- Email: [email protected]
Keratinophilic fungi associated with birds
[ 241
]
Trans. Br. mycol. Soc. 54 (2), 241-250 (1970) Printed in Great Britain
KERATINOPHILIC FUNGI ASSOCIATED WITH BIRDS II. PHYSIOLOGICAL STUDIES
By G. J. F. PUGH AND MARY D. EVANS Department of Botany, University
ofNottingham
(With 9 Text-figures) The growth of the keratinophilic fungi isolated from birds is inhibited by temperatures of 40 °C; the fungi are restricted on the birds to the outer contour feathers. Spore germination of conidial Arthroderma uncinatum and Ctenomyces serratus does not occur below 90% r.h, and reaches a maximum at 100% r.h, Where associations have been shown between keratinophilic fungi and individual bird species, feather fats from these birds stimulate, or do not affect the growth of the fungi. A decrease in growth of a fungus in the presence of feather fats from birds was associated with the infrequent occurrence of that fungus on the birds. The feather fats thus playa large part in determining the occurrence of keratinophilic fungi on birds.
In surveys of the occurrence of keratinophilic fungi on birds (Pugh, 1965, I966a; Pugh & Evans, 1970) associations have been recorded
between individual fungi and related groups of birds. At the same time it has been pointed out (Pugh, 1966 b; Pugh & Evans, 1970) that the spectrum of keratinophilic fungi in nests differs from that which is present on the birds. The present work was undertaken to investigate some aspects of these associations and differences. Amongst the factors examined were the ability of the fungi to survive and grow in a range of temperatures and at different relative humidities, and in the presence of fats and oils which occur on the feather surface. MATERIALS AND METHODS
Isolates of Arthroderma curreyi (IMI 138270), conidial A. quadrifidum (IMI 138268), conidial A. uncinatum (IMI I38~64), Chrysosporium keratinophilum (IMI 138265) and Ctenomyces serratus (1M! 138266), were tested using single spore cultures. The isolates were grown on Sabouraud's Dextrose Agar (SDA) at a range of temperatures using radial extension as a measurement of growth. Conidial A. uncinatum and Ctenomyces serratus were used in a preliminary survey of the effect of relative humidity (r.h.) on the germination of conidia. Spore suspensions were prepared from fresh and z-month-old dry cultures and the spores anchored to the slides by the gelatine technique reported by Snow (1949). Solutions of sucrose (Davidson & Gregory, 1934) and sulphuric acid (Solomon, 1951) were used in two separate experiments to maintain atmospheres of known r.h. in desicr6
MYC
54
242
Transactions British Mycological Society
cators. The desiccators were incubated at 25° for ro days and duplicate slides were examined at 24 h intervals. Further slides were maintained for up to 4 weeks. The latent period of spore germination and the percentage germination were recorded (Tomkins, 1932). Oils were collected by refluxing wool or feathers in ether and were recovered by evaporation of the ether. The oils and fats were sterilized at 160° for 2 h and redissolved in ether, which had been sterilized by membrane filtration. The ether-soluble extracts obtained from the feathers were recorded as a percentage of the dry weight of the feathers. It is appreciated that these extracts are a complex of various fatty acids and alcohols, which will be referred to as feather fats in this paper. A preliminary examination of the effect of starling feather fats on the five test fungi was made, using a plate technique. An ether solution ofthe fat or oil was added to SDA at pouring heat to give a known final concentration of additive. Growth was assessed by measurement of radial extension. Shake cultures were grown in a basic mineral medium which contained 0-4 g K 2HP04, 0'05 g MgS04.7H20, 0'01 g NACI, 0'01 g FeCI 3 , I I distilled water. Glucose (2 %) and peptone (I %) were added to form the control medium. All fungi were also grown with 1% NH4N03 forming the nitrogen source and olive oil or wool fat, at various concentrations, as the sole carbon source. Feather fats were added to the glucosepeptone medium at a range ofconcentrations up to 0·8 %weight by volume. Medium (20 ml) was added to each roo ml Erlhenmyer flask before addition of fats or oils and autoclaved at I bar gauge pressure for 30 min and sterile ether solutions of oils and fats were added. The flasks were shaken vigorously to disperse the oils or fats and were then incubated on reciprocating shakers for 14-16 days at 25°. Growth was recorded as mgj roo ml of medium. The results obtained from each experiment were compared statistically using analysis of variance. If the treatments had a significant effect then the differences between the treatments were assessed using the t-test. RESULTS
Temperature The radial extension growth rates for selected keratinophilic species recorded after 13 days growth are shown in Fig. I. In each case, maximum growth occurred at 25°, and there was virtually no growth at 40°. After incubation at 40°, the inocula did not resume growth when they were transferred to fresh media and incubated at 25°. Relative humidity, desiccation andgermination After incubation at 25 0 for 24 h at roo
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
243
the range go-IOO % r.h, but Ctenomyces serratus showed a consistently lower percentage of germination even at IOO % r.h. To allow a direct comparison, the observed results for both species have been corrected on the basis of the percentage recorded at IOO % r.h, being 100% germination for each species. 60 E
.§. 40
.'" a: 20 :>
"U
20
30
30
40
Fig. I. Temperature growth curves after thirteen days incubation on SDA. b,.-6" Arthroderma curr~i; ...- ... ) conidial A. quadrifidum; x - x )conidial A. uncinatwn; . - . , Ckrysosporium keratinophilum; 0 - - - 0, Ctenomyces serratus.
