GASTROINTESTINAL MICROFLORA OF MUTTON BIRDS (PUFFINUS TENUIROSTRIS) IN RELATION TO "LIM IY" DISEASE
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GASTROINTESTINAL MICROFLORA OF MUTTON BIRDS (PUFFINUS
TENUIROSTRIS) IN RELATION TO "LIM\IY" DISEASE
ROSE MUSHIN AND FRANCES M. ASHBURNER
School of Bacteriology, University of Melbourne, Victoria, Australia
Received for publication December 21, 1961
ABSTRACT parents cease to feed the chicks, and during the
MUSHIN, ROSE (University of Melbourne, starvation period the youngsters leave their
burrows in search of food. At the end of April and
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Victoria, Australia) AND FRANCES M. ASH-
BURNER. Gastrointestinal microflora of mutton beginning of May, the mutton birds leave the
birds (Puffinus tenuirostris) in relation to "limy" island to migrate to the northern Pacific.
disease. J. Bacteriol. 83:1260-1267. 1962.-The In the course of the biological study of this
aerobic bacterial intestinal flora of mutton birds, species, some of the chicks were found to be
healthy and affected with "limy" disease, was affected by an intestinal disorder called "limy"
investigated, mainly in birds from Great Dog disease by the local commercial birders. As part
Island, Bass Strait, Australia. A total of 1,274 of an investigation of the etiology of the disease,
specimens from gut on post-mortem and from carried out by Officers of the Wildlife Section of
cloacal swabs was examined. No indication was the Commonwealth Scientific and Industrial Re-
obtained of a pathogenic bacterial species con- search Organization, we were invited to study
cerned in "limy" disease. A study was made of the bacterial intestinal flora of healthy and
the types of organisms and their distribution in diseased birds. The results are presented in this
the gut of mutton birds. The sparsity, or occa- communication.
sional absence, of aerobic bacterial flora in some The term "limy" is derived from the grayish-
birds, especially adults, was noticed. The "limy" yellow appearance of the intestinal contents and
birds, by comparison with normal chicks, had a excreta of the diseased mutton birds. The feces
richer flora more evenly distributed through the and the regurgitated food have a strong repug-
alimentary canal. A higher ratio of gram-negative nant odor which can be recognized at a distance
to gram-positive bacterial types was recorded in from the rookeries. Autopsy examinations showed
chicks; adult birds had a lower or an even numeri- the muscles to be pale instead of the usual dark
cal ratio. The comparatively frequent occurrence red color, and the fat yellowish brown instead of
of Proteus species, except P. mirabilis, and of ir- the normal white. Evidence was obtained of ad-
regular and intermediate coliform types was vanced pathological changes in the lower part of
evident. Escherichia coli type I was not a constant the intestine and in organs such as liver and
inhabitant of the gut. The gram-positive flora spleen. The cloaca was usually packed with con-
consisted mostly of Staphylococcus saprophyticus, cretions which could be felt on palpation and
S. lactis, and Enterococcus. The possibility that which seemed to be responsible for lesions and
"limy" disease may be caused by physiological occasional perforations in the cloacal wall. On
factors is discussed. opening the cloaca the concretions retained the
shape of the cloaca in which they were formed.
Preliminary examination indicated that the
An intensive study of the ecology of the Tas- concretions were of sodium urate. (The foregoing
manian mutton bird, Puffinus tenuirostris (Tem- description of "limy" disease was kindly provided
minck), was carried out in Bass Strait, Australia by R. Mykytowyez.)
(Serventy, 1958). This species is of commercial
value to the inhabitants of the Furneaux group of MATERIALS AND METHODS
islands, and its uncommon habits make it a Specimens. The material for the study was ob-
source of interest to naturalists. The birds arrive tained by R. Mykytowycz. The specimens, unless
at Bass Strait at the end of September, egg-laying otherwise indicated, came from various rookeries
begins in the third week of November, and the on Great Dog Island of the Furneaux group of
chicks are hatched in January. In April the islands in the Bass Strait, Australia. Except for a
12601962] GASTROINTESTINAL FLORA OF MUTTON BIRDS 1261
few samples obtained in the middle of February, sucrose, and 1 % salicin-peptone-water was ob-
specimens were collected between about the mid- served. For the Bethesda-Ballerup group, growth
dle of March and the last week in April, 1960. in KCN and failure to liquefy gelatin was re-
The study group consisted of 42 "limy" chicks, corded, and the Vi agglutination test was per-
40 healthy and 8 experimentally starved young formed. Proteus was subdivided into species, and
birds, and 33 normal adult birds. Twenty-one P. rettgeri and the Providence group were con-
autopsy operations were done in the laboratory firmed by a positive tryptophan ferric chloride
and all others on the island. Specimens were test (Falkow, 1957). P. rettgeri strains were
taken from the proventriculus, upper and lower further grouped on the basis of rhamnose, salicin,
intestine, and cloaca; 16 samples were from other and arabinose fermentation (Kauffmann, 1954).
