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The normal flora of the mouths of macropods (Marsupialia: Macropodidae)
THE NORMAL FLORA OF THE MOUTHS OF MACROPODS (MARSUPIALIA: MACROPODIDAE) SAMUEL
J. L.
Departments of Veterinary Pathology and Public Health. University of Queensland. St. Lucia, Queensland 4067, Australia
Summary-From swabs obtained from the mouths of 59 captive macropods and cultured for aerobic and anaerobic bacteria and fungi, the most frequent isolates were streptococci and aerobic Gram-negative rods. Other isolates, in order of diminishing frequency, were Grampositive non-sporulating rods and filaments, staphylococci/micrococci, aerobic Gram-negative cocci, fungi, anaerobic Gram-positive cocci, anaerobic Gram-negative rods and filaments, species of Bacillus and Clostridium, and members of the Simonsiellaceae. No anaerobic Gramnegative cocci were isolated, and spirochaetes were seen only in one animal with periodontal pockets. Smears of material from the mouths of 5 wild wallabies showed organisms resembling those found in the captive animals.
INTRODUCIION
A frequent cause of death in captive macropods (kangaroos and wallabies) is a progressive, necrotizing infection of the bones and soft tissues of the face, which arises in the mouth or jaws. A great many different bacteria have been isolated from lesions, the most frequently reported being Fusobacterium necrophorum and nocardiae, but the source of the bacteria and the pathogenesis of the disease are still disputed (Arundel, Barker and Beveridge, 1977). Mouquet (1923) who attributed the disease to F. necrophorum (“bacille de la &rose”) stated that this organism was part of the oral flora of domestic animals whereas Poelma, (1964) who regarded the disease as actinomycosis, suggested that actinomycetes might be common in the mouths of kangaroos. Irwin and Cameron (1962) isolated staphylococci, streptococci, neisseriae and a corynebacterium from lesions and considered that these are all likely to be part of the normal nasal or oral flora. However, any discussion about the source of either primary pathogens of the disease or secondary invaders must be based largely on conjecture, as little is known about the composition of the normal orat flora of macropods; the only studies reported have been examinations of plaque from the incisors: Beighton and Miller (1977) found considerable variation in the flora of 12 macropods of 9 species, but overall found that Gram-positive filamentous pleomorphic rods made up over 80 per cent of the cultivable flora; they also isolated Micrococcaceae, enterococci, strictly aerobic Gram-negative cocci and coccobacilli, and facultatively anaerobic Gram-negative rods; they detected no anaerobic Gram-negative organisms. In contrast, Dent (1979) , who included 3 macropods in a survey of 33 captive wild mammals, found that 40.7 per cent of cultivable organisms from the macropods were Gram-negative rods including fusobacteria, and only 33.8 per cent were Gram-positive rods; she also isolated streptococci other than enterococci. Our aim was to gain a broader picture of the flora of the mouth, by examining organisms present in
saliva and on mucosal surfaces as well as in plaque. The intention was to detect as wide a range of oral organisms as possible, including rare or transient inhabitants, rather than to determine their relative numbers. The samples were taken from the vicinity of the cheek teeth, which is where the majority of infections appear to arise.
MATERIALS AND METHODS
Material for culture was obtained from 59 macropods, from 3 States of Australia and comprising 10 species. Details of species and source are shown in Table 1. The majority, 52, were held in 11 estabhshments, under a variety of husbandry regimes; for example enclosure size ranged from a few square metres to 20 hectares. Of the remaining 7, at the University of Queensland Veterinary School Clinic, 6 were pets and one was a wild animal. All animals were mature enough to be eating solid food, although 9 had not reached sexual maturity. The sex was recorded for 47 animals; 32 female and 15 male. The wild animal and 4 of the pets had injuries to the trunk and limbs, an animal from H was suffering from a disease of the nervous system, and one of the animals from J was sampled immediately after its sudden death from unexplained causes; all the other animals appeared healthy and normal. In addition, 5 wild rock-wallabies (Petrogale penicillata puella), living in the Hughenden region of north Queensland, were sampled during a live-trapping project for an ecological study. Only smears were obtained from these animals, no material being taken for culture. All the animals from D were anaesthetized before sampling, using sodium methohexital given intravenously into the tail. Two of the animals at the University were sampled after having been anaesthetized for other reasons, and 2 more were sampled immediately after being killed; intravenous barbiturates were used in all 4 cases. All the other animals were restrained physically. As far as possible, all samples were taken from the same site---the lateral gingival
141
J. L. Samuel
142
Table 1. Sources and species of macropods from which samples the oral flora
were obtained
for culture
of
Sources South Australia Species
ABCDEFGHIJ
Macropus rufis M. giganteus M. fuliginosus M. robustus M. parryi M. wfogriseus M. antilopinus M. eugenii M. parma Wallabia bicolor Unidentified Totals
2
5
1
3 2
--
1
_
1 1 ~ 10
N.S.W.
Queensland K
-
1
1
~
5
-
1
_
~ ~
~--
1 I
1
~
~-
~
_
-
_
~
._
_ 1
_
~
._
~._
~.~
-
~
-
--~
_
~
3
-
-~
_.
_
2
_ -
_ ~_
1 _ ~~
3
_
__
-
--
1
~
-
I
7
8
--1 21--25 4 2
6
5
2
10
~_
~
~_
-
2 ~
1
~
Total
3
4
II 9 4 3 4
1 fj
~
-
-
3
3
7
A, public zoological garden, small enclosures. B, collection of native fauna, open to public, large enclosures. C, private collection of native fauna, not open to public, small enclosures. D, E and J, collections of native fauna attached to research institutes, small enclosures, public access. F, G and H, privately owned zoos, predominantly native fauna, mostly small enclosures. I, privately owned zoo, predominantly native fauna, large enclosure. K, presented at University of Queensland Veterinary School Clinic.
