Influence of Feeding Fermented Colostrum and Lactobacillus acidophilus on Fecal Flora of Dairy Calves1

Influence of Feeding Fermented Colostrum and Lactobacillusacidophilus on Fecal Flora of Dairy Calves 1 D. K. E L L I N G E R 2. L, D. M U L L E R 3, and P. J. G L A N T Z 4 The Pennsylvania State University University Park, 16802 ABSTRACT

Twenty Holstein calves were assigned alternately at birth to diets of 1) fermented colostrum, 2) colostrum treated with 1% propionic acid, 3) whole milk, or 4) whole milk treated with Lactobacillus acidopbilus (frozen concentrate culture) at 5 X 108 organisms per litter. Diets were fed once daily for 3 wk at 10% of birth weight as the sole source of nutrients. Fecal samples were collected at 0, 7, 14, and 21 days of age and analyzed for coliform and lactobacilli numbers. Fermented colostrum diets did not alter coliform counts in feces of healthy calves. Fecal coliform counts of calves fed L. acidopbilus decreased with time. Average fecal lactobacilli counts were lower for the colostrum diets than milk diets. The apparent lowered incidence of scours frequently reported in calves fed fermented colostrum diets was not reflected in major changes in fecal microflora under the conditions of this study. INTRODUCTION

Fermented colostrum for young calves is an excellent source of nutrients (6). In addition to the nutritional and economic advantages, fermented colostrum has decreased the incidence of diarrhea in young calves from whole milk (10, 21, 27) and milk replacer (4, 26). Reasons for decreased diarrhea are not clear. One hypothesis is that lactobacilli in fermented

Received July 9, 1979. t Authorized for publication 9/11/78 as paper No. 5601 in the journal series of The Pennsylvania Agricultural Experiment Station. School of Veterinary Medicine, University of Pennsylvania, Philadelphia, 19104. 3Department of Dairy and Animal Science. 4 Department of Veterinary Medicine. 1980 J Dairy Sei 63:478--482

colostrum adhere to and colonize the intestinal tract and, thereby, inhibit coliform growth either through bactericidal properties, the production of acids, or some other mechanism. The primary cause of diarrhea in calves is infection by E. coli (7). The value of lactobacillus, specificially L. acidopbilus, for humans was proposed originally by Metchnikoff (12). Recent reviews (20, 22, 24) have emphasized some of the potential benefits of lactobacillus, including a suppression of coliform growth, implantation in the intestinal tract, and decreased gastrointestinal problems and diarrhea. Administration of a nonfermented milk product containing L. acidophilus increased numbers of lactobaciUi in the feces of healthy men (8). The value and use of acidophilus food products is based on some of these potential benefits (23, 24). The use of L. acidophilus for livestock has not been studied extensively or with conclusive results. Feeding a dried commercial preparation (25) or a viable culture (1) of L. acidophilus reduced the incidence of diarrhea in dairy calves. Findings have been similar with pigs (15, 16). Feeding cultured milk prepared by inoculating pasteurized milk with L. acidopbilus and L. lactis did not alter growth or health of calves compared to a control diet (13). Bruce et al. (3) recently reported that feeding L. acidopbilus cultures of human or calf intestinal origin increased the numbers of lactobacilli in feces of calves and reduced the numbers of coliform in feces. Since it is difficult to obtain intestinal samples for microbial analyses, enumeration of fecal microbial flora has been used as an indirect method of determining bacteria inhabiting the intestinal tract (3, 8, 15, 17, 19). The fecal flora is assumed to represent only the luminal flora and not that associated with mucosal epithelial surfaces and is assumed to vary with different types of diets (2). The objective of this study was to observe

478

TECHNICAL NOTE changes in fecal flora of calves fed excess colostrum, naturally fermented and acid preserved, and milk treated with a L. acidopbilus culture of human origin.