organs, usually the liver and spleen. In addition, A detailed study was made of coliform bacteria,
38 cloacal swabs were obtained from living nor- and four to six lactose-fermenting colonies from
mal chicks, 82 swabs from experimentally starved deoxycholate agar were biochemically examined.
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chicks, and 12 samples from regurgitated food. These strains were subdivided into types on the
From other localities, mostly from Montagu basis of biochemical reactions, according to Wil-
Island, New South Wales, and occasionally from son et al. (1935) with the omission of the gelatin-
Phillip Island, Victoria, came a total of 44 cloacal liquefaction test. The set of five tests used was
swabs, which were collected at various times from labeled IMVEC. (This formula is modified
normal adult and young birds. from the one representing a quartet of tests and is
Biochemical and serological tests. The routine used in the School of Bacteriology, University of
procedure was to collect the material into a pre- Melbourne.) The letter "E" denotes Eijkman's
servative gel according to Stuart's (1956) method, test. No distinction was made between Interme-
but specimens from birds sent to the laboratory diate type I and Irregular IV, and coliforms with
were examined directly. IMVEC reactions - + - - + were classified as
After examining Gram-stained smears of the Intermediate type I. For the indole (I) test, a
material, cultures were prepared as follows. For xylol extract was used. The methyl red (M) and
gram-negative bacteria, deoxycholate agar with a Voges-Proskauer (V) tests were performed on
low deoxycholate and citrate content (Leifson, cultures incubated for 4 days at 37 C (Kauff-
1935) and SS agar (Difco) were used; preliminary mann, 1954), using the spot test on a white tile
enrichment was in sodium selenite, followed by for the first, and adding creatine according to
subculturing to SS agar. For gram-positive bacte- O'Meara's modification for the second reaction.
ria, horse-blood agar with 10% "dispersol" Eijkman's (E) test was based on lactose fermen-
(Imperial Chemical Industries product), which tation at 44 C (Wilson et al., 1935), and citrate
prevents swarming of Proteus, was employed. As (C) utilization was observed in Koser's liquid
this study was concerned with the search for in- medium. When the IMVEC reading indicated
testinal pathogenic bacteria, the cultures were an irregular or intermediate strain, the culture
incubated aerobically at 37 C. A few samples were was plated on nutrient agar and a single colony
cultivated anaerobically on blood agar at 37 C was retested. The IMVEC formula for the four
and on nutrient agar at 25 C. The degree of most commonly occurring biochemical types was
growth on primary isolation was assessed as as follows: Escherichia coli type I (+ + - + -),
abundant, moderate, or sparse. Irregular I (+ + - - -), Intermediate type I
Unless otherwise indicated, the bacteria were (- + - - and Intermediate type II
classified according to Bergey's Manual (Breed, (+ + --+). E. coli type I strains were further
Murray, and Smith, 1957). For the identification examined serologically according to Kauffmann's
of gram-negative nonlactose-fermenting bacteria, (1954) technique, using two sets of diagnostic
representative (average, 6) colonies from each sera. The sera for the first set were prepared with
positive specimen were picked from bile salt 14 enteropathogenic E. coli strains found in
media and examined for biochemical reactions in association with infantile diarrhea. These sera
Kligler's lactose-glucose ferrous sulfate agar were diluted and pooled into three polyvalent
(Difco), mannitol-peptone-water, urea agar, and sera as follows:
in the indole test. For the differentiation of Group A: 026:B6, 055:B5, 086:B7, 0111 :B4
Paracolobactrum (paracolon) species resembling Group B: 018:B21, O119:B14, 0125:B15,
Salmonella, fermentation of 5% lactose, 5% 0127 :B8, 0128 :B121262 MUSHIN AND ASHBURNER [voL. 83
TABLE 1. Frequency of isolation (aerobically at 37 C) of bacterial types from the gut* of mutton birds
L~~~~
No. of isolations of bacterial types
No. and type No. of Positive Total Ratio of
speci- no. of Gram-negative types (I) Gram-positive types (II) groups
of bird mens specimens solations I to II
Para-
Proteus colon Coli-
form Staphy- Entero-
Total lococcust coccus Other Total
no. %
40ONormal 149 107 72 229 57 20 54 131 36 52 10 98 1.3:1
chicks
8 Starved 32 32 100 74 20 4 35 59 8 3 4 15 3.9:1
chicks
42 "Limy" 164 152 93 443
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165 35 144 344 36 54 9 99 3.5:1
chicks
33 Normal 130 35 27 52 9 0 6 15 16 16 5 37 0.4:1
adults
*
Proventriculus, upper and lower intestine, and the cloaca.