margin of the upper cheek teeth; material was taken from the saliva, gingiva, buccal mucosa, buccal surfaces of the teeth and, in some cases, the tongue. Sterile cotton-wool swabs (Sterile Serum Swabs, Johns Professional Products, Victoria) were used in all animals except the dead kangaroo from which pieces of plant fibre which were lodged in periodontal pockets were withdrawn and samples taken from them. Two swabs were taken from most animals, one for culture and one for immediate preparation of an air-dried smear; in a few cases, a second smear was prepared and fixed with formalin and heat in order to look for spirochaetes. All swabs for culture which could not be plated within 30min were placed in Amies Transport Medium (Lennette, Spaulding and Truant, 1974). A maximum of 3 h elapsed between taking and plating of samples from animals in Queensland; however, because of the distances involved, samples from New South Wales were held for 2472 h; those from South Australia were held for up to 2 wk. Each swab was plated on 2 plates of 10 per cent sheep blood agar (Blood Agar Base B45, Difco Laboratories Ltd, Detroit, Mich., U.S.A.) and on one each of the following: MacConkey agar (CM1 15, Oxoid Australia, P.O. Box 174, Hurstville, N.S.W.), tomato juice agar (Oxoid CM113), modified Edwards medium (Oxoid CM27) with 10 per cent sheep blood, mannitol salt agar (Oxoid CM85), Sabouraud dextrose agar (Oxoid CM41) and neomycin blood agar prepared by adding neomycin sulphate to the molten blood agar base to give a concentration of 200 pg per ml. Part-way through the survey, the use of mannitol salt agar was discontinued because of the smali numbers of colonies which grew on this medium and the ease with which colonies of micrococci or staphy-
4 2 59
no
locci could be recognized on blood agar. One of the blood plates plus the neomycin blood agar plate were incubated anaerobically and the remainder were incubated in air, all at 37°C. Anaerobic incubation was in an atmosphere of 90 per cent hydrogen and 10 per cent carbon dioxide, produced either by introducing the gases into a Baird and Tatlock jar fitted with a cold catalyst or in plastic bags using the method of Barton and Winzar (1973). All aerobic plates were examined at 24 h and again at 48 h: the blood agar and Sabouraud plates were incubated for at least 7 days before being discarded, in order to detect slow-growing actinomycetes or fungi. Anaerobic plates were first examined at 2-5 days and were kept for at least 2 wk to allow for development of characteristic pigments. Certain groups of bacteria could be readily recognized by their colonial morphology, pigmentation, haemolytic effect or reaction on selective media; most of these were recorded without further confirmation. All other colonial types were subcultured on blood agar and a smear of each was made for Gram-staining using the modification by Preston and Morrell(l962). Two subcultures of each of the colonies from anaerobic plates were made, one aerobic and one anaerobic. The criterion for classifying an organism as an anaerobe was failure to grow on 3 aerobic subcultures, provided that the strain was still viable as shown by simultaneous anaerobic cultures. Smears were made of the confluent growth on those primary aerobic and anaerobic blood plates on which growth was heavy; if these revealed morphological types which had not been isolated, the growth was subcultured. Strains were classified into broad groups on the basis of morphology, cultural characteristics and, for aerobically
.
Normal oral bacterial flora of macropods growing organisms, a small number of biochemical tests described by Cowan and Steel (1965). All aerobes were tested for catalase activity using a slide test (Lennette et al., 1974, p. 931). Gram-negative aerobes were tested for oxidase using the method of Cowan and Steel (1965, pp. 148 and 150); organisms which produced an acid reaction on mannitol salt agar were tested for coagulase using a slide test (third method of Cowan and Steel, 1965). The smears from the mouths were Gram-stained and the organisms recorded as to Gram reaction, morphology, estimated dimensions, presence of spores and arrangement. The formalin-fixed smears were stained with concentrated carbol fuscin and examined for spirochaetes. In the smears and on the primary plates, the relative abundance of each type of organism or colony was estimated, using an exponential scale from 1 to 4. Results of all cultures were combined so as to give 2 values for each group of organisms isolated: frequency and abundance. These terms were defined as follows: Frequency--the percentage of samples from which the organisms were isolated. For example, if streptococci were isolated from 3 out of 10 samples, the frequency of isolation was 30 per cent. Ahundunce--- the mean of the relative abundances of an organism in the samples from which it was isolated. For example, if in the 3 samples cited above, streptococci were estimated on an exponential scale at 1. 4 and 2 respectively, the abundance of streptococci in the 10 samples would be 1 + 4 + 2/3 = 2 (to the nearest whole number). Similarly, frequency and abundance were calculated for each type of organism seen in smears. . REX LTS A heavy, mixed growth was obtained from almost every sample on both aerobic and anaerobic blood plates. A wide range of microorganisms was isolated, presenting an almost continuous gradation from cocci to filaments, both Gram-positive and Gram-negative, and from aerobiosis to strict anaerobiosis. Smears v?ere obtained from 44 of the 59 animals sampled; some morphological forms were seen in smears but were not obtained in culture. Figure 1 shows the frequency with which each group of microorganisms was detected within cultures and smears, together with its abundance. The 5 wild rock-wallabies from which only smears were taken are not included. The most frequently isolated groups, and the most abundant, were aerobes. Gram-positive cocci and/or coccobacilli were found in every animal in the smear or in cultures or, in most cases, in both. Aerobically-growing cocci were divided on the basis of their morphology, colonial appearance and catalase reaction into either the Micrococcaceae or the streptococci. Streptococci, which were isolated from 96 per cent of samples, included many strains which were micro-aerophilic and only grew aerobically after 1 or 2 subcultures. The streptococci were subdivided according to the type of haemolysis produced, the cc-haemolytic strains being the most frequently isolated; the /%haemolytic strains were the least frequent and the least abundant. Aesculin hydrolysis occurred in approximately half of the strains
143
isolated on modified Edwards medium. Many of the catalase-positive cocci isolated on blood agar did not grow on mannitol salt agar. Approximately half of those that grew gave an acid reaction, but none of these was positive for coagulase; none produced haemolysis on blood agar. The anaerobic Gram-positive cocci were less frequent and abundant than the aerobic. They could be subdivided into either small, massed cocci or larger coccobacilli which formed chains; many of the coccobacilli had a distinctive morphology: small, opaque, pale greenish-cream colonies, non-haemolytic, usually growing as satellites of other colonies, and elevated above the surrounding growth. Gram-negative rods were also present in every animal. The rods in smears, and also the anaerobic rods isolated, were arbitrarily divided into rods (maximum length not greater than 10pm) and filaments (longer than 10pm); organisms with typical fusiform morphology were grouped with the filaments regardless of length. By this definition, anaerobic rods were isolated from 30 per cent of samples and filaments from 21 per cent. Anaerobic rods whose colonies developed black pigment were classed as Bacteroides melaninogenicus; these were isolated from 21 per cent of samples. Fusiforms were isolated from only 2 samples (4 per cent) although they were seen in 14 per cent of smears. None of the isolates resembled Fusohactrrium necrophorum in cellular or colonial morphology. One morphological form seen in 25 per cent of smears but never isolated, consisted of thick filaments with tapered ends, approximately 2 pm in diameter and up to 25 pm in length. Aerobic Gram-negative rods were isolated from all but one animal, often in large numbers. They were subdivided according to their ability to grow on MacConkey agar, designated as MAC+ve or MAC-ve; the MACfve rods were further subdivided on their ability to ferment lactose rapidly, as indicated by an acid reaction on this medium: designated as LF (lactose-fermenting) or NLF. The NLF rods were present in 92 per cent of samples, in greater abundance than the LF rods. Most samples yielded both oxidase-positive and oxidase-negative strains, some of the oxidase-positive strains showing the colonial morphology and pigment production characteristic of Pseudomonas aeruginosa. The MAC-ve rods formed a heterogeneous group, varying in morphology from short rods to filaments; their colonial morphology, pigmentation and oxidase and catalase reactions also varied. Whereas the MAC+ ve rods grew on all of the selective media except mannitol salt agar, the MAC-ve rods were grown on blood plates only. Rods with spores, or large Gram-positive squareended rods, were present in low numbers in smears from 11 per cent of animals, from all of which either Bacillus species or clostridia were isolated; most of the clostridia displayed swarming growth on blood agar. All other Gram-positive rods were grouped together as non-sporulating rods. This was an extremely heterogenous group and could be subdivided on only the most arbitrary criteria. It was frequently diffiult to isolate them from over-growing Gramnegative rods; moreover, when they were isolated, many strains were subcultured several times before they could be grown in air or they formed filaments.
