EXPERIMENTAL MYCOLOGY 13, 27-37(1989)

Survival of Anaerobic Fungi in Feces, in Saliva, and in Pure

ANDREW MILNE,* MICHAEL K. THEODOROU,‘/‘ MATTHEW G.C.JORDAN,* CATHERINE KING-SPoONER,t AND ANTHONY P. J. TRINCI"

*Microbiology Group, Department of Cell and Structural Biology, School of Biological Sciences, Stopford Building, University of Manchester, Manchester Ml3 9PT, and iThe AFRC Institute for Grassland and

Animal Production, Hurley, Maidenhead, Berkshire SL6 5LR, United Kingdom

Accepted for publication July 11, 1988

MILNE, A., THEODOROU, M.K., JORDAN, M.G.C., KING-SPOONER,C.,ANDTRINCI, A.P. %. 1989. Survival of anaerobic fungi in feces, in saliva, and in pure culture. Experimental Mycology 13, 27-37. Anaerobic fungi were isolated from the feces of British sheep, the feces of Ethiopian sheep and Ethiopian cattle, and the feces (collected at London Zoo) of 11 other herbivorous mammals (Arabian oryx, Asian elephant, bactrian camel, black rhinoceros, bongo, common zebra, greater kudu, gaur, llama, roan antelope, and vicuna). Anaerobic fungi could not be isolated from moist sheep feces (kept in plastic bags) that had been stored in air at 20 or 39°C for 1 day or longer. However, they were isolated from dried sheep feces (dried in air at 20 or 39°C) that had been stored in air at 20 or 39°C up to 128 days, and from sun-baked and dry feces of Ethiopian sheep and cattle. Anaerobic fungi were also isolated from sheep saliva that had been stored in air at 39°C up to 8 h. When Neocallimastix sp., isolate Rl (an anaerobic fungus isolated from the rumen of sheep), was grown in anaerobic culture at 39”C, it remained viable for 5 days in medium containing glucose and for 15 days in a medium containing wheat straw. Cultures of Neocallimastix sp., isolate Rl, grown anaerobically in glucose-containing defined medium for 3 days remained viable up to 14 h after they had been aerated and then stored in air at 39°C. The Rl isolate also survived up to 18 h in colonized straw particles that had been removed from anaerobic cultures and then dried and stored in air at 20°C. The results are discussed in relation to the survival of obligately anaerobic fungi in nature and the transfer of anaerobic fungi between animals. o 1989 Academic PESS, IIIC.

INDEX DESCRIPTORS: Neocallimastix; anaerobic fungi; rumen fungi; feces; saliva; sheep; her- bivorous mammals.

All food eaten by adult ruminants is sub- ject to digestion in the rumen by bacteria, protozoans) and obligately anaerobic, chytrid fungi. Microbial populations also function to aid digestion in the cecum and hind gut of monogastric herbivores. Anaer- obic fungi in the rumen were first reported in 1975 (Orpin) and are now considered to be normal members of the rumen micro- flora (Mountfort, 1987), although Bauchop (1979) reports that they are most abundant in ruminants receiving highly fibrous diets. They have been found in sheep, cattle, red deer, reindeer, impala, kangaroos, horses, swamp buffalo, and elephants (Bauchop, 1979, 1980, 1983; Ho et al., 1988; Or-pin, 1981; Orpin et al., 1985). In sheep, anaero- bic fungi are not present in newly born lambs but develop even before these ani-

mals start to eat solid food (Fonty et al,, 1987). No detailed study had been made of the survival of anaerobic fungi outside the rumen or how rumen fungi are transferred between animals.

A major way in which bacterial fa~natio~ of young animals occurs is when t licked or groomed by their mothers and thereby come into contact with saliva and digesta containing rumen mic~o~rg~nism~ ~ Although most rumen bacteria are obligate anaerobes, at least some can survive a pe- riod of exposure to air (unp~biis~e~ work by S. 0. Mann repor ied by Robso Mann (1963) isolated Ruminoco Veillonella sp., and Eubacterium air samples collected in a co corded up to 389 bacteria m fore, transfer of these microorg~~ism§ in

27 0147-5975189 $3.00 Copyright 0 1989 by Academic Press, !nc. AU rights of reproduction in any form reserved.

