microbial ecology of sheep fleece

ELSEVIER Agriculture, Ecosystems and Environment 49 (1994) 103-112

Agriculture Ecosystems & Environment

Microbial ecology of sheep fleece

E.W. Lyness*, D.E. Pinnock, D.J. Cooper University of Adelaide Waite Institute, Glen Osmond, S.A. 5064, Australia

Abstract

Normal fleece from sheep run on commercial propert ies was sampled for the presence of Bacillus spp., fleece- rot bacteria and other microorganisms. Bacillus cereus was always isolated, Bacillus thuringiensis (Bt) was recovered from 92% of samples and Pseudomonus spp. from 88% of fleece samples. Of 120 Bt isolates, 27 were larvicidal to the sheep blowfly Lucilia cuprina. Of 14 Bt isolates serotyped, ten were var. thuringiensis, one was var. toumanoffi, one was vat. morrisoni and two were vat. kurstaki. Larvicidal Bt var. thuringiensis jetted onto merino sheep provided protection against flystrike for 11 weeks in the presence of significant numbers of fleece- rot bacteria. Pseudomonas spp. and Bt were present on the skin in similar numbers throughout most of the 12 week trial period.

1. Introduction

The Australian sheep blowfly Lucilia cuprina causes epidermal myiasis of sheep (flystrike) which results in major annual economic losses to the Australian wool industry. Topical application of the triazine insect growth regulator cy- romazine (Hart et al., 1982) is currently the most widely used means of flystrike control. Flystrike occurs when gravid females are attracted to areas of the fleece and skin with high water activity and where bacteria are metabolically active, releas- ing volatile metabolic end products. Antago- nistic interactions between microorganisms on the skin surface, production of exoenzymes, bac- teriocins and antibiotics result in certain bacteria, particularly Pseudomonas spp. and Staphy- lococcus spp. becoming predominant. Oviposition takes place in regions of the fleece where bacteria are present in high numbers. If

*Corresponding author.

left untreated, many flystruck sheep will die from the effects of bacterial activity (systemic toxae- mia), caused by the lesions made by the feeding larvae. Few sites other than sheep skin and fleece are used by L. cuprina for reproduction and the larvae are almost obligate ectoparasites of sheep.

Certain strains of Bacillus thuringiensis (Bt) were found to be larvicidal to L. cuprina (Cooper et al., 1985 ). A Bt bioinsecticide offers the pros- pect of long-term protection from flystrike with- out the problem of insecticide residues. A crucial factor in the efficiency of a bacterial insecticide for L. cuprina control is the successful colonisation of the fleece and skin by the applied bacteria. Sampling of fleece from commercial sheep stations was conducted to investigate the colonisation of fleece by naturally occurring Bt and Pseudomonas spp. This was done in order to determine the likelihood of successful long-term colonisation of fleece by entomopathogenic Bt under field conditions. The following factors are important to the success of the Bt larvicide.

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104 E. W. L yness et aL /Agriculture, Ecosystems and Environment 49 (1994) 103-112

( 1 ) Initial colonisation of the fleece and skin by Bt applied as a formulated spore preparation.

(2) Successful competition of the larvicidal bacteria against naturally occurring microorganisms of the skin and fleece.

(3) Maintenance of the population of Bt required to maintain protection against flystrike for extended periods. A trial oflarvicidal Bt was car- ried out to assess the effectiveness of protecting sheep from flystrike by jetting with formulated spore preparation.

2. Materials and methods

2.1. Media

Nutrient agar was used to derive a total mi- croorganism count and for viable spore estima- tion after heat treatment (65°C for 10 min) of samples. Pseudomonas isolation agar (Difco) was used to derive a total Pseudomonas spp. count. Vogel Johnson agar was used to isolate Staphylococci spp. and the yeasts Rhodotorula and Cryptococcus were isolated on this medium. Pseudomonas Isolation Agar (Oxoid) including

(a) 10

• "- 8 0 v

0

5 10 15

[] total cfu

[] Bt spores o Pseudomonas

I[] I

20

$

I

25

f leece sample

E. W. Lyness et aL / Agriculture, Ecosystems and Environment 49 (1994) 103-112

B.cereus (b) Proportion [] B.thuringiensis

1.0

105

0.8

0.6

0.4

0.2

0.0

fleece sample

Fig. 1. (a) Total B. thuringiensis and Pseudomonas count from fleece. (b) Ratio ofB. thuringiensis to B. cereus (0-1).

