Ind ian Journal of Experi menta l Bio logy Vol. 37, December 1999, pp. 12 18- 1222

Influence of fowl uropygial gland and its secretory lipid components on the growth of skin surface fungi of fowl

An urad ha 8andyopad hyay & S P B haltac haryya*

Department of Zoo logy, Uni versi ty or K~ l yan i . Kalyani 74 I 235. India

Received 5 Febnrary /999; rel'isl'd 24 A llgllsl /999

Funga l species, which were shown to colonize consistent ly on the skin su r face of the breast region or :Idu lt (I year old) whi te leghorn rowl , were identi t1ed as Aspergilllls srdoll 'ii, A. lamarii , A. m gll /oSils and Ahsidi(l ('O /Tll i bi/;' /'{i. 0 1' til e:,..:. A. sydowii and A. talllarii were the dominant rorms. Two species or ru ngi , namely, A.I'perg i /ill.l' lI iger and ScofJlI/(lri()/I.I'i.l' brevica ll lis were shown to be present in the cultures or the scrubbings from breast sk in ~urface alter 60 cbys or capti vit y or the fowls. Extirpation or the uropygia i gland resu lted in encouragement of the i ll I'ilm population growtil or all 'pecic> nr rungi except th::lt or A. m gllio.ws. The effec t was fou nd to be very conspicuous for A. sw/olI 'ii and A. 1(IIII(lui. panicul arly after 60 days of gland removal. Addition or to tal l ipids and the wax dies ter component o f free- Ilowing uropygi:11 secreti on a, 0.2% suspension in Sabouraud 's agar med ium or individua l fungal iso lates caused marked suppression or tile PO(1l1l :ltiol! growth or A. srdOl vii , A. lalllarii, Ab.l'idia corl'lI/ /;ijcm and to some exten t or S. hrel'ic{l lIii.l'. Other components or senct(lry lipids, slI ch as wax alcohols (2.3-a lkane-dio ls), wax acids, trlglycerides and hydrocarhons (i ncluding squaicne) II'hell supplemented separately to culture mediulll or individual fungi at identical concentration, were abo sil()\\'n tn C,IU\L'

inhihition of the growth or 1110, t of fungal species at different degrees.

The uropygial (preen) gland , present at the base of tai l feathers in fl yi ng birds, sec retes waxy materials that exh ibit so me uniqueness and enormous divers ities in thei r structuresl - -I, A generall y agreed opin ion is that uropygial secretion provides water-proof coating ove r plumages and maintai ns plumage texture l. 2 Few preliminary stud ies have reported inh ibitory effec ts of feather lipids of some birds on the growth of certain forms of saprophytic and ke ratinophilic fun gi U,6 . But the funct ional 'ignificance of the major lipid components of uropygi al secretion, particularly the ones that ex hi bit unusual features in their chemical make-up7, remains yet unexp lored. Effects of sevei'a l components of fowl uropygial gland I ipids on the growth of the popul ation of bacterial spec ies exi st in g on the sk in surface of fow l have been reported by us recentl l . Tn the present study we attempted to exami ne the inf luence of fow l Vropygial gland and the lipid components of its free-flowing secretion on the growth of fu nga l species occurri ng on the breast sk in surface of the same bird. Interes tingly, the uropygial gland of chicken and other ga lli fo rl1l birds so fa r examined, are fo und to secrete primarily the diester

. I 1 <) waxes of un usual 2, 3-a lkane-dl ols .. .

M aterials and Methods Experimental birds-Adu lt (20 weeks old) white

*Correspondcnt author

leghorn fow ls ( = 14) were procurecl frolll W. Bengal State Govern ment poul try farm . Fro ll1 7 fow l ~

uropygial glands were carefu ll y cxtirpated . Detai led procedures of gla nd removal and post -ope rat i ve measures taken ha ve been menti oncd in our previous reports8

.IO

. Glandless fow ls and tlie remaining 7 hirds with intact gland: (norma l) were housed so litar il y in separate cages and vere maintained for 60 days wi th standard chick feed (obtai ned from Stat e Govcrnment poul try farm) and water.