MATERIALS AND METHODS Experimental Diets and Animal Handling

Twenty Holstein calves (11 males and 9 females) were separated from their dam immediately after birth and hand-fed approximately 2.5 kg of their dam's colostrum. The calves were fed an additional 2.5 kg of dam's colostrum within the next 12 to 24 h and then moved to individual solid-walled pens in a heated and mechanically ventilated calf barn. Calves were assigned alternately at birth to one of four diets with five calves per diet. Diet 1 was naturally fermented colostrum (FC) composed of the first six milkings postpartum from Ayrshire, Holstein, and Brown Swiss cows. Diet 2 was the same colostrum treated with 1% by weight daily of propionic acid (PA). Calves on diets 1 and 2 were fed 3 kg of the respective diets diluted with 1.5 kg warm water daily. Diet 3 was whole milk (WM) drawn daily from the bulk cooler. Diet 4 was whole milk treated with L. acidolphilus (LA). A commercial, viable human L. acidopbilus product (Fargo 606 frozen concentrate by Microlife Technics) was added to warm whole milk immediately prior to feeding at a concentration of 1.06 x 106 organisms per liter. This dosage was based on a 108 dosage for humans (8). Calves on diets 3 and 4 were fed 4 kg of their respective diets daily. All diets were fed once daily by nipple pail for 21 days as the sole source of nutrientS. A few calves were fed twice daily for 2 to 3 days until adjusted to diets, then once daily. Water was available free choice. Colostrum for diets 1 and 2 were stored in 80-liter plastic containers for at least 7 days and not longer than 21 days prior to feeding. Colostrum was added daily to the containers until they were full; then they were covered tightly. Colostrum was stirred only after additions, before feeding and before samples were collected. Each calf was fed from one specific container of colostrum. The trial was from January to May during which the temperature of the calf barn was maintained at approximately 10 C.

479

Sampling Procedures

Sampling (200 to 300 ml) of each liquid diet was weekly for laboratory analysis. Also, 5 ml of each diet were collected into sterile tubes and mixed with 5 ml of a sterile buffered glycerol-saline solution adjusted to pH 7.4 for subsequent microbial analyses. Fecal samples were obtained at birth and 7, 14, and 21 days of age. Samples obtained from the rectum with sterile gloves were placed into sterile, plastic graduated centrifuge tubes. The fecal samples were diluted with five parts of the sterile buffered glycerol solution to one part feces and mixed. The buffered glycerol-saline solution has been a widely used medium from preservation of fecal samples (5). Since immediate analysis of liquid and fecal samples was not possible, samples were frozen for later analyses. Freezing of fecal samples in the buffered glycerol solution has yielded coliform counts nearly 80% of those from a fresh, unfrozen sample, while samples frozen without the buffered glycerol yielded counts of only 10% of the fresh samples (9). Fecal lactobacilli counts were similar to those reported by Bruce et a l . (3) for fresh fecal samples, thus suggesting that freezing in buffered glycerol solution did not alter fecal lactobacilli counts. Fecal samples were thawed and centrifuged at 2000 × g to obtain a wet packed volume of feces, and results were recorded as log per milliliter wet packed feces. This fecal precipitate was diluted 1:10 with .85% sterile saline solution. Serial dilutions then were with .85% sterile saline solution. Serial dilutions then were with .85% sterile saline in test tubes. Violet Red Bile agar (VRB) BBL and Lactobacillus Selective agar (LBS) BBL were used to enumerate coliforms and lactobacilli, respectively. Pour plates were made with the VRB agar and spread plates, with a glass spreading bar, were made on the LBS agar. Dilutions were 10 -3 to 10 -7 . The VRB plates were incubated at 37 C for 24 h. The LBS plates were incubated at the same temperature for 48 h in a GasPak BBL anaerobic system with two disposable hydrogen and carbon dioxide generator envelopes. Colony counts were done manually with numbers over 300 designated as too numerous to count (TNTC). Plates that contained the 10 -7 dilution and were TNTC were assigned arbitrarily the number of 1 x 10 l° for analytical purposes. Journal of Dairy Science Vol. 63, No. 3, 1980

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ELLINGER ET AL.