100
,~
f(~
/f
80
(!
g c
..
0 .;:;
60
J/
e
'E...
40
o'"
20
~>e/ lC
0
75
80
85
",0
90
95
100
Relative humidity (%)
Fig. 2, Germination of conidia of Arthroderma uncinatum and Ctenomyces serratus at various relative humidities at 25 "C. X - x, A. uncinatum; 0 --- 0, C. serratus.
Superficial oils andfats In each case there was a reduction in radial extension of the test fungus in the presence of increasing concentrations of starling feather fats over the experimental range (Fig. 3). Preliminary experiments using spore production as a measure ofgrowth, showed a marked increase in sporulation of Chrysosporium keratinophilumf unit area of surface growth but that conidial Arthroderma quadrifidum, conidial A. uncinatum and Ctenomyces serratus were significantly if only slightly inhibited. 16-2
Transactions British Mycological Society
244
Pheasant feather fats were used at a range of concentrations in shake cultures to test the effect on all five fungi. Other feather fats were not available in sufficient quantities and were tested against selected fungi. With each fungus growth was also measured in the presence of wool fats and of olive oil. Oils and fats in general supported a very limited amount of growth when used as the sole carbon source .
.-._ 20
~
'-
::s v
«
15
.--
01 v
c ~
; 10
v v
~
..--
o III
...l?f
.--
C III
... V
III
0..
'" 0
E ... "5'"
II r--
'"
~
15 2 tIII
v
C III
...... :> t-
a
III
bO
o
1·0 2·0 3·0 4·0 Log,o cone. feather fat (mg/I) Fig. 3
r--
C
D
r--
u u
OL.-_-L._ _L-_....L-_--I
......
...'"
......
C III
... V
~
0
A
B
E
Fig. 4
Fig. 3. The effect of starling feather fat on radial growth of keratinophilic fungi on SDA at 25°C. /:0.-/:0., Arthroderma currryi; A-A, conidial A. quadrifidum; x-x, conidial A. uncinatum; e-e, Chrysosporium keratinophilum; 0 --- 0, Ctenomyces serratus. Fig. 4. Comparison between I, the percentage of birds of different species with Arthroderma currryi on the feathers, and II, the percentage of feather fats. A, blackbird; B, pheasant; C, partridge; D, pigeon; E, starling.
Arthroderma curreyi was recorded on thirteen different species of bird and was most often found on blackbirds and pheasants. It was infrequently isolated from partridges and starlings and was not recorded on pigeons. In Figs. 4, its frequency of occurrence on these bird is compared with the
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
245
relative amount offeather fats obtained from the birds. It can be seen that there is an inverse relationship between the amount of the feather fats and the frequency of occurrence of this species. At the concentrations used experimentally, however, the feather fats obtained from blackbird and pheasant were found to stimulate the growth of this species, (Fig. 5), while those from the partridge had no marked effect. Growth in the presence of the pigeon feather fats appeared to be slightly inhibited, although
E ::l
1: 600 E
E
o o
~ 400
5
-----~--~~-o ~
E ::l
eE 200 ..,
..r:::
I>C
'4j ~
0
~
c
,
.......~_~_~~___ __...&.._ _.L'_ _......_ ...... 4·0 0 1·0 2·0 3·0 4·0 Log, O cone. lipid source (mg/l)
~_
0
Fig. 5. Growth of Arthroderma curreyi in shake culture after 16 days incubation at 2 5 °C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (P = 0'001); blackbird featber fats, ~ -~ (P = 0'001); partridge feather fats, 0---0 (not significant); pheasant feather fats, /::,-/::, (P= 0'001) and pigeon feather fats, (not significant).
e-e
E ::l
1500
[
...
"'0
E
E 1200
~
0 0
....
--
1>0
S
900
E ::l
Q; u
600
>..
..
E
..r:::
1>0
300
'OJ
~ :>.. L-
Q
0
0
1·0
2·0
3·0
4·0 0
1·0
2·0
3·0
4·0
Log,o cone, lipid source (mg/l) Fig. 6. Growth of conidial Arthroderma quadrijidum in shake culture after 14 days incubation at 25°C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (not significant); blackbird feather fats, ~-~ (no effect) and pheasant feather fats, /::,-/::, (no effect),
246
Transactions British Mycological Society
this was not statistically significant because of a high variability between replicates, while radial extension was significantly reduced in the presence of starling feather fats (Fig. 3). Conidial A. quadrifidum showed no significant alteration of growth rate in the presence of blackbird, pheasant and wool fats (Fig. 6). With olive oil there was a significant increase in growth at the higher concentrations used.
E::>
2000
.,
"0
E
E o o
!
1600 1200 . - . -.....~
E ::>
Qi.
800
.s::
400
...E 00
'iii
~ >.