J. L. Samuel
An G+ve
“on-
G + “*
SP or square -ended
rods
6aclllus
COCCI
haem
,treptococc,
G-w
MAC-w
sp
Clortridaa
Aer
b--l
OtherAn G-w
An
G-w
Aer G-w
rods
rods
falaments flloments
Funq, 36
9 Fungi 1 100
:
: 60 Psrc*ntoge
I
I
I
I1 40
60 frequency
I”
1
: 20
:
sm~orr
0
I 20
.
Psrcentope
1 40
It
frequency
1 60 I”
I
.I 60
1
I too
EUltUres
Fig. 1. Frequency and abundance of different groups of organisms within smears and cultures from the mouths of 59 normal macropods. Abundance of organisms in shaded boxes was 2, and in unshaded boxes was 1. G+ve, Gram-positive; G-ve, Gram-negative; Aer, aerobic; An, anaerobic; haem, haemolytic; sp, sporulating. Frequency and abundance defined in text.
Arbitrary division of these organisms in smears into short (up to 5 pm) and long (greater than 5 pm) gave frequencies of 59 and 36 per cent respectively; overall they were present in 70 per cent of smears. They were cultured from 83 per cent of animals, 63 per cent yielding filamentous forms which showed metachromatic granules, irregular outlines and sometimes branching, with colonies that were dense, white, rough and frequently crumb-like. Some strains appeared to be obligate anaerobes, many grew well in either hydrogen or air plus 10 per cent carbon dioxide, and some grew in air on primary isolation. None of the aerobic strains produced the aerial hyphae typical of Nocardia. All the Gram-negative cocci isolated grew in air, most failing to grow anaerobically. All were oxidase-
positive and resembled neisseriae in morphology, but approximately half were catalase-negative. The Gramnegative cocci in 75 per cent of smears all resembled neisseriae, being large and paired with adjacent sides flattened. In 70 per cent of smears, organisms were seen which appeared to belong to the Simonsiellaceae (Buchanan and Gibbons, 1974), broad filaments made up of apposed flattened cells which often appeared to be attached to epithelial cells. These organisms were grown from only 2 samples; they formed small haemolytic colonies of coiled filaments. Fungi grew from 36 per cent of samples; most were yeasts but, in 9 per cent of samples, including 7 out of the 8 taken at establishment E, branching hyphae and aerial spores were present.
145
Normal oral bacterial flora of macropods No spirochaetes were seen in any of the formalinfixed smears, but they were seen in Gram-stained smears from one animal, the dead kangaroo from establishment J in which samples had been taken from periodontal pockets. All 5 of the smears taken from the wild P. penicilkm yielded Gram-positive cocci and non-sporulating rods, Gram-negative rods or filaments, and organisms resembling Simonsirllaceae (Table 2). Gram-negative cocci were seen in 4 of these smears and yeasts in one, but neither sporulating rods nor the thick Gramnegative filaments seen in some of the captive macropods were detected. The frequencies of the different groups of organisms were compared in animals from the 3 different States; significant differences were calculated by the chisquare test. Several groups of organisms were isolated more frequently @ < 0.01) from animals in Queensland, the anaerobic Gram-positive cocci and Gramnegative rods and filaments, and the aerobic Gramnegative cocci and Gram-negative MAC-ve rods. On the other hand, fungi were more frequently isolated from animals in New South Wales, because all samples from establishment E yielded fungi. There were some differences in frequencies of organisms in smears from the 3 States, but these differences were not significant. DISCUSSION
As the object was to obtain a broad picture of the normal oral flora of macropods, it was desirable to sample a large number of animals of several species and from several locations. A compromise had to be reached between the thoroughness with which samples were examined and the number of samples taken. For this reason, simple techniques were employed for culture and isolates were not classified beyond broad groups. It is possible that there were many groups of organisms present in the mouths which, because of extreme sensitivity to oxygen or requirements for complex nutrients, remained undetected. Whether more sophisticated culture techniques would have increased the yield of anaerobes is doubtful because much of the superiority of such techniques is believed to lie in the methods of obtaining and
transporting samples, rather than in the method of incubation itself (Rosenblatt, Fallon and Finegold, 1974). As most of the samples from macropods were taken in the field, it would have been impossible to avoid exposure to oxygen or to process them immediately; under these circumstances, more rigorous exclusion of oxygen during plating and incubation would probably not have greatly increased the number of anaerobes isolated. That loss of some groups of organisms was effected by the time lapse before plating is suggested by a comparison of isolations from animals from the 3 different states; frequencies of isolation of several groups were greatest from the Queensland samples, which were plated within a few hours. Moreover, the evidence from the smears is that some classes of organisms failed to grow in culture: the thick, tapered Gram-negative filaments were never seen in cultures and several other groups were rarely isolated although frequently seen in smears. In addition, the abundance of certain morphological types within smears was greater than their abundance within cultures, suggesting that either growth was inhibited or some strains of that morphological type failed to grow at all. Thus, it is probably not valid to interpret differences in frequency and abundance of isolation of different groups of bacteria as necessarily reflecting either their frequency of occurrence in the macropod population or their numbers within the mouth. The limitations of the measurement of abundance must be borne in mind: its calculation from estimated values, and the method of calculation which took into account only those cases from which the organism in question was isolated. An organism might have been represented as of low frequency and high abundance when in fact it had been present in all or most mouths but in numbers too low to be detected. Nevertheless, differences between groups of organisms with similar oxygen sensitivities and growth requirements probably represent the actual situation; for example the low frequency and abundance of the /I-haemolytic streptococci compared with the r*-haemolytic. Of the groups of bacteria which are regarded as usual components of normal oral flora in man (Hardie and Bowden, 1974; Burnett and Scherp. 1976) all were represented in the mouths of the macropods,
Table 2. Frequency and abundance of morphological types of organisms seen in smears from mouths of 5 wild rock-wallabies (Petrogale pmicillutcl) Frequency Gram tve cocci Gram + ve sporulating/square-ended rods Gram +ve non-sporulating rods <5 pm Gram + ve non-sporulating rods > 5 pm Gram -ve cocci Gram - ve rods (< 10 pm long) Gram - ve filaments (> 10 pm long) Simonsiella-like filaments Fungi Frequency
and abundance
defined in text
loo 0 100 100 80 100 80 100 20
Abundance 2
2 1 2 2
1 1 3
146
J. L. Samuel
