THE CANADIAN VETERINARY JOURNAL LA REVUE VETERINAIRE CANADIENNE Volume 18

No. 5

May-mai 1977

ACUTE UNDIFFERENTIATED NEONATAL DIARRHEA OF BEEF CALVES: THE PREVALENCE OF ENTEROTOXIGENIC E. COLI, REO-LIKE (ROTA) VIRUS AND OTHER ENTEROPATHOGENS IN COW-CALF HERDS S. D. Acres, J. R. Saunders and INTRODUCTION THE

ETIOLOGY OF NEONATAL CALF DIARRHEA

is

complex and involves a variety of infectious, nutritional, immunological and environmental factors. Although many infectious agents have been isolated from the feces and tissues of diarrheic calves the relative importance of each one has not been established. Recently, infections of individual calves and herds with multiple enteropathogens have been demonstrated (1, 2, 4, 10, 18, 19, 20, 31). Calves in one beef herd were infected with enteropathogenic Escherichia coli (EEC) and Citrobacter, neonatal calf diarrhea Reo-like (NCD-R), Corona-like (NCD-C) and bovine virus diarrhea (BVD) viruses (1). The EEC and Citrobacter were isolated from diarrheic calves one to seven days old whereas the three viruses were isolated from calves five to ten days old. The syndromes associated with the presence of the various infectious agents cannot be differentiated clinically and routine diagnostic procedures for many enteropathogens are not yet available. Therefore it is difficult for veterinarians to recommend the use of specific vaccines or antibacterial therapy for the prevention and treatment of neonatal diarrhea. Because of these problems we felt that it would *Department of Veterinary Clinical Studies (Acres and Radostits) and Department of Veterinary Microbiology (Saunders), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO. Present address of senior author: Veterinary Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, Saskatchewan. 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 Agriculture Canada EMR Grant 7111. JOUR.,

vol. 18,

no. 5,

May, 1977

M. Radostits*

be useful to establish the prevalence of several enteropathogens which are known to occur in Western Canada and to further define their age distribution in calves. MATERIALS AND METHODS Collection of Samples During the 1973 spring calving season private practitioners and veterinarians at the diagnostic laboratories in Alberta and Saskatchewan were asked to nominate herds for study in which the morbidity or mortality from neonatal diarrhea was unusually severe. Crude fecal samples, rectal and nasal swabs and blood were collected from 222 calves from 59 beef herds. All except 14 of the calves were under 30 days of age, and the oldest calf was 44 days of age when sampled. Forty-six herds were visited and one or more diarrheic, and normal calves when possible, were sampled in each herd. Sixteen calves from 11 other herds were sampled at veterinary clinics where they had been taken for treatment, and samples from one herd were submitted directly to the laboratory. Samples were also taken from the intestines of eight calves which died of severe diarrhea. Twelve normal calves in one herd in

which there had never been diarrheic calves also sampled. The modified-live Reo-like virus vaccine, which became available in 1974, had not been used in any of the herds. The clinical syndrome present in most of the calves was typical of acute undifferentiated neonatal diarrhea and was characterized by acute, profuse, watery diarrhea accompanied by anorexia, dehydration, muscular weakness, collapse and death if untreated. However, three herd outbreaks were seen in which severe diarrhea was not the predominant clinical feature. Calves in these three herds were from one to six weeks old when they developed were

L13 CAN. VET.

0.

CANADIAN VETERINARY JOURNAL

signs referrable to the gastrointestinal and respiratory systems. Convalescent serum was obtained from calves in two of these herds three to four weeks following the initial occurrence of the disease. Demonstration of Enteropathogenic Agents Rectal or intestinal samples for bacterial isolation were taken using commercial swabs which contained Amies transport medium with charcoal' and were kept refrigerated until processing in the laboratory. Samples were streaked onto blood agar and MacConkey's agar plates and were also inoculated into selenite broths which, following incubation for 24 hours, were subcultured onto Brilliant-green agar and Hektoen-enteric agar for isolation of Salmonella species. Four isolated colonies of E. coli were picked from each blood agar plate, serogrouped and stored on trypticasesoy slants. Colonies were classified as mucoid if a strand of mucus-like material adhered to a wire inoculating loop when it was withdrawn from a colony grown overnight on blood agar. Serogrouping was done using antisera prepared in rabbits against the following 26 0 group antigens: 1, 2, 5, 8, 9, 15, 17, 20, 21, 26, X28, 35, 45, 55, 73, 78, 86, 88, 101, 114, 115, 117, 119, 126, 137 and 145. Antisera against E. coli K antigens were not available. One, two, or occasionally three strains of E. coli from each calf were examined for enterotoxin production (enterotoxigenic E. coli (ETEC)) using the ligated intestinal segment technique (LIST) in calves as previously described (1). Crude fecal samples and nasal swabs were collected as outlined previously (1) and were stored frozen in dry ice until arrival at the laboratory where they were held at -700C. Feces were examined for the presence of NCD-R virus by the fluorescent antibody technique (FAT).2 Nasal swabs were examined for the presence of the infectious bovine rhinotracheitis (IBR), BVD and the parainfluenza-3 (PI3) viruses by the methods described earlier (1). Blood samples were centrifuged within six hours following collection and the serum was stored in dry ice until arrival at the laboratory. IBR neutralizing (SN) antibody titers were determined by incubating 100 TCID0o of IBR virus with varying dilutions of serum in microtiter plates containing confluent monolayers of bovine embryonic kidney cells grown in Eagle's medium (MEM) supplemented with