I-
0
0
0
1·0
2·0
t
.....J 2·0 4·0 0 1·0 3·0 Log,. cone. lipid source (mgjl)
, 3·0
4·0
Fig. 7. Growth of conidial Arthroderma uncinatum in shake culture after r6 days incubation at zg DC in the presence of various concentrations of olive oil, 0-0 (p= 0'001); wool fats, x-x (P= o-oor j j pheasant feather fats, D,-D, (P= o-oor ) and pigeon feather fats, . - . (P= o·oor).
E ::1
.,
1500
L.
"0
E
E
1200
0 0
....
bo 900 ~ E
::>
Qi.
600
.s::
300
...E
bO
'iii
~ >..
...
0
0 0
1·0
2'0
3·0
4·0
Fig. 8. Growth of Chrysosporium keratinophilum in shake culture after 14 days incubation at 25°C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (P = 0'001); blackbird feather fats, j.- j . (not significant); partridge feather fats, 0- - -0 (not significant) and pheasant feather fats, D,-D, (not significant).
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
247
Conidial A. uncinatum was strikingly stimulated by pheasant feather fats and inhibited by those of pigeon (Fig. 7). The controls also showed a contrast in growth, with olive oil stimulating, and wool fat inhibiting growth. Chrysosporium keratinophilum was not significantly affected by blackbird, partridge and pheasant feather fats (Fig. 8). Olive oil stimulated growth, while wool fat brought about an increase in growth up to a concentration of o· I %, after which growth diminished. Ctenomyces serratus was not significantly affected by partridge feather fats, but growth was greatly reduced in the presence of pheasant feather fats (Fig. 9), as it was with both controls.
g 1000 "E
E 800 o o
.....
Oii
5
-----0---,;-----0-
E
"\
:l
]
>.
E
a...
6_
>..
00"_ _L-_-:-'-"::
3·0
4·0
Log,o cone. lipid source (mgjl)
Fig. g. Growth of Ctenomyces serratus in shake culture after 16 days incubation of olive oil, 0-0 (p= 0'001); wool fats, x-x (P= 0'001); partridge feather fats, 0---0 (not significant) and pheasant feather fats, L,-L, (P= 0'001).
DISCUSSION
When the results of these experiments are related to the observations of Pugh & Evans (1970), they help to explain the patterns of occurrence which were found on the birds. The keratinophilic fungi which were isolated from the birds were all found to be mesophilic. Consequently they would occur on the outer contour feathers rather than on the inner down feathers, where the temperature would be higher. The generally high body temperature of birds would also tend to preclude the occurrence of dermatophytes from those parts of the body surface which are insulated by feathers. The records of dermatophytes on birds, when a site of infection is mentioned, normally relate to the head and particularly to the comb (e.g, Metianu, Lucas & Drouhet, 1966). There is little information available on the r.h. within the plumage of birds. Pugh (1965) suggested that the humidity which allows lice to grow
248
Transactions British Mycological Society
on birds at least approaches a level suitable for fungal growth. The occurrence of A. curreyi and Ctenomyces serratus within freshly collected surface-sterilized feathers indicates that these species grew while the feathers were on the birds, thus confirming this suggestion. Some constituents of the fatty secretions on hair and skin of man and other animals have been shown to be effective against dermatophytes in vitro by many workers while the factors concerned in the self-sterilization of the human epidermis have been reviewed by Burtenshaw (1948). The relative importance of these factors is still debated: Rothman & Lorincz (1963) reported that fatty acid fractions are inhibitory, while Kligman (1963) found that in the presence of a keratinous substrate this is not necessarily so. On birds, the origin of the feather fats is subject to some speculation (Bolliger & Varga, 1960, 1961; Spearman, 1966), but the secretions of the preen gland contribute to the general ethersoluble fraction. The secretions from a variety of water birds have been analysed in detail by Odham (1967). Arthroderma curreyi has been shown to be frequently associated with birds of the genus Turdus (Pugh, 1965) and especially with T. merula (blackbirds) by Pugh (1964) and Pugh & Evans (1970). Apart from pheasants from a hatchery, this fungus was relatively uncommon on the other birds sampled. Its general level of occurrence on birds is inversely proportional to the quantity of fats present on the feathers. The results obtained in liquid cultures show that the growth of A. curreyi was stimulated both by blackbird and pheasant feather fats. Although it was not significantly affected by those of the partridges and pigeons, this species was not found on pigeons, and was recorded on only 4 out of 49 partridges. Presumably, on these birds other factors affect its occurrence. The low frequency of occurrence on starling may be correlated with the adverse effect of increasing amounts of feather fats of this bird on radial extension of growth. Conidial A. quadrijidum was uncommon on birds except when it was the only species present. This was in marked contrast