t According to the classification by Shaw et al. (1951), also in Tables 2, 4, and 5.
Group C: 025:11L, 044:74L, 0112:B11, the Enterococcus group, and were identified by
0124:B17, 0126:B16 growth in 6.5% sodium chloride broth and on
Slide agglutinations were performed with 40% bile blood agar. Gram-positive rods were
living organisms on large glass slides, marked in classified as Bacillus spp. on the basis of a positive
squares. As none of the strains isolated in the catalase test, spore formation, and motility. The
present study reacted with these sera, there was gram-negative nonsporing rods appearing on
no need for confirmatory tube agglutinations. blood agar plates, when confirmed as tolerant to
The second set of sera was used to detect 20 bile salt by subculture on deoxycholate agar,
somatic antigens which represent coliforms occa- were not further investigated.
sionally found in association with intestinal disor- Additional tests. Gastrointestinal segments
ders of animals (Orskov, personal communication). from 12 mutton birds were tested for pH value
These sera were diluted and pooled into the with British Drug Houses indicator papers.
following groups: As many specimens from mutton birds yielded
Group W: 02, 03, 025, 086, 0117 poor growth of intestinal flora, the antibacterial
Group X: 01, 04, 06, 08, 09 activity of contents from gut and samples of oil
Group Y: 015, 018, 021, 078, 0115 were tested, using gram-negative intestinal
Group Z: 05, 045, 051, 075, 0114 bacteria. The following strains were examined:
The somatic suspensions for tube agglutination E. coli, Aerobacter aerogenes, three Proteus spp.,
tests at 52 C with the above sera were prepared Paracolobactrum sp., Pseudomonas aeruginosa,
by steaming saline suspensions from nutrient- and Salmonella typhimurium. A bacterial lawn
agar slopes at 100 C for 90 min. Suspensions for was prepared on nutrient agar with a 2-hr broth
agglutination with group 08 and 09 antisera culture, and either spotting or the cup technique
were heated at 120 C for 2 hr to destroy the was employed using the material previouslv
thermostable antigens sometimes found in those emulsified in saline.