28 MILNE ET AL.

aerosols, food, or common drinking water is possible. Finally, some rumen bacteria produce resistant cysts or spores which are capable of surviving passage through the di- gestive tract of animals and these bacteria can be isolated from feces (Hungate, 1966). Feces may therefore serve as a further route by which rumen bacteria are trans- ferred between animals.

Like rumen bacteria, rumen protozoans are transferred between animals in saliva and digesta during grooming (Becker -and Hsuing, 1929) and both ciliate (Eadie, 1962) and flagellate (Becker and Everett, 1930) protozoa may be transferred between ani- mals in aerosols formed from saliva and di- gesta. However, large rumen protozoans rapidly die when exposed to air, and since they do not apparently form resistant struc- tures, they cannot be isolated from feces (Hobson, 1971).

Or-pin (1981) found that anaerobic fungi from horse cecum failed to survive when cultures were aerated for 5 minutes. This result suggests that rumen fungi are killed by aerobic conditions. Nevertheless, Lowe et al. (1987b) were able to isolate anaerobic fungi from fresh feces and saliva of sheep, suggesting that these organisms are to some extent aerotolerant. Isolation of anaerobic fungi from feces has also led to the sugges- tion that they may participate in hindgut fermentations (Lowe et al., 1987b).

The purpose of the present study was to isolate anaerobic fungi from the feces of a wide range of ruminant and monogastric herbivores. The survival time and aerotol- erance of pure cultures of anaerobic fungi and of anaerobic fungi in saliva and feces were also assessed for possible routes of transfer of anaerobic fungi between ani- mals.

MATERIALS AND METHODS

Source of Organisms

Feces and saliva of sheep were obtained

from animals at the AFRC Institute for Grassland and Animal Production (IGAP, Hurley, U.K.). Feces of cattle and sheep were also imported from the International Livestock Centre for Africa (ILCA), Addis Ababa, Ethiopia, and from the ILCA sub- station at Debre Zeit, Ethiopia. These sam- ples, which were sun-baked and dry on col- lection, were brought to the United King- dom in sterile vials and at ambient temperature. Fresh feces from the animals listed in Table 1 were gathered into plastic bags from the floor of the mammal enclo- sures at London Zoo (Regents Park, Lon- don). Neocallimastix sp., isolate Rl, was isolated from the rumen of sheep at IGAP (Lowe et al., 1985) and has been the subject of previous publications (Lowe, 1986; Lowe et al., 1987a-d).

Media and Culture Conditions

Defined medium B (Lowe et al., 1985) was used as the culture medium, except that coenzyme M solution, trypticase pep- tone, and yeast extract were omitted. The medium contained 25 mM glucose, Cello- phane (3 x 5-cm strips of British Cello- phane, BT 300 per 10 ml of medium), or 10 g liter-’ of wheat straw (milled to pass through l-mm-diameter dry mesh screen) as the carbon source. Pure cultures of anaerobic fungi were grown in the absence of antibiotics. The (enrichment) medium for all other cultures, apart from those inocu- lated with saliva, contained 300 pg ml-i penicillin G (Sigma) and 200 p,g ml- * strep- tomycin sulfate (Sigma). The antibiotic so- lution of Lowe et al. (1985) was used in the (enrichment) medium for cultures inocu- lated with saliva.

The anaerobic techniques used were as described by Lowe et al. (1985, 1987a). Cultures were grown at 39°C under an at- mosphere of 100% CO, in 10 ml of medium in thick-walled glass tubes (18 x 142 mm, Bellco Glass Inc., New Jersey) or in 50 or 100 ml of medium in 60- or 125ml serum

TABL

E 1

Isol

atio

n of

Ana

erob

ic Fu

ngi

from

the

Fec

es o

f 18

Her

bivo

rous

M

amm

als

That

H

ave

Cel

lulo

se

as a

Maj

or

Die

tary

C

ompo

nent

a

Anim

al

Maj

or

diet

C

omm

on

nam

e Sp

ecifi

c na

me

Ethi

opia

n sh

eep

(fat

taile

d)

Ethi

opia

n ca

ttle

(Zeb

u ca

ttle)

Ba

ctria

n ca

mel

Vi

cuna

Ll

ama

spec

ies

(all

in s

ame

enclo

sure

)

Arab

ian

oryx

G

aur

Bong

o G

reat

er

kudu

R

oan

ante

lope

Bo

nteb

ok

Gira

ffe

Blac

kbuc

k O

kapi

As

ian

elep

hant

Braz

ilian

tapi

r C

omm

on

zebr

a G

iant

pa

nda

Ovis

sp

p.