to its frequent occurrence in nests and soils. It was most often recorded on pheasants and blackbirds, and their feather fats had no significant effect on its growth. It occurred on only two starlings and starling feather fats depressed its radial extension of growth. The non-viability of spores washed from feathers may be due to high levels offats on the feather surface proving to be lethal rather than merely inhibitory, or possibly to some other factor such as u.v, light. Chrysosporium spp. have been found universally on birds and in their nests (Pugh, 1965, 1966a, b; Rees, 1967a; Otcenasek, Hudec, Hubalek & Dvorak, 1967; Pugh & Evans, 1970). C. keratinophilum, which occurred at a low level on a wide range of bird species, was not significantly affected in liquid cultures by blackbird, partridge and pheasant feather fats, but its radial growth was adversely affected by starling feather fats. These, however, brought about an increase in sporulation. If these results apply to the other species of Chrysosporium they would help to explain the widespread and frequent occurrence of these species. Ctenomyces serratus has been isolated almost exclusively from members of
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
249
the Galliformes (Pugh, 1966a; Pugh & Evans, 1970; Rees, 1967a, b). Within this order it has been reported most often from partridges (Pugh & Evans, 1970) and domestic poultry (Rees, 1967 b). Its growth on partridge feather fats was not significantly affected, while those of pheasant brought about a marked decrease in growth in liquid culture, as did those of starlings on radial growth. From these results, the associations previously recorded (Pugh, 1964, 1965; Pugh & Evans, 1970) may be explained by the stimulation, or at least tolerance, of A. curreyi to feather fats of blackbirds, conidial A. uncinatum to those of pheasants, Ctenomyces serratus to those of partridges and Chrysosporium spp. to most feather fats tested. Conversely, the decrease in growth of these fungal species by increasing amounts of feather fats from other birds may explain their infrequent occurrence on those birds. Starlings, for example, yielded only 31 isolates of keratinophilic fungi from 126 birds, and their feather fats significantly depressed growth of all the test fungi. It would appear that the feather fats both qualitatively and quantitatively determine the distribution of keratinophilic fungi on birds in general while on any particular bird temperature and exposure to desiccation will help decide the location of the fungi. We wish to thank Dr ]. F. Peberdy for helpful discussions and Dr Rieley for advice on the statistical analyses.
J.
REFERENCES
BOLLIGER, A. & VARGA, D. (1960). Cholestanol in avian plumage. Aust. J. expo Biol. med. Sci. 38, 265-27°' BOLLIGER, A. & VARGA, D. (1961). Feather lipids. Nature, Lond. 190, 1125. BURTENSHAW,J. M. L. (1948). In Moderntrends in dermatology (ed. byR. M. B. Mackenna) London: Butterwick & Co. Ltd. DAVIDSON, A. M. & GREGORY, P. H. (1934). In situ cultures ofdermatophytes. Can. J. Res. 10, 373-393. KLIGMAN, A. M. (1963). In Advances in the biology ofthe skin. IV. The sebaceous gland (ed. by W. Montagna, R. A. Ellis and A. F. Silver). New York: Macmillan. METIANU, T., LUCAS, A. & DROUHET, E. (1966). Teigne favique du dindon due a un nouveau dermatophyte. Mycopath. Mycol. appl. 22, 175-181. ODHAM, G. (1967). Studies of feather waxes of waterfowl. Ark. Kemi. 27, 295-307. OTCENAsEK, M., HUDEC, K., HUBALEK, Z. & DVORAK, J. (1967). Keratinophilic fungi from the nests of birds in Czechoslovakia. Sabouraudia 5, 350-354. PUGH, G.]. F. (1964). Dispersal of Arthroderma curreyi by birds, and its role in the soil. Sabouraudia 3, 275-278. PUGH, G.]. F. (1965). Cellulolytic and keratinophilic fungi recorded on birds. Sabouraudia 4, 85-91. PUGH, G.]. F. (1966a). Fungi on birds in India.]. Indian bot. Soc. 45,296-303. PUGH, G.]. F. (1966b). Associations between birds' nests, their pH and keratinophilic fungi. Sabouraudia 5, 49-53· PUGH, G.]. F. & EVANS, M. D. (1970). Keratinophilic fungi associated with birds.T. Fungi isolated from feathers, nests and soils. Trans. Br. mycol, Soc. 54, 233-240. REES, R. G. (1967a). Keratinophilic fungi from Queensland. II. Isolation from feathers of wild birds. Sabouraudic 6, 14-1 B. REES, R. G. (1967b). Keratinophilic fungi from Queensland. III. Isolation from feathers of domestic fowls. Sabouraudia 6, 19-28.
Transactions British Mycological Society ROTHMAN, S. & LORINCZ, A. L. (1963). Defense mechanisms of the skin. A. Rev. Med. 14, 21 5- 2 4 2 . SNOW, D. (1949). The germination of mould spores at controlled humidities. Ann. appl.
BioI. 35, 1-13· M. E. ( 1951). Control of humidity with potassium hydroxide, sulphuric acid and other solutions. Bull. ent, Res. 42 , 543-554. SPEARMA:-<, R. I. C. (1966). The keratinization of epidermal scales, feathers and hairs. BioI. Rev. 41, 59-96. TOMKINS, R. G. ( 19 3 2) . Measuring germination. Trans. Br. mycol. Soc. 17, 147-149.