10 #g ml - 1 cetrimide, 10 #g ml - 1 fucidin and 50 #g ml-~ cephaloride was used to derive a total Pseudomonas spp. count during the trial of larvicidal Bt on sheep. Isolates were checked for pu- rity and the Gram reaction and cell morphology determined from 18-24 h cultures. The 50CHB, 20E, 20NE, STAPH and 20C API systems were used for strain identification with reference to Buchanan and Gibbons (1974) for confirma- tion. Serotyping of fleece isolates of Bt was performed by Professor H. De Barjac.

2.2. Sample processing

Samples of normal fleece free of excess moisture were stored at 4°C in plastic bags. A 0.4 g staple was cut from each sample and added to 10 ml of 0.2 M Na-phosphate buffer pH 7.2 in a 30 ml glass bottle containing 20 ( 3 mm) glass beads. The fleece preparation was vortexed for 1 min, shaken for 10 rain, vortexed again and serial dilutions prepared immediately. These were plated onto nutrient agar (Gibco, Melbourne, Vic.), Pseudomonas isolation agar (Oxoid, Hamp-

106 E.W. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112

Table I Serotype and larvicidal activity of fleece isolates of B. thuringiensis

Variety (serotype) Percentage mortality

Mean SD

Thuringiensis ( 1 ) Thuringwnsts Thurmgwnsts Thurmgwnsts Thuringwnsts Thuringwnsts Thuringwnsts Thuringtensts Thuringwnsts Thuringtensts Morrisoni ( Sab ) Toumanoffi ( 1 lab) Kurstaki ( 3ab ) Kurstaki

78 2.4 87 6.2

100 88 5.9 93 14 95 6.8 97 4.2 94 2.7

100 100 100 46 1.6 51 21

100

shire, UK), Vogel Johnson agar (Oxoid) and MacConkey agar no. 2 (Oxoid) while the re- mainder of the sample was heated to 65 ° C for 10 min. The heat-treated preparation was plated onto nutrient agar to determine the viable spore count.

2.3. Application of Bt for protection against flystrike

A trial to investigate the effect of larvicidal Bt on Merino sheep was conducted by jetting a 1 : 1 bentonite-diatomaceous earth:spore formula- tion into the fleece. Treated sheep were dosed with Bt spore suspension at 6 (high dose), 3 (medium dose) and 0.6 (low dose) g of spores per 200 cm 2 of fleece in a suspension containing 0.05% Howet (Hoechst, Frankfurt, Germany) surfactant. Control sheep were jetted with water containing 0.05% Howet surfactant. Fleece and skin scrub samples were taken from four sites per sheep prior to jetting and weekly for up to 12 weeks after application. Fleece staples were taken from four sites per sheep by cutting a 2 em plug of fleece at skin level. A 0.2 g portion of fleece was immersed in 10 ml of buffer and treated as previously described. Each denuded area of skin had a sterile cup, 2 cm in diameter, placed onto

it while 5 ml of 0.2 M Na-phosphate buffer pH 7.2 was pipetted into the cup and a standard scrub pattern performed with a sterile glass rod for approximately 20 s. The sample was then pipetted into a collection vial and serial dilutions prepared and plated onto agar media as described for fleece samples. Flystrike challenge was applied weekly by implanting recently hatched (2-4 h previously) larvae after irritating the skin area selected for implantation.

2.4. Bioassay

For incorporation into molten larval diet at 42°C, each Bt fleece isolate was grown as a shaken culture for 18-20 h at 32 °C in 5 ml of 3.7% brain heart infusion broth per 30 ml glass bottle. Colonies of L. cuprina were maintained as described by Cooper et al. ( 1985 ). Eggs of L. cuprina were surface sterilised by exposure to 0.5% HCHO, 0.05% Tween 80 for 20 min. After decantation with sterile reverse-osmosis water, approximately 50 eggs were placed aseptically onto the surface of solidified larval diet agar containing the incorporated cells. The larval diet in- cluded 2 g agar (Oxoid), 2.5 g brewers yeast and 60 ml of full cream UHT milk per 100 ml diet. Control diets contained 40 ml reverse-osmosis water and the test diets 39 ml of reverse-osmosis water plus 1 ml test Bt culture. One hundred mil- lilitres of diet were dispensed into three 65 m m × 15 mm UV sterilised diet petri dishes. The bioassay was read after 46-48 h at 32°C and the results recorded as the ratio of dead larvae to total larvae per diet expressed as percent mortality.