Bos

spp.

Ca

mel

us

bact

rianu

s Vi

cugn

a vic

ugna

La

ma

glam

u

Lam

a pa

ces

Lam

a gu

anico

e Or

yx

leuco

ryx

Bos

gaur

us

Taur

otra

gus

eury

ceru

s Tr

agel

aphu

s st

reps

icero

s Hi

ppot

ragu

s eq

uinu

s

Dam

alisc

us

dors

as

Gira

ffa

cam

elop

arda

lis

Antilo

pe

cerv

icapr

a

Oka

piu

john

ston

i El

epha

s m

axim

us

Tapi

rus

terre

stris

Equu

s bu

rche

lli Ai

luro

poda

m

elan

oleu

ca

Drie

d cl

over

ha

y D

ried

clov

er

hay

Drie

d cl

over

ha

y

Hay

D

ried

clov

er

hay

Drie

d cl

over

ha

y D

ried

clov

er

hay

Drie

d cl

over

ha

y an

d ha

y D

ried

clov

er

hay

Drie

d cl

over

ha

y D

ried

clov

er

hay

Drie

d cl

over

ha

y H

ay,

clov

er

hay,

fru

it,

and

vege

tabl

es

Drie

d cl

over

ha

y H

ay

Bam

boo,

ric

e,

min

ce,

raw

eggs

, prim

ate

pelle

ts,

and

vita

min

/min

eral

pr

emix

Preg

astri

c (P

), co

loni

c (C

), or

hi

ndgu

t (H

) fe

rmen

ter

Pres

ence

(+

) or

ab

senc

e (-)

of

an

aero

bic

fung

i

P P P P P P P P P H

H

H C

+ - + -

a Ex

cept

fo

r Et

hiop

ian

cattl

e an

d sh

eep,

the

fec

es w

ere

colle

cted

at

Lo

ndon

Zo

o.

(See

M

ater

ials

and

Met

hods

fo

r de

tails

.)

30 MILNE ET AL.

bottles, respectively (Phase Separation Ltd., Clwyd, U.K.). All cultures were sealed with butyl rubber stoppers (Bellco Glass Inc.). Roll tube cultures (Hungate, 1969) contained 3 ml of glucose medium and 0.2 ml of inoculum. They were pre- pared in thick-walled glass tubes and 18 g liter - ’ Taiyo powdered agar (Davis Gela- tine, Leamington Spa, U.K.) was used to solidify the medium.

Isolation from Saliva

The sheep was fed ad libitum a diet of hay (ca. 1.5 to 2.0 kg day- ‘) and was fitted with an esophageal fistula. Following a period of rumination, the sheep was allowed to chew on a gloved hand, so causing it to salivate. The fluid sample (here called saliva) was collected into two sterile flasks until each flask contained ca. 25 ml; it took ca. 2 min to collect each sample. After collection, flasks were closed with polyurethane foam bungs and stored in air at 20 or 39°C. Vol- umes of saliva (0.2 ml) were withdrawn at intervals and used to inoculate thick-walled glass tubes (five replicates) containing lo- ml volumes of glucose medium and antibi- otics. These enrichment cultures were in- cubated for 3 days and then 0.2-ml volumes were withdrawn and used to inoculate roll tubes containing glucose medium. Roll tube cultures were incubated for 3 days and then examined for anaerobic fungi using an in- verted microscope. In some cases, fungal colonies formed in the roll tube cultures were used to inoculate fresh roll tubes con- taining glucose medium. This subculture procedure was repeated until pure fungal cultures were obtained. Isolates were then transferred to liquid glucose medium in thick-walled glass tubes or serum bottles and were subsequently maintained on me- dium containing wheat straw but no antibi- otics.

Isolation from Feces

Moist feces of unknown age (ca. 0.5 g

fresh feces per bottle) collected from Lon- don Zoo were used to inoculate 125-ml se- rum bottles of medium containing wheat straw and antibiotics (four cultures were prepared per sample and there was an in- terval of up to 3 h between the collection of a fecal sample at the Zoo and its use as an inoculum). Similar enrichment cultures were prepared using the dried feces from Ethiopia. The enrichment cultures were in- cubated for 3 days and subsequently used to inoculate roll tubes containing glucose medium, from which anaerobic fungi were isolated as described above.

