Journal o f Chemical Ecology, Vol. 8, No. 9, 1982
M I C R O F L O R A A N D VOLATILE FATTY A C I D S P R E S E N T IN I N G U I N A L P O U C H E S OF THE WILD RABBIT, Oryctolagus cuniculus, IN A U S T R A L I A
G.C. MERRITT,
I B.S. G O O D R I C H ,
2 E.R. HESTERMAN,
a n d R. M Y K Y T O W Y C Z ,
3
3
~Division o f Animal Health, CSIRO, McMaster Laboratory Private Bag No. 1, Glebe, N.S. W. 2037, Australia 2Division o f Wildlife Research, CSIRO P.O. Box 52, North Ryde, N.S.W. 2113, Australia 3Division o f Wildlife Research, CSIRO P.O. Box 84, Lyneham, A.C.T. 2602, Australia (Received October 31, 1981; revised February 2, 1982) Abstract--Contents of inguinal pouches of the wild rabbit, Oryctolagus cuniculus, have been examined for the presence of microorganisms and volatile fatty acids. A total of 245 isolates were made from 167 samples taken from 75 rabbits, and microorganisms were obtained from 93% of individuals. The most common microorganism encountered was Staphylococcus aureus, followed by the yeast Candida kruzei. Other organisms isolated were Bacillus subtilis, Escherichia coli, and Streptococcus faecalis. Quantitative analysis of volatile fatty acids in scrapings from pouches showed considerable variation between individuals. Of the acids identified, acetic acid and isovaleric acid were the most abundant. In a liquid medium containing one of these acids, the growth of microorganisms was only slightly affected. However, growth became inhibited when these fatty acids were used in combination. Key Words--Rabbits, Oryctolagus cuniculus, inguinal pouch, microorganisms, volatile fatty acids, acetic acid, isovaleric acid, odor signals.
INTRODUCTION H u m a n s w e a t s e c r e t e d b y a p o c r i n e g l a n d s is o d o r l e s s w h e n it a p p e a r s o n t h e s k i n s u r f a c e i n t h e a x i l l a . I t is o n l y a f t e r a f e w h o u r s , i n t h e p r e s e n c e o f m i c r o o r g a n i s m s , t h a t it a c q u i r e s a n i n d i v i d u a l l y c h a r a c t e r i s t i c s m e l l ( S h e l l e y et al., 1953). T h e h a i r s i n t h e a x i l l a a c t as c o l l e c t i n g sites, n o t o n l y f o r 1217 0098-0331/82/0900-1217503.00/0 9 1982 Plenum Publishing Corporation
12t8
MERRITT ET AL.
glandular secretion and skin debris, but also for bacteria. Shaving and thorough washing of the axilla may eliminate odor for at least a few hours. Similarly in other mammals, specially modified sites on the body offer ideal conditions for the collection of glandular secretions and the growth of bacteria which are involved in their fermentation (Mykytowycz, 1970). Increasing interest in the function of the odoriferous glands in the communication of mammals stimulated a parallel concern for the role of bacteria in shaping the odor profiles. The bacteriology of the skin gland secretions has already been studied in some species; e.g., the microflora have been described in the anal sacs of the red fox, Vutpes vulpes (Ware and Gosden, 1980); in the anal and castor glands of the beaver, Castor canadensis (Svendsen and Jollick, 1978); and in the anal pockets of the Indian mongoose, Herpestes auropunctatus (Gorman et al., 1974). It has been suggested that microorganisms play an important role in shaping odor signals in mammals and that volatile fatty acids which are frequently found in skin gland secretions could be the products of their fermentation (Albone et al., 1977; Albone and Perry, 1976; Albone, 1977). The wild rabbit, Oryctolagus cuniculus, is well equipped with odorproducing skin glands which function specifically for communication purposes. The glands were the subject of prolonged interdisciplinary studies (Mykytowycz, 1968; Goodrich and Mykytowycz, 1972). The inguinal glands, which communicate identity and sex (Hesterman and Mykytowycz, 1982), secrete into bilateral pouches where the secretion and desquamated epithelium form a thick, waxy sebum which has a strong rabbity odor (Mykytowycz, 1966). These pouch characteristics seem to offer suitable conditions for microorganisms. Preliminary studies of the chemical composition of inguinal pouch contents indicated the presence of volatile fatty acids. A number of synthetic volatile fatty acids, when presented to rabbits under experimental conditions, caused changes in heart rate (Hesterman et al., unpublished). In the present study an effort has been made to gain some information on the volatile fatty acid profiles and microorganisms in the inguinal pouches and their possible interaction.
M E T H O D S AND MATERIALS
Animals Of 75 wild-type rabbits sampled, 40 were males, which included four 10-day-old kittens, and 35 were females. All rabbits sampled had been in captivity for at least three months. Nineteen of them were born in captivity, while the others were caught in the field in various localities in the vicinity of
CONTENTS OF W I L D RABBIT I N G U I N A L POUCHES
1219
Canberra. Some were kept singly in small wire cages, others lived in 2 • 4-m outdoor pens in groups consisting of three or four individuals.