with the exception of the veillonellae. Beighton and Miller (1977) also found no anaerobic Gram-negative cocci in macropods, and Dent (1979) did not find them in plaque from any of the wild animals she examined. The high frequency and abundance of the a-haemolytic streptococci, supported by the evidence of the smears, were in agreement with Burnett and Scherp (1976) who found this group the most numerous in the human mouth. On the other hand, the Gram-negative, MAC+ ve rods, which I found in high frequency and abundance, are regarded by Burnett and Scherp (1976) as either transient inhabitants of human mouths or else permanently but sparsely present. This difference may reflect contrasts in living conditions and standards of hygiene; Hardie and Bowden (1974) report higher numbers of facultative Gram-negative rods in mouths of man living under primitive conditions. Enteric bacteria must continually be introduced to the mouths of macropods when they feed from the ground, and clostridia also. Similarly, the fungus which was found in the mouths of all the macropods sampled at establishment E is likely to have been a saprophyte present in the environment and regularly introduced to the mouths. The range of organisms I detected cannot be taken as a complete list of the oral flora of macropods, not only because of the limitations of the culturing techniques, but also because not all sites in the mouth were sampled. It is probable that, in macropods as in man, the composition of the resident flora varies greatly between different sites within the mouth. This may help to account for the differences between my survey and that of Beighton and Miller (1977) who examined only the plaque from incisors; in particular their failure to isolate Gram-negative anaerobic rods or filaments, which in my survey were found in 45 per cent. However, Dent (1979), who also examined plaque from incisors, isolated anaerobic Gram-negative filaments from 2 out of 3 macropods. As I found spirochaetes only in one sample taken from deep in the periodontal pocket of a kangaroo, it is interesting that Soames and Davies (1974) found spirochaetes only deep within the gingival sulcus of a dog. The failure to isolate either F. necrophorum or species of Nocardiu does not necessarily mean that these were absent from the mouths examined; they may have been present in sites which were not sampled, or in small numbers which failed to grow or were overgrown by more numerous organisms; certainly the morphology of some of the Gram-negative filaments seen in smears was consistent with that of F. necrophorum. Differences in the oral flora of macropods from different sources and differences between species might be expected. However, any such differences in the flora would have been masked by the variations in handling of samples; thus the differences between States were probably attributable to the differing lengths of time before plating. Even when results of smears are compared, the effects of species and sources cannot be separated because species were not evenly distributed; for example all the 9 M. fuliyino~us were from South Australia. The smears from the S wild P. penicillata were included in order to detect any major differences between wild and captive animals; again there was a species difference. The morphological types seen in these smears, and their rela-
tive proportions, resembled those obtained from captive animals. The oral flora of the one wild animal from which a sample was obtained for culture, the M. rujbgriseus presented to the Veterinary School Clinic, likewise resembled that of captive animals. Obviously. greater numbers of animals, and possibly more detailed examination of samples, would be necessary to detect differences between species of macropod or environments. Acknowirdyemenrs--This work was undertaken with the help of a Postgraduate Studentship from the Commonwealth Scientific and Industrial Research Organisation. It was carried out within the Department of Veterinary Pathology and Public Health, and I am indebted to the Head of the Department, Professor J. Francis, for his support, to Dr A. J. Frost for his help and advice. and to the technical staff for their assistance. I also wish to thank the many people who owned or cared for macropods and who allowed and helped me obtain samples from their animals; in particular Dr M. E. Christian, Mr W. R. Gasking, Dr W. E. Lancaster, Dr W. E. Poole and Professor G. B. Sharman. The swabs from the wild wallabies were collected by Dr W. Davies. to whom I am very grateful. REFERENCES Arundel J. H., Barker I. K. and Beveridge I. 1977. Diseases of marsupials. In: Tire Bioloy~ oJ’ Marsupiuls (Edited by Stonehouse B. and Gilmour D.) pp. 141-154. Macmillan, London. Barton A. P. and Winzar J. A. 1973. Simple economic anaerobiosis. J. clin. Path. 26, 2388239. Beighton D. and Miller W. A. 1977. A microbiological study of normal flora of macropod dental plaque. J. dent. Rex 56, 995-1000. Buchanan R. E. and Gibbons N. E. 1974. Beryrj’s Man& $Determinative Buc’ferio/ogg, 8th edn, pp. 116~118. William and Wilkins, Baltimore, Md. Burnett G. W. and Scherp H. W. 1976. OTLIIMicrobioloy) and Injectious Disease, 4th edn, Chap. 17, pp. 219-258. William and Wilkins, Baltimore. Md. Cowan S. T. and Steel, K. J. 1965. Mama/for the ldentijication qj’Medicul Bucteriu. Cambridge Univ. Press, London. Dent V. E. 1979. The bacteriology of dental plaque from a variety of zoo-maintained mammalian species. Archs oral Eiol. 24, 217-282.
Hardie J. M. and Bowden G. H. 1974. The normal microbial flora of the mouth. In: The Normal Microbial FIora of Man (Edited by Skinner F. A. and Carr J. G.) See. uppl. Bact. Swap. Ser. No. 3, pp. 47783. Academic Press, London. Irwin D. H. G. and Cameron C. 1962. A note on oral infection in the kangaroo (Macrops rujis) and wallaby (Macropus
Lennette
r$coili.s).
.ll S. ,4fr. ret. med. Ass. 33, 231-232.
E. H. and Truant J. P. 1974. 2nd edn, p. 890. Am. Sot. Microbial. . Washington, D.C. Mouquet A. 1923. Maladie de Schmorl chez un kangourou. Bull. Sot. cent. Med. ret. 76, 419-423. Poelma F. G. 1964. Diseases in marsupials in captivity. Tijdschr. Diergeneesk 89. Suppl. No. 1, 149-155. Preston N. W. and Morrell AI -1962. Reproducible results with the Gram strain. J. Prrth. Bact. 84. 241-243. Rosenblatt J. E.. Fallon A. and Finegold S. M. 1974. Comparison of methods for isolation of anaerobic bacteria. In: Anaerobic Bacteria: Role in Disease (Edited by Balows A.. DeHaan R. M., Dowell V. R. and Guze L. B.) pp. 21-36. Thomas, Springfield, 111. Soames J. V. and Davies R. M. 1974. The distribution of spirochaetes in the gingival crevice of a Beagle dog. J. tmu// Anim. Prwt. 15, 529-533. Mumud
E. H., Spaulding qf Clinical
Microbiology,
J. L.
Departments of Veterinary Pathology and Public Health. University of Queensland. St. Lucia, Queensland 4067, Australia
Summary-From swabs obtained from the mouths of 59 captive macropods and cultured for aerobic and anaerobic bacteria and fungi, the most frequent isolates were streptococci and aerobic Gram-negative rods. Other isolates, in order of diminishing frequency, were Grampositive non-sporulating rods and filaments, staphylococci/micrococci, aerobic Gram-negative cocci, fungi, anaerobic Gram-positive cocci, anaerobic Gram-negative rods and filaments, species of Bacillus and Clostridium, and members of the Simonsiellaceae. No anaerobic Gramnegative cocci were isolated, and spirochaetes were seen only in one animal with periodontal pockets. Smears of material from the mouths of 5 wild wallabies showed organisms resembling those found in the captive animals.