10% fetal calf serum, 1.0% nonessential amino

acids, 100 units per ml of penicillin and 100 ,ug per ml of streptomycin. Test plates were incubated at 37°C in 5% CO2 for four days and then examined for cytopathic effects (CPE). The SN titer was defined as the reciprocal of the serum dilution which produced total inhibition of CPE.

Antimicrobial Sensitivity Testing The antimicrobial drug sensitivity patterns of ETEC were determined on Mueller-Hinton agar plates using the Kirby-Bauer, Sherris standardized single disc susceptibility test for rapidly growing pathogens3 (3). The following antibiotic sensitivity discs were used: gentamycin 10 ,ug, nifuraldezone 100 MAg, polymixin B 300 units, chloramphenicol 30 ,ug, kanamycin 30 ,ug, neomycin 30 ug, ampicillin 10 ug, cephalothin 30 Mug, tetracycline 30 Mug, triple sulfa 250 ug, penicillin 10 units, dihydrostreptomycin 10 ,g. The dry matter content (DMC) of feces was determined by drying approximately 10 g to a constant weight. Feces were classified as diarrheic, abnormal, or normal when the DMC was 14% respectively. RESULTS Herd Prevalence Rates of ETEC and NCD-R Virus Seventy-eight normal and diarrheic calves in 35 herds were examined for the presence of ETEC and NCD-R virus (Table I). Eightyone other calves in the same herds were examined only for the presence of ETEC, and 22 calves were examined only for NCD-R virus. One or both of these enteropathogens were found in 53 of 181 (29.3%) calves in 29 of the 35 (82.9%) herds. Thirty-seven calves in 20 other herds were examined only for the presence of ETEC; ten calves (27.0%) in ten of these herds (50.0%) were positive (Table II). Six of the ten calves were sampled at veterinary clinics where they were being treated with intravenous fluids for acute, profuse watery diarrhea. Four calves from four additional herds were examined only for the presence of NCD-R virus and all were negative. Salmonellae were not isolated from any calves in the 59 herds.

Age-Specific Prevalence Rates of ETEC and

NCD-R Virus Fifty-nine of the 78 calves which were examined for the presence of both enteropatho-

lPrecision Dynamics Corp., Burbank, California. 2Samples kindly processed by Dr. C. A. Mebus, Department of Veterinary Science, University of

Nebraska, Lincoln, Nebraska.

3Difco Laboratories, Detroit, Michigan. 114

NEONATAL DIARRHEA

TABLE I PREVALENCE RATESa OF ENTEROTOXIGENIC E. coli (ETEC) AND NCD-R VIRUS IN HERDS EXAMINED FOR THE PRESENCE OF BOTH ENTEROPATHOGENS. ALBERTA AND SASKATCHEWAN, 1973 Number of calvesb examined for Result

ETEC + NCD-R

Pos. ETEC Pos. NCD-R Pos. ETEC + NCD-R Negative Total

17 18 3 40 78

ETEC 6

75 81

NCD-R

9 13 22

Total calvesb

Herds

23 (14.5%)a 27 (27.0%) 3 (3.8%) 128 181

11 (31.4%)" 13 (37.2%) 5 (14.3%) 6 (17.1%) 35 (100.0%)

positive for enteropathogen x aPrevalence rate = number number examined for enteropathogen 100% bIncludes diarrheic and nondiarrheic calves. See text and Table III.

TABLE II PREVALENCE RATE" OF ETEC IN HERDS EXAMINED ONLY FOR THE PRESENCE OF ETEC. WESTERN CANADA, 1973 No. Calves

Positive Negative Total

"Prealence rate

10 27 37 =

(27.0%)a

No. Herds 10 10 20

(50.0%)"

