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
                                                  1260
1962]                  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 :B12
1262                                   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

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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
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                                                                                                   1264
1962]                  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" disease
1962]                  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

                                                                                                             Downloaded from http://jb.asm.org/ on March 23, 2021 by guest
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
"limy" disease. (iii) The pattern of intestinal            Mag. 12:327-332.
flora could be an indicator or result of a physio-     SHAW, C., J. M. STITT, AND S. T. COWAN. 1951.
logical stress which leads to "limy" disease.              Staphylococci and their classification. J. Gen.
(iv) The type of diet (e.g., high in protein) may          Microbiol. 5:1010-1023.
contribute to the formation of a mechanical            SIEBURTH, J. M. 1959. Gastrointestinal microflora
blockage, possibly in the form of sodium urate             of antarctic birds. J. Bacteriol. 77:521-531.
concretions. When an obstruction is established,       SIEBURTH, J. M. 1960. Acrylic acid, an "antibio-
it is reasonable to assume that the bacterial              tic" principle in phaeocystis blooms in antarc-
flora above that point would proliferate to a              tic waters. Science 132:676-677.
greater extent, as was demonstrated in human           STUART, R. D. 1956. Transport problems in public
                                                           health bacteriology. Can. J. Public Health
cases (Bishop and Allcock, 1960). However, the             47:114-122.
reason for the initial accumulation and blockage       WILSON, G. S., R. S. TWIGG, R. C. WRIGHT, C. B.
is not understood. It is difficult to assess cause         HENDRY, M. P. COWELL, AND I. MAIER. 1935.
and effect in intestinal disorders, that is, whether       The bacteriological grading of milk. Medical
the upset balance of the intestinal flora precedes         Research Council (Brit.) Spec. Rep. Ser. No.
or follows pathological conditions.                        206.
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