Sampling Five bacteriological samplings were carried out at irregular intervals covering a period of 19 months and altogether 167 samples were taken. In the first three samplings only aerobic cultures were made. Both aerobic and anaerobic methods were used for the last two samplings. The frequency of sampling of the pouch contents f r o m individuals was also irregular, i.e., 84, 197, 231, and 560 days following first sampling. Material was taken once from 30 rabbits, twice from 22, three times f r o m 4, four times f r o m 14, and five times from 5 animals. On one occasion only, swabbings were made of the back and the belly areas of I 1 rabbits of different sexes.
Bacteriological Procedures Collection of Samples. During sampling rabbits were restrained manually. Since inguinal sebum is thick, waxy, and insoluble in water, irrigation by pipet or syringe and needle was impractical. Sterile cotton-wool swabs moistened immediately before use with sterile distilled water were employed to collect samples from pouches. For all samples, blood agar, serum agar, and Mycosel agar plates, sown immediately from the swabs, were incubated aerobically at 37 ~ C. During the last two samplings, a duplicate set of plates for each sample was incubated anaerobically at 37 ~ C within 5 rain of collection using the GAS P A K method and a self-heating catalyst. Following incubation, colonies were counted, and organisms were checked for morphology and G r a m reaction. All organisms were immediately subcultured onto horse blood agar plates and subsequently identified by conventional methods. Inhibition of Growth of Microogranisms by Volatile Fatty Acids. Dilutions of acetic acid or isovaleric acid from 1 : 10 to 1 : 1 0 6 (V/V) were made in nutrient broth. To each tube was added one standard drop (0.025 ml) of a 24-hr broth culture in the case of Staphylococcus, or a 48-hr-broth culture in the case of Candida. The cultures were then incubated at 37~ for seven days with occasional shaking.
Volatile Fatty Acid Analysis Sample Preparation. Samples of sebum from the inguinal pouches of individual animals (18 males and 18 females) were collected and stored 3-6 months at - 2 5 ~ prior to use. The scrapings (10-20 mg) were triturated with a mixture of 0.5 ml ether and 0.5 ml of 0.1 M hydrochloric acid, and the organic layer was separated
1220
MERRITT ET AL.
out. The aqueous layer was extracted a further three times with 0.5 ml ether and the combined ether extracts shaken twice with 0.5 ml of 0.05 M sodium hydroxide. The alkaline extract was acidified with 0.4 ml of 5 M sulfuric acid and then steam distilled into 2 ml of 0.05 M sodium hydroxide unitl 15 ml of distillate had been collected. The distillate was reduced to a small volume on a rotary evaporator, transferred to a small glass vial and evaporated to dryness at 40~ under a stream of nitrogen. Just prior to gas chromatographic analysis, 20 #1 of water was added to the sample, followed by 30 #1 of 7 M hydrochloric acid. Gas Chromatography. A Varian Aerograph model 204B equipped with flame ionization detectors was used for the analysis of volatile fatty acids in the inguinal pouch scrapings. The test sample (0.5 #1) was injected through a Teflon-faced septum onto the front portion of a nickel column (2 m • 2 m m ID) packed with Chromosorb 101,100-120 mesh (Johns-Manville). Temperatures were: injectors 220 ~C; detectors, 250 ~C; column, 180 ~ C. Gas flow rates were: N2 carrier gas, 25 ml/min; H2, 25 ml/min; air, 150 ml/rain. Tentative identification of the acids in the samples was based on comparisons of their retention times with those of the authentic compounds. Quantitative determination of the acids in sample solutions was based on comparisons of their peak areas to those of the standard solutions.
RESULTS
One or more species of microorganisms were isolated from the inguinal pouches of 70 (93%) of the 75 rabbits sampled. Cultures from the pouches of two males (5%) and three females (9%) failed to grow microorganisms. Bacteria were found in the inguinal pouches of all the 10-day-old nestlings. Microorganisms were isolated from 155 (93%) of the total 167 samples. Altogether 245 isolates were cultured, representing six genera with the two most c o m m o n being the facultative aerobes Staphylococcus and Candida. In addition to these organisms, Bacillus subtilis, Escherichia coli, and Streptococcusfaecalis were occasionally isolated, with Corynebacterium ovis found only once. Most frequently only one genus was cultured from a single sample (76%). Two different genera were found in 20% of samples and three in 4%. There were no obvious sex differences in this respect. Repeated sampling revealed that the microflora in the inguinal pouches of an individual did not remain constant throughout the period of the study. Thus from the 19 females which were sampled at least twice, only three (16%) carried the same microorganism, and of the 22 males which were sampled at least twice, the same microorganisms were found in only seven (32%). Of the 22 samples cultured from the back and belly of 11 rabbits, a total of only 12 colonies grew.