Feces from British sheep (fed ad libitum as above) were collected from the rectum directly into plastic bags. These feces were stored in air at 20 or 39°C in lidless glass Petri dishes or sealed plastic bags. At inter- vals after the onset of storage, a fecal pellet was used to inoculate 10 ml of glucose me- dium containing antibiotics. The contents of the tube were mixed vigorously for 2 min and these cultures were then incubated for 3 days: three pellets were sampled for each storage treatment. Roll tube cultures in glu- cose medium were prepared from the en- richment cultures (two per enrichment cul- ture) and fungal isolates were purified as described above.

To determine changes in the fresh weight of fecal pellets during storage, five Petri dishes and five plastic bags, each contain- ing 20 fecal pellets, were stored at both tem- peratures and weighed at intervals up to 30 h after the commencement of storage.

Survival and Aerotolerance of Pure Cultures

Cultures of Neocallimastix sp., isolate Rl, were grown in 10 ml of medium con- taining glucose or wheat straw; these cul- tures were inoculated with l-ml volumes of the supernatant of a stationary-phase anaerobic culture grown on medium con- taining Cellophane as the carbon source (Lowe et al., 1987a). Periodically, l-ml vol-

SURVIVAL OF ANAEROBIC FUNGI 31

TABLE 2 Survival of Anaerobic Fungi in Sheep Fecal Pellets Stored at 20 and 39°C in Sealed Plastic Bags and in

Lidless Petri Dishes”

Number of roll tube cultures prepared from pellets containing

anaerobic fung?’

Period since Plastic bags Lidless Petri dishes collection

O.Ws) 20°C 39°C 20°C 39°C

B 0 0 2 5 2 0 0 4 5 4 0 0 2 1 8 0 0 0 2

16 0 0 2 0 32 0 0 0 2 64 0 0 0 0

128 0 0 4 2

(1 At each sample time, enrichment cultures were prepared from three pellets from each treatment and each enrichment culture was used as inoculum to pre- pare two roll tube cultures (total of six roll tube cul- tures per treatment at each temperature).

b Maximum = 6.

umes of culture supernatant or loopfuls of culture solids were removed and used to inoculate tubes of fresh glucose medium. These subcultures were incubated for 3 days and observed for fungal growth using an inverted microscope.

To test for sensitivity to aerobic condi- tions, 3-day-old cultures of Neocallimastix sp., isolate RI, grown on glucose medium were aerated for 1 min by removing the stoppers and vigorously pumping sterile air (400 cm3 air min- ‘) into them via gassing needles attached to membrane filters (0.45km pore diameter). During aeration the resazurin indicator in the medium changed from colorless to pink, and stayed this color during the incubation period, in- dicating that the medium had become oxy- genated. The cultures were then reincu- bated aerobically at 39°C and periodically 2-ml volumes of culture supernatant and loopfuls of culture solids were used to in- oculate thick-walled glass tubes of fresh glucose medium. These subcultures were

incubated under anaerobic conditions for 3 days and the culture supernatant was use to inoculate roll tubes containing glucose medium. The roll tube cultures were in bated for 7 days and then observed growth of anaerobic fungi using an inverted microscope.

Fungi isolated from feces and Meocallh- mastix sp., isolate Rl, were grown on wheat straw medium for 4 days in 125ml serum bottles. Culture supernatants and colonized straw particles were separated by filtration and the solids were stored in air in lidless glass Petri dishes at 39°C. At inter- vals ca. 0.5 g (fresh weight) of solids was removed (two replicates) from the dishes and used to inoculate medium con- taining wheat straw in 60-ml serum bottles. These cultures were incubated for 4 days and then observed for the presence of via- ble zoospores using light microscopy. To determine if drying out induced the forma- tion of resistant structures, coloniz wheat straw was removed from a tally grown cultures and allowed to in air.