Milk samples also were analyzed for c o l i f o r m and lactobacilli numbers. Serial dilutions were m a d e directly f r o m the 1:1 dilution of milk and b u f f e r e d glycerol. The dilutions that were used were 2 x 10 -1 to 2 x 10 - s . The agars and m e t h o d s described for the fecal analyses were identical to those for milk samples e x c e p t for the difference n o t e d above. Statistical Analyses

The test for determining significant differences a m o n g t r e a t m e n t effects was the F-ratio from the analyses of variance analyzing for log of bacterial counts. An orthogonal c o m p a r i s o n t e c h n i q u e was used to determine differences between means. RESULTS A N D DISCUSSION

Results of the chemical and microbial analyses for the four liquid diets are in Table 1. The chemical c o m p o s i t i o n generally agrees with published c o m p o n e n t s for colostrum and whole milk (6). Numbers of c o l i f o r m s in both FC and PA diets were similar. N u m b e r s in f e r m e n t e d c o l o s t r u m agree with o t h e r reports but in PA were higher than r e p o r t e d (6). Numbers of lactobacilli were c o m p a r a b l e to those in (11, 14). Results of fecal microbial analyses are in Table 2, and Figure 1 and 2. All fecal samples at birth c o n t a i n e d less than 10 3 lactobacilli and less than 10 3 coliforms per milliliter of wet packed feces and are n o t reported. No differences were significant in the numbers of coli-

forms in feces for all sampling times (Table 2), although c o u n t s t e n d e d to be lower from calves fed the f e r m e n t e d diets and the L. acidopbilus s u p p l e m e n t e d diet. Calves fed the FC diet tended t o w a r d less coliforms for the three sampling times (Figure 1). Calves fed the L A diet had a linear decrease in c o l i f o r m s (Figure 1), suggesting an antagonistic action t o w a r d coliforms or possible i m p l a n t a t i o n of lactobacilli in the intestinal tract. This response is similar to studies with pigs (17). Bruce et al. (3) r e p o r t e d a greater decrease in c o l i f o r m s f r o m feces of calves fed L. acidopbilus for the first 2 wk o f age c o m p a r e d to a control diet. Differences were small b e t w e e n L. acidopbilus cultures of h u m a n or calf origin (3). The largest decrease in fecal flora was at 3 wk for calves fed the LA diet (P<.05). No other study is available for c o m p a r i s o n since Bruce et al. (3) did n o t measure fecal counts after 2 wk. Calves fed the milk diets had significantly m o r e lactobacilli per milliliter feces than calves fed c o l o s t r u m diets. The lactobacilli c o u n t s from feces of calves fed the WM diet was u n e x p e c t e d and not readily explainable but persisted for all 3 wk (Figure 2). Bruce et al. (3) r e p o r t e d a greater increase in lactobacilli f r o m feces of calves fed the L. acidopbilus culture. A possible e x p l a n a t i o n for the lower lactobacilli from feces o f calves fed the c o l o s t r u m diets (FC and PA) is that lactobacilli in the diets were colonizing the small intestine. Only those organisms in excess were r e m o v e d via the feces, resulting in little change in lactobacilli. Perhaps

~i'ABLE 1. Chemical composition and microbial analysis of liquid diets, fermented colostrum (FC), FC treated with propionic acid (PA), whole milk (WM), and WM treated with L. acidopbilus (LA). Item

FC

PA

WM

LA

Total solids a (%) SE Fat (%) SE Protein (%) SE Coliforms b SE Lactobacilli b SE

15.50 .28 4.92 .25 4.90 .16 3.81 .07 5.56 .27

15.80 .65 5.65 .25 5.01 .16 4.20 .32 5.82 .22

12.18 .18 3.00 .21 2.81 .10 < 1.30

12.18 .18 3.00 .21 2.81 .10 < 1.30

2.66 .10

3.18 .04

a14 samples for each of the FC and PA diets; 9 samples for each of the WM and LA diets. bLogl0 o f organisms/ml liquid diet. Journal of Dairy Science Vol. 63, No. 3, 1980

TECHNICAL NOTE

481

TABLE 2. Mean fecal microbial counts averaged across the 7, 14, and 21 day sampling period for all calves on diets fermented colostrum (FC), FC treated with propionic acid (PA), whole milk (WM), and WM treated with L. acidopbilus (LA). Diet

Coliformsa Lactobacillia L:C ratio b

FC

PA

WM

LA

7.03 6.93c .99:1 e

7.28 7.34c 1.01:1 e

7.33 8.60 d 1.17:1 f

6.86 8.15d 1.19:1 f

aLogt0 of organisms/ml wet packed feces. bLactobacilli to coliform ratio. c'dwM and LA>FC and PA (P<.O1). e'fwM and LA>FC and PA (P<.05).