2.5. Microscopy

Isolated colonies from selective media were subcultured for subsequent staining by the Gram method and examination at × I000 magnifica- tion. For presumptive identification of Bacillus cereus and identification of Bt, viable spores ger- minating on nutrient agar were counted and in- dividual colonies with the matt, irregular spreading appearance characteristic of Bacillus spp. selected at random for transfer to nutrient agar plates with 56 marked positions. Heat-fixed

E. W.. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112 107

F

10 -

6 - "

4

2

- ' ~ high dose ~ c o n t r o {

detection limit

/

'\

0 1 2 3 4 5 6 7 8 9 10 11 12 13

week

Fig. 2. Pseudomonas total count on skin, high B. thuringiensis dose and control.

smears were prepared from a number of typical colonies and Gram stained to confirm the cells as gram positive. One spot transfer plate was prepared per fleece sample. After 3 days at 32 ° C, wet mounts of each colony were examined by phase contrast microscopy at × 1000 magnifica- tion. Colonies with endospores and free spores only were recorded as presumptive B. cereus, and those with endospores, parasporal bodies and free spores plus crystals (typically bipyramidal in shape) were identified as Bt.

3. Results and discussion

All fleece samples from sheep stations in South Australia and Western Australia contained B. cereus. Bt was isolated from 92% of samples and comprised up to 3% of the total nutrient agar

count with the B. cereus to Bt ratio highly vari- able (Fig. l (a ) and (b)) . Of 120 isolates of Bt from normal fleece, 27 were larvicidal to L. cuprina. Of the 14 selected for serotyping, ten were var. thuringiensis, one was var. morrisoni, one was var. toumanoffi and two were vat. kurstaki (Table 1 ). The presence of larvicidal Bt in nor- real fleece may explain the natural resistance of some sheep to flystrike. Pseudomonads were isolated from 88% of fleece samples and are often the predominant organisms in cases of fleece rot (Bun'ell, 1985; London and Griffith, 1984). In most fleeces, there is a low population of fleece rot organisms present which can readily prolif- erate under warm, wet conditions to produce fleece rot conditions attractive to ovipositing L. cuprina. Larvicidal Bt is frequently present in low numbers on fleece in the presence of other normal fleece microorganisms.

108 E. W. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112

"U

10 ~ high dose

control + detection limit

// I/' L ~ \ /

~ - I I ~ 1 1 ~1 ~1 ~ l r7 ~ -

0 1 2 3 4 5 6 7 8 9 10 11 12 13

week

Fig. 3. Total Pseudomonas count on fleece, high B. thuringiensis dose and control.

The flystrike protection trial showed a large increase in total pseudomonad and spore (Bacil- lus spp.) counts after jetting. The Pseudomonas spp. count on skin increased 5 log(cfu cm -2) and 7 log(efu cm -2) on the control and high dose sheep respectively, with a sharp subsequent decline (Fig. 2). The corresponding increase in Pseudomonas spp. count on fleece was less dra- matic with no post-jetting decline (Fig. 3). Ba- cillus spore counts increased from 3 log(cfu cm -2) to 7.1 log(cfu cm -2) and 3.7 log(cfu cm-2), respectively for the high dose and control sheep on skin 5 days after jetting (Fig. 4). Spore counts on fleece peaked at 9.4 log (efu g- 1 ) for the high dose 3 days after jetting and at 8.1 log (efu g-~ for B.1 log(cfu g- l ) for the control sheep 5 days after jetting (Fig. 5 ). Colonisation of skin by Bt occurred over a 5 day period and

10 -

E ¢..3

-~- spores q~-control ~o- detection limit

0 1 2 3 4 5 6 7 8 9 10 11 12 13

week

Fig. 4. Spore count on skin, high dose.

the count on fleece peaked in 3 days and then declined to 8.1 log(cfu g- 1 ) 1 week after jetting. Spore counts for the control sheep subsequently declined after the post-jetting peak to a level 2-3 log units below that of the high dose sheep for both skin and fleece counts. The high dose provided protection against flystrike until Week 12, when complete breakdown occurred. With the medium dose there was partial breakdown in protection during Weeks 4, 10 and 11 and complete breakdown in Weeks 3 and 12. The low dose sheep had partial flystrike protection in Weeks 5 and 11 and complete breakdown of protection in Weeks 2, 4, 7 and 12. Control sheep were affected, 'flystruck', by larval implants with each weekly challenge during the 12 week trial period (Cooper et al., 1991 ). Breakdown in flystrike protection in Weeks 2, 3 and 4 occurred when midday temperatures were relatively high (in