RESULTS AND DISCUSSION

Isolation of Anaerobic Fzmgi from Feces

Anaerobic fungi were isolated from feces

h-+--kkl I ’ ’ 8 24 26 28 30

Time since oK+et 0‘ dVi”ci (h)

FIG. 1. Change in fresh weight of sheep fecal pellets stored at 25°C (circles) and 39°C (quares) in sealed plastic bags (0, q ) and lidless Petri dishes (0, n). Each Petri dish or sealed plastic bag contained 20 pel- lets and there were five replicates of each treatment.

32 MILNE ET AL.

of 12 of the 18 herbivorous mammals sur- veyed (Table 1). Failure to isolate anaero- bic fungi from some of the animals sur- veyed may have been because the feces used were too old (see below) rather than because the animals lacked anaerobic fungi. Anaerobic fungi were also isolated from 9 of the 14 samples of cattle feces and 3 of the 10 samples of sheep feces collected in Ethiopia.

All of the fecal isolates grew on wheat straw medium and the majority were of the Neocallimastix type, i.e., they formed zoosporangia and an extensive network of branched, tapering rhizoids , However, pre- liminary observations suggest that these isolates represent more than one species. Anaerobic fungi have previously been iso- lated from the feces of sheep (Lowe et aE., 1987b) and horse and elephant (Bauchop, 1981). Lowe et al. (1987b) isolated two dif- ferent fungi from sheep feces; one was a Neocallimastix-like organism and the other resembled Sphaeromonas communis, an anaerobic fungus described by Orpin

(1976). ~though anaerobic fungi were not obtained from all fecal enrichment cultures, the present findings confirm and extend previous reports and suggest that anaerobic fungi are probably common inhabitants of the digestive tracts of many animals.

Survival of Anaerobic Fungi in Feces and Saliva

Feces of cattle and sheep in Ethiopia were sun-baked and dry on collection and anaerobic fungi were isolated from these fe- ces 4 days later in the United Kingdom (Ta- ble 1). Anaerobic fungi were also isolated from fresh feces of British sheep, but not from fecal pellets which were stored for 1 day in sealed plastic bags in air at 20 or 39°C (Table 2). However, if fecal pellets from British sheep were allowed to dry out at 20 or 39°C in air, anaerobic fungi could be iso- lated from them for at least 128 days (Table 2). Fungal isolates obtained from fresh and dried feces of British sheep resembled Neo- callimastix spp., producing spherical zoo-

FIG. 2. Anaerobic fungus isolated from a sheep fecal pellet stored in air for 1 day at 20°C in a lidless Petri dish. Zp, z~spomn~om; R, rhizoid.

SURVIVAL OF ANAEROBIC FUNGI 33

TABLE 3 Survival of Anaerobic Fungi in Sheep Saliva

Stored in Air at 20 and 39°C

Period for Number of roll tube cultures

which saliva prepared from saliva containing

was stored in anaerobic fung?

air (h) 20°C 39°C

1 1 2 2 1 3 4 0 0 8 0 1

16 0 0 32 0 0 64 0 0

’ At each sample time, five enrichment cultures were prepared at each temperature and these were used as inocula to prepare five roll tube cultures per treatment. Anaerobic fungi were isolated from 3 of 10 enrichment cultures prepared from freshly collected saliva (0 time sample).

b Maximum = 5.

sporangia with an extensive rhizoidal sys- tem (Figs. 2 and 3). These isolates were also similar to the Fl and Sl isolates obtained from the feces and saliva of sheep by Lowe

et al. (1987b). Sheep fecal pellets store lidless Petri dishes dried out rapidly i whereas those stored in plastic bags out more slowly (Fig. 1). At 39°C for ex- ample, fecal pellets stored in open dis lost ea. 47% of their fresh weight within of the onset of storage, whereas those stored in sealed plastic bags lost only El% of their fresh weight within the same time pe- riod. Anaerobic fungi were also isolate days after collection, drying, and stora (in air at 20°C) of feces from the llama an bongo enclosures at London Zoo.

The observation that ~e~~~~~~~~~~~~~ type anaerobic fungi survive dcsiccat~~~ and aerobic conditions in feces suggests that the storage conditions may formation of resistant structures. spherical bodies (resting cysts?) observed in pure cultures of a fungal is (Hl) obtained from horse cecum fluid pin, 1981). Other anaerobic fungi have not been observed to form such structures, al- though resistant zoosporangia and “cystsl’ are formed by aerobic chytrids (~a~~i~

FIG. 3. Anaerobic fungus isolated from a sheep fecal pellet stored in air for 2 days at 39°C in a lidless Petri dish. Zp, zoosporangium; R, rhiioid.

