MEDREK, T. F. & BARNES, E. M. (1962). J . nppl. Bart. 25 (2), 159-168.

THE DISTRIBUTION OF GROUP D STREPTOCOCCI IN CATTLE AND SHEEP

BY T. F. MEDREK AND ELLA M. BARNES Low Temperature Research Station, Cambridge

SUMMARY: Numbers and types of' Lancefield group D streptococci have been deter- mined in samples from the colons of 17 cattle and 9 sheep. Mean total streptococcal counts of 8 x 104/g in cattle and 2 x 106/g in sheep were obtained. Streptococcus bovis was found in every sample and was the predominant species in 16 of the cattle and 6 of the sheep. Other group D streptococci (Strep. faecaZis, Strep. faeciurn and Strep. durans) were rare in cattle, but in sheep they formed a significant proportion of the population. Of 60 Strep. faecium, Strep. durans and related strains, 51 fermented raffinose. Many of the strains of Strep.faeciurn were also atypical in that they fermented sorbitol and appreciably reduced tetrazolium in broth at pH 6.0.

Atrep. bowis remained the predominant streptococcus in faeces samples from 4 dairy cows when they were tested again after an interval of 17 and 18 months.

WITH THE IMPROVED methods now available for the isolation and identification of faecal streptococci, interest has once more been focussed upon the distribution of these organisms in various animals. In early studies on ruminants, Winslow & Palmer (1910) observed that raffinose fermenting strains of streptococci appeared to be more abundant in bovine than in human faeces and that mannitol fermenting strains, which constituted a quarter of human faecal streptococci, were very rare in the faeces of cattle. Orla-Jensen (1919) described the species Streptococcus bovis and considered it to be the most frequently occurring faecal streptococcus in cattle, an opinion later snpported by Ayres & Mudge (1923) and by Higginbottom & Wheater (1954). On the other hand Cooper & Ramadan (1955), Buttiaux (1958) and Wilssens & Buttiaux (1958) found this species only irregularly in cattle faeces and very seldom in sheep. Kjellander (1960) developed a selective medium for counting faecal strepto- cocci which also differentiated the sorbitol fermenters, the inference being that these latter were mainly Strep. faecalis and its varieties liquefaciem and zynwgenes. He examined faecal samples from 14 cattle, 10 calves and 10 sheep and found that the average (geometric mean) total streptococcal counts were 74,00O/g for cows, 12O,OOO/g for calves and 23O,OOO/g for sheep, whereas the counts of sorbitol fermenting (faecalis) strains were 420/g, 8,OOO/g, and 3,4OO/g, respectively. Wilssens & de Vleeechauwer (1959) found a higher Strep. faecalis count in young caIves than in adult cattIe and suggested that this was due to the milk diet of the young calf. When the milk diet ceased, Strep. faecalis tended to disappear. Smith (1961) also found that young animals tend to have a higher proportion of Strep. faecalis strains in their faeces than do adult animals.

I n the experiments described in this paper, the relative proportions of the different faecal streptococci have been determined in cattle and sheep, and the strains isolated have been examined to compare their properties with those of strains from other

I 60 T . F. Medrek and Ella M . Barnes

animals. The effects of time and the change from winter to summer feeding have also been determined in a single herd of cattle.

MATERIALS AND METHODS

Origin of samples At a local abattoir samples were withdrawn aseptically from the colons of freshly slaughtered cattle and sheep at intervals over a period of one year. Other samples of freshly voided faeces were taken as aseptically as possible from 4 dairy COWS

of the herd belonging to the Cambridge University School of Veterinary Medicine.

Bacteriological examination Enumeration of organisms

Preparation of dilutions. A sample of 1 or 2 g of faecal maberial was weighed into a wide necked 1 oz bottle containing 15 glass beads and emulsified with 9 or 18 ml of quarter-strength Ringer's solution. Decimal dilutions were then prepared using the same diluent and 0.025 ml of each of four consecutive dilutions was spread in duplicate over one quarter of Petri dishes containing the required agar medium.