INTRODUCIION
A frequent cause of death in captive macropods (kangaroos and wallabies) is a progressive, necrotizing infection of the bones and soft tissues of the face, which arises in the mouth or jaws. A great many different bacteria have been isolated from lesions, the most frequently reported being Fusobacterium necrophorum and nocardiae, but the source of the bacteria and the pathogenesis of the disease are still disputed (Arundel, Barker and Beveridge, 1977). Mouquet (1923) who attributed the disease to F. necrophorum (“bacille de la &rose”) stated that this organism was part of the oral flora of domestic animals whereas Poelma, (1964) who regarded the disease as actinomycosis, suggested that actinomycetes might be common in the mouths of kangaroos. Irwin and Cameron (1962) isolated staphylococci, streptococci, neisseriae and a corynebacterium from lesions and considered that these are all likely to be part of the normal nasal or oral flora. However, any discussion about the source of either primary pathogens of the disease or secondary invaders must be based largely on conjecture, as little is known about the composition of the normal orat flora of macropods; the only studies reported have been examinations of plaque from the incisors: Beighton and Miller (1977) found considerable variation in the flora of 12 macropods of 9 species, but overall found that Gram-positive filamentous pleomorphic rods made up over 80 per cent of the cultivable flora; they also isolated Micrococcaceae, enterococci, strictly aerobic Gram-negative cocci and coccobacilli, and facultatively anaerobic Gram-negative rods; they detected no anaerobic Gram-negative organisms. In contrast, Dent (1979) , who included 3 macropods in a survey of 33 captive wild mammals, found that 40.7 per cent of cultivable organisms from the macropods were Gram-negative rods including fusobacteria, and only 33.8 per cent were Gram-positive rods; she also isolated streptococci other than enterococci. Our aim was to gain a broader picture of the flora of the mouth, by examining organisms present in
saliva and on mucosal surfaces as well as in plaque. The intention was to detect as wide a range of oral organisms as possible, including rare or transient inhabitants, rather than to determine their relative numbers. The samples were taken from the vicinity of the cheek teeth, which is where the majority of infections appear to arise.
MATERIALS AND METHODS
Material for culture was obtained from 59 macropods, from 3 States of Australia and comprising 10 species. Details of species and source are shown in Table 1. The majority, 52, were held in 11 estabhshments, under a variety of husbandry regimes; for example enclosure size ranged from a few square metres to 20 hectares. Of the remaining 7, at the University of Queensland Veterinary School Clinic, 6 were pets and one was a wild animal. All animals were mature enough to be eating solid food, although 9 had not reached sexual maturity. The sex was recorded for 47 animals; 32 female and 15 male. The wild animal and 4 of the pets had injuries to the trunk and limbs, an animal from H was suffering from a disease of the nervous system, and one of the animals from J was sampled immediately after its sudden death from unexplained causes; all the other animals appeared healthy and normal. In addition, 5 wild rock-wallabies (Petrogale penicillata puella), living in the Hughenden region of north Queensland, were sampled during a live-trapping project for an ecological study. Only smears were obtained from these animals, no material being taken for culture. All the animals from D were anaesthetized before sampling, using sodium methohexital given intravenously into the tail. Two of the animals at the University were sampled after having been anaesthetized for other reasons, and 2 more were sampled immediately after being killed; intravenous barbiturates were used in all 4 cases. All the other animals were restrained physically. As far as possible, all samples were taken from the same site---the lateral gingival
141
J. L. Samuel
142
Table 1. Sources and species of macropods from which samples the oral flora
were obtained
for culture
of
Sources South Australia Species
ABCDEFGHIJ
Macropus rufis M. giganteus M. fuliginosus M. robustus M. parryi M. wfogriseus M. antilopinus M. eugenii M. parma Wallabia bicolor Unidentified Totals
2
5
1
3 2
--
1
_
1 1 ~ 10
N.S.W.
Queensland K
-
1
1
~
5
-
1
_
~ ~
~--
1 I
1
~
~-
~
_
-
_
~
._
_ 1
_
~
._
~._
~.~
-
~
-
--~
_
~
3
-
-~
_.
_
2
_ -
_ ~_
1 _ ~~
3
_
__
-
--
1
~
-
I
7
8
--1 21--25 4 2
6
5
2
10
~_
~
~_
-
2 ~
1
~
Total
3
4
II 9 4 3 4
1 fj
~
-
-
3
3
7
A, public zoological garden, small enclosures. B, collection of native fauna, open to public, large enclosures. C, private collection of native fauna, not open to public, small enclosures. D, E and J, collections of native fauna attached to research institutes, small enclosures, public access. F, G and H, privately owned zoos, predominantly native fauna, mostly small enclosures. I, privately owned zoo, predominantly native fauna, large enclosure. K, presented at University of Queensland Veterinary School Clinic.
