Ethanol and Torulopsis glabrata in the rumens of milk-replacer fed farm calves: A survey

Livestock Production Science, 4 (1977) 335--342

335

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

E T H A N O L A N D T O R U L O P S I S G L A B R A T A IN T H E R U M E N S O F M I L K R E P L A C E R FED FARM C A L V E S : A S U R V E Y

R.W. WHITE

A.R.C., Institute of Animal Physiology, Biochemistry Department, Babraham, Cambridge CB2 4AT (Great Britain) (Received 3 November 1976)

ABSTRACT White, R.W., 1977. Ethanol and Torulopsis glabrata in the rumens of milk-replacer fed farm calves: a survey. Livest. Prod. Sci., 4: 335--342. Samples of stomach contents from 150 calves on farms and in veal units were examined for the presence of Torulopsis glabrata yeast and ethanol. Ninety-one animals contained the yeast, and 94 ethanol. Out of 10 calves having in excess of 30 rag/100 ml ethanol and counts of more than 104/ml of yeasts in their tureens, 7 were receiving the only milk replacer demonstrably containing large amounts of carbohydrate fermentable by T. glabrata. Twelve animals also had Candida albicans in their rumens. A separate group of 14 calves, receiving one milk replacer not containing carbohydrate fermentable by T. glabrata, were similarly examined twice at a 7-day interval; 12 contained low levels of ethanol and small numbers of yeasts in their rumen contents.

INTRODUCTION In a previous r e p o r t (White, 1 9 7 4 ) the feeding o f farm calves c o n t a i n i n g in their s t o m a c h s t h e resident y e a s t Torulopsis glabrata, with a milk replacer c o n t a i n i n g f e r m e n t a b l e c a r b o h y d r a t e (glucose or f r u c t o s e ) , was a c c o m p a n i e d b y an increase in t h e level o f e t h a n o l in t h e r u m e n w h e n samples o f r u m e n c o n t e n t s were e x a m i n e d o n t w o successive occasions. T h o s e limited observations led t o t h e c a r r y i n g o u t o f a survey, e x a m i n i n g single samples o f r u m e n c o n t e n t s t a k e n f r o m calves o n f a r m s in D e v o n , S o m e r s e t a n d A y r s h i r e a n d in veal units in Somerset. All the calves were receiving milk replacer e x c e p t f o r 19 animals o n fresh c o w milk. A d d i t i o n a l l y , a g r o u p o f 14 calves receiving a single milk replacer k n o w n n o t t o c o n t a i n glucose was also e x a m i n e d o n t w o successive occasions, as a d i r e c t c o n t r a s t t o t h e earlier r e p o r t (White, 1974). MATERIALS AND METHODS

Sampling. R u m e n c o n t e n t s were o b t a i n e d t h r o u g h a plastic sampling t u b e passed d o w n t h e o e s o p h a g u s . Samples were o b t a i n e d f r o m t h e a n a t o m i c a l l y

336 distinct rumen, whether or n o t the animal was sufficiently developed for this to be functioning. On the day that the single samples were taken, cultures were first made for determining yeast numbers, and the residual samples were immediately fixed for subsequent analytical procedures on return to the base laboratory. The duplicate samples from the small group of 14 calves were dealt with for both microbiological and analytical procedures on the same day they were received.

Cultures and media. These were as described previously (White et al., 1972). Analytical procedures. Ethanol estimations were carried o u t by gas liquid chromatography as described elsewhere (White, 1977a) either on freshly drawn samples, or on samples which had been fixed on the day they were taken by the addition of formaldehyde solution (37--41% HCHO w/v) at the rate of 0.15 ml to 20 ml of sample. An ethanol standard solution containing 80 mg ethanol/100 ml to which was added formaldehyde in the same concentration as that used to fix the samples, was also run on the gas-liquid chromatograph. A n u m b e r of the samples taken in the field were also preserved for an enzymic ethanol determination b y adding 0.5 ml of fresh sample to 2 ml of 5% w/v perchloric acid solution. Enzymic estimations of ethanol were by the m e t h o d of Bernt and G u t m a n (1970): they confirmed the gas liquid chromatography results b o t h qualitatively and quantitatively. TESTING AND RESULTS

In vitro tests These were carried o u t on any milk replacers encountered in use, and which had n o t already been tested, in the manner described earlier (White, 1974), using the author's strain 1A2 o f T. glabrata ATCC 32936. The results are set out in Table I, which includes earlier published figures where these refer to products being fed to calves from which the samples were currently obtained. Milk replacer 'B', which supports the production of a similar quantity of ethanol to that produced from the substrate containing 2% added glucose, was a recently introduced product, containing microbial protein. Since T. glabrata ferments only glucose, fructose and trehalose, this is evidence for the presence of glucose and/or fructose in this p r o d u c t (trehalose is an unlikely component). Diet 'G', on which only four of the calves were fed, is n o t a milk replacer b u t a calf gruel, and forms a starch-like gel when heated in water. Replacer ' F ' is the same one as No. 6 in the earlier report (White, 1974).