two groups. When positive results were recorded
with pooled sera, further tests were performed RESULTS
with the appropriate single-factor sera. Table 1 shows the frequency of isolation of
Gram-positive cocci, isolated on blood agar various types of bacteria from the gut of mutton
plates, were tested for catalase production on birds which were examined in the laboratory or on
nutrient-agar slopes. Staphylococcus (Micrococ- the island. The alimentary canal of normal chicks
caceae) strains were coagulase-tested, and the was colonized by a variety of species, both gram-
negative strains were subdivided according to the positive and gram-negative, with a slight pre-
classification of Shaw, Stitt, and Cowan (1951). dominance of the first group. Both "limy" and
Streptococcus strains belonged predominantly to starved chicks showed a high percentage of posi-A\nt%
19/j;62
I
(GASTROINTESTINAL FLORA OF MUTTON BIRDS 1263
TABLE 2. Frequency of isolation (aerobically at 37 C) and identification of bacterial types from the gut of
mutton birds
No. of isolations of bacterial types
Proteus Para- Coliform Staphy- Entero- Other gram-positive
colon Coiomlococcuts coccus bacteria
N o. and type Tota-of
of bird n.o
isolations
> >
_
_
_
__
_ _ O O
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40ONormal 229 27 281 1 1 19 26 8 8 10 2 22 14 52 6 0 0 4
chicks
8 Starved 74 9 6 5 0 3 1 0 24 0 11 0 6 2 3 0 1 2 1
chicks
42 "Limy" 443 57 53 39 16 20 15 35 10 36 56 7 18 18 54 3 4 2 0
chicks
33 Normal 52 3 6 0 0 0 0 3 0 1 1 1 4 12 16 3 1 1 0
adults
TABLE 3. Frequency of isolation (aerobically at 37 C) of bacterial types from gastrointestinal segments of
mutton birds
No. and type No. of gram- No. of gram-
Organ No. of Positive No. of negative positive Ratio of
of bird g specimens specimens isolations bacterial bacterial grouns
types (I) types (II) I to I
no. %
40 Normal Proventriculus 35 23 66 39 17 22 0.8:1
chicks Upper intestine 40 24 60 48 27 21 1.3:1
Lower intestine 40 28 70 59 34 25 1.4:1
Cloaca 34 32 94 83 53 30 1.8:1
8 Starved Proventriculus 8 8 100 13 12 1 12:1
chicks Upper intestine 8 8 100 12 9 3 3:1
Lower intestine 8 8 100 21 17 4 4.3:1
Cloaca 8 8 100 28 21 7 3:1
42 "Limy" Proventriculus 41 36 88 101 72 29 2.5:1
chicks Upper intestine 41 34 83 83 62 21 3:1
Lower intestine 41 41 100 121 99 22 4.5:1
Cloaca 41 41 100 138 111 27 4:1
33 Normal Proventriculus 32 4 13 6 2 4 0.5:1
adults Upper intestine 33 0 0 0 0 0 0
Lower intestine 33 11 33 19 5 14 0.4:1
Cloaca 32 20 63 27 8 19 0.4:1
tive specimens and a comparatively abundant quency of isolation of various types of bacteria
flora, with a higher ratio of gram-negative types from the gut of "limy" and other mutton birds.
of bacteria. Specimens from adult birds often In normal chicks, some organisms, namely P.
gave no growth or the growth was sparse, with a morganii, P. mirabilis, and Bethesda were seldom
predominance of gram-positive types of encountered; other types, such as P. rettgeri, P.
organisms. vulgaris, E. coli type I, Staphylococcuss, and
In Table 2 a comparison is shown of the fre- Enterococcus, showed a higher incidence. In-t
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12641962] GASTROINTESTINAL FLORA OF MUTTON BIRDS 1265
starved chicks, the absence of E. coli type I and TABLE 6. Biochemical classification* of 143 Proteus
the presence of coliforms Irregular I and Inter- rettgeri cultures isolated from mutton birds
mediate type II was noted. In the diseased chicks, 4865 4555 4806 A 5
Proteus, showing the highest incidence, was repre- "Sugar" Group Group Group Group
I II ILL IV
v
sented by four species, of which P. rettgeri and P.
vulgaris were the most commonly encountered. Rhamnose .... - + - +
The comparatively frequent presence of Bethesda Salicin ......... + - + + +
strains was recorded. Coliforms were often iso- Arabinose .... _ + +
lated, and amongst them E. coli type I was con-
siderably less frequent than the total of Irregular No. of strains
I, Intermediate type I, and Intermediate type II. isolated ...... 80 57 4 1 1
In adult birds, the absence of P. morganii, P.
* According to Kauffmann (1954).
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mirabilis, and paracolon bacilli was noted. In all
birds, gram-positive cocci were represented
mainly by Staphylococcus saprophyticus, S. lactis, birds from various localities, and from chicks
and Enterococcus; gram-positive rods were with "limy" disease.
seldom isolated. However, "limy" birds yielded Amongst Proteus species, P. rettgeri and P.
no aerobic bacterial species which would be con- vulgaris showed the highest incidence and P.
spicuous by its presence in all pathological ma- mirabilis the lowest. The total incidence of
terial and its absence in normal specimens. paracolons was similar in normal and "limy"
The distribution of bacterial flora in various birds. Amongst coliforms, E. coli type I strains
parts of the gastrointestinal tract in "limy" and were less frequently recorded than the total of
other mutton birds is presented in Table 3. The other types. The incidence of gram-positive
most consistent distribution of organisms bacterial types was higher in normal and starved
throughout the length of the gut was recorded in birds than in "limy" chicks. As previously
"limy" and starved chicks, with a higher inci- observed, there was no indication of the presence
dence of gram-negative than gram-positive types. of a specific bacterial strain in the diseased chicks.
