search in VeterinaryScience 1983, 35,4246

lacterit31 overgrowtt1 assoc:iated writh a n

nteropathy inI the German shephcerd do1

K. M. HA I I , uepanment of Verennaryt'athology, University of Liverpool, J . R. NEEDHAM,

Division of ComparativeMedicine, MRC Clinical Research Centre, Harrow, Middlesex, M. W . CARTER,
Department of VeterinaryPathology, University of Liverpool, Liverpool

tuanuiauve add qurnliauve uauteriological studie-s

ere performed on duodenal juiice from l!9 dogs wit1h
- .-malities affecting the brush borders, lysosomes
~LJIIUII
and endoplasmic reticulum. The four anirr~alsin
ironic small intestinal disease. Bacterial c~vergrowtl h group 2 had specific biochemical abnormalitic:S con-
as demonstrated in seven Gem a n.-she1 - herd dogs
ith minimal histological but specitic biochemicaII
. . to the brush borders and villous atropkiy was
fined
evlden~tonly in biopsies from cases 3 and 4. In group
bnormalities in the jejunal mucosa. Most frequent1y 3 (six(jogs) there were lysosomal changes and specific
is overgrowth comprised bacteria of the norma11 brush border abnormalities (Batt et al 1982) distinct
om, particularly Escherichia coli and enterococcii, from 'those observed in groups 1 or 2. Histological
ut these wlere occasic~nallyaccompanied by bacteri a changes were m:inimal apart from villous atrophy in
rely presesat in the proximal small intestine of th e case I1. The bic~ p s ychanges in the eight dogs in
ormal do!g, particu~larly Clostridium species. 11
lost cases"tllw
:.I .
.
.
u r s l x I ~ ~was
.
..
a
,.
-
t h associated with raise1d
" group 4 were vsiriable, with cases 12 and 15 having
Ibarulba ;
some Faotr.rz.r in common with group 3 and the
serum folalte and re(luced sentm vitami n B,, con ling cases resemblinig those in ggroup 2.
centrations, a finding consistenlt with the capacity o ylose absorption test indicated reduced absorp-
many entenic bacteriaI to synthcesise folatle and binc 1 cases 1, 5 a n d 9 t cI 12, whilet in all ca:ses the
vitamin B,,. --- -h I -rAaA test
nral
.
"
.
-
.
-A . . aemonstrated
. - .-..,
normal~
el:ncrine
--

pancrc:atic function (Batt and Mann 1981). R

ations of serum folate and vltamln B, haems tol logy showed neutrophilia 0 1 1 . 5 >
Dns has be1en shown to assist the detectioln litre) in cases 5, 10 and 12 and lymphopenia (<
-+,.+:A- AF .
IU u l r c l p l s r a r l u r l UI chronic small intestinal abnor -
1m/1:tre) in cases 1 and 11. Routine clinic&, ,
1" ,111 ,-
~alitiesin the dog (Batt and h![organ 196t2). In thaa chemi:stry revealed no abnormalities. Faecal ex;mina-
udy it was suggested that hi]gh serum folate ancd tion dlemonstrated Toxocara species ova in cast:9 and
:duced serum vitamin B12concentration s might be
..
ldicative of bacterial overgrown in rne proxima11
. .. cysts Iof Giardia species in cases 7 and 8. Barium-
. .st, radiographic studies performea
contra -
.I - - .--..
on c:ases I
-
nall intestineof theclog and th is possibility has bee1n to 12 revealed no gross a1latomica1 abnormalities in
ursued in the present study. the stc~machor small intest:ine.
Coritrol animals for ttiis study consisted of six
-. .
llly normal adult dogs, three greyhoundIs, two
Materials ana methoas an shepherd dogs a nd one rrlongrel, writh no
able histological or biochemic:a1 abnornnalities
Animals jejunal mucosa.
Nineteen dogs wit:h small intestinal disease had Invest igations
een referred for an investigation of chronic
iarrhoea w4th
.. ..or .wit1 . lout loss of weight. These dogs11 The.I concentr
. . vitamins in the serum of
have been dlvlded lnto four groups on the basis 0" tasted anlmas were estimated using Lactobacillus
the serum folate and vitamin B12 concentration.s casei ;as test organism for the folate assay (Waters
(Table 1). Jejunal biopsies from animals within each and N Iollin 1961) and the Z strain of Euglena gracilis
of the first three groups revealed the characteristi c for thct assay of vitamin BI2(Anderson 1964). Jejunal
biochemical changes described r (Batt an1d biopsies were cjbtained r,erorally 1kom a si:te just
Morgan 19812). distal to the duodenal-jeju nal flexurc:using a m~ultiple
Case 1 (1group 1) klad an en1reropatny resemoling biopsjr capsule (:Batt 1979)
-:,...I a...,.,*
chronic troblca , :I. I1
J V I U G 111 IIIUI d a t t et a1 1983) with a
,, fnr+ A..
l o r r v u r a u u odenal
.,,...,.l
. .Aft1cr a r a a r df appr0Xilrrarsry
mo.. n r