a f a c t o r is in f e r m e n t e d c o l o s t r u m and n o t in w h o l e milk w h i c h facilitates c o l o n i z a t i o n in t h e intestinal tract. Several researchers (8, 18) have suggested t h a t feeding lactobacilli m a y n o t be effective

8•5 l ~

unless stress is applied. Gilliland et al. (8) fed L.

acidopbilus to h e a l t h y h u m a n males and f o u n d an increase in t h e n u m b e r o f lactobacilli in t h e i r feces b u t f o u n d n o e f f e c t o n c o l i f o r m n u m b e r s . T h e y suggested t h a t this r e s p o n s e m a y have b e e n related to t h e test subjects being h e a l t h y and n o t e x p e r i e n c i n g intestinal disorders. In o u r s t u d y , diarrhea and h e a l t h disorders w e r e n o t

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Figure 1. Average number of coliforms in the feces of calves on all diets for individual sampling periods. (Vertical lines above the bars are standard errors). FC, fermented colostrum; PA, propionic acid treated FC; WM, whole milk ; LA, L. acidophilus treated WM.

7

14

21

DAYS

Figure 2. Average number of lactobacilli in the feces of calves on all diets for individual sampling periods. (Vertical lines above the bars are standard error. C~ indicates WM and LA>FC and PA, P<.05.) FC, fermented colostrum; PA, propionic and treated FC; WM, whole milk; LA, L. acidopbilus treated WM. Journal of Dairy Science Vol. 63, No. 3, 1980

482

ELLINGER ET AL.

present and may account for the absence of s i g n i f i c a n t c h a n g e s in c o l i f o r m n u m b e r s . T h e r e s u l t s o f this s t u d y s u g g e s t a p o s s i b l e relationship between the numbers of lactobacilli a n d c o l i f o r m s in feces, p a r t i c u l a r l y w i t h calves f e d a L. a c i d o p b i l u s c u l t u r e . T h e L : C r a t i o ( T a b l e 2) is c o n s i s t e n t w i t h t h a t in h e a l t h y , n o n s c o u r i n g pigs (17). T h e L : C r a t i o i n c r e a s e d m o s t w i t h age f o r t h e L. a c i d o p b i l u s s u p p l e m e n t d i e t , a s i m i l a r i n c r e a s e w i t h age as r e p o r t e d (3). A d d i t i o n a l s t u d i e s are n e e d e d t o d e t e r m i n e if L. a c i d o p b i l u s a d m i n i s t r a t i o n c a n c o n t r o l e n e r t i c p a t h o g e n s a n d to d e t e r m i n e m i c r o b i a l survival in t h e i n t e s t i n a l t r a c t a n d attachment to the intestinal mucosa. The a p p a r e n t l o w e r e d i n c i d e n c e o f d i a r r h e a freq u e n t l y r e p o r t e d in calves f e d f e r m e n t e d c o l o s t r u m d i e t s (4, 6, 10, 21, 26, 27) w a s n o t r e f l e c t e d in m a j o r c h a n g e s in fecal m i c r o f l o r a u n d e r t h e c o n d i t i o n s o f this s t u d y , a l t h o u g h a t r e n d e x i s t e d f o r l o w e r fecal c o l i f o r m s f r o m calves f e d f e r m e n t e d c o l o s t r u m c o m p a r e d t o the control. REFERENCES

1 Bechman, T. J., J. V. Chambers, and M. D. Cunningham. 1977. Influence of Lactobacillus acidopbilus on performance o f young dairy calves. J. Dairy Sci. 60 (Suppl. 1):74. (Abstr.) 2 Bornside, G. tl. 1978. Stability of human fecal flora. Am. J. Clin. Nutr. 31:5141. 3 Bruce, B. B., S. E. Gilliland, L. J. Bush, and T. E. Staley. 1979. Influence of feeding cells o f Lactobacillus acidopbilus on the fecal flora of young dairy calves. Page 207 in Oklahoma Anita. Sci. Res. Rep. 4 Daniels, L. B., J. R. l tall, (2. R. l lornsby, and J. A. Collins. 1977. Feeding naturally fermented, cultured, and direct acidified colostrum to dairy calves. J. Dairy Sci. 60:992. 5 Edwards, P. R., and W. 11. Ewing. 1972. Identification o f enterbacteriaceae. 3rd ed. Burgess Publ. Co., Minneapolis, MN. 6 Foley, J. A., and D. Otterby. 1978. Availability, storage, treatment, composition, and feeding value o f surplus colostrum: A review. J. Dairy Sci. 61:1033. 7 Frey, I1. 1972. Colibacillosis in calves. Verlag Hans l luber, Bern, Switzerland. 8 Gilliland, S. E., M. L. Speck, G. F. Nauyok, Jr., and F. G. Giesbrect. 1978. Influence of consuming non-fermented milk containing Lactobacillus acidopbilus in fecal flora o f healthy males. J. Dairy Sci. 61:1. 9 Glantz, P. J. 1948. Studies on the etiology of calf scours. M. S. thesis, The Pennsylvania State University, University Park.