In normal chicks, the intestinal segments showed Sixteen specimens, obtained from "limy"
a slightly higher ratio of gram-negative, and the birds from sources other than the gastrointestinal
proventriculus of gram-positive bacterial types. tract, gave no growth, or gave organisms of the
In normal adult birds, the proventriculus occa- same species as those encountered in the bird's
sionally yielded growth, but the upper intestine gut.
possessed no cultivable aerobes; in the proventric- P. rettgeri. The comparatively high frequency
ulus, lower intestine, and cloaca the bacterial of isolation of P. rettgeri prompted a further
flora from these birds was predominantly gram- study of biochemical types. Of the 165 P. rettgeri
positive. strains isolated, 143 strains were examined and
Table 4 sets out the incidence and identity of subdivided into five biochemical groups on the
aerobic bacterial types obtained from cloacal basis of rhamnose, salicin, and arabinose
swabs. The number of specimens yielding growth fermentation (Kauffman, 1954), as shown in
was higher than from the gut of mutton birds Table 6. Groups I and II contained a total of
(Table 1). The cloacal swabs from adult birds 137 strains, group I being predominant; the
gave a poorer growth than those from chicks; three remaining groups were represented by
some samples had no gram-negative organisms, only six strains. Group V, which had one strain,
and none yielded Proteus. In young birds, the was not listed by Kauffman (1954). The flora of
predominance of gram-negative bacterial types both "limy" and normal birds contained strains
and, in adults, an even ratio of gram-negative to of groups I and II, and therefore no specific
gram-positive types were noted. association with the disease was indicated.
Samples of regurgitated food showed similar E. coli type I. Amongst the coliform strains,
types of bacteria to those found in the gut, with a 85 E. coli type I were found (7% of the total
slight predominance of gram-positive cocci. isolations). Of the 14 enteropathogenic serotypes
Table 5 shows the total frequency and per- associated with infantile diarrhea, only one E.
centage of isolation of bacterial types from coli 0128 B12 strain was cultivated from a chick
normal and starved chicks and normal adult from Montagu Island. With the use of 20 addi-1266 MUSHIN AND ASHBURNER [VOL. 83
TABLE 7. Determination of pH of specimens from Antibiotic tests. Gastrointestinal material
the gastrointestinal tract of 12 normal mutton birds
occasionally showed a weak antibacterial activity
No. of Proven- Upper Lower against some gram-negative strains. Of the 15
birds
tested
Age triculus intestine intestine specimens tested, 1 acted slightly on E. coli
type I and paracolon spp., 1 on E. coli type I
9 Adult 4.3to5.5 7.Oto7.3 7.Oto7.3 and P. mirabilis, and 1 on P. mirabilis. However,
3 Chick 2.5to5.8 7.lto7.2 7.1to7.2 this activity did not seem to be uniform and
significant. One of the four oil samples tested
had a pigment-promoting action on P. aeruginosa.
tional diagnostic sera, E. coli 018 was isolated
DISCUSSION
on autopsy, eight times from specimens obtained
from five adult birds in one locality and once The present investigation failed to disclose
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from a chick from another locality, both on particular aerobic bacteria etiologically related
Great Dog Island. Further, E. coli 08 and 09 to "limy" disease of mutton birds, Previous work
serotypes were found in cloacal swabs from one eliminated viruses (Mykytowycz, Dane, and
chick each on Montagu Island. Therefore, Beech, 1955) and endoparasites (Mykytowycz,
typing with 34 diagnostic coliform sera indicated personal communication).