Cullure of skill Sill/ace .filllgi- Arler .iO- and 60 days of exti rpati on or the uropygia l gland, dermatophytes from 4 cm2 area or the breast :-.k ill surface of the left and the ri ght sides res pective ly or each normal and gland less row l were co ll ected hy ' scrub method ,x. lo.11 using buffered Trit on X-I OO. Serial 10-fold dilutions (upto 10(' ) or the inoc uluill were prepared with 0.075 M phos pil at e' buffe r (pH 7.9) ,10 and were plated on sterile pet ri dishes 1'0 1'

culture of fun gi in Sabouraud's Agar (SA) mediul1l '·('. After 7 days of incubation at n oc ind ividua l co lony fo rming units (c.f.u.) were counted. Representative iso lates of-all funga l fo rms we re ass igned to genera or broad groups on the bas is or mo rph ology and co lour of the colonies as well as by exa mi ning their reproducti ve structures aft er rou tine st aining with cotton bl ue I:! . Colo nies were ma intained sepa rate ly in appropriate agar slants at 28°C. Indi vidu al i ~o l at es

-t-

BANDYOPAD HYA Y & BH ATIACHAR YYA: INFLUENCE OF FOWL UROPYGIAL GLAND 12 19

were then sent to C. A. B. International Mycological Institute at Kew (Surrey, England) and to the Micro­biology Section of the Department of Botany of the University of Kal yani for identificati on of spec ies.

Collection of II ropygia I secretion and separation of

major lipid componen ts- Free-fl owing secreti ons fro m the glands of 20 adult white leghorn chicken were collected by inserting sterili zed glass canulae th rough the duct ori fice and appl ying mild pressure on the glands2

.8

-lo. Tota l I ipids from the secreti on were

extracted with ch loroform-methanol-water8.lo. u.

Extracts were filtered and lipid materi als were recovered after slow evaporation of so lvents between 45° and 50°C. Separation of individual lipid classes was achi eved by part ition chromatography employ ing the solvent grad ients as reported earlier8

.1:1 .14.

Homogenei ty of hydrocarbons (HY) (plus squa lene), diester waxes (DW) , triglyceri des (TG) and free fatty acids (FFA) was examined by co-chromatography on silica ge l G-coated plates wi th the fo ll owing standard (TLC grade) reference lip ids app lied on separate lane. These were: eicosane, squalene, triole in , pa lmitoleic acid (Sigma Chemical Co., USA) and highl y puri fied 2,3-d iol wax diesters recovered from the uropygial secretion of Japanese quail (Coturnix cntll rnix J . ) ~. IO I:; Ch d i d apol1lca '. romatograms were eve ope successively in mu ltipl e solvent systems: (a) hexane; (b) benzene and (c) hexanc-diethy lether-acetic acid glacial (70 : 30 : 1)1).16 . Wax acids (WA) and wax alcohols (W-OL) (i. e., alkane-2,3-diols) were separated by saponificati on of the DW fracti onl7, which was elu ted from glandular lipids by co lumn chromatography as mentioned above. The identity of W-OL thus recovered was further examined by TLCI~.

Effects of uropygia/ lipids (lnd individual lipid

components on fill/ga l population - Samples (20 mg) of secretory total lipids and each indiv idual lipid component (Tab le I ) were suspended in mini mum quantity (0.2 ml ) of di ethyl ethcr and were added to the cu lture tubes that contained 10 ml of SA medium in order to make 0.2% concent rat ion as per Baxter and Trotler6

. To the med ium in the tubes marked 'control', only 0.2 ml of diethylether was added. The materi als were then dispensed homogeneously and the solvent was removed by co nstant shaking at 50°C in a metabol ic shaker. At 28°C tu bes containing SA broth with or without (control) uropygial lipid components were inocu lated with 0 .1 ml of spore suspension (one loop-full spores of each fun ga l species was suspended in 10 ml of sterile water and fro m th is 0. 1 ml was

used as inoculum). The fun ga l spec ies were al lowed to grow for 7 days in the medium. Co loni es ,ve re then separated and dri ed for 24 hI'. Weights of dry co lonies were recorded. The weights of individual funga l colonies obtained from cultures after Llclditio n of secretory total lipids and indi vidual li pid components to the mediu 'll were compared with the weights of dry colonies of same spec ies of fu ngi obta ined from cultures in control medium .