C O N T E N T S OF W I L D R A B B I T I N G U I N A L P O U C H E S
1221
Staphylococcus aureus. This was the most c o m m o n isolate. It was cultured from 110 samples (66% of total), and occurred in 83% of all males and 77% of females. Primary culturing on serum agar produced most frequently a moderate growth, i.e., less than 100 colonies per plate. The same organism was also found in 73% of the anaerobic cultures. Candida kruzei. This facultative aerobic yeast was the second most c o m m o n organism encountered, being found in 60 samples (39%). It occurred in 50% of males and 60% of females. During primary culturing moderate growth was produced in 86% of the positive samples. F r o m a total of 67 samples cultured anaerobically, 23 (34%) produced C. kruzei. Bacillus subtilis. This organism was found only in nine (23%) males. It was isolated from 11 (7%) of total samples. The cultures yielded a light growth of this organism, except in one case where there was moderate growth. Escherichia coli. This organism was isolated only during the last two samplings, and only from five females. It appeared in six (4%) of all samples, producing only light growth. Streptococcus faecalis. This organism was found on only four occasions throughout the sampling period, twice in males and twice in females. Corynebacterium ovis. This organism was found only in scrapings taken from one male with only three colonies isolated. Volatile Fatty Acids. The relative quantities of eight volatile fatty acids in scrapings from 18 males and 18 females are shown in Table 1. These are expressed as a range because of the wide fluctuation of results between individuals. Acetic acid was the most common, constituting on the average approximately 80% of total volatile fatty acids in males and 74% in females. The second most abundant was isovaleric acid (approximately 16% of total in males and 23% in females). Six other acids occurred in much smaller quantities. The two most abundant acids--acetic and isovaleric--had an effect on the in vitro growth of the most c o m m o n organisms, S. aureus and C. kruzei. Acetic and isovaleric acids when used together at dilutions down to 103 prevented the growth of the two most c o m m o n isolates. Staphylococcus and Candida. C. kruzei appeared to be more affected by the mixture of two acids as there was some effect on growth at dilutions of 104. The two acids when used separately were less effective in inhibiting growth of these microorganisms (Table 2).
DISCUSSION
When the pattern of social behavior of the rabbit is considered (Mykytowycz, 1968), it is possible that more selective sampling could reveal a pattern of group, colony, or geographical distribution of specific microflora.
79,7 (16.5-98.9) 73.8 (13.1-99.1)
2.3 (0.3-9.3) 0,6 (0.1-3,9)
Propionic (C3) 1,1 (0.1-8.3) 1.0 (0.1-7.6)
lsobutyric (iC4) 0.3 (0.1-2.0) 0.1 (0.1-O,4)
n-Butyric (nC4) 16.1 (0.7-84,0) 23.0 (0.1-85.7)
Isovaleric (iCs)
0.1 (0J-2.1) 0,9 (0.2-11.9)
n-Valeric (nCs)
aThe mean (and range) concentrations of C2-C 6 acids in samples from 18 male and 18 female rabbits are shown.
Females (N = 18)
Males (N = 18)
Acetic (C2)
Composition (mot %)
Oryctolagus cuniculus a
0,2 (0.1-1.9) 0,5 (0,4-3.3)
Isocaproic (iC6)
0.3 (0.2-4.4) 0,2 (0,3-1,9)
n-Caproic (nC6)
TABLE 1, VOLATILEFATTY ACIDCOMPOSITION OF INGUINAL POUCH SCRAPINGS FROMMALEAND FEMALE RABBITS,
t'~ ~4
~q
t.# b.)
1223
CONTENTS OF WILD RABBIT INGUINAL POUCHES
TABLE 2. I N H I B I T O R Y E F F E C T OF ACETIC ACID ( C 2 ) AND I S O V A L E R I C A C ID ( i C s ) ON BROTH C U L T U R E S OF S. aureus AND C. k r u z e i I S O L A T E D FROM I N G U I N A L POUCHES OF W I L D RABBITS
D i l u t i o n factor
Micro0rganism ~. attreug
C. kruzei
Acid
10- 2
10- 3
10- 4
10- 5
10 - 6
C2 iC 5
_a +
+ +
+ +
+ +
+ +
C2 + iC5
-
-
+
+
+
C2
+
+
+
+
+
iCs C2 + iCs
+ -
+ -
+ -+
+ +
+ +
a+, growth; +-, retarded growth; -, no growth.