Calf tests, single samples Samples of rumen contents were taken on farms and in veal units from 150 calves, of which 126 were Friesians or Friesian crosses, 16 were Ayrshires,

337

TABLE I In vitro fermentation of unheated calf milk replacers in the presence of 1000 units/ml of penicillin by T. glabrata Milk replacer

Ethanol produced (mg/100 ml)

A B C D E F G H I Cadbury's Marvel alone Cadbury's Marvel with 2% glucose

0 625 7 3* 25* 93* 38O No sample available 40 0 672

* Previously published values (White, 1974). 'B' contains hydrolyzed microbial protein. 'G' is not a true milk replacer, but a calf gruel. Cadbury's Marvel is a commercial fat-free dried milk powder suitable for human and animal consumption.

7 were Charolais crosses, and 1 was a H e r e f o r d cross. T h e results are summarised in T a b l e II. O f 9 4 animals in w h i c h e t h a n o l was present, n o yeasts w e r e f o u n d in 29. O f 91 animals in w h i c h T. glabrata was present, n o e t h a n o l was f o u n d in 46. T h e highest single values f o u n d w e r e 65 m g / 1 0 0 ml o f e t h a n o l and 106/ml viable c o u n t o f yeasts. T h e r e w e r e n o o b v i o u s d i f f e r e n c e s e i t h e r b e t w e e n areas visited, o r b e t w e e n farm calves and t h o s e in veal units, and n o evidence was e n c o u n t e r e d o f clinical d i s t u r b a n c e associated specifically w i t h the p r e s e n c e o f e t h a n o l or o f T. glabrata in the rumens. In Table III t h e same results are g r o u p e d a c c o r d i n g t o t h e milk r e p l a c e r being fed, and e x p a n d e d t o s h o w t h e d i s t r i b u t i o n o f levels o f e t h a n o l c o n c e n trations, and t h e n u m b e r s o f viable yeasts f o u n d . T h e r e p l a c e r 'B' is singled o u t b y t h e increased i n c i d e n c e o f animals in w h i c h h i g h e r r u m e n c o n c e n t r a tions o f e t h a n o l and n u m b e r s o f t h e yeasts are r e c o r d e d ; this w o u l d be expected f r o m t h e results in T a b l e I. T h e r e is little d i f f e r e n c e b e t w e e n c o w ' s milk and a n y o f t h e o t h e r feeds, w i t h the possible e x c e p t i o n o f replacer 'I', w h e r e the r u m e n s c o n t a i n low levels o f e t h a n o l and y e t it s u p p o r t s v e r y little ethanolic f e r m e n t a t i o n in vitro ( T a b l e I). In samples f r o m 12 f a r m animals, nine o f w h i c h w e r e in A y r s h i r e and six o f these o n o n e farm, a t y p e o f y e a s t s u b s e q u e n t l y identified b y R.W. Buhagiar at the F o o d Research I n s t i t u t e , N o r w i c h , England as an a t y p i c a l Candida albicans ( R o b i n ) , B e r k o u t was f o u n d . T h e same organism has b e e n i d e n t i f i e d occasionally f r o m t h e r u m e n s o f e x p e r i m e n t a l calves and lambs, and has b e e n

15 4 14 7

40' 522 463 45

Age ( d a y s ) from to

61

22 24 11 4 33

8 4 17 4 37

14 14 4 5 54

11 11 17 15

>I

<1

<10

>I0

(log10 c o u n t s / m l )

Ethanol (mg/100 ml)

T. glabrata

N u m b e r o f calves c o n t a i n i n g

29

46

16 12 6 12

ethanol --

T. g l a b r a t a -

14 12 2 1

T. glabrata +

Ethanol +

single a n i m a l 51 days old h a v i n g n o e t h a n o l or T. glabrata. a n i m a l s over 6 0 d a y s old, o f w h i c h 3 h a d e t h a n o l w i t h T. glabrata a n d o n e h a d T. glabrata only. a n i m a l s over 8 0 days old n e i t h e r h a v i n g e t h a n o l , o n e h a v i n g T. glabrata p r e s e n t . calves n o e t h a n o l was f o u n d a n d in 59 calves n o T. glabrata.