Results and Discussion Out of 6 spec ies of fungi iso lated from the ski n of

the breast region of fow ls, 4 spec ies namely, Aspergillus sydowii (Fig. I ) , A. tallw rii (Fig. 2). Absidia corymb!fem (Fig. 3) and Aspergillus rugillosus were fo und to co loni ze consistentl y on the sk in surface . A. sydowii and A. t{fl/wrii we re the predominant forms (Fig. 6). Two species. A.I'l'('I'gilllfS niger (Fig. 4) and Scop lf/a riopsis /m'I'i(,([ lf li.1 (Fig. :'i )

were found to be absent in the cul tures of the sc rubs from normal fow ls initi al ly and aft er 30 days of capti vity of the birds. In few cu lt ures. ~I small population of Candida albic([l/s was identified . Since , it s presence was very inconsis tent. it was not considered subsequently for examination 0 (' the effects of uropygial li pid co mponent s. It may h.e ment ioned that the numbe r of species of fungi existing on feathers and the skin surface of birds includ ing the chi cken may be variab le. Becau se. Illan y fun gal species mi grate from feed . soi I, hu mous and

h d · . I I ') 1() d I' I ot er ecay rng materi a s '- . an co onl ze t le~e structures. ln addition , diffe rences III bod y temperature, mo isture content of the epidermis. specialized features of plu mages as well as relative humid ity may also be some or the det ermi nin ~ fac tors)' 11 .2 1. -

Resu lts of the present stud y amp ly demonstrate that the uropygial gland and its secreto ry components ha ve important ro le in the regul at ion of the occurrence and growth of fungal dermatophytes of the skin surface or adult fowl. Ablation of the gland ca used increase in the popul ation of all the funga l fo rms except A.

ruguloslIs, the growth of whic h seemed to be completely suppressed after gland remova l (Fig. 6). Promotion of popul ati on growth of A . .I'rc!{!I\ ·ii anc! A. tamarii was found to be remarkab le. pa rticularly ai'ter 60 days of gland remova l (F ig. 6). Two spec ies of pathogenic fungi (A. I/iger and S. bn' l'ic{flfli.l' j, which did not appear in the cultures from normal fowls until 60 days of capti vity, were fo und to occur in the cultures from glandless fowls after 30 days of

1220 IND IAN J EXP BIOL, DECEMBER 1999

extirpati on of the uropygial gland and their popu lation also exhibited a moderate ri se after 60 days (Fig. 6).

From the data presented in Table I it is c lear that add ition of total lipids of free- fl owin g sec retion frolll

Table I - Effects of to tal li pid material and it s major co mponents from free- Il owin g sec re ti on o f fow l uropyg i ~ 1 g land on ill "i,m gnmth of fun gi isolated from breas t skin surface o f adult fowls

I Values indi cate mean dry weight s ± SE of the popu lat ion of 5 cultures o f individual fun gi I

Fungal species Control val ues Values a ft e r addi tion of secretory lipid compone nt s Total lipids Di es ter wax Wax alco hol Wax acids Tri g lyce rides Il yd rocarho ll ,