The seemingly high incidence of S. aureus and C. kruzei found may be peculiar to the experimental colony involved in this study. If microorganisms such as Staphylococcus, Candida, etc., are indeed frequently carried by the rabbit in its natural environment, it could play some role in the epidemiology of diseases caused by these species in domestic animals. At this stage one can only speculate that fermentation of the skin gland secretion is necessary for the formation of functional od or signals because the experimental data are inadequate to substantiate such a contention. In fact, observations during the present study and views on the anal glands of the fox (Albone and Perry, 1976) and the bacteriology of the beaver's scent glands (Svendsen and Jollick, 1978), suggest that the microbial populations, and consequently the fermentation of secretions, may not be sufficiently consistent to form a basis for the production of olfactory signals. It is possible, however, that conditions of captivity were responsible for the seemingly high incidence of S. aureus and C. kruzei and for frequent changes in the microflora profiles of animals used in these studies. On the other hand, in some instances periodic alterations of the quality of personal odor may even be essential to communicate changes in the behavioral or physiological status of an individual. Thus microbial populations in the human vagina change in response to endocrine factors at puberty and menopause (Brown, 1978). Fluctuation of estrous cycles have been shown to influence short-term changes in microbial flora in the vagina of rats and rhesus monkeys, Macaca rnulatta, and in the latter may also affect the quality and rate of production of copulins (Bonsall and Michael, 1980). The mongoose Herpestes auropunctatus can be trained to differentiate between mixtures of volatile fatty acids (Gorman, 1976). This finding reinforces the
1224
MERRITT ET AL.
speculation that different mixtures of acids in an animal's odor could be at least partly responsible for individual recognition. The synergistic inhibitory effect of acetic and isovaleric acids on the growth of microorganisms suggests the existence of an additional mechanism for shaping the odor profiles of the inguinal pouches. Inclusion of other acids may increase the titer of the inhibitory effect. To test more fully the importance of this mechanism, it is necessary to study systematically the fluctuations in levels of the different acids in the same animal in relation to the density of populations of different microorganisms. However, there is a need first of all to establish whether the volatile fatty acids are produced in the glands or are a product of microbial fermentation. In addition, other constituents of the pouch and their contribution to the final odor signals would need to be investigated. Acknowledgments--We thank S. Gambale, C.C. Reece, and T. Williams for their diligent and skillful assistance.
REFERENCES ALBONE, E. 1977. Ecology of mammals--new focus for chemical research. Chem. Br. 13:92-112. ALSONE, E.S., and PERRY, G.C. 1976. Anal sac secretion of the red fox, Vulpes vulpes; volatile fatty acids and diamines: Implications for a fermentation hypothesis of chemical recognition. J. Chem~ Ecol. 2:101-111. ALl,ONE, E.S., GOSDEN, P.E., and WARE, G.C. 1977. Bacteria as a source of chemical signals in mammals, pp. 35-43, in D. Miiller-Schwarze and M.M. Mozell (eds.). Chemical Signals in Vertebrates. Plenum Press, New York. BONSALL,R.W., and MICHAFL,R.P. 1980. The externalization of vaginal fatty acids by the female rhesus monkey. J. Chem. Ecol. 6:499-509. BROWN, W.J. 1978. Microbial ecology of the normal vagina, pp. 407-422, in E.S.E. Hafez and T.N. Evans (eds.). The Human Vagina. Elsevier, North Holland, Amsterdam. GOODRICH, B.S, and MV~YTOWYCZ, R. 1972. Individual and sex differences in the chemical composition of pheromone-like substances from the skin glands of the rabbit, Oryctolagus cunieulus. J. Mammal. 53:540-548. GORMAN, M.L. 1976. A mechanism for individual recognition by odour in Herpestes auropunetatus (Carnivora: Viverridae). Anita. Behav. 24:141-145. GORMAN, M.L., NEDWELL,D.B., and SMITH,R.M. 1974. An analysis of the contents of the anal scent pockets of Herpestes auropunctatus (Carnivora: Viverridae). d. ZooL 172:389-399. HESTERMAN, E.R., and MYKYTOWYCZ,R. 1982. Misidentification by wild rabbits, Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion, t. Experiments with all-male groups. J. Chem. Ecol. 8:419-427. MYKYTOWYCZ, R. 1966. Observations on odoriferous and other glands in the Australian wild rabbit, Oryctolagus euniculus (L.), and the hare, Lepus europaeus: II. The inguinal glands. CSIRO Wildl. Res. 11:49-64. MYKYTOWYCZ, R. 1968. Territorial marking by rabbits. Sci. Am. 218:116-119, 123-126. MYKYTOWYCZ, R. 1970. The role of skin glands in mammalian communication, pp. 327-360, in J.W. Johnston, D.G. Moulton, and A. Turk (eds.). Advances in Chemoreception, Vol. I, Communication by Chemical Signals. Appleton-Century-Crofts, New York.
CONTENTS OF WILD RABBIT INGUINAL POUCHES
1225
SHELLEY,W.B., HURLEY,H.J., and NICHOLS,A.C. 1953. Axillary odor; experimental study of the role of bacteria, apoerine sweat and deodorants. Arch. Dermatol. Syphilol. 68:430-446.
SVENDSEN,G.E., and JOLLICK,J.D. 1978. Bacterial contents of the anal and castor glands of beaver (Castor canadensis). J. Chem. Ecol. 4:563-569.
WARE,G.C., and GOSDEN,P.E. 1980. Anaerobic microflora of the anal sac of the red fox (Vulpes vulpes). J. Chem. Ecol. 6:97-102.