E. W. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112 109

o

10 - T . . . . . . o4~spores

~ c o n t r o l o- detection limit

,

0 1 2 3 4 5 6 7 8 9 10 11 12 13

10

o 4

E ¢.3

"5 8

o ~

I i

4~ total count ~,3 spore count ,:, detection limit

iJ

2

0 1 2 3 4 5 6 7 8 9 10 11 12 13

week week

Fig. 5. Spore count on fleece, high dose and control.

Week 4 fleece temperatures exceeded 40°C). Vegetative cells of Bt are sensitive to heat treatment for 10 rain at 45-46°C and above under laboratory conditions, a finding which supports the idea that vegetative cells in the fleece would be affected by exposure to high temperature. Protection against flystrike is effective in the presence of similar overall counts for Bt and for Pseudomonas spp. on skin (Figs. 2 and 4) and on the fleece fibres (Figs. 3 and 5 ).

In dry conditions, the population of Bt was present on treated sheep primarily as spores, al- though vegetative cells comprised up to 98% of the Bt present during wet conditions. Following the peak count after jetting, the total species count (Fig. 2) declined by 6 log(cfu cm -2), with the Bt count declining by 4 log (cfu era- 2 ) at the skin surface (Fig. 4) over the 12 week trial period. This shows a movement of bacterial cells

Fig. 6. Total Bacillus spp. count and spore count on skin for high dose.

and spores away from the skin surface. On fleece the Pseudomonas spp. count showed no obvious trend with time (Fig. 3), but the Bt count declined by almost 2 log(cfu g- ~ ) over the 12 week period (Fig. 5). The microorganisms listed as isolates from normal fleece (Appendix 1 ) were also recovered from the fleece and/or skin of the treated sheep during the course of the flystrike protection trial. Staphylococcus cohnii and Staphylococcus xylosus were frequently isolated from the treated and control sheep, particularly in the last 4 weeks. Staphylococci produce alkaline phosphatase activity which may affect Bt strains in which the primary mode of larvicidal action is by thuringiensin. In the 12 week trial period, protection was effective in the presence of Staphylococcus spp. which were present at up to log 2 (cfu cm -2) skin. Reisolates of Bt from

110 E.W. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112

60 • precipitation mm

50

40

30

20

10

I I l i I I h I I I I

1 2 3 4 5 6 7 8 9 10 11 12

week

Fig. 7. Rainfall during 12 week flystrike protection trial.

the dosed sheep were toxic to L. cuprina larvae in laboratory bioassay. It is evident that the effective dose oflarvicidal Bt on the skin is the major factor in protection against flystrike. A flystrike is formed by congregations of larvae feeding cooperatively which penetrate the skin barrier. In a natural flystrike the skin is weakened by wetting and the action of bacterial exoenzymes (Buffell et al., 1982; London and Griffith, 1985; Raadsma, 1989). In this trial, the bacteria largely contributing to this condition, Pseudomonas spp. and Staphylococcus spp., were present in sub- stantial numbers despite the absence of significant fleece rot.

It is evident that a major episode of germination of the Bt spore population occurred during the period from Weeks 4 to 6 (Fig. 6). This cor- related with a period of significant rainfall (Fig. 7). If the water activity and nutritional condi-

tions are sufficient to support germination, then Bt can compete successfully with the Pseudo- monas population. Despite the increase in total Pseudomonas spp. count on skin and fleece during the wet period (Weeks 4, 5 and 6) (Figs. 2 and 3) protection from flystrike was effective with the high dose. When a general breakdown in protection against flystrike at Week 12 oe- cuffed with all three dose levels, the spore counts on skin were 3.6 log(efu cm-2), 3.2 log(cfu cm- 2 ) and 2 log (cfu era- 2 ) for the high, medium and low dose sheep respectively. Protec- tion against flystrike was effective up to Week 12 for the high dose of Bt spore preparation despite the reduction in spore count from an initial 7.1 to 4 log(efu cm -2) by Week 6 (Fig. 4). The total Pseudomonas count was 1.2 log(cfu era-2), 1.4 log(efu em -2) and 1.7 log(cfu cm -2) for the high, low and medium Bt dose respectively, on skin at the end of the 12 week period. Protection against flystrike failed when the Pseudomonas count was more than 1 log unit lower than the spore count for the high and medium dose, indi- cating that pseudomonads were not a significant cause of the breakdown in protection at Week 12.