Aspergilills sydOlvii I 10.05 ± 6. 10 56 .30 ± 3.30 36.01 ± 0.52 91.28 ± 2.80 39.63 ± 0 .. 0 46. 15 ± 2...+1 3-1 0) ± 2.37 Aspergilills lall1arii I 17.55 ± 5.4 1 73.45 ± 2.42 54.63 ± 0 .80 45.35 ± 3.80 4 1.65 ± 1.60 65 J, 5 ± I.eLI -16.65 ± 5 .1 7 Absidia corYll1bifera 126.63 ±4.7 1 74.25 ± 2.3 1 42.05 ± 1.1 3 91.25 ± 3. 18 88.60 ±3. IS l) 1.50 ± ). \J 36.30 ± O.XX Aspergilllls m gllloslls 132.60 ± 3.73 138.00 ± 5.39 142. 30±4. IO 132.50 ± 5. 10 125 .00 ± 2. sn 137.:l.'i ± 7. IO 1.\(1.60 ± X.03 Aspergilills Iliger 77.62 ± 2.4 3 52 .04 ± 2.50 72.34 ± 3. 10 46.00 ± 30 1 24 .34 ± 2. 12 BO.60 ± 3.23 7(J.30 ± S.2()

Scopulariopsis brevicalliis 16 1.00 ± 5.21 127.60± 4. 12 80.90 ± 2.6 1 125.04 ±H9 124.65 ± 8.06 148.37 ± 1.(1.\ ! -1-1 .-10 ± :1.20

L _____ -'

... .4

50 f-lm 50 JJm

;A..l.r.

'fl

' . III I 50 jJm -1 :;

Fi gs 1-5 - Photomicrographs of some fun gi isolated from the cul tures of breast skin su rface scrubbings 01' ad ult fow ls. Lacto phcno l cotton blue stain ing. Magnili cation s, as shown by the scale on figures. 1- Aspergillus sydOlvii. 2- A. /(I/llarii showi ng d istinct vesicle and globose spores. 3 - Ahsidia cornllill/e /"{{ with hyalinc sporangiophores and globose sporangia (-7). 4 - Aspergillus niger. 5 - ScoPlllariopsis brevicalliis showing short hvph ~lC and thick­walled, pyriform conid ia.

BANDYOPADHYAY & BHATIAC HARYY A: INFLUENCE OF FOWL UROPYGIAL GLA 0 1221

240,-------------------------------------,

200

:;; 120 a.

" ...: u 1r0 c o .. ~

40

o

-I .~

0

" ~ <il

~ '.

=\ ~I (; E .9

~I ~I

030 days after gland removal ~50 days otter gland removal o Corresponding control values

C:L.LL cO:£J ~ : ..

II e VI 2 :; .D 0 E u ;:- '> 0 ~

<il u .D

<il VlI

Fig. 6 - Changes in fungal population from breast ski n surf<lce of fowl after 3()- and 60 days of uropygial gland ex ti rpation . Gland removal is shown to result in the increase of colony growth of al l Ihe forms except A. m gll/o.w s. The effect is spectacu lar for A. sydoll' ii and A. Iallwrii.

the uropygial gland to the culture broth of individual fu ngi caused in most cases suppress ion of fungal growth , the magnitude of which was marked for A. sydowii (49.3%), A . tamarii (37 .6%) and Absidia cOIymbifera (41.3%) . OW also produced similar (in some cases, moderately greater) inhibitory effects on the populati on growth of these fungi (Table !). This was not unexpected, since uropygial secretion of domestic ch icken was fou nd to contain as much as 97% of OW22

.

When W-OL and WA, recovered by the hydro lys is of OW, were supplemented separate ly to the culture medium of indi vidual fungi , considerable degree of inhibition in the co lony growth of A. sydowii, A . lamarii, A. niger, Absidia corymb!fera and S. brevicaulis was observed . As W-Ols from the uropygial glands of all the ga lliform birds so far examined , have been identi fied as the unusual 2,3-I k I· I I-,??? , I I f I a ane-( 10 s .. --.-. , t le resu ts 0 t lC present study

seem to be signi fica nt. From the result s it is also clear that alkane di ols from the glands of domestic chicken were equ all y effective against fungal growth in both free- and esterified states (Table I). The inhibitory ac tion of WAs on the prolifcration of the co lon ies of A. sydowii and A. niger from breast skin surface of fowl seemed to be of greater magnitude than the effects of W-OL on these spec ies (Table I). In thi s context it may be mentioned that WAs of the secretory lipids from uropygial gland of fowl have

been found to exert considerable suppression or the ill vitro growth of skin surface bacteria of the same bi rcr