SOLOMON,
(Accepudfor publication 3 October 1969)
]
Trans. Br. mycol. Soc. 54 (2), 241-250 (1970) Printed in Great Britain
KERATINOPHILIC FUNGI ASSOCIATED WITH BIRDS II. PHYSIOLOGICAL STUDIES
By G. J. F. PUGH AND MARY D. EVANS Department of Botany, University
ofNottingham
(With 9 Text-figures) The growth of the keratinophilic fungi isolated from birds is inhibited by temperatures of 40 °C; the fungi are restricted on the birds to the outer contour feathers. Spore germination of conidial Arthroderma uncinatum and Ctenomyces serratus does not occur below 90% r.h, and reaches a maximum at 100% r.h, Where associations have been shown between keratinophilic fungi and individual bird species, feather fats from these birds stimulate, or do not affect the growth of the fungi. A decrease in growth of a fungus in the presence of feather fats from birds was associated with the infrequent occurrence of that fungus on the birds. The feather fats thus playa large part in determining the occurrence of keratinophilic fungi on birds.
In surveys of the occurrence of keratinophilic fungi on birds (Pugh, 1965, I966a; Pugh & Evans, 1970) associations have been recorded
between individual fungi and related groups of birds. At the same time it has been pointed out (Pugh, 1966 b; Pugh & Evans, 1970) that the spectrum of keratinophilic fungi in nests differs from that which is present on the birds. The present work was undertaken to investigate some aspects of these associations and differences. Amongst the factors examined were the ability of the fungi to survive and grow in a range of temperatures and at different relative humidities, and in the presence of fats and oils which occur on the feather surface. MATERIALS AND METHODS
Isolates of Arthroderma curreyi (IMI 138270), conidial A. quadrifidum (IMI 138268), conidial A. uncinatum (IMI I38~64), Chrysosporium keratinophilum (IMI 138265) and Ctenomyces serratus (1M! 138266), were tested using single spore cultures. The isolates were grown on Sabouraud's Dextrose Agar (SDA) at a range of temperatures using radial extension as a measurement of growth. Conidial A. uncinatum and Ctenomyces serratus were used in a preliminary survey of the effect of relative humidity (r.h.) on the germination of conidia. Spore suspensions were prepared from fresh and z-month-old dry cultures and the spores anchored to the slides by the gelatine technique reported by Snow (1949). Solutions of sucrose (Davidson & Gregory, 1934) and sulphuric acid (Solomon, 1951) were used in two separate experiments to maintain atmospheres of known r.h. in desicr6
MYC
54
242
Transactions British Mycological Society
cators. The desiccators were incubated at 25° for ro days and duplicate slides were examined at 24 h intervals. Further slides were maintained for up to 4 weeks. The latent period of spore germination and the percentage germination were recorded (Tomkins, 1932). Oils were collected by refluxing wool or feathers in ether and were recovered by evaporation of the ether. The oils and fats were sterilized at 160° for 2 h and redissolved in ether, which had been sterilized by membrane filtration. The ether-soluble extracts obtained from the feathers were recorded as a percentage of the dry weight of the feathers. It is appreciated that these extracts are a complex of various fatty acids and alcohols, which will be referred to as feather fats in this paper. A preliminary examination of the effect of starling feather fats on the five test fungi was made, using a plate technique. An ether solution ofthe fat or oil was added to SDA at pouring heat to give a known final concentration of additive. Growth was assessed by measurement of radial extension. Shake cultures were grown in a basic mineral medium which contained 0-4 g K 2HP04, 0'05 g MgS04.7H20, 0'01 g NACI, 0'01 g FeCI 3 , I I distilled water. Glucose (2 %) and peptone (I %) were added to form the control medium. All fungi were also grown with 1% NH4N03 forming the nitrogen source and olive oil or wool fat, at various concentrations, as the sole carbon source. Feather fats were added to the glucosepeptone medium at a range ofconcentrations up to 0·8 %weight by volume. Medium (20 ml) was added to each roo ml Erlhenmyer flask before addition of fats or oils and autoclaved at I bar gauge pressure for 30 min and sterile ether solutions of oils and fats were added. The flasks were shaken vigorously to disperse the oils or fats and were then incubated on reciprocating shakers for 14-16 days at 25°. Growth was recorded as mgj roo ml of medium. The results obtained from each experiment were compared statistically using analysis of variance. If the treatments had a significant effect then the differences between the treatments were assessed using the t-test. RESULTS
Temperature The radial extension growth rates for selected keratinophilic species recorded after 13 days growth are shown in Fig. I. In each case, maximum growth occurred at 25°, and there was virtually no growth at 40°. After incubation at 40°, the inocula did not resume growth when they were transferred to fresh media and incubated at 25°. Relative humidity, desiccation andgermination After incubation at 25 0 for 24 h at roo
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
243
the range go-IOO % r.h, but Ctenomyces serratus showed a consistently lower percentage of germination even at IOO % r.h. To allow a direct comparison, the observed results for both species have been corrected on the basis of the percentage recorded at IOO % r.h, being 100% germination for each species. 60 E
.§. 40
.'" a: 20 :>
"U
20
30
30
40
Fig. I. Temperature growth curves after thirteen days incubation on SDA. b,.-6" Arthroderma curr~i; ...- ... ) conidial A. quadrifidum; x - x )conidial A. uncinatwn; . - . , Ckrysosporium keratinophilum; 0 - - - 0, Ctenomyces serratus.