150

Totals

Includes a 2Includes 4 3 Includes 2 Note. I n 56

40 47 37 26

Devon (farms) Ayrshire (farms) S o m e r s e t (farms) S o m e r s e t (veal u n i t s )

(total)

Number o f calves

Field survey of T. glabrata a n d e t h a n o l in single s a m p l e s o f t u r e e n c o n t e n t s f r o m calves

T A B L E II

O0

339

T A B L E III Single samples o f r u m e n c o n t e n t s f r o m f a r m a n d veal u n i t calves s h o w i n g e t h a n o l a n d T. glabrata c o n c e n t r a t i o n s g r o u p e d a c c o r d i n g t o m i l k replacers fed Milk

Total

replacer

N u m b e r s o f calves

T. glabrata

Ethanol (mg/100 ml) trace

(log,0 c o u n t s / m l )

10--30

30+

1

2

4

4+

8 10 1 1 2 0 0 0 0

0 7 0 1 0 0 0 0 0

8 4 5 1 3 2 2 0 4

9 4 3 0 2 1 2 1 3

1 7 0 0 2 0 0 0 0

0 7 0 0 0 0 0 0 0

2

2

5

6

6

3

F a r m calves A B C D E F G H Cow's milk

25 37 14 6 9 8 4 2 19

13 11 10 3 2 4 0 2 11

Veal u n i t calves I

26

4

found in the rumens of experimental Soay lambs at up to 10S/ml viable cells (White, unpublished observations, 1975). The organism ferments only glucose in vitro, producing ethanol, b u t it has never been found regularly associated with elevated levels of ethanol in the stomach, nor was there any evidence of such an association in these animals. On the farm where the group of six animals harboured this yeast, there was a history of an unspecified 'indigestion' among calves for some years, for which no specific cause had been found; the animals sampled showed no clinical signs of illness at the time.

Single milk replacer, duplicate samples A group of 14 Friesian calves, all on milk replacer 'A' were sampled twice, the first time at 11--14 days of age, after being on the feed for 3--6 days, and the second time 7 days later. At the first sampling the calves were being fed twice daily, and by the time of the second sampling, once daffy. The samples of rumen contents were taken b y Mr. Norman Day at the West of Scotland Agricultural College, Auchincruive, Ayr, Scotland. The samples were dispatched unfixed by night passenger train on the day they were taken and were approximately 18 hours old when tested in the author's laboratory. In 10 of the 12 animals where ethanol was present, the level fell between the first and second samples: in only t w o samples was the level greater than 30 mg/100 ml (Table IV).

340

T A B L E IV T w o samples o f r u m e n c o n t e n t s at 7-day interval from 14 calves receiving milk replacer ' A ' Calf

A121 A122 A123 A124 A125 A226 A127 A128 A221 A222 A224 A225 A227 A228

E t h a n o l ( m g / l O 0 ml)

T. glabrata (log~o c o u n t / m l )

Sample 1

Sample 2

Sample 1

Sample 2

4 3 43 0 0 6 0 4 79 7 0 24 13 7

0 0 0 2 0 3 0 8 35 8 3 0 8 6

2 1 0 1 2 0 0 2 4 1 1 2 1 0

1 0 0 0 0 1 2 1 2 2 2 1 1 0

Calves were aged 11--14 days at first sampling.

T. glabrata was present in relatively small numbers in 12 animals, varying by only one log value except in one animal containing 104/ml of yeast. This calf, No. A221, yielded rumen samples containing higher levels of ethanol. No experimental reason for this single finding could be discovered. DISCUSSION

These results, together with the findings among a small number of calves on farms in Suffolk earlier reported (White, 1974) confirm the widespread occurrence of Torulopsis glabrata in the alimentary tract of young calves. The association of this yeast and the presence of sufficient glucose (or fructose) in a milk replacer diet has been demonstrated clearly to results in yeast populations increased from 10/ml to 10'°/ml in the rumen and ethanol levels of 400--800 mg/100 ml in blood plasma and rumen contents when milk fat is withheld from the diet (White et al., 1972; White, 1977a). Under natural conditions this potential for ethanol production in milk diets is held in check by the presence of milk fat, but it has been shown that this control is not necessarily exerted by other dietary fats or oils commonly incorporated in commercial milk replacers (White, 1977a). Only relatively small amounts of ethanol were produced in the calves studied here, in some cases small enough t o b e accounted for by possible bacterial fermentation, not necessarily from glucose (White, 1977b). However, some control may have been exerted, particularly in the younger animals, by the presence in the replacer diet of fats

341

'effective' (White, 1977a) in controlling ethanol production. The oil or fat used in any particular batch of milk replacer will have depended on the cost and availability at the time of manufacture, and it was not possible to ascertain precisely which oil had been used in these particular cases. The duplicate samples of stomach contents examined from the calves at Auchincruive (Table IV) made a test precisely similar to that described in the earlier report (White, 1974), but it was carried out with a replacer which does not support ethanolic fermentation by the specific yeast, so that the earlier findings of rising levels of ethanol with increases in the yeast population in the presence of fermentable carbohydrate were, understandably, absent. ACKNOWLEDGEMENTS