margin of the upper cheek teeth; material was taken from the saliva, gingiva, buccal mucosa, buccal surfaces of the teeth and, in some cases, the tongue. Sterile cotton-wool swabs (Sterile Serum Swabs, Johns Professional Products, Victoria) were used in all animals except the dead kangaroo from which pieces of plant fibre which were lodged in periodontal pockets were withdrawn and samples taken from them. Two swabs were taken from most animals, one for culture and one for immediate preparation of an air-dried smear; in a few cases, a second smear was prepared and fixed with formalin and heat in order to look for spirochaetes. All swabs for culture which could not be plated within 30min were placed in Amies Transport Medium (Lennette, Spaulding and Truant, 1974). A maximum of 3 h elapsed between taking and plating of samples from animals in Queensland; however, because of the distances involved, samples from New South Wales were held for 2472 h; those from South Australia were held for up to 2 wk. Each swab was plated on 2 plates of 10 per cent sheep blood agar (Blood Agar Base B45, Difco Laboratories Ltd, Detroit, Mich., U.S.A.) and on one each of the following: MacConkey agar (CM1 15, Oxoid Australia, P.O. Box 174, Hurstville, N.S.W.), tomato juice agar (Oxoid CM113), modified Edwards medium (Oxoid CM27) with 10 per cent sheep blood, mannitol salt agar (Oxoid CM85), Sabouraud dextrose agar (Oxoid CM41) and neomycin blood agar prepared by adding neomycin sulphate to the molten blood agar base to give a concentration of 200 pg per ml. Part-way through the survey, the use of mannitol salt agar was discontinued because of the smali numbers of colonies which grew on this medium and the ease with which colonies of micrococci or staphy-
4 2 59
no
locci could be recognized on blood agar. One of the blood plates plus the neomycin blood agar plate were incubated anaerobically and the remainder were incubated in air, all at 37°C. Anaerobic incubation was in an atmosphere of 90 per cent hydrogen and 10 per cent carbon dioxide, produced either by introducing the gases into a Baird and Tatlock jar fitted with a cold catalyst or in plastic bags using the method of Barton and Winzar (1973). All aerobic plates were examined at 24 h and again at 48 h: the blood agar and Sabouraud plates were incubated for at least 7 days before being discarded, in order to detect slow-growing actinomycetes or fungi. Anaerobic plates were first examined at 2-5 days and were kept for at least 2 wk to allow for development of characteristic pigments. Certain groups of bacteria could be readily recognized by their colonial morphology, pigmentation, haemolytic effect or reaction on selective media; most of these were recorded without further confirmation. All other colonial types were subcultured on blood agar and a smear of each was made for Gram-staining using the modification by Preston and Morrell(l962). Two subcultures of each of the colonies from anaerobic plates were made, one aerobic and one anaerobic. The criterion for classifying an organism as an anaerobe was failure to grow on 3 aerobic subcultures, provided that the strain was still viable as shown by simultaneous anaerobic cultures. Smears were made of the confluent growth on those primary aerobic and anaerobic blood plates on which growth was heavy; if these revealed morphological types which had not been isolated, the growth was subcultured. Strains were classified into broad groups on the basis of morphology, cultural characteristics and, for aerobically
.
Normal oral bacterial flora of macropods growing organisms, a small number of biochemical tests described by Cowan and Steel (1965). All aerobes were tested for catalase activity using a slide test (Lennette et al., 1974, p. 931). Gram-negative aerobes were tested for oxidase using the method of Cowan and Steel (1965, pp. 148 and 150); organisms which produced an acid reaction on mannitol salt agar were tested for coagulase using a slide test (third method of Cowan and Steel, 1965). The smears from the mouths were Gram-stained and the organisms recorded as to Gram reaction, morphology, estimated dimensions, presence of spores and arrangement. The formalin-fixed smears were stained with concentrated carbol fuscin and examined for spirochaetes. In the smears and on the primary plates, the relative abundance of each type of organism or colony was estimated, using an exponential scale from 1 to 4. Results of all cultures were combined so as to give 2 values for each group of organisms isolated: frequency and abundance. These terms were defined as follows: Frequency--the percentage of samples from which the organisms were isolated. For example, if streptococci were isolated from 3 out of 10 samples, the frequency of isolation was 30 per cent. Ahundunce--- the mean of the relative abundances of an organism in the samples from which it was isolated. For example, if in the 3 samples cited above, streptococci were estimated on an exponential scale at 1. 4 and 2 respectively, the abundance of streptococci in the 10 samples would be 1 + 4 + 2/3 = 2 (to the nearest whole number). Similarly, frequency and abundance were calculated for each type of organism seen in smears. . REX LTS A heavy, mixed growth was obtained from almost every sample on both aerobic and anaerobic blood plates. A wide range of microorganisms was isolated, presenting an almost continuous gradation from cocci to filaments, both Gram-positive and Gram-negative, and from aerobiosis to strict anaerobiosis. Smears v?ere obtained from 44 of the 59 animals sampled; some morphological forms were seen in smears but were not obtained in culture. Figure 1 shows the frequency with which each group of microorganisms was detected within cultures and smears, together with its abundance. The 5 wild rock-wallabies from which only smears were taken are not included. The most frequently isolated groups, and the most abundant, were aerobes. Gram-positive cocci and/or coccobacilli were found in every animal in the smear or in cultures or, in most cases, in both. Aerobically-growing cocci were divided on the basis of their morphology, colonial appearance and catalase reaction into either the Micrococcaceae or the streptococci. Streptococci, which were isolated from 96 per cent of samples, included many strains which were micro-aerophilic and only grew aerobically after 1 or 2 subcultures. The streptococci were subdivided according to the type of haemolysis produced, the cc-haemolytic strains being the most frequently isolated; the /%haemolytic strains were the least frequent and the least abundant. Aesculin hydrolysis occurred in approximately half of the strains
143
isolated on modified Edwards medium. Many of the catalase-positive cocci isolated on blood agar did not grow on mannitol salt agar. Approximately half of those that grew gave an acid reaction, but none of these was positive for coagulase; none produced haemolysis on blood agar. The anaerobic Gram-positive cocci were less frequent and abundant than the aerobic. They could be subdivided into either small, massed cocci or larger coccobacilli which formed chains; many of the coccobacilli had a distinctive morphology: small, opaque, pale greenish-cream colonies, non-haemolytic, usually growing as satellites of other colonies, and elevated above the surrounding growth. Gram-negative rods were also present in every animal. The rods in smears, and also the anaerobic rods isolated, were arbitrarily divided into rods (maximum length not greater than 10pm) and filaments (longer than 10pm); organisms with typical fusiform morphology were grouped with the filaments regardless of length. By this definition, anaerobic rods were isolated from 30 per cent of samples and filaments from 21 per cent. Anaerobic rods whose colonies developed black pigment were classed as Bacteroides melaninogenicus; these were isolated from 21 per cent of samples. Fusiforms were isolated from only 2 samples (4 per cent) although they were seen in 14 per cent of smears. None of the isolates resembled Fusohactrrium necrophorum in cellular or colonial morphology. One morphological form seen in 25 per cent of smears but never isolated, consisted of thick filaments with tapered ends, approximately 2 pm in diameter and up to 25 pm in length. Aerobic Gram-negative rods were isolated from all but one animal, often in large numbers. They were subdivided according to their ability to grow on MacConkey agar, designated as MAC+ve or MAC-ve; the MACfve rods were further subdivided on their ability to ferment lactose rapidly, as indicated by an acid reaction on this medium: designated as LF (lactose-fermenting) or NLF. The NLF rods were present in 92 per cent of samples, in greater abundance than the LF rods. Most samples yielded both oxidase-positive and oxidase-negative strains, some of the oxidase-positive strains showing the colonial morphology and pigment production characteristic of Pseudomonas aeruginosa. The MAC-ve rods formed a heterogeneous group, varying in morphology from short rods to filaments; their colonial morphology, pigmentation and oxidase and catalase reactions also varied. Whereas the MAC+ ve rods grew on all of the selective media except mannitol salt agar, the MAC-ve rods were grown on blood plates only. Rods with spores, or large Gram-positive squareended rods, were present in low numbers in smears from 11 per cent of animals, from all of which either Bacillus species or clostridia were isolated; most of the clostridia displayed swarming growth on blood agar. All other Gram-positive rods were grouped together as non-sporulating rods. This was an extremely heterogenous group and could be subdivided on only the most arbitrary criteria. It was frequently diffiult to isolate them from over-growing Gramnegative rods; moreover, when they were isolated, many strains were subcultured several times before they could be grown in air or they formed filaments.