100
,~
f(~
/f
80
(!
g c
..
0 .;:;
60
J/
e
'E...
40
o'"
20
~>e/ lC
0
75
80
85
",0
90
95
100
Relative humidity (%)
Fig. 2, Germination of conidia of Arthroderma uncinatum and Ctenomyces serratus at various relative humidities at 25 "C. X - x, A. uncinatum; 0 --- 0, C. serratus.
Superficial oils andfats In each case there was a reduction in radial extension of the test fungus in the presence of increasing concentrations of starling feather fats over the experimental range (Fig. 3). Preliminary experiments using spore production as a measure ofgrowth, showed a marked increase in sporulation of Chrysosporium keratinophilumf unit area of surface growth but that conidial Arthroderma quadrifidum, conidial A. uncinatum and Ctenomyces serratus were significantly if only slightly inhibited. 16-2
Transactions British Mycological Society
244
Pheasant feather fats were used at a range of concentrations in shake cultures to test the effect on all five fungi. Other feather fats were not available in sufficient quantities and were tested against selected fungi. With each fungus growth was also measured in the presence of wool fats and of olive oil. Oils and fats in general supported a very limited amount of growth when used as the sole carbon source .
.-._ 20
~
'-
::s v
«
15
.--
01 v
c ~
; 10
v v
~
..--
o III
...l?f
.--
C III
... V
III
0..
'" 0
E ... "5'"
II r--
'"
~
15 2 tIII
v
C III
...... :> t-
a
III
bO
o
1·0 2·0 3·0 4·0 Log,o cone. feather fat (mg/I) Fig. 3
r--
C
D
r--
u u
OL.-_-L._ _L-_....L-_--I
......
...'"
......
C III
... V
~
0
A
B
E
Fig. 4
Fig. 3. The effect of starling feather fat on radial growth of keratinophilic fungi on SDA at 25°C. /:0.-/:0., Arthroderma currryi; A-A, conidial A. quadrifidum; x-x, conidial A. uncinatum; e-e, Chrysosporium keratinophilum; 0 --- 0, Ctenomyces serratus. Fig. 4. Comparison between I, the percentage of birds of different species with Arthroderma currryi on the feathers, and II, the percentage of feather fats. A, blackbird; B, pheasant; C, partridge; D, pigeon; E, starling.
Arthroderma curreyi was recorded on thirteen different species of bird and was most often found on blackbirds and pheasants. It was infrequently isolated from partridges and starlings and was not recorded on pigeons. In Figs. 4, its frequency of occurrence on these bird is compared with the
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
245
relative amount offeather fats obtained from the birds. It can be seen that there is an inverse relationship between the amount of the feather fats and the frequency of occurrence of this species. At the concentrations used experimentally, however, the feather fats obtained from blackbird and pheasant were found to stimulate the growth of this species, (Fig. 5), while those from the partridge had no marked effect. Growth in the presence of the pigeon feather fats appeared to be slightly inhibited, although
E ::l
1: 600 E
E
o o
~ 400
5
-----~--~~-o ~
E ::l
eE 200 ..,
..r:::
I>C
'4j ~
0
~
c
,
.......~_~_~~___ __...&.._ _.L'_ _......_ ...... 4·0 0 1·0 2·0 3·0 4·0 Log, O cone. lipid source (mg/l)
~_
0
Fig. 5. Growth of Arthroderma curreyi in shake culture after 16 days incubation at 2 5 °C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (P = 0'001); blackbird featber fats, ~ -~ (P = 0'001); partridge feather fats, 0---0 (not significant); pheasant feather fats, /::,-/::, (P= 0'001) and pigeon feather fats, (not significant).
e-e
E ::l
1500
[
...
"'0
E
E 1200
~
0 0
....
--
1>0
S
900
E ::l
Q; u
600
>..
..
E
..r:::
1>0
300
'OJ
~ :>.. L-
Q
0
0
1·0
2·0
3·0
4·0 0
1·0
2·0
3·0
4·0
Log,o cone, lipid source (mg/l) Fig. 6. Growth of conidial Arthroderma quadrijidum in shake culture after 14 days incubation at 25°C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (not significant); blackbird feather fats, ~-~ (no effect) and pheasant feather fats, /::,-/::, (no effect),
246
Transactions British Mycological Society
this was not statistically significant because of a high variability between replicates, while radial extension was significantly reduced in the presence of starling feather fats (Fig. 3). Conidial A. quadrifidum showed no significant alteration of growth rate in the presence of blackbird, pheasant and wool fats (Fig. 6). With olive oil there was a significant increase in growth at the higher concentrations used.
E::>
2000
.,
"0
E
E o o
!
1600 1200 . - . -.....~
E ::>
Qi
800
.s::
400
...E 00
'iii
~ >.