number positive X 100% number examined

gens were diarrheic at the time of sampling (Table III). ETEC were isolated only from diarrheic calves, whereas NCD-R virus was demonstrated in five calves which were not diarrheic. However, the four NCD-R virus infected calves in which the fecal DMC was between 10 and 14% had been diarrheic one to two days prior to sampling. Similarly, two of three calves which were infected with both enteropathogens had abnormal feces when sampled, but both calves had previously been diarrheic. The age-specific prevalence rates of ETEC and NCD-R virus are shown in Table IV. Calves in which the fecal DMC at the time of sampling was between 10 and 14% are included in the table because they had been diarrheic one to two days previously. The overall prevalence rates of ETEC and NCD-R virus in diarrheic calves were very similar (23.9% vs. 22.5%). However, the agespecific prevalence rates of these two enteropathogens differed markedly. Calves which were infected with ETEC became diarrheic at a younger age than calves which were infected with NCD-R virus. Thirteen of 16 (81%) ETEC-infected calves, but only two of 15 (13%) NCD-R virus-infected calves became diarrheic before five days of age. The prev-

alence rates of ETEC and of NCD-R virus in calves which became diarrheic before five days of age were 62% and 10% respectively, whereas in calves which became diarrheic between five and ten days of age the prevalence rates were 8% and 40% respectively. Calves infected with ETEC and NCD-R virus became diarrheic at average ages of 3.9 and 9.8 days respectively (P < 0.01). Furthermore, ETEC were isolated only from calves one to 11 days old whereas NCD-R virus was found in calves from three to 30 days old. All three of the calves infected with ETEC and NCD-R virus became diarrheic before five days of age. Eighty of the other 118 calves (81 in Table I plus 37 in Table II) which were examined only for the presence of ETEC had fecal DMC's under 14% when sampled (Table V). Thirteen of 22 (59%) calves which became diarrheic before five days of age, as compared to only one of 22 (5%) calves which became diarrheic at from five to ten days of age, were infected with ETEC. The average age at the onset of diarrhea in ETEC infected calves in this group was 2.5 days. Four of the other nine calves which were infected with NCD-R virus (Table I) became diarrheic at four, eight, 12 and 15 days of age (average 9.8 days). The age at onset was unknown for four other calves and one calf was normal (fecal DMC 25%) when sampled. Prevalence and Serogrouping of ETEC A total of 49 of 285 (17.2%) strains of E. coli which were isolated from 33 of 196 (16.8%) calves were enterotoxigenic (Table VI). These ETEC were found in calves from 26 of 55 (47.3%) herds examined (Tables I and II). One strain of ETEC was isolated from each of 11 herds, and two, three and four strains were isolated from each of ten, two and three herds respectively. For each of 23 out of 33 calves found to be infected with 115

CANADIAN VETERINARY JOURNAL

TABLE I I I PREVALENCE RATESa OF ETEC AND/OR NCD-R VIRUS IN CALVES EXAMINED FOR THE PRESENCE OF BOTH ENTEROPATHOGENS, CLASSIFIED ACCORDING TO FECAL DRY MATTER CONTENT (DMC). ALBERTA AND SASKATCHEWAN, 1973 Fecal Dry Matter Content Result

10%

10-14%

14%

Total Calves

Pos. ETEC 17 (29%)a 0 (0%) 17 (21.8%)c 0 (0%) 4 (33%) Pos. NCD-R 12 (20%) 1 (20%) 17b (23.1%) Pos. ETEC + NCD-R 1 (2%) 2 (17%) 3 (3.8%) 0 (0%) Negative 29 (49%) 6 (50%) 4 (80%) 39b (51.3%) Total 12 (100%) 5 (100%) 76 (100%) 59 (100%) aPrevalence rate _ number of calves in this fecal DMC group infected with enteropathogen X 100% number of calves in this fecal DMC group examined bFecal DMC of one case unknown. cPrevalence rate calculated using total number of calves infected with this enteropathogen and total number of calves (i.e. 78) examined for both enteropathogens. TABLE IV AGE-SPECIFIC PREVALENCE RATES" OF ETEC AND/OR NCD-R VIRUS IN CALVES EXAMINED PRESENCE OF BOTH ENTEROPATHOGENS. ALBERTA AND SASKATCHEWAN, 1973

FOR THE

Age at Onset of Diarrheab (Days)

Result

10

Total Calves

Pos. ETEC Pos. NCD-R Pos. ETEC + NCD-R Negative Total

13 (62%) 2 (8%) 1 (5%) 160 (23.9%)e 2 (10%) 10 (40%) 3 (16%) 15c (22.5%) 3 (14%) 3 (4.3%) 0 (0%) 0 (0%) 3 (14%) 13 (52%) 31d (49.3%) 15 (79%) 21 (100%) 25 (100%) 19 (100%) 65 (100.0%) of calves positive in age group x 100% "Age-specific prevalence rate =number total calves examined in age group bIncludes all calves with fecal DMC

Acute undifferentiated neonatal diarrhea of beef calves: the prevalence of enterotoxigenic E. coli, reo-like (rota) virus and other enteropathogens in cow-calf herds.

THE CANADIAN VETERINARY JOURNAL LA REVUE VETERINAIRE CANADIENNE Volume 18 No. 5 May-mai 1977 ACUTE UNDIFFERENTIATED NEONATAL DIARRHEA OF BEEF CALVE...
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