M I C R O F L O R A A N D VOLATILE FATTY A C I D S P R E S E N T IN I N G U I N A L P O U C H E S OF THE WILD RABBIT, Oryctolagus cuniculus, IN A U S T R A L I A
G.C. MERRITT,
I B.S. G O O D R I C H ,
2 E.R. HESTERMAN,
a n d R. M Y K Y T O W Y C Z ,
3
3
~Division o f Animal Health, CSIRO, McMaster Laboratory Private Bag No. 1, Glebe, N.S. W. 2037, Australia 2Division o f Wildlife Research, CSIRO P.O. Box 52, North Ryde, N.S.W. 2113, Australia 3Division o f Wildlife Research, CSIRO P.O. Box 84, Lyneham, A.C.T. 2602, Australia (Received October 31, 1981; revised February 2, 1982) Abstract--Contents of inguinal pouches of the wild rabbit, Oryctolagus cuniculus, have been examined for the presence of microorganisms and volatile fatty acids. A total of 245 isolates were made from 167 samples taken from 75 rabbits, and microorganisms were obtained from 93% of individuals. The most common microorganism encountered was Staphylococcus aureus, followed by the yeast Candida kruzei. Other organisms isolated were Bacillus subtilis, Escherichia coli, and Streptococcus faecalis. Quantitative analysis of volatile fatty acids in scrapings from pouches showed considerable variation between individuals. Of the acids identified, acetic acid and isovaleric acid were the most abundant. In a liquid medium containing one of these acids, the growth of microorganisms was only slightly affected. However, growth became inhibited when these fatty acids were used in combination. Key Words--Rabbits, Oryctolagus cuniculus, inguinal pouch, microorganisms, volatile fatty acids, acetic acid, isovaleric acid, odor signals.
INTRODUCTION H u m a n s w e a t s e c r e t e d b y a p o c r i n e g l a n d s is o d o r l e s s w h e n it a p p e a r s o n t h e s k i n s u r f a c e i n t h e a x i l l a . I t is o n l y a f t e r a f e w h o u r s , i n t h e p r e s e n c e o f m i c r o o r g a n i s m s , t h a t it a c q u i r e s a n i n d i v i d u a l l y c h a r a c t e r i s t i c s m e l l ( S h e l l e y et al., 1953). T h e h a i r s i n t h e a x i l l a a c t as c o l l e c t i n g sites, n o t o n l y f o r 1217 0098-0331/82/0900-1217503.00/0 9 1982 Plenum Publishing Corporation
12t8
MERRITT ET AL.
glandular secretion and skin debris, but also for bacteria. Shaving and thorough washing of the axilla may eliminate odor for at least a few hours. Similarly in other mammals, specially modified sites on the body offer ideal conditions for the collection of glandular secretions and the growth of bacteria which are involved in their fermentation (Mykytowycz, 1970). Increasing interest in the function of the odoriferous glands in the communication of mammals stimulated a parallel concern for the role of bacteria in shaping the odor profiles. The bacteriology of the skin gland secretions has already been studied in some species; e.g., the microflora have been described in the anal sacs of the red fox, Vutpes vulpes (Ware and Gosden, 1980); in the anal and castor glands of the beaver, Castor canadensis (Svendsen and Jollick, 1978); and in the anal pockets of the Indian mongoose, Herpestes auropunctatus (Gorman et al., 1974). It has been suggested that microorganisms play an important role in shaping odor signals in mammals and that volatile fatty acids which are frequently found in skin gland secretions could be the products of their fermentation (Albone et al., 1977; Albone and Perry, 1976; Albone, 1977). The wild rabbit, Oryctolagus cuniculus, is well equipped with odorproducing skin glands which function specifically for communication purposes. The glands were the subject of prolonged interdisciplinary studies (Mykytowycz, 1968; Goodrich and Mykytowycz, 1972). The inguinal glands, which communicate identity and sex (Hesterman and Mykytowycz, 1982), secrete into bilateral pouches where the secretion and desquamated epithelium form a thick, waxy sebum which has a strong rabbity odor (Mykytowycz, 1966). These pouch characteristics seem to offer suitable conditions for microorganisms. Preliminary studies of the chemical composition of inguinal pouch contents indicated the presence of volatile fatty acids. A number of synthetic volatile fatty acids, when presented to rabbits under experimental conditions, caused changes in heart rate (Hesterman et al., unpublished). In the present study an effort has been made to gain some information on the volatile fatty acid profiles and microorganisms in the inguinal pouches and their possible interaction.
M E T H O D S AND MATERIALS
Animals Of 75 wild-type rabbits sampled, 40 were males, which included four 10-day-old kittens, and 35 were females. All rabbits sampled had been in captivity for at least three months. Nineteen of them were born in captivity, while the others were caught in the field in various localities in the vicinity of
CONTENTS OF W I L D RABBIT I N G U I N A L POUCHES
1219
Canberra. Some were kept singly in small wire cages, others lived in 2 • 4-m outdoor pens in groups consisting of three or four individuals.