I-
0
0
0
1·0
2·0
t
.....J 2·0 4·0 0 1·0 3·0 Log,. cone. lipid source (mgjl)
, 3·0
4·0
Fig. 7. Growth of conidial Arthroderma uncinatum in shake culture after r6 days incubation at zg DC in the presence of various concentrations of olive oil, 0-0 (p= 0'001); wool fats, x-x (P= o-oor j j pheasant feather fats, D,-D, (P= o-oor ) and pigeon feather fats, . - . (P= o·oor).
E ::1
.,
1500
L.
"0
E
E
1200
0 0
....
bo 900 ~ E
::>
Qi
600
.s::
300
...E
bO
'iii
~ >..
...
0
0 0
1·0
2'0
3·0
4·0
Fig. 8. Growth of Chrysosporium keratinophilum in shake culture after 14 days incubation at 25°C in the presence of various concentrations of olive oil, 0-0 (P = 0'001); wool fats, x - x (P = 0'001); blackbird feather fats, j.- j . (not significant); partridge feather fats, 0- - -0 (not significant) and pheasant feather fats, D,-D, (not significant).
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
247
Conidial A. uncinatum was strikingly stimulated by pheasant feather fats and inhibited by those of pigeon (Fig. 7). The controls also showed a contrast in growth, with olive oil stimulating, and wool fat inhibiting growth. Chrysosporium keratinophilum was not significantly affected by blackbird, partridge and pheasant feather fats (Fig. 8). Olive oil stimulated growth, while wool fat brought about an increase in growth up to a concentration of o· I %, after which growth diminished. Ctenomyces serratus was not significantly affected by partridge feather fats, but growth was greatly reduced in the presence of pheasant feather fats (Fig. 9), as it was with both controls.
g 1000 "E
E 800 o o
.....
Oii
5
-----0---,;-----0-
E
"\
:l
]
>.
E
a...
6_
>..
00"_ _L-_-:-'-"::
3·0
4·0
Log,o cone. lipid source (mgjl)
Fig. g. Growth of Ctenomyces serratus in shake culture after 16 days incubation of olive oil, 0-0 (p= 0'001); wool fats, x-x (P= 0'001); partridge feather fats, 0---0 (not significant) and pheasant feather fats, L,-L, (P= 0'001).
DISCUSSION
When the results of these experiments are related to the observations of Pugh & Evans (1970), they help to explain the patterns of occurrence which were found on the birds. The keratinophilic fungi which were isolated from the birds were all found to be mesophilic. Consequently they would occur on the outer contour feathers rather than on the inner down feathers, where the temperature would be higher. The generally high body temperature of birds would also tend to preclude the occurrence of dermatophytes from those parts of the body surface which are insulated by feathers. The records of dermatophytes on birds, when a site of infection is mentioned, normally relate to the head and particularly to the comb (e.g, Metianu, Lucas & Drouhet, 1966). There is little information available on the r.h. within the plumage of birds. Pugh (1965) suggested that the humidity which allows lice to grow
248
Transactions British Mycological Society
on birds at least approaches a level suitable for fungal growth. The occurrence of A. curreyi and Ctenomyces serratus within freshly collected surface-sterilized feathers indicates that these species grew while the feathers were on the birds, thus confirming this suggestion. Some constituents of the fatty secretions on hair and skin of man and other animals have been shown to be effective against dermatophytes in vitro by many workers while the factors concerned in the self-sterilization of the human epidermis have been reviewed by Burtenshaw (1948). The relative importance of these factors is still debated: Rothman & Lorincz (1963) reported that fatty acid fractions are inhibitory, while Kligman (1963) found that in the presence of a keratinous substrate this is not necessarily so. On birds, the origin of the feather fats is subject to some speculation (Bolliger & Varga, 1960, 1961; Spearman, 1966), but the secretions of the preen gland contribute to the general ethersoluble fraction. The secretions from a variety of water birds have been analysed in detail by Odham (1967). Arthroderma curreyi has been shown to be frequently associated with birds of the genus Turdus (Pugh, 1965) and especially with T. merula (blackbirds) by Pugh (1964) and Pugh & Evans (1970). Apart from pheasants from a hatchery, this fungus was relatively uncommon on the other birds sampled. Its general level of occurrence on birds is inversely proportional to the quantity of fats present on the feathers. The results obtained in liquid cultures show that the growth of A. curreyi was stimulated both by blackbird and pheasant feather fats. Although it was not significantly affected by those of the partridges and pigeons, this species was not found on pigeons, and was recorded on only 4 out of 49 partridges. Presumably, on these birds other factors affect its occurrence. The low frequency of occurrence on starling may be correlated with the adverse effect of increasing amounts of feather fats of this bird on radial extension of growth. Conidial A. quadrijidum was uncommon on birds except when it was the only species present. This was in marked contrast