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Concept:
Microbial Community Profiling and Characterisation (MCPC) A
comparison with other methods for the diagnosis of bacterial
overgrowth in the duodenum of broiler chickens
H.
Panneman, Dr. Van Haeringen laboratory; and J.H. van der
Stroom-Kruyswijk;
Elanco Animal Health
Summary
and Introduction
Broilers
frequently suffer from dysbacteriosis, characterised by too loose,
too thin, voluminous, bad digested and or viscous faeces. The
growth is retarded and the litter quality deteriorates. The
activity of the birds is slightly reduced. The objective of this
study was to find a correlation between detailed bacterial
profiles of the duodenal micro flora of broilers with the results
of other methods that are used as a diagnostics for
dysbacteriosis. For the comparison animals were selected from one
or more houses on different farms in Belgium and The Netherlands.
Because of this experimental design the birds formed a
highly variable group with respect to age, housing, feed, food
supplements and medication.
The birds
were initially classified on the basis of the flock faeces score.
A score of 0 is indicative for a house free of dysbacteriosis
whereas a score of 1 is considered to be affirmative for the
clinical diagnosis of dysbacteriosis. The validity of this method
was checked by a semi-quantitative test plate test, originally
developed by van Saene(1). The plate test is based on serial
dilution of a swab sample from the duodenal contents on
MacConkey and Mannitol plates, the plate-dilution swab test. The
clinical diagnosis of dysbacteriosis was considered to be
confirmed by the plate-dilution swab test when more then 25 % of
the swabs, obtained from a group of broilers from one house
showed
bacterial
growth on either the MacConkey and Mannitol plates or on both
plates. Confirmation of the diagnosis free from dysbacteriosis
was defined as no growth on the plates.
A
selection of samples obtained in the course of this study was
submitted to Dr. Van Haeringen laboratory for the determination
of bacterial profiles using the MCPC method. MCPC is a PCR based
method by which a detailed profile of a complex microbial
community is generated. In addition the MCPC method can be used
to get an indication of the identity of the species present in
the sample.
After
normalisation and grouping of the data we discovered one specific
peak that showed a clear correlation with the flock faeces score
and with growth on either or both of the plates used in the swab-dilution
test. This peak was not found in 13 control samples that were
free of dysbacteriosis. In addition some of the samples
showed a pattern that is indicative for the presence of high
amounts of at least four different Lactobacillus strains.
Another
group of samples showed only a (large) number of very small peaks,
which may be indicative for the presence of antibiotic substances
in the feed or water. This was a remarkable finding since, at
first glance, the resulting MCPC profiles were highly variable,
especially between samples obtained from different farms, which
is consistent with the high degree of variation in the origin of
the samples.
Materials
and methods
Selection
of the broilers.
Farms with
clinical dysbacteriosis in one or more houses were selected. The
clinical diagnosis was based on the flock faeces score. In
addition, four houses from farms with a history of being free
from dysbacteriosis were selected as a control group. From each
house five individuals were selected at random.
Determination
of the flock faeces score
The
clinical selection criterion for dysbacteriosis was the faecal
score of a house. For this diagnosis faecal droppings are
collected on absorbing paper placed in a specially designed
litter box. A dropping is considered humid when it shows a ring
of humidity with a diameter that is at least twice the diameter
of the dropping. When at least 3 out 10 droppings are humid the
flock faeces score 1, and this is considered to be affirmative
for the clinical diagnosis of dysbacteriosis. When less
then 3 out of 10 droppings are humid the faeces score is 0 and
the flock is considered to be clinically free of dysbacteriosis.
Additional
criterion for the samples in the control group
For the
samples in the control group, birds were selected that produced a
dry dropping at sacrifice. Effectively this means that each
individual from this group can be considered free from
dysbacteriosis.
Sampling
of the duodenum with swabs
For the
sampling sterile cervical swabs (rigid nylon brush on a metal
wire) were used. The birds were sacrificed by applying Electro-voltage,
a barbiturate or by cervical dislocation. The duodenum was
removed by cutting the duodenal loop from the gizzard until the
caudal part of the pancreas. The surface was disinfected by local
heating (hot scalpel moving over the intestinal outside, with the
speed of cutting cheese). Subsequently the duodenum was opened
with a sterile scalpel and a swab sample was taken by moving the
swab up and down over a length of 3 cm, starting 1 cm behind the
exit of the gizzard, whilst applying mild pressure from the
outside with two fingers. In this way both the
contents
and the superficial mucosa were sampled.
The
plate-dilution swab test
The swabs
were semi-quantitative cultured on two different media, MacConkey
agar (bio Trading, code K039) and Mannitol (bio Trading, code
K040). The
media were inoculated, using a four-quadrant dilution method.
Bacterial growth in only quadrant 1 was given the score 1, growth
in quadrant 1 and quadrant 2, was given score 2, a positive
finding up to quarter 3, was recorded as score 3 and up to
quarter 4, score 4.