Sampling Five bacteriological samplings were carried out at irregular intervals covering a period of 19 months and altogether 167 samples were taken. In the first three samplings only aerobic cultures were made. Both aerobic and anaerobic methods were used for the last two samplings. The frequency of sampling of the pouch contents f r o m individuals was also irregular, i.e., 84, 197, 231, and 560 days following first sampling. Material was taken once from 30 rabbits, twice from 22, three times f r o m 4, four times f r o m 14, and five times from 5 animals. On one occasion only, swabbings were made of the back and the belly areas of I 1 rabbits of different sexes.
Bacteriological Procedures Collection of Samples. During sampling rabbits were restrained manually. Since inguinal sebum is thick, waxy, and insoluble in water, irrigation by pipet or syringe and needle was impractical. Sterile cotton-wool swabs moistened immediately before use with sterile distilled water were employed to collect samples from pouches. For all samples, blood agar, serum agar, and Mycosel agar plates, sown immediately from the swabs, were incubated aerobically at 37 ~ C. During the last two samplings, a duplicate set of plates for each sample was incubated anaerobically at 37 ~ C within 5 rain of collection using the GAS P A K method and a self-heating catalyst. Following incubation, colonies were counted, and organisms were checked for morphology and G r a m reaction. All organisms were immediately subcultured onto horse blood agar plates and subsequently identified by conventional methods. Inhibition of Growth of Microogranisms by Volatile Fatty Acids. Dilutions of acetic acid or isovaleric acid from 1 : 10 to 1 : 1 0 6 (V/V) were made in nutrient broth. To each tube was added one standard drop (0.025 ml) of a 24-hr broth culture in the case of Staphylococcus, or a 48-hr-broth culture in the case of Candida. The cultures were then incubated at 37~ for seven days with occasional shaking.
Volatile Fatty Acid Analysis Sample Preparation. Samples of sebum from the inguinal pouches of individual animals (18 males and 18 females) were collected and stored 3-6 months at - 2 5 ~ prior to use. The scrapings (10-20 mg) were triturated with a mixture of 0.5 ml ether and 0.5 ml of 0.1 M hydrochloric acid, and the organic layer was separated
1220
MERRITT ET AL.
out. The aqueous layer was extracted a further three times with 0.5 ml ether and the combined ether extracts shaken twice with 0.5 ml of 0.05 M sodium hydroxide. The alkaline extract was acidified with 0.4 ml of 5 M sulfuric acid and then steam distilled into 2 ml of 0.05 M sodium hydroxide unitl 15 ml of distillate had been collected. The distillate was reduced to a small volume on a rotary evaporator, transferred to a small glass vial and evaporated to dryness at 40~ under a stream of nitrogen. Just prior to gas chromatographic analysis, 20 #1 of water was added to the sample, followed by 30 #1 of 7 M hydrochloric acid. Gas Chromatography. A Varian Aerograph model 204B equipped with flame ionization detectors was used for the analysis of volatile fatty acids in the inguinal pouch scrapings. The test sample (0.5 #1) was injected through a Teflon-faced septum onto the front portion of a nickel column (2 m • 2 m m ID) packed with Chromosorb 101,100-120 mesh (Johns-Manville). Temperatures were: injectors 220 ~C; detectors, 250 ~C; column, 180 ~ C. Gas flow rates were: N2 carrier gas, 25 ml/min; H2, 25 ml/min; air, 150 ml/rain. Tentative identification of the acids in the samples was based on comparisons of their retention times with those of the authentic compounds. Quantitative determination of the acids in sample solutions was based on comparisons of their peak areas to those of the standard solutions.
RESULTS
One or more species of microorganisms were isolated from the inguinal pouches of 70 (93%) of the 75 rabbits sampled. Cultures from the pouches of two males (5%) and three females (9%) failed to grow microorganisms. Bacteria were found in the inguinal pouches of all the 10-day-old nestlings. Microorganisms were isolated from 155 (93%) of the total 167 samples. Altogether 245 isolates were cultured, representing six genera with the two most c o m m o n being the facultative aerobes Staphylococcus and Candida. In addition to these organisms, Bacillus subtilis, Escherichia coli, and Streptococcusfaecalis were occasionally isolated, with Corynebacterium ovis found only once. Most frequently only one genus was cultured from a single sample (76%). Two different genera were found in 20% of samples and three in 4%. There were no obvious sex differences in this respect. Repeated sampling revealed that the microflora in the inguinal pouches of an individual did not remain constant throughout the period of the study. Thus from the 19 females which were sampled at least twice, only three (16%) carried the same microorganism, and of the 22 males which were sampled at least twice, the same microorganisms were found in only seven (32%). Of the 22 samples cultured from the back and belly of 11 rabbits, a total of only 12 colonies grew.