the occurrence of four serotypes, the 12 isolates The type and distribution of bacterial flora
representing 14% of the 85 strains examined. are of interest, but it must be emphasized that
Other organisms. Some specimens showed this study was predominantly concerned with
gram-positive rods and cocei in smears but did aerobic organisms. Isolations under aerobic
not yield aerobic growth on bile salt media or on conditions at 37 C seemed to show that some
blood agar at 37 C. From a few of these samples, samples were free of organisms capable of growth
organisms morphologically resembling those in on the media provided. However, direct smears
smears were cultivated either anaerobically on and cultivation of a few samples under anaerobic
blood agar or aerobically on nutrient agar at 25 conditions and at 25 C usually indicated the
C, but the remaining specimens were negative. presence of bacteria. Possibly, a larger inoculum
Determination of pH. The results of pH testing and the use of additional enrichment and selective
of material from various parts of the gut of media would show that the material was not
normal mutton birds are presented in Table 7. "bacteriologically sterile." Sieburth (1959), in
The proventricular material from the few chicks his study on antarctic birds, did not find them
examined had an acid reaction and a wider "bacteriologically sterile," although they were
range of variation than specimens from adult often devoid of typical intestinal organisms.
birds. Of the 12 samples tested, only one yielded In adult mutton birds, the intestinal flora was
growth at pH 5.5 (P. vulgaris, E. coli type I, and usually scanty and often absent, especially in the
S. lactis). The pH of the material from the upper upper gut. In healthy chicks, the microflora was
and lower intestine was nearly constant, on the absent or poor in the alimentary canal of a small
slightly alkaline side, and the specimens examined group of birds examined but more abundant and
were either negative or contained a mixed flora. varied in the intestine of others. In "limy"
Metabolic disorders
(a) High protein diet? (b) Natural starvation?
I
Concretions of ----Proliferation of certain flora and
sodium urate >-changes in distribution in gut
Mechanical
I-
-| "LIMY" DISEASE |
I
Physiological stress
FIG. 1. Biological factors in relation to "limy" disease1962] GASTROINTESTINAL FLORA OF MUTTON BIRDS 1267
birds, bacteria were found in larger numbers than No infectious agent was detected in "limy"
in normal chicks from the same locality, and the disease, but the biological factors concerned
organisms were more consistently distributed require further study.
through the upper region of the gut. The com-
paratively high incidence in mutton birds of ACKNOWLEDGMENT
P. rettgeri, and the absence or comparative
scarcity of P. mirabilis and E. coli type I strains, We wish to thank G. G. Blake for technical
is the reverse of the distribution of these assistance.
organisms in the human alimentary canal LITERATURE CITED
(Mushin, 1950). The isolation of one only entero-
pathogenic serotype, E. coli 0128 B12, does not BISHOP, R. F., AND E. A. ALLCOCK. 1960. Bacterial
indicate the mutton bird to be a significant host flora of small intestine in acute intestinal
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of these organisms. On the other hand, a few obstruction. Brit. Med. J. 1:766-770.
specific coliform serotypes belonging to groups BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH.
1957. Bergey's manual of determinative bac-
08, 09, and 018 were cultivated. teriology, 7th ed. The Williams & Wilkins Co.,
Examination of mutton birds from other Baltimore.
localities may indicate more clearly the sig- FALKOW, S. 1957. A screening method for enteric
nificance of the aerobic intestinal flora found in organisms using a ferric chloride test. Am. J.
birds from Great Dog Island. There is a Clin. Pathol. 28:99-102.
possibility of the presence of antibiotics in the KAUFFMANN, F. 1954. Enterobacteriaceae. Ejnar
food of some birds, e.g., acrylic acid (Sieburth, Munksgaard, Copenhagen.
1960), although in our experiments this was LEIFSON, E. 1935. New culture media based on
apparently not a significant factor. sodium desoxycholate for the isolation of
A few biological factors could be considered in intestinal pathogens and for the enumeration
of colon bacilli in milk and water. J. Pathol.
relation to "limy" disease, and these are indicated Bacteriol. 40:581-599.
in Fig. 1 as follows. (i) The normal starvation of MUSHIN, R. 1950. Bacteriological aspects of gas-
mutton birds may contribute to changes in troenteritis in infants. Australian J. Exptl.
bacterial flora, with the proliferation of some Biol. Med. Sci. 28:493-508.
types. (ii) Further, the numerical rise and change MYKYTOWYCZ, R., D. S. DANE, AND M. BEECH.
in bacterial flora and wider distribution in the 1955. Ornithosis in the petrel, Puffinus ten-
gut in "limy" than in normal chicks may be an uirostris (Temminck). Australian J. Exptl.
indicator of metabolic disorders which in turn Biol. Med. Sci. 33:629-636.
may influence further changes predisposing to SERVENTY, D. L. 1958. Recent studies on the
Tasmanian mutton-bird. Australian Museum
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