34 MILNE ET AL.

1977). There are two reasons why anaero- bic fungi may survive longer in dried feces than in moist feces. First, the drying pro- cess may induce the formation of resistant structures by anaerobic fungi. In the ab- sence of rapid drying and the formation of resistant structures, fecal microorganisms may die rapidly on exposure to air and re- duced temperatures outside the animal. It has been shown, for example, that zoo- spores and immature plants of Neocalli- mastix sp., isolate Rl, in anaerobic culture rapidly lose viability when transferred from 39 to 20°C (Lowe et al., 1987b, c). Second, continued bacterial activity in moist feces may in some way inhibit the anaerobic fun- gal population.

Table 3 shows that anaerobic fungi could be isolated from sheep saliva after it had been stored in air for up to 2 h at 20°C and up to 8 h at 39°C. The Neocallimastix-like fungi isolated from saliva (Fig. 3) were mor- phologically similar to those isolated from feces (Fig. 4) and the rumen (Lowe et al., 1987a).

These observations show that feces and

saliva may provide routes for the transfer of anaerobic fungi between animals. Cer- tainly, the saliva results indicate that anaer- obic fungi can be transferred between ani- mals during grooming. Becker and Everett (1930) observed that direct contact between isolated calves or lambs was not necessary for transfer of Chilomustix frontalis ( = N. frontalis), suggesting the transfer of organ- isms in aerosols formed from saliva. Re- sults with feces suggest a further possible route for transfer of anaerobic fungi be- tween the same or different species of her- bivorous mammals; although most mam- mals are not coprophilic, accidental contact with feces may occur. Furthermore, if anaerobic fungi form aerotolerant, resistant structures, these may be distributed to fresh forage which may be subsequently eaten by other animals.

Survival of Neocallimustix sp., Isolate Rl, at 39°C in Anaerobic Cultures Grown on Glucose or Wheat Straw and in Cultures Exposed to Air

Table 4 shows that Neocullimustix sp.,

FIG. 4. Anaerobic fungus isolated from sheep saliva stored in air at 20°C for 2 h. Zo, zoospores; Zp, zoosporangium; R, rhizoid.

SURVIVAL OF ANAEKOBIC FWNGI 35

TABLE 4 Survival of Neocallimastix sp., Isolate RI, at 39°C in

Anaerobic Cultures Grown on Medium Containing (a) 25 mM glucose or (b) 10 g Milled Wheat Straw

liter-’ as the Carbon Source”

Number of subcultures

Culture age containing anaerobic fungi

(days) and Inoculated Inoculated carbon source with culture with culture

in medium supernatant biomass

Medium a 0 1 3 5 7

Medium b 0 5

10 11 I2 13 14 15 16 -

Q At each sample time, l-ml volumes of the super- natant and part of the solid portion (biomass) of the culture were used separately to inoculate three lo-ml volumes of glucose medium; these subcultures were incubated anaerobically at 39°C for 3 days and then observed for fungal growth.

isolate Rl , survived up to 5 days in medium containing glucose as the carbon source, but up to 15 days in medium containing wheat straw as the carbon source. This dif- ference in survival time is probably related to the period taken for the cultures to attain stationary phase; cultures grown on glu- cose have a shorter lag phase and a faster specific growth rate than cultures grown on wheat straw. The results obtained indicate that Neocallimastix sp., isolate Rl, does not form a resistant structure (a resistant zoosporangium or some other resistant structure) under the culture conditions em- ployed.

Table 5 shows that Neocallimastix sp., isolate Rl, remained viable in stationary- phase (3 days) cultures up to 14 h after they

had been aerated and stored in air at 39°C; there was no appreciable difference in the survival of Neocallimastix sp., isolate in the supernatant (presumably as zoo- spores) and in the solid portion of the cul- ture. The difference in survival ti tween anaerobic fungi in saliva (up to 8 h) and Neocallimastix sp., isolate RI, in aer- ated cultures (up to 14 h) may result from (a) strain differences, (b) differences in t composition of suspending medium, or (c) activity of other rumen microorganisms in the saliva. In particular, it may be sign& cant that, unlike saliva, the culture rned~~~ used contains a reducing agent (cysteine sulfide) ~