Spores must provide an inoculum sufficient for germination in the flystrike microsites at the skin surface so that growth in the serum exudate gen- erated at the strike is sufficient to support toxin production. An important question is the fate of ingested spores: do they germinate and grow in the larval gut producing a lethal dose of toxin in- side the larva or is effective toxin production the result only of growth outside the larvae on regions of the sheep's skin with a sufficiently high water activity and the presence of required nutritional factors? Culture supernatant of strains of larvicidal Bt var. thuringiensis grown in brain heart infusion broth is larvicidal to L. cuprina in the absence of vegetative cells or spores (E.W. Lyness and D.E. Pinnoek, unpublished data, 1990). The effect of wetting, bacterial prolifera- tion and feeding activity of the L. cuprina larvae leads to the release of serum which further mois- tens the skin and provides protein for the bacterial population to utilise. Larvicidal Bt is able to compete with other bacteria in the fleece-rot plus flystrike situation and prevent the establishment

E. IV. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112 111

of flystrike for 11 weeks at the high dose level used in this trial. Further investigation is under- way with the objective of defining the conditions required on or near the skin surface for control of flystrike over extended periods.

Acknowledgements

Serotyping of Bt isolates was performed by Professor H. de Barjac of the Institut Pasteur, France. Thanks are expressed to Jane Scarbor- ough, Jill Wise and Karen Barry for their tech- nical assistance. Cultures of L. cuprina were maintained by Cathy Smallridge and Jenny Fewster. Funds for this investigation were provided by the Australian Wool Corporation.

References

Buchanan, R.E. and Gibbons, N.E., 1974. Bergeys Manual of Determinative Bacteriology, 8th edn. Williams and Wil- liams, Baltimore, MD.

Burrell, D.H., 1985. Immunization of sheep against experi- mental Pseudomonas aeruginosa dermatitis and fleece rot associated with body strike. Aust. Vet. J., 62(2): 55-57.

Burrell, D.H., Merrin, G.C., Watts, J.W. and Walker, K.H., 1982. The role of Pseudomonas aeruginosa in pathogen- esis of fleece-rot and the effect of immunization. Aust. Vet. J., 58: 34-35.

Cooper, J.D.J., Greatrex, T., Lyness, E.W. and Pinnock, D.E., 1991. Bacillus thuringiensis as a larvicide for control of the Australian sheep blowfly Lucilia cuprina. 1 st Int. Conf. on Bacillus thuringiensis, St. Catherine's College, Oxford. (Abstr.)

Cooper, D.J., Pinnock, D.E. and Were, S.T., 1985. Bacterial pathogens of the Australian Sheep Blowfly Lucilia cuprina. In: R.B. Chapman (Editor), Proc. 4th Australasian Conf. on Grassland Invertebrate Ecology, Lincoln Col- lege, Canterbury, 13-17 May 1985, Caxton Press.

Hart, R.J., Cavey, W.A., Ryan, K.J., String, M.B., Thomas, P.L., Boray, J.C. and von Orelli, M., 1982. CGA-42662 a new blowfly insecticide. Aust. Vet. J., 59: 104-109.

London, C.J. and Griffith, I.P., 1984. Characterization of Pseudomonads isolated from diseased fleece. Appl. En- viron. Microbiol., 47(5) 993-997.

Raadsma, H.W., 1989. Fleece rot and body strike in Merino sheep III. Significance of fleece moisture following exper- imental induction of fleece rot. Aust. J. Agric. Res., 40: 907-912.

Appendix 1

Microorganisms present in healthy fleece

Bacillus cereus Isolated from all fleece samples, gram + ve en-

dospore forming rod. Isolates utilised D-glucose, D-fructose, often mannose, n-acetyl-glucosamine, arbutine, salicine, cellobiose, maltose, trehalose and glycogen. Some isolates utilise amyg- daline. Esculin is hydrolysed (fl-glucosidase activity). Phospholipase C is produced, en- zymes lytic to bacterial cells and proteolytic en- zymes are produced, nitrate is reduced to nitrite.