Coli-aerogenes bacteria. These were counted on MacConkey's agar (Oxoid no. 3) which was incubated at 37" for 1 day.

Faecal streptococci (Lance$eld group D). The thallous acetate-tetrazolium agar medium of Barnes (1956a) was used. It is important that the pH ofthe basal medium should not be below 6.0, as a t pH 5-8 no growth occurs with certain strains of Strep. bovis and other faecal streptococci.

The plates were incubated aerobically for 24 hr a t 37". Colonies of Xtrep. faecalis and its varieties are generally medium sized (1-1-5 mm diameter) with red centres and white peripheries, but may also be dark red without the white periphery. Xtrep. faecium, Strep. durans and certain strains related to these two species form small to medium sized colonies varying from white through pink centred to uniformly pink. Strep. bovis strains characteristically form minute dark red colonies, but aome strains classified as Strep. bovis form medium sized white colonies (Medrek & Barnes, 1958).

Selection and puri$;fication of isolates The choice of strains for detailed study was based upon the appearance and size

of colonies. Representatives of each colony type were taken from the highest dilution in which it appeared.

For the purification of isolates transfers were made into 0.1 % thallous acetate broth (Barnes, 1956a) incubated for 24 hr a t 37", then on to tetrazolium-glucose agar (without thallous acetate) and incubated for 24 hr. The colonies were examined microscopically and were also tested for catalase to eliminate micrococci ; but these were seldom found. All colonies, except those which were probably Strep. bovis, were subcultured in Hartley's digest broth; for colonies resembling those of Xtrep. bovis brain-heart infusion broth (Difco) was used.

Group D streptococci in cattle and sheep 161

Identi3cation of species Except for the following additions and modifications the methods used were those

described by Barnes, Ingram & Ingram (1956). Haemolysis. Hartley's digest agar was overpoured with a layer of digest agar

containing 5% of horse blood. The plates were streaked with the organisms and examined after incubation a t 37" for 24 and 48 hr. Final observations were made after overnight storage a t 1".

Aesculin hydrolysis. Medium B of Barnes et al. (1956) containing 0.1 yo of aesculin and 0.01% of ferric citrate was used. The aesculin and ferric citrate solutions were sterilized separately a t 5 Ib/in2 €or 30 min. The plates were incubated for 5 days and a positive reaction was indicated by blackening of the medium.

Slime production. Sucrose-gelatin agar plates were prepared as described by Niven, Smiley & Sherman (1941). Two sets of plates were streaked and incubated for 18 hr a t 37", one in an atmosphere of H, plus 10% of CO, and the other in air.

Starch hydrolysis. Medium B without glucose but containing 0.2% of soluble starch was used. The starch solution was sterilized separately by autoclaving a t 5 lb/in2 for 30 min. The plates were incubated for 5 days and zones of hydrolysis detected by flooding the plates with a 1 in 5 dilution of Gram's iodine.

Carbohydrate fermentation. Peptone water containing 1 % of carbohydrate and Andrade's indicator was used.

Tetrazolium reduction at p H 6.0. The method of Barnes (19566) was used, but the percentage of reduction to red formazan was recorded as follows: +, <lo; + +, 10-20; +++, 20-30; ++++, >30.

Geelatin liquefaction. Cultures were incubated a t 20" and examined after 14 days. Those strains which failed to grow a t 20" (i.e. Strep. bovis) were incubated for 4 days at 37" and then chilled to reveal liquefaction.

Growth at 45". Cultures were examined daily for up to 3 days.

RESULTS

The numbers and presumptive types of faeml streptococci In order to determine both the total counts of faecal streptococci in cattle and sheep and the relative proportions of the main species present, thallous acetate-tetrazolium agar (TlTG) was used. Although Strep. bovis does not grow as well on this as on some ofthe other selective media, the colonies being very small, it has the considerable advantage of permitting an initial differentiation between species on the basis of colony size and tetrazolium reduction (see above). Seventeen samples taken from the colons of freshly slaughtered cattle and 9 from sheep were examined. At the same time coli-aerogenes bacteria were counted on a proportion of the samples.