This work was partially supported by a grant from the Wellcome Foundation, 1 Park Square, London, NW8 4LJ. Thanks are due to Mrs. M.E. Black and Mr. P. Barker for technical assistance, to Mr. J.L. Paxman, M.R.C.V.S. of Volac Ltd., and Mr. G. Fraser of Quantock Veal Ltd., for access to calves in veal units and to the veterinary practitioners concerned for access to farm calves. Laboratory facilities for initial cultures in the field were made available by Mr. J.G. Jenkinson (Seale Hayne Agricultural College, Newton Abbot, Devon), Dr. D.C. Harfoot (Hannah Research Institute, Ayr, Scotland) and Mr. Norman Dickie (Somerset College of Agriculture, Cannington, Somerset). REFERENCES Bernt, H. and Gutman, I., 1970. Section 4. Two and one carbon compounds. In: H.U. Bergmeyer, Methoden der Enzymatischen Analyse. Vol. 3, 2nd edition, Verlag Chemie, Weinheim, p. 1499. White, R.W., Lindsay, D.B. and Ash, R.W., 1972. Ethanol production from glucose by Torulopsis glabrata occurring naturally in the stomachs of new born animals. J. Appl. Bacteriol., 35: 631--646. White, R.W., 1974. In vivo and in vitro ethanolic fermentation of milk replacers by the naturally occurring yeast Torulopsis glabrata. J. Agric. Sci., Cambridge, 83: 189--191. White, R.W., 1977a. In vivo inhibition by specific lipids of ethanol production from glucose by the yeast Torulopsis glabrata. J. Agric. Sci., Cambridge, 88: 493--501. White, R.W., 1977b. Microbial ethanol yeasts and coliform bacteria in the stomachs of milk fed calves. J. Appl. Bacteriol., 42: 423--427.

Rl~SUMl~ White, R.W., 1977. Enqu~te sur la presence d'alcool et de Torulopsis glabrata, dans le rumen des veaux recevant des aliments d'allaitement. Livest. Prod. Sc~, 4 : 3 3 5 - - 3 4 2 (en anglais). On a recherch~ la presence de la levure Torulopsis glabrata et d'~thanol dans des ~chantillons de contenu de rumen de 150 veaux ~lev~s dans les fermes ou dans des ateliers: 91 contenaient des levures et 94 de l'~thanol. Parmi les 10 veaux qui pr~sentaient plus

342

de 30 mg/100 ml d'~thanol et plus de 104 levures par ml, 7 recevaient le seul aliment d'allaltement qui contenait une grande quantit~ de glucides fermentescibles par T. glabrato. Douze animaux avaient aussi Candida albicans dans leur rumen. On a examin~ de la m~me fa¢on deux lois ~ 7 jours d'intervalle, un groupe s~par~ de 14 veaux recevant un aliment d'allaitement ne contenant pas de glucides fermentescibles par T. glabrata; 12 ont pr(~sent~ une faible teneur en alcool et un petit nombre de levures dans le contenu de leur rumen.

KURZFASSUNG White, R.W., 1977. Athanol und Torulopsis glabrata in den Pansen yon K~lbern, die mit Milchaustauschern gef~ttert wurden. Eine Ubersicht. Livest. Prod. Sc~, 4 : 3 3 5 - - 3 4 2 (in Englisch). Von 150 K~bern, welche auf landwirtschaftlichen Betrieben und in K~lberst~llen standen, wurden Proben des Mageninhalts entnommen, um das Vorhandensein des Hefepilz Torulopsis glabrata sowie yon ..Athanol zu pr~fen. Einundneunzig Tiere enthielten den Hefepilz.;. 94 Tiere enthielten Athanol. Von 10 K~lbern, welche in ihren Pansen 30 mg/ 100 ml Athanol und 104/ml Hefe im Uberschuss hatten, erhielten 7 Tiere den einzigen Milchaustauscher, der offensichtlich grosse Mengen an Kohlehydraten enthielt, die durch T. glabrata fermentiert werden kSnnen. In den Pansen yon 12 Tieren befand sich auch Candida albicans. Eine getrennte Gruppe yon 14 K~lbem, die einen Milchaustauscher erhielten, welcher nicht das durch T. glabrata fermentierbare Kohlehydrat enthielt, wurden zweimal in einem Abstand yon 7 Tagen in ~hnlicher Form untersucht. Der Panseninhalt yon 12 Tieren enthielt niedrige Mengen an Athanol sowie geringe Mengen an Here.