Bacillus thuringiensis Isolated from 92% of fleece samples. Charac-

teristics as for B. cereus using the API50CHB system but in addition the production of a protein parasporal body and often an extracellular toxin active toward Dipteran larvae.

Acinetobacter calcoaceticus Very common on skin and fleece, short gram

- v e rod. Nutritionally versatile, capable of hy- drolysing fats and waxes. May hydrolyse gelatin. Isolates utilised caprate, adipate and phenyl acetate. Cytochrome oxidase -ve . Nitrate not reduced. Fleece isolates did not hydrolyse gelatin. CDC Gr. VE2: frequently isolated gram - ve rod. Malate, citrate, D-glucose, arabinose, mannose and mannitol were assimilated. Nitrate not reduced, oxidase - ve.

Pseudomonas aeruginosa Isolated from fleece showing green discolora-

tion, not isolated from healthy fleece. A gram - ve rod. Lipolytic, produces phenazine pigment (pyocyanin) which has antimicrobial activity. Arginine dihydrolase and urease activity produced. Gelatin hydrolysed, D-glucose, D-mannitol, n-acetyl-glucosamine, gluconate, caprate, adipate, malate and citrate utilised. Oxidase + ve, nitrate reduced.

112 E. W. Lyness et al. / Agriculture, Ecosystems and Environment 49 (1994) 103-112

Pseudomonas fluorescens Occasionally isolated, lipolytic, antimicrobial

activity produced, water-soluble fluorescent pigment produced, nitrate not reduced, oxidase - ve. Arginine dihydrolase activity produced. D- glucose, arabinose, mannose, mannitol, n-acetyl- glucosamine, gluconate, caprate, malate and citrate assimilated. Some isolates hydrolyse esculin and gelatin.

Pseudomonas putida Occasionally isolated, gram - v e rod. Water-

soluble pigments produced, adipate not utilised, growth of the organism produces a strong fetid odour. Arginine dihydrolase activity produced. Esculin and gelatin not liquefied, oxidase -ve . D-glucose, gluconate, caprate malate, citrate, phenyl acetate assimilated.

sucrose, melibiose amygdalin and arabinose fermented. Nitrate reduced, no oxidase activity.

Staphylococcus cohnii Frequently isolated gram + ve cocci. Acid pro-

duction from utilisation of D-glucose, D-fructose, maltose, D-trehalose. Alkaline phosphatase activity produced. Nitrate not reduced. Hydrol- ysis of a wide range of fats and proteins hippur- ate and arginine, salt tolerant.

Staphylococcus xylosus II Frequently isolated gram +ve cocci. Reac-

tions as for S. cohnii, but in addition fermenta- tion of lactose; a majority of isolates reduce nitrate.

Escherichia vulneris Occasionally isolated, gram - v e rod. Cap-

sules formed, nitrates reduced. Arginine dihydrolase and ~galactosidase activity produced. D- glucose mannitol, rhamnose, amygdalin and arabinose fermented. Cytochrome oxidase activity produced.

Enterobacter amnigensis Occasionally isolated, gram - v e rod. Argi-

nine dihydrolase, p-galactosidase and ornithine decarboxylase activity produced. Acetoin produced. Citrate utilised. D-glucose, mannitol, sorbitol, rhamnose melibiose, amygdalin and arabinose fermented. Oxidase -ve , nitrate reduced, may hydrolyse gelatin.

Serratia plymuthica Occasionally isolated, gram - ve rod, capsules

formed. May hydrolyse gelatin./~-galactosidase activity produced. D-glucose, mannitol, inositol,

Cryptococcus terreus Always isolated. D-glucose, 2-keto D-glucon-

ate, D-xylose, sorbitol, n-acetyl-D-glucosamine, cellobiose and trehalose utilised as the sole source of carbon.

Cryptococcus albidus Always isolated. Does not utilise n-acetyl-D-

glucosamine or L-arabinose but utilises a-Me-D- glucoside, maltose, sucrose, melezitose, and raffinose in addition to the substrates utilised by C. terreus as the sole source of carbon.

Rhodotorula rubra Always isolated. Utilises D-glucose, L-arabi-

nose, D-xylose, adonitol, xylitol, galactose, sorbitol, maltose, sucrose, trehalose, melezitose and raffinose as the sole source of carbon.

microbial ecology of sheep fleece

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