Definition
of positive swab scores
No growth
was defined as negative. Growth on the MacConkey and / or the
Mannitol plate was considered to be positive for dysbacteriosis.
If more then 25 % (in this case two or more out of five) of the
swabs originating from animals from one house are positive, the
clinical diagnosis dysbacteriosis is confirmed.
MCPC
The basic
technique that is used to establish an MCPC profile is generally
referred to as 16S rRNA T-RFLP or 16S ribosomal RNA Terminal
Restriction Fragment Length Polymorphism (2,3). This technique is
based on the selective amplification of the 16S rRNA gene from
bacteria by PCR (Polymerase Chain Reaction). The 16S rRNA genes
from a large number of different bacteria can be amplified in a
single PCR reaction. The length of the resulting PCR product is
variable and strain dependant. However for a large number of
strains the same length of PCR product will be found. Therefore
the PCR products are cut into smaller pieces using restriction
enzymes. These enzymes will cut at different positions in the PCR
products from different strains. The resulting fragments exhibit
a much larger variation in length and will thus result in a
higher esolution. A product of a specific length is
indicative for a very limited number of micro organisms.
The final products of the reaction are separated on an Applied
Bio systems ABI Prism 3100 Genetic Analyser that allows very
accurate determination the length of the products.
Using the
fact that a large number of 16s RNA sequences are publicly
available we have generated a database containing the theoretical
length of the products for a large number of known micro
organisms. With this database we are able to get an indication of
the species that are present in a sample.
Results
and Discussion
MCPC
profiles were established for 130 samples that were obtained from
a total of 26 flocks, reared at 20 different farms. The MCPC
profiles were highly variable, especially between different
flocks. The profiles of the control flocks showed a small number
of large peaks. Based on our database these peaks are likely to
represent four to five different Lactobacillus species. Even in
the other samples, where these peaks were less pronounced the
same group of peaks is often dominant. The latter is not true
though for a group of 53 samples that exhibited an extremely low
total peak height. In most of the samples from this group only a
few peaks were present. Fifty of these samples were obtained from
a group 14 flocks originating from 8 different farms. These
observations indicate that these flocks may have received
antibiotic treatment or supplements
with
antibiotic activity. For only two farms this could actually be
confirmed. Superficially there is no specific profile that is
correlated with either the flock faeces score or the plate-dilution
swab tests. The data were therefore analysed in more detail by
converting the peak patterns into a table. In the table each peak
is represented by its height. The data were normalised by
dividing the height of each peak in a sample by the sum of the
peak heights of that sample. Furthermore the samples that
exhibited an extremely low total peak height were excluded from
the analysis and are not taken into the consideration in the next
section. The normalised data were then grouped based on different
experimental
parameters.
Based on this normalised partial data set we found that the
presence of a peak with a length of approximately 473 nucleotides
shows a positive correlation with a positive flock faeces score,
and a positive score on the either or both of the plates used in
the plate-dilution swab test. The strongest correlation is found
for those samples where both the flock faeces score and the
mannitol plate test are positive. This peak with a length of 473
nucleotides was not found in any of the samples that combined a
negative flock faeces score with negative plat-dilution swab test.
These results corroborate our findings in an earlier research
project (3), which also indicated that the microorganism(s)
responsible for a peak with a length of approximately 473
nucleotides might be associated with the clinical diagnosis of
dysbacteriosis. According to our database there are only two
possible microorganisms that can be responsible for a peak of
this length, Clostridium paradoxum and Clostridium
thermoalcaliphilum. Whether this indication is correct and
whether they play a role in the development of dysbacteriosis in
will need to be established by further research.
Conclusions
Using the
MCPC technique we found a clear correlation between a peak with a
length of 473 nucleotides in the bacterial profile and the
combination of a positive flock faeces score and a positive on
either or both plates in the plate-dilution swab test. For those
samples where a positive flock faeces score was combined with a
negative mannitol plate in the plate-dilution swab test this peak
was found in only three out of nine samples. Further analysis of
the different bacterial profiles in correlation with feed and
farm factors will in the future improve the understanding and
solution of dysbacteriosis.
References
1) HKF van
Saene, AP Tometzki, SJ Fairclough, JH Rommes, AJ Petros. 1999.
Carriage, colonisation and infection. In: J.Giar, editor.
Internal Care Medicine. BMJ Books, London. 23:272-285
2) Marsh T.L.
1999. Terminal restriction fragment length polymorphism (T-RFLP):
an emerging method for characterizing diversity among homologous
populations of amplification products. Curr. Op. Microbiol. 2:323-327
3)
Panneman H. 2001. Microbial community profiling and
characterisation helps the vet. World Poultry (17) 4:34-36
The
Elanco Global Enteritis Symposium July 9-11, 2002 Concept:
Microbial Community Profiling
_ A
comparison with other methods for the diagnosis of bacterial
overgrowth in the duodenum of broiler chickens,
E-3 to
E-7