nlarked vilious atropl

. nvestigaticsn was ot
 iergrowth in an enter 'thedog
'ABLE 1: Clinical details Iand bacterialIflora in the duodenal juice of dogs H - 9 ..
Serum Serum Case Weight Age at Viable bacteria in duodenal
folate vitamin 812 number Breed 14ge Sex (kg) onset juice:logro per ml (types)
Low Low 31 :Pp sp)
Low Normal BC 4.4(Ec) 4..2 (El
L 4.8 (Ha) 4. 6 (Cl sp)
- -
IS 4.3 (St e) 3. " --",
5 IS <3.0 (no isolates)

3 High Low or 6 GS
normal 7 GS
0 CC

4 Normal Normal 12 GS 1y M 31 7m 6.3(Sf)5.:

13 GS llm M 29 5m 3.9(Ec)3.5(Sta) 3,4(Cb)
3.3 (El 3.0 16s) 3.0 (Gal
14 n <3.0 (no is,olates)
15 33 <3.0 (no isolates)
16 38 <3.0 (no i solates)
17 23 <3.0 (no is1dates)
18 25 .- - Inn
<3.0 ,. .- i+i
.- nlntpsl
19 IS <3.0 (no is1
BC Border collie, GS German shepherd, IS Irish setter, L Labrador
Aa Acinetobacier anitratus, Bf sp Bifidobacteriurn spa, Bs Bacillus
E Enterococcus, Ec Escherichiacoli, Ga sp Gaffkya sp, Ha Hafnia a r m
bm bovis, CI
- - - ~ -~
. .
t a Siaphyloc i,St c Staph) *us, St e Siaphylococ~usepiderrnidis, Stm sp str re^
Obligate ana

...
losltlonea .
hrough the tubing I
.. ..
..a
wtn tne
psy. capsu:
. le.
tlp just proxlma to the duodenal . a1nimals wit h small in
the isease and shows
the control ranges descnbed previously (Batt and
lexure. Prior sterilisation of the tubing was achieved Morgan 1982). In group 1 both serum folate and
ly filling it with 70 per cent (v/v) ethanol in water for vitamin B,, concentrations were reduced, but in
t least 20 minutes, subsequently flushing with 50 ml animals of group 2 a reduced serum folate was
terile isotonic saline and finally with air. The first 2 accon~paniedby a normal concentration of vitamin
o 3 ml of ' juice asr,irated was discarded to avoid Bll. Ety contrast, the serum folate concentrations in
lotential clontaminat ion with I~rganismsof oral c)r anlmi11s of group 3 were increased to values up to
astric origin. four t imes that of the highest control, while in all but
Bacteriological study of a sample of duodenal juice- --.
one case the vitamin B,, conc i were
from each dog was either begun immediately or the reducied. In grolup 4 the le:vels of thf IS were
juice was diluted 10-I in transport medium (Crowther norm;a1.
1971) and stored at -20°C for UD to four davs .-:--A:--
U ~ -_,
E X Z U I I I I I ~UIL Iuuoaena .~-.-rrom rlve of the
julce -

efore examination. IFor bacter ial countil~g dilutionIS
f 10-2and in per,tone water were prelpared and a
. ... .c~f
1 ml aliquot of eaclI was-spre;ad over thle surface
single blood-agar plate. Pour plates of each dllut~o n
rere incubated at 37OC for 48 hours, two aerobically
nd two anaerobically in an anaerobic jar with a gae
enerating pack (Oxoid). After incubation th e
umber of colonies of each bacterial species wa.W;
:corded. Representative colonies were then sut-
ultured on appropriate media and identified ae
etailed by Cowan (1974) or An-Ident (Oxoid).
PI^ 1 detals the serum vitamln concentrations In
contrc11 animals revealed no bacteria; hence the total
viable counts were less than 1.0 x 1@ organisms per
ml. InI the sixth dog 1.9 x 10d of Escherichia coli per
o r-ew aerobically. The bacteriql
.A
ml b.
.a.. I(U +-I--
11u1a in the
duodc:nal juice of the animals wittI small inttestinal
abnor malities are summarised in Fig 1 and det;filed in
Table 1. When compared to the higtlest contra11value
.... elevated in
tnctutal viable counts (Fig 1) were sl~gnt~y
.L-.- .
the ar~imalin group 1 and in two of the animals in
group 2. In contrast all animals from group 3 and
one animal from group 4 had a marked bacterial
overgrowth (>1.0 x 1 8 organisms per ml) in the
proximal small intestine. The counts in group 3 were
significantly elevated (Mann-Whitney U test)
(P<O.01) when compared not only with the c:ontrol
 K . M. Butt. J. K . Needham, M. W . Cartor
re) 1