C O N T E N T S OF W I L D R A B B I T I N G U I N A L P O U C H E S
1221
Staphylococcus aureus. This was the most c o m m o n isolate. It was cultured from 110 samples (66% of total), and occurred in 83% of all males and 77% of females. Primary culturing on serum agar produced most frequently a moderate growth, i.e., less than 100 colonies per plate. The same organism was also found in 73% of the anaerobic cultures. Candida kruzei. This facultative aerobic yeast was the second most c o m m o n organism encountered, being found in 60 samples (39%). It occurred in 50% of males and 60% of females. During primary culturing moderate growth was produced in 86% of the positive samples. F r o m a total of 67 samples cultured anaerobically, 23 (34%) produced C. kruzei. Bacillus subtilis. This organism was found only in nine (23%) males. It was isolated from 11 (7%) of total samples. The cultures yielded a light growth of this organism, except in one case where there was moderate growth. Escherichia coli. This organism was isolated only during the last two samplings, and only from five females. It appeared in six (4%) of all samples, producing only light growth. Streptococcus faecalis. This organism was found on only four occasions throughout the sampling period, twice in males and twice in females. Corynebacterium ovis. This organism was found only in scrapings taken from one male with only three colonies isolated. Volatile Fatty Acids. The relative quantities of eight volatile fatty acids in scrapings from 18 males and 18 females are shown in Table 1. These are expressed as a range because of the wide fluctuation of results between individuals. Acetic acid was the most common, constituting on the average approximately 80% of total volatile fatty acids in males and 74% in females. The second most abundant was isovaleric acid (approximately 16% of total in males and 23% in females). Six other acids occurred in much smaller quantities. The two most abundant acids--acetic and isovaleric--had an effect on the in vitro growth of the most c o m m o n organisms, S. aureus and C. kruzei. Acetic and isovaleric acids when used together at dilutions down to 103 prevented the growth of the two most c o m m o n isolates. Staphylococcus and Candida. C. kruzei appeared to be more affected by the mixture of two acids as there was some effect on growth at dilutions of 104. The two acids when used separately were less effective in inhibiting growth of these microorganisms (Table 2).
DISCUSSION
When the pattern of social behavior of the rabbit is considered (Mykytowycz, 1968), it is possible that more selective sampling could reveal a pattern of group, colony, or geographical distribution of specific microflora.
79,7 (16.5-98.9) 73.8 (13.1-99.1)
2.3 (0.3-9.3) 0,6 (0.1-3,9)
Propionic (C3) 1,1 (0.1-8.3) 1.0 (0.1-7.6)
lsobutyric (iC4) 0.3 (0.1-2.0) 0.1 (0.1-O,4)
n-Butyric (nC4) 16.1 (0.7-84,0) 23.0 (0.1-85.7)
Isovaleric (iCs)
0.1 (0J-2.1) 0,9 (0.2-11.9)
n-Valeric (nCs)
aThe mean (and range) concentrations of C2-C 6 acids in samples from 18 male and 18 female rabbits are shown.
Females (N = 18)
Males (N = 18)
Acetic (C2)
Composition (mot %)
Oryctolagus cuniculus a
0,2 (0.1-1.9) 0,5 (0,4-3.3)
Isocaproic (iC6)
0.3 (0.2-4.4) 0,2 (0,3-1,9)
n-Caproic (nC6)
TABLE 1, VOLATILEFATTY ACIDCOMPOSITION OF INGUINAL POUCH SCRAPINGS FROMMALEAND FEMALE RABBITS,
t'~ ~4
~q
t.# b.)
1223
CONTENTS OF WILD RABBIT INGUINAL POUCHES
TABLE 2. I N H I B I T O R Y E F F E C T OF ACETIC ACID ( C 2 ) AND I S O V A L E R I C A C ID ( i C s ) ON BROTH C U L T U R E S OF S. aureus AND C. k r u z e i I S O L A T E D FROM I N G U I N A L POUCHES OF W I L D RABBITS
D i l u t i o n factor
Micro0rganism ~. attreug
C. kruzei
Acid
10- 2
10- 3
10- 4
10- 5
10 - 6
C2 iC 5
_a +
+ +
+ +
+ +
+ +
C2 + iC5
-
-
+
+
+
C2
+
+
+
+
+
iCs C2 + iCs
+ -
+ -
+ -+
+ +
+ +
a+, growth; +-, retarded growth; -, no growth.