Cattle A summary of the total numbers and probable distribution of species of faecal

streptococci from the 17 colon samples from cattle is given in Table 1. The total streptococcal count varied between 4x 103 and 3 x 10s/g (geometric mean, 8x 104/g).

I 62 T . F. Medrek and Ella M. Barnes

Strep. boviR was found in all the samples and was the predominant faecal strept,o- coccus in 15 of them. Except for a single isolate a t low dilution, Strep. fapcalis and its varieties were not found. Strep. faecium and related strains were found in 8 samples only and in low numbers.

Five of the samples were tested for coli-aerogenes organisms, the counts being between <2x los and 5 x 104/g (geometric mean, <lO4/g).

Sheep The results obtained with the 9 samples from the colons of sheep are also given

in Table 1. The total streptococcal count varied between 1.5 s lo5 and 2 x 107/g (geometric mean, 2 x l O B / g ) . Strep. bovis was again found in all the samples, and was predominant in 6 of them. Strep. faecalis and/or its varieties were found in 2 samples, and in one of these it was the predominant faecal streptococcus. Strep. faecium and related organisms were found in all the samples examined, often in significant numbers (geometric mean, c. 105/g). Judging from the variety of colony types the streptococcal flora appeared to be very heterogeneous.

Table 1. The occurrence and probable numbers of the different species of faecal streptococci in the colons of cattle and sheep, as determined

by counts and colonial appearance on thallous acetate- tetrazolium agar

Source Colony No. of Log,, (count/g wet wt) s a m p 1 e s v ?-.----h-- and type

no. of on TlTG positive Mini- Maxi- Geometric samples agar mum mum mean*

17 3.2 G.5 4.8 Minute red

(Strep. boais)

Cattle

17

Small to medium,

(Strep. faecium type) Medium, red centred

white to pink 8 <“.3 6.2 <%-9

(Strep. .faecaZia and 1 (2.5 2.3 <“.3

Total colonies 3.6 6.5 4.9 . variants)

(Strep. bovis) 9 5.2 6.9 8.9 Minute red c Small to medium,

(Strep. faecium type) white to pink 9 2.4 7.3 5.1

Medium, red centred (Strep. faecaZis and 2 (1.3 6.7 (2.0 variants)

Total colonies 5.2 7.3 6.3 I

* Calculated on the basis of the total number of samples.

Coli-aerogenes bacteria were found in all the 9 samples, the counts varying between 1 x lo5 and 3 x 107/g (geometric mean, 4 x IOa/g).

Group D streptococci in cattle and sheep 163

ConJirmatory ident iwt ion of streptococcal isolates Representative strains of the different types of faecal streptococci from all the cattle and sheep samples were identified as far as possible using both serological and bio- chemical tests. The results are given below.

Strep. bovis and related strains All the strains selected which formed minute red colonies on TlTG agar were found

to conform with the Characteristics offitrep. bovis as described by Orla-Jensen (1919). Altogether 152 of these isolates from cattle and sheep were examined physiologically and 148 serologically; they formed a remarkably homogeneous collection (Medrek & Barnes, 1962). One strain which appeared on TlTG agar as a small white colony was also identified as Strep. bovis but differed in a number of characteristics.

Table 2. The differentiation of 60 strains of Strep. faecium and Strep. durans (and unrelated strains) isolated from cattle and sheep into groups

Character Nos. of samples and strains* and reactionst of

Haemolysis of horse blood

Growth at 45"

Growth at p H 9.6

Survival at 60" for 30 min

Sucrose

Mannitol Sorbitol Arabinose

Raffinose

Litmus milk

Tetrazolium reduction in broth at pH 6.0s

Colony size

Strep. faecium, group Related, group Strep. durans, group . . . .