Group
- 2 3
Group
FIG 1: Serum folate and vitamin 012 wncenrrarlon: iarlu IWWI wr;Iarlal cuunrs in duodenal juice rrorn
animals with small iritestinal dise;a&. Dotted Iines define ciontrol ranges1 for the vitarnin results arld
upper c:ontrol value for total baaterial counts. Open circle denotes aninnal given par'enteral vitamin
812

roup but also with me orner animius wlrn sma11 proxllm a smal inresrlne or me normal dog, F--'---IanlCU-
itestinal disease. The organisms responsible for thle larly (Zlostridium species. The remaining anim als had
vergrowth varied (Table 1). but enterococci (incluc1- count s of less than 1(Y organisms per ml of ' juice,
ig Streptococcus faecalis), E coli and Clostridiurn emph
. .
asising that bacterial overgrowth is Ilot an
pecies were most frequently isolated. inevitable consequence of small intestinal disease in
the dc
In I umbers an is both
alter In vmous structural and functional dl!sorders
The use of an open-ended tube to quantify enteriic that result in intestinal stasis, conditions collc:ctively
bacteria has been validated in man and in experi- called the 'blind-loop syndrome' (Tabaqchldi and
mental dogs (Tabaqchali and Booth 1%7, Gorbach Boott1 1967, Draser and Shiner 1969, Gorbalch and
et al 1967). The multiple biopsy capsule used her.e Tabac - .. . .~chali1969, Gorbach 1971, Banwell et a1 1981).
ras particularly convenient, as a single intubation VlabIe counts frequently range fromI 106 toas high &
rovided not only duodenal juice but also intestinal 10I1/rnl of juice and comprise a complex flora,
iopsies. The quantitative bacteriological findings in which contains particularly coliforrns and a~bligate
-.--.
le control dogs agree with two previous studie,, ana~robessuch as Bacteroides specles and PIn r- -.,
rith mean viable counts of less than 104 organismu tridium species. Disordered motility could be I.espon-
er ml in juice from the proximal jejunum of normid sible for the bacterial overgrowth describec1 here.
ogs (Gelbart et al 1976, Greenlee et al 1977). In bot h Howe!ver, affected dogs had no obvious anal.omical
lese studies E coli, enterococci and lactobacilli we1.-c aurtur
-I----
malities in the small intestine, the bacrer~al --.--1-1

redominant and only small numbers of obligate count s were generally lower than in the 'blind-loop
1

naerobes were isolated. Ihasz et al(1976) also found syndrlome' and in three of the seven dogs no obligate
I

