the upper small intestine. The evidence that the strains of E. coli isolated during this study were an important cause of diarrhea in those calves from which they were isolated includes the following: 1) they dilated ligated gut loops in calves, 2) they reproduced diarrhea when fed to young lambs, 3) there was a high correlation between the presence of these strains and the occurrence of diarrhea, and 4) the majority of these strains belong to 0 serogroups previously shown to include enteropathogenic strains. Twenty-two of 91 (24.2%) fecal samples examined for the presence of Reo-like virus were either positive or suspect positive using the fluorescent antibody technique. Reo-like virus was detected in the feces as early as six days before and as late as seven days after the onset of diarrhea. The im-

129

portance of collecting fecal samples within a few hours after the onset of diarrhea in the diagnosis of field outbreaks of diarrhea due to this virus has been emphasized (19, 24), however it appears as though the virus may be present for longer periods of time than was originally thought. In calves experimentally exposed to the Reo-like agent, viral particles were detected in the feces for up to 210 hours after oral exposure to the virus and at 100 hours after the onset of diarrhea (13). In this study 91% of positive or suspect positive demonstrations of Reo-like virus were in calves five to ten days of age. Other reports have given the peak incidence of infection with this virus as being under 96 hours of age (19). Corona-like virus was demonstrated in three calves six and seven days old which agrees with the findings of other workers (19, 36). Enteropathogenic E. coli were isolated from diarrheic calves one to seven days old and Reo-like virus was demonstrated in diarrheic calves five to ten days old. In all four calves which excreted both enteropathogenic E. coli and Reo-like virus the E. coli were isolated from the feces prior to the demonstration of Reo-like virus. The presence of an enteropathogenic strain of E. coli and Reo-like virus on the same day was shown in only one calf. These results do not support the theory that Reo-like virus is an initiating factor in enteric colibacillosis which is caused by the presence in the small intestine of large numbers of E. coli which have the ability to produce enterotoxin. It is possible that enteropathogenic strains of E. coli could proliferate secondary to viral infection in some outbreaks of diarrhea but this remains to be shown. It is also possible that other types of E. coli, which either do not possess the properties mentioned above or do not possess them in sufficient magnitude, could proliferate to very high numbers in the small intestine following an initial viral infection (24, 30, 38) and contribute to the pathogenesis of diarrhea by some as yet undefined mechanism. None of the potential enteropathogens could be detected in 11 of 32 (34%) diarrheic calves. There are three explanations which must be considered: 1) there may have been other enteropathogens present which were not detected; for example only four samples were examined for the presence of Corona-like virus, 2) one or more

130

of the designated potential enteropathogens may have been present and not detected because of inaccuracies in the methods used for demonstrating them. Loop dilating strains of E. coli may have been missed because it was difficult to examine more than one, and rarely more than two strains from each rectal swab by the ligated gut loop technique. Reo-like virus detection by the fluorescent antibody technique is dependent on the presence of virus containing epithelial cells which are not always present in high enough numbers to give reliable results and 3) there is the possibility that diarrhea in these calves was not caused by infectious agents but rather was due in part to nutritional factors (1). An enteropathogenic strain of E. coli and Reo-like virus were demonstrated in one calf which remained normal throughout the study period and Reo-like virus was demonstrated in five other calves prior to the onset of diarrhea. Demonstration of the presence of Reo-like virus in the feces of non-diarrheic calves has also been reported by others (4). Eight of 21 (38%) diarrheic calves in this study group excreted multiple potentially enteropathogenic bacteria and/or viruses in their feces. Multiple mixed viral infections in calves (5, 19, 29, 39) and humans (10) and mixed bacterial and viral infections in calves (41) and in humans (10) have been reported. In one recent study it was possible to reproduce diarrhea in calves only when a bacteria and the Reo-like virus were administered simultaneously. Diarrhea did not occur when either the bacteria or the virus were given separately to calves (41). These observations, as well as the finding that calves can be infected with low numbers of enteropathogenic E. coli without manifesting diarrhea, point out the need for further investigation of the factors which affect the distribution and balance of microorganisms in the gastrointestinal tract. Vaccines are becoming available for some of the enteropathogens found in this study (26, 27). There are several problems which may make their use under field conditions non-specific: 1) the clinical syndrome which occurs is very similar in calves which are infected with a variety of enteropathogens, 2) the techniques which are necessary to demonstrate the presence of some enteropathogens, such as E. coli, remain too laborious and expensive to use for routine diagnostic purposes. Those enteropathogens

Can. J. comp. Med.

which are most easily demonstrated will be looked for but it should be remembered that isolation of either a bacterial or a viral enteropathogen from a diarrheic calf is insufficient evidence to attach etiological significance to such an agent unless all other possible pathogens are excluded (14), 3) several different enteropathogens can be found in the same calf or a group of calves and 4) enteropathogens can be recovered from non-diarrheic calves (27, 33, 41). For these reasons, the etiology of many individual cases and herd outbreaks of acute neonatal diarrhea in calves will remain undifferentiated. Available vaccines offer a solution to part of the problem, however, the variety of infectious agents capable of causing diarrhea will probably not allow the development of a single preventive measure which will be effective under all circumstances. The observation that enteropathogens can be recovered from normal calves is an indication that factors other than the mere presence of infectious agents are important in causing overt clinical disease. In the future more attention must be given to the epizootiological conditions which surround herd outbreaks of the calf scours syndrome. Maximum prevention may be obtained only by the judicious use of vaccines in combination with selected changes in management.

ACKNOWLEDGMENTS The authors acknowledge and support the cooperation of Dr. B. Schiefer, Dr. J. Neitzke, Dr. J. Greenfield and Dr. C. H. Bigland; and the technical assistance of V. Lacroix, F. Shearer and B. Laing. The support and collaboration of Dr. C. M. Williams, M. C. Johnson, and the University of Saskatchewan, Department of Animal Science Feedlot staff are acknowledged.

REFERENCES 1. ACRES, S. D., B. T. ROUSE and 0. M. RADOSTITS. Acute undifferentiated neonatal diarrhea in beef calves II. The nutrient composition of colostrum and milk and its effect on the occurrence of diarrhea. Can. J. comp. Med. In press. 1975. 2. ALDASY, P., L. CSONTOS and A. BARTHA. Pneumoh-enteritis in calves caused by adenoviruses. Acta. vet. hung. 15: 167-175. 1965.

Vol. 39

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April, 1975

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