MAS MARS' 'MSAR: 1 2 Atypical Typical (2, 2) ( 5 , 16) (6, 16) (2, 2) (8, 19) (2, 4) (1, 1)

+ +

A A

A A

A A

- A

- -

rA (9) rAC { RA (6)

RAC (2)

- +++

rA RA rA (18) (1) rA

Medium Small-

White Pink medium

Medium Medium Small Small

White White White White

Medium

White-pink Appearance on '

* These are given at the head of each column of reactions, in brackets, with the no. of samples first. t Reactions: +, positive; -, negative. Blood agar: g, greening; y, no haemolysis; p, beta

haemolysis. Fermentations: A, acid; r, slight reduction; R , strong reduction; C , clot. The figures in brackets indicate the nos. of strains with the specified reaction. The letters indicate the carbohydrates fermented: M, mannitol; A, arabinose; R, raffinose; S, sorbitol.

TlTG agar

8 Tetrazolium reduction: -, none; +, ~ 1 0 % ; + +, 10-30%; +++, 20-30%.

’64 T. F. Medrek and Ella M. Barnes

Strep. faecium, Strep durans and related strains Sixty strains, which by their colonial appearance on tetrazolium agar were probably

Xtrep. faecium, Strep. durans or related strains, were examined using the methods given above. They all belonged serologically to Lancefield’s group D, and all grew on 40% bile agar, hydrolyzed aesculin and fermented lactose, glucose and salicin. They failed to ferment or hydrolyze starch, to produce typical growth on potassium tellurite-blood agar, and to liquefy gelatin; all grew at 10” and 45” (except for 2 strains) and in the presence of 6.5% salt. They could be differentiated into 7 main groups as shown in Table 2, mainly by differences in fermentation pattern and blood agar reactions. Three of the groups were designated Strep. faecium MA, MAR and MSAR, the letters indicating the carbohydrates fermented. Out of a total of 34 strains only 2 were typical (Strep. faecium MA, being strains which fermented mannitol and arabinose but not sorbitol or raffinose). Of the other 32 strains 31 fer- mented raffinose and 16 (Strep. faecium MSAR) also fermented sorbitol. Furthermore 1030% of the tetrazolium was reduced to formazan by the majority of these Strep. faecium strains when they were grown in tetrazolium broth. The 2 strains designated ‘related group 1’ were similar to the ‘unclassified type 1’ strains described by Barnes et al. (19561, which differ from Xtrep. faecium in their failure to ferment arabinose. The 19 strains designated ‘related, group 2’ were separated from group 1 on their ability to ferment raffinose. Only one strain of a typical P-haeniolytic Strep. durans was isolated. Four strains did not produce P-haemolysis and were differentiated from ‘unclassified type 1’ strains only by their failure to ferment sucrose and to grow at pH 9.6.

Strep. faecalis and varieties liquefaciens and zymogenes In all, 7 strains which resembled Xtrep. faecalis or its varieties were isolated, 1 from

cattle and 6 from sheep. None of these was typical. The single isolate from cattle was identified as Strep. faecalis var. liquefaciens and was serologically type H69D5 (Lancefield, 1941). Of the 6 isolates from sheep, all of which were Lancefield’s type D76, 2 were identified as Strep. faecalis var. liquefaciens and 4 as Xtrep. faecalis var. zymogenes.

Distribution of the different species in cattle and sheep Tables 3 and 4 give the origin and numbers of all the species (including varieties and related strains) of group D streptococci which were isolated from faecal material obtained from the colons of cattle and sheep. Strep. bovis was the predominant faecal streptococcus in both the cattle and sheep samples, though the numbers in the bovine samples were only one-tenth of those in the sheep.

It will be seen that only 8 out of the 17 cattle samples contained strains which fulfilled the criteria of Sherman (1937) for the ‘enterococci’ (i.e. Strep. faecalis, Strep. faecium, Strep. durans and related strains), and as only 17 organisms in all were isolated and examined it is diEcult to draw any conclusions about the distribution; but all t h e e of the Strep. faecium groups described above were found as well as both groups of the related strains.