: coli as the most frequent organism iin duodenid anaer,obes were isolated. Achlorhydria has also been
lice from normal dogs although mean counts we1.e associ~atedwith intestinal colonisation in mim and
lightly higher (10l/m this dlefective secretion of gastric acid was tho1ught to
In the present stud: d have been responsible for the overgrow1I with
ne animal from group 4 had a marred overgrowtt1, aerobes and anaerobes described in a baseriji dog
with total viable counts greater than 10l bacteria pf :r (Strornbeck et a1 1981). However this explanation is
ml of duodenal juice. Most frequently this oveir- unlikc:ly since people with an uncomplicated gastric
growth comprised bacteria present in the norm;d muco sal atrophy have jejunal counts of less than 104
.
flora., ~articularlvE coli and enterococci. but in somle ornanisms Der ml. and overgrowth in other achlor-
ases orgariisms werce isolated that are. rare in th :subjects appears nlost likely to be reli
 vergrowth
concurrent intestinal stasis (Drasar et al 1969, Drasar
and Shiner 1969, Gorbach and Tabaqchali 1969).
This is supported by a study in experimental dogs
in which varying degrees of vagal denervation il., .A
gastric resection were performed (Greenlee et al
1977). All these procedures achieved comparable
reductions in gastric acidity but the highly selective
parietal-cell vagotomy did not result in jejunal over-
growth, a consequence of all the more extensive
surgical operations. The third group of diseases
associated with bacterial overgrowth in man are the
immunodeficiency disorders (Arnent et al 1973,
Webster 1976). Interestingly there are many features
in common with the animals in the present study
since in immunodeficient states viable counts are
generally lower than in the 'blind-loop syndromc'Z
ropathy oj'the dog
(Donaldson 1962, Donaldson et al 1962, Giannella et
al 19171, 1972) so that it becomes unavailable for
transport by the enterocyte. This effect may be
,.,,,;rated
mnrl. by intrinsic factor (IF), which can
compete for vitamin B,, at the bacterial surface and
also form an IF-B,, complex which inhibits microbial
binding and hence subsequent uptake of vitamin BIZ
(Donaldson 1962, Giannella et al 1972). Yamaguchi
et al (1969a) indicated that the dog does not secrete
IF so that although other BIZ-bindersof gastric origin
(Yamaguchi et al 1969b) might afford some protec-
tion, the dog could be particularly vulnerable to the
adverse effects of bacteria on vitamin B,, absc~rption.
More recently, evidence for IF siecretion ;ind IF-
,
mediated absorption of vitamin B by the (dog has
L---
VFFIl presented; nevertheless only a re~a~ively low
--.-A:-.

aerobic rather than anaerobic organisms frequenltly
predominate, and infection with Giardia species is
not uncommon. At present, however, the cause of
the overgrowth in these dogs is unknown
The high serum folate and low serum vitamin E$12
.
concentrations in dogs of group 3 wer.e .consistent
with the bacterial colonisation of the proxlma~small
intestine in these animals. Many enteric bacteria can
synthesise folate (Doig and Girdwood 1960, Klipstein
and Samloff 1969, Hoffbrand et al 1971) and an
association between bacterial overgrowth and
elevated serum folate concentrations has been
demonstrated in man (Hoffbrand et a1 1966, 197I).
However, this only occurs when the overgrowth is in
the proximal rather than the distal small intestinP
(Hoffbrand et a1 1971) since folate is poorly absorb
from the ileum in man (Hepner et al 1968). Similar1
in experimental dogs bacterial overgrowth in surg
ally constructed blind loops only resulted in increased
conccentration of IF appears to be secreted into
gastric juice (Marcoullis and Rothenberg 1981). In
man, malabsorption of vitamin B,, due to bacterial
overgrowth appears to be related to the number
rather than the specific types of bacteria colonising
the small intestine (Gorbach and Tabaqchali 1969)
since vitamin B,, uptake is a property of a broad
spectrum of bacteria (Giannella et a1 1971, King and
Toskes 1979). Sfaecalis appears to be atypical in not
binding vitamin B,, (Giannella et al 1971) and,
interestingly, this was the red om in ant organism
respcmsible foir the oveirgro&h iln case 12 of the
prese:nt study, an aninlal with a normal serum
vitan in BIZconicentratiorI.
le autnors are grateful to the wellcome Trust,
the BSAVA Clinical Studies Trust Fund r--' UIU A-
L11G

serum folate concentrations when the loops we:re
constructed in the jejunum but not the ileu~rn
.
(Bernstein et a1 1972). Case 12 (Table 1,) emphasis;es
I-
that an elevation in serum folate level IS nut an--A
inevitable sequel to bacterial colonisation in t
proximal small intestine of the dog. Indeed, in m
folate concentrations can be normal or occasiona
reduced, and this level appears to depend partly (
the capacity of particular microorganisms to sy
thesise or consume folate, and partly on wheth
mucosal damage has affected folate transpc
(Hoffbrand et a1 197
There is a direct hip betwe:en bacter
overgrowth and defectwe a mxption of ' vitamin I
.- - --. .- ---- ,--I--
resulting in vitamin BIZdeficlrncy In rrlarl [~auia
chali and Booth 1967. Donaldson 1970, Neale et al
1972). Malabsorption of vitamin BIZhas also been
observed in experimental animals following the
surgical construction of blind loops (Strauss et 9 1
1961, Donaldson 1962). TInis interfierence wi
absorption is proba.bly due t~D the ability of ma
bacteria to bind and subsequer~ t l yabsort) vitamin I
German Shepherd Dog L eague for financial support.
.
We also thank Mr D. G Altman of the division of
.. ,...
computing and statistics, Clinical Research Centre,
. .
for statistical advice and Mrs r. M. Laws for typing
lanuscript
Rece,ived forpz September-3, 1982
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