It seems pertinent to cite earl y reports concernin g the inhibitory effects of human sebum on a nu mber of pathogenic fun gi that coloni ze on the sk in surface or man24

. Baxter and Trotter6 also observed considerable degree of suppress ion of ill I'ilm growth or hielw­phylon mellfag rophytes, T. ({ielloi, M icro'\'/J0/'01I audouinii and Penicilliul/1 ./i'eqll({lI t({II S after add iti on of 0.2% hu man hair fat in the culture medi um. In these cases, the effects were attributed to the inhibitory actions of short- and long chained rree fatty acids, . which are formed of even- and odd ca rbon numbers. Furthermore, beta-hyd roxy fa tty acids, whi ch are present in the plumage lipids of common pi geon and are main constituent s of the uropyg ial gland secretions of Europcan starl i ng and wood pigeon, have also been implicated in the suppress ion of the population of man y saprophyt ie, kerat i nophil ic and non-keratinophi I ic funga l derlllatophytes ex ist i ng on plumages I. 25. 26 .

Addition of 0.2% TG and HY fracti ons to culture med ium resulted In marked inhibiti on of the population of A. -"ydo ll 'ii , A. w nwrii and Ahsidi({ corymbifera , and to a little exte nt or S. hrel'ical/lis (Table I ). As TG is present in th e uropyg ial sec reti on of fowl in very low concent rat i on ~.1 , it is not certa in to what extent this component from the uropygial gland is in volved in the regul ati on of the fun ga l populati on of plumages and sk in surface of fowl. Howcver. TG from the epidermal source in birds, part icularl y or the corneocytic domain , where it is present in hi gh concentrati on27

, may have some in volvement in thi s regard ; and such inhibitory acti on Illay bc p ~lrtl y

attributed to free fatty acids released a~ a result or hydrolys is ofTG by epidermal and bactcriallipa:-es ' .

The suppress ive acti on of HY fraction , which al so included squal ene (SQ), on the populati on of run gi from fowl skin surface is not unex pected. as st rong growth inhibitory effect of SQ at low concentrations on some forms of pathogenic fun gi rrom hUlllan sk in

f d I () ox TI' ,.. I' sur ace was reporte ear y . - . li S Inc In g seems to be noteworthy, since HY and SQ have been reported to be major constituents of the uropyg ial lipids or some birds2'). 30.

Last ly, from the results of the present study it Illay be concluded that the uropygial gland of fowl as well as it s secretory lipids and the individual lip id components play a role in the regulati on or the population growth of fun ga l spec ies whi ch occur on the sk in surface of thi s bird. III vilro growth inhibiti on

1222 IND IAN J EX P BIOL, DECEMBER 1999

has been recorded when secretory lipids or individua l components were supple mented to the culture medium, a lthough the magnitude of responses of fu ng i was found to vary. Aspergillus rugu/osus may be cons idered as an exception, since its growth was found to be complete ly suppressed after extirpation o f uropygia l g land and it did not show sensitivity to the lipid classes of uropyg ial sec ret ion.

Acknowledgement T hi s invest igation was financ ia ll y supported by a

grant from UGC, New De lhi . Authors are grateful to C. A . B. Inte rnat iona l In stitute of Mycology at Kew (Surrey) , E ngland for carefull y identifying the fungal species and to Dr. (Mrs.) Mira Sen and Prof. S. P. Sen, Emeritu s Professor of the Department of Botany, Kalyan i Unive rs ity for the ir inte res t in thi s work and for va luable suggesti ons and he lp.

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