In contrast to the results with cattle, ‘enterococci’ were found in all the sheep colon samples, not only in significant numbers but also with usually more than one

Group D streptococci in cattle and sheep 1 65

species in a given sample. Strep. faecium was present in all but one sample and related strains of group 2 were regularly found. The single typical isolate of Strep. durans came from a sample (S-5) which also yielded atypical strains of this species

Table 3. The numbers and distribution of the different kinds of group D streptococci* isolated from the colons of cattle

Sample No. of

(~og10

no. Strep. bovis

countlg)

1 4.4 2 3.9 3 4.9 4 4.0 5 4.5 6 4.7 7 3.7 8 3.9 0 3.2

10 4.1 11 6.5 12 4.7 13 5.8 14 4.8 16 4.3 16 4.3 17 5.2

4.8t

Strep. faecium and related strains

Total no.

count/g) (log,,

<2.3 <2.3

6.2 t 2 . 3 <2.3

3.1 2.3 2.3 3 4

<2.3 4.4

<2.3 <2.3 (2.3

2.9 4.1

<2.3 <2.9t

Total strains

No. § of strains of groups r-------* 7

Strep. faeciu?n$ Related - - MA MAR MSAR 1 2

5

2 1

1 2

2

1 2

1 5 3 1 6

* Sample 8 alono yielded 1 strain of Strep. faemlis or a variety thereof. It was not typical and the logarithm of its colony count was 2.3. t Geometric mean. $ See Table 2.

Q For clarity of presentation zeros have been omitted.

Table 4. The numbers and distribution of the diflerent kinds of group D streptococci isolated from the cololzs of sheep

Sample no.

1 2 3 4 5 0 7

9 a

No. of Strep. bOUi8 (lo&,

count / g )

6% 5.8 0.9 5.7 5-9 5.3 6.1 5.3 5.3 5.9*

r Total no.

(lOK,o COUnt/g)

5.9 4.1 6.9 4.3 7.3 3.8 4.0 5.6 2.4 5.1.

Total strains

Stres. faeeiunt and related strains

No.1 of strains of groups -- strep. fueciumt Related Strep. duruns

rAT 7-7 MA MAR MSAR 1 2 At,ypical Typical

6 4

5 2 1 1 4

2 2 2

3

6 2 1

1 1 1

1

1 11 13 1 13 4 1

Strep. faeealia and varieties

T Z G - z (10Kio of

count/g) strain8

< 1.3 < 1.3

< 1.3 < 1.3 < 1.3 < 1-3

c 1.3 c 2.01

2.0 2

0.7 4

6

* Qeometric mean. 7 See Table 2. 1 For clarity of presentation zeros have been omitted.

I 66 T . F. Medrek and Ella M . Barnes

and a number of 'related, group 2' strains. All the strains from this sample were typed serologically, using the methods and typing sera of Sharpe & Fewins (1960), and whereas the six 'related, group 2' strains proved to be serological type 39, the two atypical Strep. duram strains were serological type 37. When the p-haemolytic strain of Xtrep. durans was tested it was also serological type 37, but had lost its p-haemolytic property.

Effect of diet Preliminary studies were made to see whether there was any change in the numbers and kinds of faecal streptococci in the faeces of four dairy cows in a single herd following the change from summer to winter diets. Unfortunately more than a year elapsed between the first and second of the two tests, whereas there was only a difference of one month between the winter diet and the transitional diet. The results on the four cows (Susan, Prune, Proxy and Nautch) are given in Table 5.