J. L. Samuel
An G+ve
“on-
G + “*
SP or square -ended
rods
6aclllus
COCCI
haem
,treptococc,
G-w
MAC-w
sp
Clortridaa
Aer
b--l
OtherAn G-w
An
G-w
Aer G-w
rods
rods
falaments flloments
Funq, 36
9 Fungi 1 100
:
: 60 Psrc*ntoge
I
I
I
I1 40
60 frequency
I”
1
: 20
:
sm~orr
0
I 20
.
Psrcentope
1 40
It
frequency
1 60 I”
I
.I 60
1
I too
EUltUres
Fig. 1. Frequency and abundance of different groups of organisms within smears and cultures from the mouths of 59 normal macropods. Abundance of organisms in shaded boxes was 2, and in unshaded boxes was 1. G+ve, Gram-positive; G-ve, Gram-negative; Aer, aerobic; An, anaerobic; haem, haemolytic; sp, sporulating. Frequency and abundance defined in text.
Arbitrary division of these organisms in smears into short (up to 5 pm) and long (greater than 5 pm) gave frequencies of 59 and 36 per cent respectively; overall they were present in 70 per cent of smears. They were cultured from 83 per cent of animals, 63 per cent yielding filamentous forms which showed metachromatic granules, irregular outlines and sometimes branching, with colonies that were dense, white, rough and frequently crumb-like. Some strains appeared to be obligate anaerobes, many grew well in either hydrogen or air plus 10 per cent carbon dioxide, and some grew in air on primary isolation. None of the aerobic strains produced the aerial hyphae typical of Nocardia. All the Gram-negative cocci isolated grew in air, most failing to grow anaerobically. All were oxidase-
positive and resembled neisseriae in morphology, but approximately half were catalase-negative. The Gramnegative cocci in 75 per cent of smears all resembled neisseriae, being large and paired with adjacent sides flattened. In 70 per cent of smears, organisms were seen which appeared to belong to the Simonsiellaceae (Buchanan and Gibbons, 1974), broad filaments made up of apposed flattened cells which often appeared to be attached to epithelial cells. These organisms were grown from only 2 samples; they formed small haemolytic colonies of coiled filaments. Fungi grew from 36 per cent of samples; most were yeasts but, in 9 per cent of samples, including 7 out of the 8 taken at establishment E, branching hyphae and aerial spores were present.
145
Normal oral bacterial flora of macropods No spirochaetes were seen in any of the formalinfixed smears, but they were seen in Gram-stained smears from one animal, the dead kangaroo from establishment J in which samples had been taken from periodontal pockets. All 5 of the smears taken from the wild P. penicilkm yielded Gram-positive cocci and non-sporulating rods, Gram-negative rods or filaments, and organisms resembling Simonsirllaceae (Table 2). Gram-negative cocci were seen in 4 of these smears and yeasts in one, but neither sporulating rods nor the thick Gramnegative filaments seen in some of the captive macropods were detected. The frequencies of the different groups of organisms were compared in animals from the 3 different States; significant differences were calculated by the chisquare test. Several groups of organisms were isolated more frequently @ < 0.01) from animals in Queensland, the anaerobic Gram-positive cocci and Gramnegative rods and filaments, and the aerobic Gramnegative cocci and Gram-negative MAC-ve rods. On the other hand, fungi were more frequently isolated from animals in New South Wales, because all samples from establishment E yielded fungi. There were some differences in frequencies of organisms in smears from the 3 States, but these differences were not significant. DISCUSSION
As the object was to obtain a broad picture of the normal oral flora of macropods, it was desirable to sample a large number of animals of several species and from several locations. A compromise had to be reached between the thoroughness with which samples were examined and the number of samples taken. For this reason, simple techniques were employed for culture and isolates were not classified beyond broad groups. It is possible that there were many groups of organisms present in the mouths which, because of extreme sensitivity to oxygen or requirements for complex nutrients, remained undetected. Whether more sophisticated culture techniques would have increased the yield of anaerobes is doubtful because much of the superiority of such techniques is believed to lie in the methods of obtaining and
transporting samples, rather than in the method of incubation itself (Rosenblatt, Fallon and Finegold, 1974). As most of the samples from macropods were taken in the field, it would have been impossible to avoid exposure to oxygen or to process them immediately; under these circumstances, more rigorous exclusion of oxygen during plating and incubation would probably not have greatly increased the number of anaerobes isolated. That loss of some groups of organisms was effected by the time lapse before plating is suggested by a comparison of isolations from animals from the 3 different states; frequencies of isolation of several groups were greatest from the Queensland samples, which were plated within a few hours. Moreover, the evidence from the smears is that some classes of organisms failed to grow in culture: the thick, tapered Gram-negative filaments were never seen in cultures and several other groups were rarely isolated although frequently seen in smears. In addition, the abundance of certain morphological types within smears was greater than their abundance within cultures, suggesting that either growth was inhibited or some strains of that morphological type failed to grow at all. Thus, it is probably not valid to interpret differences in frequency and abundance of isolation of different groups of bacteria as necessarily reflecting either their frequency of occurrence in the macropod population or their numbers within the mouth. The limitations of the measurement of abundance must be borne in mind: its calculation from estimated values, and the method of calculation which took into account only those cases from which the organism in question was isolated. An organism might have been represented as of low frequency and high abundance when in fact it had been present in all or most mouths but in numbers too low to be detected. Nevertheless, differences between groups of organisms with similar oxygen sensitivities and growth requirements probably represent the actual situation; for example the low frequency and abundance of the /I-haemolytic streptococci compared with the r*-haemolytic. Of the groups of bacteria which are regarded as usual components of normal oral flora in man (Hardie and Bowden, 1974; Burnett and Scherp. 1976) all were represented in the mouths of the macropods,
Table 2. Frequency and abundance of morphological types of organisms seen in smears from mouths of 5 wild rock-wallabies (Petrogale pmicillutcl) Frequency Gram tve cocci Gram + ve sporulating/square-ended rods Gram +ve non-sporulating rods <5 pm Gram + ve non-sporulating rods > 5 pm Gram -ve cocci Gram - ve rods (< 10 pm long) Gram - ve filaments (> 10 pm long) Simonsiella-like filaments Fungi Frequency