The seemingly high incidence of S. aureus and C. kruzei found may be peculiar to the experimental colony involved in this study. If microorganisms such as Staphylococcus, Candida, etc., are indeed frequently carried by the rabbit in its natural environment, it could play some role in the epidemiology of diseases caused by these species in domestic animals. At this stage one can only speculate that fermentation of the skin gland secretion is necessary for the formation of functional od or signals because the experimental data are inadequate to substantiate such a contention. In fact, observations during the present study and views on the anal glands of the fox (Albone and Perry, 1976) and the bacteriology of the beaver's scent glands (Svendsen and Jollick, 1978), suggest that the microbial populations, and consequently the fermentation of secretions, may not be sufficiently consistent to form a basis for the production of olfactory signals. It is possible, however, that conditions of captivity were responsible for the seemingly high incidence of S. aureus and C. kruzei and for frequent changes in the microflora profiles of animals used in these studies. On the other hand, in some instances periodic alterations of the quality of personal odor may even be essential to communicate changes in the behavioral or physiological status of an individual. Thus microbial populations in the human vagina change in response to endocrine factors at puberty and menopause (Brown, 1978). Fluctuation of estrous cycles have been shown to influence short-term changes in microbial flora in the vagina of rats and rhesus monkeys, Macaca rnulatta, and in the latter may also affect the quality and rate of production of copulins (Bonsall and Michael, 1980). The mongoose Herpestes auropunctatus can be trained to differentiate between mixtures of volatile fatty acids (Gorman, 1976). This finding reinforces the
1224
MERRITT ET AL.
speculation that different mixtures of acids in an animal's odor could be at least partly responsible for individual recognition. The synergistic inhibitory effect of acetic and isovaleric acids on the growth of microorganisms suggests the existence of an additional mechanism for shaping the odor profiles of the inguinal pouches. Inclusion of other acids may increase the titer of the inhibitory effect. To test more fully the importance of this mechanism, it is necessary to study systematically the fluctuations in levels of the different acids in the same animal in relation to the density of populations of different microorganisms. However, there is a need first of all to establish whether the volatile fatty acids are produced in the glands or are a product of microbial fermentation. In addition, other constituents of the pouch and their contribution to the final odor signals would need to be investigated. Acknowledgments--We thank S. Gambale, C.C. Reece, and T. Williams for their diligent and skillful assistance.
REFERENCES ALBONE, E. 1977. Ecology of mammals--new focus for chemical research. Chem. Br. 13:92-112. ALSONE, E.S., and PERRY, G.C. 1976. Anal sac secretion of the red fox, Vulpes vulpes; volatile fatty acids and diamines: Implications for a fermentation hypothesis of chemical recognition. J. Chem~ Ecol. 2:101-111. ALl,ONE, E.S., GOSDEN, P.E., and WARE, G.C. 1977. Bacteria as a source of chemical signals in mammals, pp. 35-43, in D. Miiller-Schwarze and M.M. Mozell (eds.). Chemical Signals in Vertebrates. Plenum Press, New York. BONSALL,R.W., and MICHAFL,R.P. 1980. The externalization of vaginal fatty acids by the female rhesus monkey. J. Chem. Ecol. 6:499-509. BROWN, W.J. 1978. Microbial ecology of the normal vagina, pp. 407-422, in E.S.E. Hafez and T.N. Evans (eds.). The Human Vagina. Elsevier, North Holland, Amsterdam. GOODRICH, B.S, and MV~YTOWYCZ, R. 1972. Individual and sex differences in the chemical composition of pheromone-like substances from the skin glands of the rabbit, Oryctolagus cunieulus. J. Mammal. 53:540-548. GORMAN, M.L. 1976. A mechanism for individual recognition by odour in Herpestes auropunetatus (Carnivora: Viverridae). Anita. Behav. 24:141-145. GORMAN, M.L., NEDWELL,D.B., and SMITH,R.M. 1974. An analysis of the contents of the anal scent pockets of Herpestes auropunctatus (Carnivora: Viverridae). d. ZooL 172:389-399. HESTERMAN, E.R., and MYKYTOWYCZ,R. 1982. Misidentification by wild rabbits, Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion, t. Experiments with all-male groups. J. Chem. Ecol. 8:419-427. MYKYTOWYCZ, R. 1966. Observations on odoriferous and other glands in the Australian wild rabbit, Oryctolagus euniculus (L.), and the hare, Lepus europaeus: II. The inguinal glands. CSIRO Wildl. Res. 11:49-64. MYKYTOWYCZ, R. 1968. Territorial marking by rabbits. Sci. Am. 218:116-119, 123-126. MYKYTOWYCZ, R. 1970. The role of skin glands in mammalian communication, pp. 327-360, in J.W. Johnston, D.G. Moulton, and A. Turk (eds.). Advances in Chemoreception, Vol. I, Communication by Chemical Signals. Appleton-Century-Crofts, New York.
CONTENTS OF WILD RABBIT INGUINAL POUCHES
1225
SHELLEY,W.B., HURLEY,H.J., and NICHOLS,A.C. 1953. Axillary odor; experimental study of the role of bacteria, apoerine sweat and deodorants. Arch. Dermatol. Syphilol. 68:430-446.
SVENDSEN,G.E., and JOLLICK,J.D. 1978. Bacterial contents of the anal and castor glands of beaver (Castor canadensis). J. Chem. Ecol. 4:563-569.
WARE,G.C., and GOSDEN,P.E. 1980. Anaerobic microflora of the anal sac of the red fox (Vulpes vulpes). J. Chem. Ecol. 6:97-102.