Table 5. The inJEuence of diet and passage of time on the numbers of three species of faecal streptococci in the faeces of cows

Date Diet* Group D No., as log,, (count/g wet wt of faeces), sampled species from animal no.:

c 1 2 3 4

5.2 5.7 6.1 6.1 6.2 6.7 6.6 5.1 6.4 5.9

2.0 <1.7 <1.7 <1.7 <1.7 <1.7 (1.7 <1.7 t 1 . 7 <1.7 <1.7 <1*7 < l . 7 < 1 . 7 < 1 . 7 (1.7 < 1 . 7 <1.7 (1.7 <1.7

Oct. '57 Mar. '59 Apr. '59 Oct. '57 Mar. '59 Apr. '59

t l . 7 <1.7 <1.7 <1.7

Oct. '57 Mar. '59 Apr. '59

* Diets: a, summer (grass and grain); b, winter (hay, silage and grain); c, transition

t Determined from the appearance of colonies on thallous acetate-tetrazolium agar

1 Identities of animals: 1, Susan; 2, Prune; 3, Proxy; 4, Nautch.

Strep. { ;:: bovist

{ Strep. f a e c i m

Strep.

!I typet

9 faemlist

between summer and winter.

at pH 6.0.

The samples were counted as previously, using thallous acetate-tetrazolium agar, and it was found that Xtrep. bovis continued to be the predominant faecal strepto- COCCUS; Strep. faecalis and its varieties were not found and Strep. faecium or related strains were found in one sample only, that from Susan on a winter diet.

DISCUSSION

By using a differential medium it has been possible to analyze the minor as well as the major components of the faecal streptococci flora in cattle and sheep. Strep. bovis has been confirmed as the dominant species in both animals; details of the strains isolated have been given by Medrek & Barnes (1962). When considering Strep. faecium and related strains, one of the most interesting observations was the large number (50 out of 60) of raffinose fermenting strains present. The observation

Group D streptococci i n cattle and sheep 167

of Winslow & Palmer (1910) of a high proportion of raffinose fermenting strains in the faeces of cattle could therefore apply to these organisms as well as to Strep. bovis. The Strep. faecium strains were also remarkable in that 16 out of 60 fermented sorbitol as well as raffinose. Similar strains were not found by Barnes & Ingram (1955) in pigs nor have any been found by them in human faeces (unpublished data), but among strains sent by Dr. H. Williams Smith three gave similar reactions and were obtained from the faeces of a dog and a rabbit and off the back of a piglet. Wilssens & de Vleeschauwer (1959) reported an aberrant sorbitol fermenting strain of Strep. faecium from their cattle but did not state whether i t also fermented raffinose. A number of the Strep. faecium strains were much more reducing in tetrazolium broth than usual and in addition reduced litmus milk. Although some of the Strep. faecium strains isolated from pigs by Barnes & Ingram (1955) actively reduced litmus milk they did not reduce the tetrazolium broth a t pH 6.0 when retested recently (unpublished data).

The difficulties of classifying Strep. durans and related strains are once more illustrated in this work, and the whole relationship of Strep. durans to Strep. faecium will be clarified only when more information is available on the serology and physiology of a large number of isolates from different sources.

The Strep. faecalis strains isolated were of interest in that they were of the same serological types (H69D5 and D76) as those which are so frequently found in man. The low numbers of Strep. faecalis strains found, particularly in cattle, confirm the findings of others that these organisms are rare once the animals are old enough to be on a normal diet of grass, hay or grain.

Very little difference was found in the samples from cattle whether the animals were on summer, winter, or transitional diets. It is probable that a very considerable dietary change is necessary before a change will occur in either the numbers or types of faecal streptococci present, as for example the change from the milk diet of young animals to adult food.

The authors are indebted to the Cambridge University School of Veterinary Medicine for facilities for sampling the School's herd of cattle. The sera for typing strains of Strep. faecalis and Strep. faecium were kindly supplied by Dr. S. D. Elliott and Dr. M. E. Sharpe. One of us (T.F.M.) was in receipt of a Fulbright Award while doing this work.

The present address of T. F. Medrek is: Microbiology Experiment Station, University of Massachusetts, Amherst, Mass., U.S.A.

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(Received 19 January, 1962)