and abundance
defined in text
loo 0 100 100 80 100 80 100 20
Abundance 2
2 1 2 2
1 1 3
146
J. L. Samuel
with the exception of the veillonellae. Beighton and Miller (1977) also found no anaerobic Gram-negative cocci in macropods, and Dent (1979) did not find them in plaque from any of the wild animals she examined. The high frequency and abundance of the a-haemolytic streptococci, supported by the evidence of the smears, were in agreement with Burnett and Scherp (1976) who found this group the most numerous in the human mouth. On the other hand, the Gram-negative, MAC+ ve rods, which I found in high frequency and abundance, are regarded by Burnett and Scherp (1976) as either transient inhabitants of human mouths or else permanently but sparsely present. This difference may reflect contrasts in living conditions and standards of hygiene; Hardie and Bowden (1974) report higher numbers of facultative Gram-negative rods in mouths of man living under primitive conditions. Enteric bacteria must continually be introduced to the mouths of macropods when they feed from the ground, and clostridia also. Similarly, the fungus which was found in the mouths of all the macropods sampled at establishment E is likely to have been a saprophyte present in the environment and regularly introduced to the mouths. The range of organisms I detected cannot be taken as a complete list of the oral flora of macropods, not only because of the limitations of the culturing techniques, but also because not all sites in the mouth were sampled. It is probable that, in macropods as in man, the composition of the resident flora varies greatly between different sites within the mouth. This may help to account for the differences between my survey and that of Beighton and Miller (1977) who examined only the plaque from incisors; in particular their failure to isolate Gram-negative anaerobic rods or filaments, which in my survey were found in 45 per cent. However, Dent (1979), who also examined plaque from incisors, isolated anaerobic Gram-negative filaments from 2 out of 3 macropods. As I found spirochaetes only in one sample taken from deep in the periodontal pocket of a kangaroo, it is interesting that Soames and Davies (1974) found spirochaetes only deep within the gingival sulcus of a dog. The failure to isolate either F. necrophorum or species of Nocardiu does not necessarily mean that these were absent from the mouths examined; they may have been present in sites which were not sampled, or in small numbers which failed to grow or were overgrown by more numerous organisms; certainly the morphology of some of the Gram-negative filaments seen in smears was consistent with that of F. necrophorum. Differences in the oral flora of macropods from different sources and differences between species might be expected. However, any such differences in the flora would have been masked by the variations in handling of samples; thus the differences between States were probably attributable to the differing lengths of time before plating. Even when results of smears are compared, the effects of species and sources cannot be separated because species were not evenly distributed; for example all the 9 M. fuliyino~us were from South Australia. The smears from the S wild P. penicillata were included in order to detect any major differences between wild and captive animals; again there was a species difference. The morphological types seen in these smears, and their rela-
tive proportions, resembled those obtained from captive animals. The oral flora of the one wild animal from which a sample was obtained for culture, the M. rujbgriseus presented to the Veterinary School Clinic, likewise resembled that of captive animals. Obviously. greater numbers of animals, and possibly more detailed examination of samples, would be necessary to detect differences between species of macropod or environments. Acknowirdyemenrs--This work was undertaken with the help of a Postgraduate Studentship from the Commonwealth Scientific and Industrial Research Organisation. It was carried out within the Department of Veterinary Pathology and Public Health, and I am indebted to the Head of the Department, Professor J. Francis, for his support, to Dr A. J. Frost for his help and advice. and to the technical staff for their assistance. I also wish to thank the many people who owned or cared for macropods and who allowed and helped me obtain samples from their animals; in particular Dr M. E. Christian, Mr W. R. Gasking, Dr W. E. Lancaster, Dr W. E. Poole and Professor G. B. Sharman. The swabs from the wild wallabies were collected by Dr W. Davies. to whom I am very grateful. REFERENCES Arundel J. H., Barker I. K. and Beveridge I. 1977. Diseases of marsupials. In: Tire Bioloy~ oJ’ Marsupiuls (Edited by Stonehouse B. and Gilmour D.) pp. 141-154. Macmillan, London. Barton A. P. and Winzar J. A. 1973. Simple economic anaerobiosis. J. clin. Path. 26, 2388239. Beighton D. and Miller W. A. 1977. A microbiological study of normal flora of macropod dental plaque. J. dent. Rex 56, 995-1000. Buchanan R. E. and Gibbons N. E. 1974. Beryrj’s Man& $Determinative Buc’ferio/ogg, 8th edn, pp. 116~118. William and Wilkins, Baltimore, Md. Burnett G. W. and Scherp H. W. 1976. OTLIIMicrobioloy) and Injectious Disease, 4th edn, Chap. 17, pp. 219-258. William and Wilkins, Baltimore. Md. Cowan S. T. and Steel, K. J. 1965. Mama/for the ldentijication qj’Medicul Bucteriu. Cambridge Univ. Press, London. Dent V. E. 1979. The bacteriology of dental plaque from a variety of zoo-maintained mammalian species. Archs oral Eiol. 24, 217-282.
Hardie J. M. and Bowden G. H. 1974. The normal microbial flora of the mouth. In: The Normal Microbial FIora of Man (Edited by Skinner F. A. and Carr J. G.) See. uppl. Bact. Swap. Ser. No. 3, pp. 47783. Academic Press, London. Irwin D. H. G. and Cameron C. 1962. A note on oral infection in the kangaroo (Macrops rujis) and wallaby (Macropus
Lennette
r$coili.s).
.ll S. ,4fr. ret. med. Ass. 33, 231-232.
E. H. and Truant J. P. 1974. 2nd edn, p. 890. Am. Sot. Microbial. . Washington, D.C. Mouquet A. 1923. Maladie de Schmorl chez un kangourou. Bull. Sot. cent. Med. ret. 76, 419-423. Poelma F. G. 1964. Diseases in marsupials in captivity. Tijdschr. Diergeneesk 89. Suppl. No. 1, 149-155. Preston N. W. and Morrell AI -1962. Reproducible results with the Gram strain. J. Prrth. Bact. 84. 241-243. Rosenblatt J. E.. Fallon A. and Finegold S. M. 1974. Comparison of methods for isolation of anaerobic bacteria. In: Anaerobic Bacteria: Role in Disease (Edited by Balows A.. DeHaan R. M., Dowell V. R. and Guze L. B.) pp. 21-36. Thomas, Springfield, 111. Soames J. V. and Davies R. M. 1974. The distribution of spirochaetes in the gingival crevice of a Beagle dog. J. tmu// Anim. Prwt. 15, 529-533. Mumud
E. H., Spaulding qf Clinical
Microbiology,