to its frequent occurrence in nests and soils. It was most often recorded on pheasants and blackbirds, and their feather fats had no significant effect on its growth. It occurred on only two starlings and starling feather fats depressed its radial extension of growth. The non-viability of spores washed from feathers may be due to high levels offats on the feather surface proving to be lethal rather than merely inhibitory, or possibly to some other factor such as u.v, light. Chrysosporium spp. have been found universally on birds and in their nests (Pugh, 1965, 1966a, b; Rees, 1967a; Otcenasek, Hudec, Hubalek & Dvorak, 1967; Pugh & Evans, 1970). C. keratinophilum, which occurred at a low level on a wide range of bird species, was not significantly affected in liquid cultures by blackbird, partridge and pheasant feather fats, but its radial growth was adversely affected by starling feather fats. These, however, brought about an increase in sporulation. If these results apply to the other species of Chrysosporium they would help to explain the widespread and frequent occurrence of these species. Ctenomyces serratus has been isolated almost exclusively from members of
Fungi on birds. II. G. J. F. Pugh and M. D. Evans
249
the Galliformes (Pugh, 1966a; Pugh & Evans, 1970; Rees, 1967a, b). Within this order it has been reported most often from partridges (Pugh & Evans, 1970) and domestic poultry (Rees, 1967 b). Its growth on partridge feather fats was not significantly affected, while those of pheasant brought about a marked decrease in growth in liquid culture, as did those of starlings on radial growth. From these results, the associations previously recorded (Pugh, 1964, 1965; Pugh & Evans, 1970) may be explained by the stimulation, or at least tolerance, of A. curreyi to feather fats of blackbirds, conidial A. uncinatum to those of pheasants, Ctenomyces serratus to those of partridges and Chrysosporium spp. to most feather fats tested. Conversely, the decrease in growth of these fungal species by increasing amounts of feather fats from other birds may explain their infrequent occurrence on those birds. Starlings, for example, yielded only 31 isolates of keratinophilic fungi from 126 birds, and their feather fats significantly depressed growth of all the test fungi. It would appear that the feather fats both qualitatively and quantitatively determine the distribution of keratinophilic fungi on birds in general while on any particular bird temperature and exposure to desiccation will help decide the location of the fungi. We wish to thank Dr ]. F. Peberdy for helpful discussions and Dr Rieley for advice on the statistical analyses.
J.
REFERENCES
BOLLIGER, A. & VARGA, D. (1960). Cholestanol in avian plumage. Aust. J. expo Biol. med. Sci. 38, 265-27°' BOLLIGER, A. & VARGA, D. (1961). Feather lipids. Nature, Lond. 190, 1125. BURTENSHAW,J. M. L. (1948). In Moderntrends in dermatology (ed. byR. M. B. Mackenna) London: Butterwick & Co. Ltd. DAVIDSON, A. M. & GREGORY, P. H. (1934). In situ cultures ofdermatophytes. Can. J. Res. 10, 373-393. KLIGMAN, A. M. (1963). In Advances in the biology ofthe skin. IV. The sebaceous gland (ed. by W. Montagna, R. A. Ellis and A. F. Silver). New York: Macmillan. METIANU, T., LUCAS, A. & DROUHET, E. (1966). Teigne favique du dindon due a un nouveau dermatophyte. Mycopath. Mycol. appl. 22, 175-181. ODHAM, G. (1967). Studies of feather waxes of waterfowl. Ark. Kemi. 27, 295-307. OTCENAsEK, M., HUDEC, K., HUBALEK, Z. & DVORAK, J. (1967). Keratinophilic fungi from the nests of birds in Czechoslovakia. Sabouraudia 5, 350-354. PUGH, G.]. F. (1964). Dispersal of Arthroderma curreyi by birds, and its role in the soil. Sabouraudia 3, 275-278. PUGH, G.]. F. (1965). Cellulolytic and keratinophilic fungi recorded on birds. Sabouraudia 4, 85-91. PUGH, G.]. F. (1966a). Fungi on birds in India.]. Indian bot. Soc. 45,296-303. PUGH, G.]. F. (1966b). Associations between birds' nests, their pH and keratinophilic fungi. Sabouraudia 5, 49-53· PUGH, G.]. F. & EVANS, M. D. (1970). Keratinophilic fungi associated with birds.T. Fungi isolated from feathers, nests and soils. Trans. Br. mycol, Soc. 54, 233-240. REES, R. G. (1967a). Keratinophilic fungi from Queensland. II. Isolation from feathers of wild birds. Sabouraudic 6, 14-1 B. REES, R. G. (1967b). Keratinophilic fungi from Queensland. III. Isolation from feathers of domestic fowls. Sabouraudia 6, 19-28.
Transactions British Mycological Society ROTHMAN, S. & LORINCZ, A. L. (1963). Defense mechanisms of the skin. A. Rev. Med. 14, 21 5- 2 4 2 . SNOW, D. (1949). The germination of mould spores at controlled humidities. Ann. appl.
BioI. 35, 1-13· M. E. ( 1951). Control of humidity with potassium hydroxide, sulphuric acid and other solutions. Bull. ent, Res. 42 , 543-554. SPEARMA:-<, R. I. C. (1966). The keratinization of epidermal scales, feathers and hairs. BioI. Rev. 41, 59-96. TOMKINS, R. G. ( 19 3 2) . Measuring germination. Trans. Br. mycol. Soc. 17, 147-149.
SOLOMON,
(Accepudfor publication 3 October 1969)