To determine the effect of desiccation on

TABLE 5 Survival of Neocallimastix sp., Isolate RI, in Cultures Grown for 3 Days at 39°C in Glucose

Medium (Stationary-Phase Cultures) Aerated for 1 min, and Then Incubated at 39°C under

Aerobic Conditions”

Number of roll tube cultures containing anaerobic fungib

Time after aeration (h)

Inoculated Inoculded with culture with culture supernatant biomass

0 6 6 1 6 6 2 6 6 4 6 4 8 6 4

12 6 4 14 2 0 16 0 0 18 0 0 24 6 0 32 0 0

n At each sample time, 2-ml volumes of the culture supematant and part of the solid portion (biomass) of the culture were used separately to inoculate l&ml volumes of defined medium B; these subcultures were incubated anaerobically at 39°C for 3 days at which time anaerobic roll tube cultures (six per treatment) were prepared using the supernatant of the subcultures as inoculum. Neocallimastix sp., isolate Rl, was iso- lated from 11 out of 12 cultures inoculated with culture supematant from a 3-day culture of Neocallimastix sp., isolate Rl. grown at 39°C (nonaerated control).

’ Maximum = 6.

36 MILNE ET AL.

TABLE 6 Survival of Anaerobic Fungi on Dried Culture Solids”

Source of fungal Survival time since onset of drying (h) isolate used to test

for survival 0 2 4 6 8 10 12 14 16 18 20 22 24

Sheep rumen (Rl) + + + + + + -t + + - - - - Gaur feces + + + + + + + + + - - - - Llama feces + + + + + + + + + + - - - Ethiopian cattle feces + + + + + + + + + + - - -

a Neocallimastix sp., isolate RI, and anaerobic fungi isolated from the feces of Llama species, gaur (30s gaurus), and Ethiopian cattle were incubated for 4 days on medium containing wheat straw. Culture solids (re- sidual straw and adherent fungal biomass) were separated from culture supernatants and dried in air at 39°C. At each sample time, culture solids (ca. 0.5 g fresh wt) were removed and used to inoculate 50-ml volumes of medium containing wheat straw; these cultures were incubated anaerobically at 39°C for up to 14 days and ob- served for fungal growth. + , growth; - , no growth of anaerobic fungi.

the survival of anaerobic fungi, Neocalli- ma&ix sp., isolate Rl, and three other iso- lates from feces (Table 6) were grown for 4 days in medium containing wheat straw. Residual straw and adherent fungal bio- mass were separated from culture superna- tant and dried in air at 39°C. The rate of drying of culture solids (results not shown) was similar to that obtained for feces of sheep in lidless Petri dishes (Fig. 1). Table 6 shows that anaerobic fungi survived in har- vested solids up to 18 h after the onset of drying. Similar results were obtained when cultures of Neocallimastix sp., isolate Rl, were allowed to dry out at 39°C under anaerobic conditions (S. E. Lowe, M. K. Theodorou, and A. P. J. Trinci, unpub- lished results).

Although the survival times of anaerobic fungi in aerated cultures (12-14 h), dried culture solids (18 h), moist feces (~1 day), and saliva (2-8 h) were similar (Tables 2, 3, 5, and 6), they were considerably shorter than those obtained in desiccated feces (at least 128 days; Table 2). Thus, anaerobic fungi displayed limited survival under most conditions, but they were also capable of surviving for much longer periods, presum- ably due the formation of cysts or spores. Desiccation alone, however, was not suffr- cient to induce long-term survival in colo- nized wheat straw (Table 6).

ACKNOWLEDGMENTS

The authors thank London Zoo and the Interna- tional Livestock Centre for Africa for provision of fe- cal samples, and Dr. Irene Mueller-Harvey for collect- ing and transporting the Ethiopian samples. The Min- istry of Agriculture, Fisheries and Food provided a license (AHZ/533kr8715) to import the samples.

REFERENCES

BAUCHOP, T. 1979. The rumen anaerobic fungi: Colo- nizers of plant fibre. Ann. Rech. Vet. 10: 246-248.

BAUCHOP, T. 1980. Scanning electron microscopy in the study of microbial digestion of plant fragments in the gut. In Contemporary Microbial Ecology (D. C. Ellwood, J. N. Hedger, M. J. Latham, J. M. Lynch, and J. H. Slater, Eds.), pp. 305-326. Aca- demic Press, New York/London.