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10 Jenny, B. F., S. E. Mills, and G. D. O'Dell. 1977. Dilution rates of sour colostrum for dairy calves. J. Dairy Sci. 60:942. 11 Jenny, B. F., G. D. O'Dell, and M. G. Johnson. 1977. Microbial and acidity changes in colostrum fermented by natural flora at low and high ambient temperatures. J. Dairy Sci. 60:453. 12 Metchnikoff, E. 1908. Prolongation o f life. G. P. Putmam's Sons, New York, NY. 13 Morrill, J. L., A. D. Dayton, and R. Mickelsen. 1977. Cultured milk and antibiotics for young calves. J. Dairy Sci. 60:1105. 14 Muller, L. D., and J. Smallcomb. 1977. Laboratory evaluation o f several chemicals for preservation of excess colostrum. J. Dairy Sci. 60:627. 15 Muralidhara, K. S., W. E. Sandine, P. R. Elliker, and D. C. England. 1972. Effect o f feeding lactobacillus concentrates on coliform excretion and scouring in swine. J. Dairy Sci. 55:661. (Abstr.) 16 Muralidhara, K. S., W. E. Sandine, D. C. England, and P. R. Elliker. 1973. Colonization of Escbericbia coil and lactobacillus in intestines o f pigs. J. Dairy Sci. 56:635. (Abstr.) 17 Muralidhara, K. S., G. G. Sheggeby, P. R. Elliker, D. C. England, and W. E. Sandine. 1977. Effect of feeding lactobacilli on the coliform and lactobacillus flora o f intestinal tissue and feces from piglets. J. Food Protect. 40:288. 18 Parker, R. B. 1975. Applications o f lactobacillus feeding in swine and other livestock. Page 38 in Proc. AFMA Nutr. Council. 19 Redmond, tt. E., and R. W. Moore. 1965. Biologic effect of introducing Lactobacillus acidopbilus into a large swine herd experiencing enteritis. The Southwestern Vet. 18 : 387. 20 Reiter, B. 1978. Review of the progress of dairy science: antimicrobial systems in milk. J. Dairy Res. 45:131. 21 Rindsig, R. B., and G. W. Bodah. 1977. Performance o f calves fed colostrum natur',dly fermented, or preserved with propionic acid o f formaldehyde. J. Dairy Sci. 60:79. 22 Sandine, W. E., K. S. Muralidhara, P. R. Elliker, and D. C. England. 1972. Lactic acid bacteria in food and health: a review with special reference to enteropathogenic Escbericbia coil as well as certain enteric diseases and their treatment with antigiotics and lactobacilli. J. Milk Food Technol. 35:691. 23 Speck, M. J. 1975. Market outlook for acidophilus food products. Cult. Dairy Prod. J. 10:8. 24 Speck, M. L. 1976. Interactions among lactobacilli and man. J. Dairy Sci. 59:338. 25 Thomas, R. O., R. C. tlatch, and W. V. Thayne. 1974. Effect o f Lactobacillus acidopbilus as an additive to the feed of baby calves. West Virginia Agric. Forest. 5:15. 26 White, R. W., D. ft. Yungblut, J. L. Albright, B. W. Crowl, and F. J. Babel. 1974. Colostrum and nutritive value of fermented colostrum for feeding dairy calves. J. Dairy Sci. 57:643. 27 Yu, Y., J. B. Stone, and M. R. Wilson. 1976. Fermented colostrum for ltolstein replacement calf rearing. J. Dairy Sci. 59:936.