BAUCHOP, T. 1983. The gut anaerobic fungi: Coloniz- ers of dietary tibre. In Fibre in Human and Animal Nutrition (G. Wallace and L. Bull, Eds.), pp. 143- 148. Royal Society of New Zealand, Wellington.

BECKER, E. R., AND EVERETT, R. C. 1930. Compara- tive growth of normal infusoria-free lambs. Amer. J. Hyg. II: 362.

BECKER, E. R., AND HSUING, T. S. 1929. The method by which ruminants acquire their fauna of infusoria and remarks concerning experiments on the host specificity of protozoa. Proc. Nat. Acad. Sci. USA 15: 684.

EADIE, J. M. 1962. The development of rumen micro- bial populations in lambs and calves under various conditions of management. J. Gen. Microbial. 29: 563-578.

FONTY, G., GOUET, P., JOUANY, J. P., AND SENAND, J. 1987. Establishment of the microflora and anaer- obic fungi in the rumen of lambs. J. Gen. Microbial. 133: 1835-1843.

SURVIVAL OF ANAEROBIC FUNGI 37

Ho, Y. W., ABDULLAH, N., AND JALALUDIN, S. 1988. Penetrating structures of anaerobic rumen fungi in cattle and swamp buffalo. J. Gen. Microbial. 134: 177-181.

HOBSON, P. N. 1971. Rumen micro-organisms. Progr. Znd. Microbial. 9: 42-77.

HUNGATE, R. E. 1966. The Rumen and Its Microbes,

pp. 10-11. Academic Press, New York. HUNGATE, R. E. 1969. A roll-tube method for cultiva-

tion of strict anaerobes. Methods Microbial. 3B: 117-132.

KARLING, J. S. 1977. Chytriomycetarum Zcono-

graphia. J. Cramer, Monticello, NY/Lubrecht & Cramer.

LOWE, S. E. 1986. The Physiology and Cytology of an Anaerobic Rumen Fungus. Ph.D. thesis, University of Manchester.

LOWE, S. E., GRIFFITH, G. G., MILNE, A., THE- ODOROU, M. K., AND TRINCI, A. P. J. 1987a. The life cycle and morphology of an anaerobic rumen fungus. J. Gen. Microbial. 133: 1815-1827.

LOWE, S. E., THEODOROU, M. K., AND TRINCI,

A. P. .I. 198% Isolation of anaerobic fungi from sa- liva and faeces of sheep. J. Gen. Microbial. 133: 1829-1834.

LOWE, S. E., THEODOROU, M. K., AND TRINCI, A. P. J. 1987~. Growth of an anaerobic rumen fun- gus on various carbon sources and the effects of temperature on development. Appl. Environ. Micro-

biol. 53: 121&1215.

LOWE, S. E., THEODOROU, M. K., AND TRINCI~ A. P. J. 1987d. Cellulases and xylanases of an anaerobic rumen fungus grown on wheat straw ho- locellulose, cellulose and xylan. Appi. Environ. Mi- crobiol. 53: 12161223.

LOWE, S. E., THEODOROU, M. K., TRINCI, A. P. J., AND HESPELL, R. B. 1985. Growth of an anaerobic rumen fungus on defined and semi-defined media lacking rumen fluid. J. Gen. Microbial. 131: 2225- 2229.

MANN, S. 0. 1963. Some observations on the airborne dissemination of rumen bacteria. J. Gen. Microbial.

33: ix. MOUNTFORT, D. 0. 1987. The rumen anaerobic fungi.

FEMS Microbial. Rev. 48: 401-408.

ORPIN, C. 6. 1975. Studies on the rumen flagellate Neocallimastix frontalis. .I. Gen. Microbial. 91: 249-262.

ORPIN, C. G. 1976. Studies on the rumen flagellate Sphaeromonas communis. J. Gen. MicrobiooE. 94z

270-280. OWIN, C. G. 1981. Isolation of celluloytic phyco-

mycete fungi from the cecum of the horse. J. Gen. Microbial. 123: 287-296.

ORPIN, C. G., MATHIESEN, S. D., GREENWOOD, Y., AND BLIX, A. S. 1985. Seasonal changes in the ru- men microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus). Appl. Environ.

Microbial. 50: 144-151.