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Molecular epidemiology of BDD |
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We assumed BDD-associated Treponema spp. bacteria
to be the aetiological ‘necessary cause’ of BDD; we performed
detailed microbiological studies investigating the diversity of these
organisms.
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We obtained 23 cultured isolates from tissue biopsies
taken from BDD lesions on nine farms. Three distinct antigenic groups were
consistently identified when performing electron microscopy, 16S rRNA sequence
analysis and flagellin gene analysis, enzyme activity profiling, and serology.
The groups were closely phylogenetically related to T. medium, T.
phagedenis and T. denticola, three human treponemes (to view
phylogenetic trees in a new window, click here). These findings were
consistent with, albeit much more detailed than, other microbiological studies
performed in the past ten years (Choi et al., 1997; Collighan and Woodward,
1997; Collighan et al., 2000; Stamm et al., 2002; Trott et al., 2003; Edwards
et al., 2003). We cannot yet relate the isolates to specific clinical findings
or farm-level morbidity of BDD. Trott et al. (2003) postulate about the
relative importance of the treponemal groups as ‘primary’ and
‘secondary’ invaders, but as we currently lack information on
predominating treponemal morphotypes and possible sequential shifts of
different species over the course of the infection (Edwards et al., 2003b), we
are unable as yet to make any inferences. Research is ongoing: the development
of phylotype-specific PCRs is uncovering further antigenic diversity in the
cultured isolates. These results have not yet been
presented.
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Fig. W3. a) Electron micrograph of negatively stained cells of T320A
(T. phagedenis-like) isolate, b) showing 11 flagellar attached
subterminally (indicated by arrows). Bars, 0.5µm |
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T. phagedenis-like treponemes were most commonly
identified; T. denticola-like treponemes were found less frequently, and
T. medium-like treponemes were identified on only one farm. More than
one treponemal species was cultured from biopsies on four of the nine farms;
this suggests that mixed populations are common on farms. Furthermore, isolates
from two species were cultured from single lesion biopsies from two different
animals on two different farms, indicating that simultaneous infections with
treponemes from different species are possible. We do not yet know whether all
treponemes have pathological significance.
Although treponemes were successfully cultured from BDD lesion
tissue, there is no information on the distribution of the BDD-associated
Treponema spp. in the farm environment. There were indications that
these bacteria may have been present on the clinically negative farm included
in the cross-sectional study (seroconversion of clinically negative animals),
but until we can identify and sample from reservoirs of infection, this cannot
be confirmed. Although the treponemes are closely related to treponemal species
infecting humans, it seems likely that dairy cattle are the only host species
or ‘target population’ (although contagious ovine digital dermatitis,
CODD, appears to have a common or shared aetiology (Dhawi et al., 2005)). It
therefore seems likely that either the environment constitutes a reservoir, or
the target population is itself the source population, or both.
Long-term persistence of the
treponemes in the environment seems unlikely due to their fastidiously
anaerobic nature. Over the summer grazing period, the cubicle houses are
usually cleaned out; yet within a short period of re-housing, the incidence of
clinical infection rises. This evidential information suggests that the source
of infective treponemes is more likely to be animal-related. Various locations
could be suggested; further advances in microbiological techniques may provide
more information in the near future. Such information would enable more
accurate epidemiological modelling of the disease to be performed. Exploratory
work performed by us has failed to locate treponemes related to those
associated with BDD in any part of the gastrointestinal tract. The skin or
urinary tract are other locations we have sampled, but have consistently failed
to identify treponemes in. BDD lesions are currently the only known reservoirs
of the treponemes; our current hypothesis therefore is that transmission occurs
by dissemination of treponemes into the underfoot environment and ‘foot to
foot’ infection.
In order to
apply the ELISA for routine analysis of serum samples taken on the study farms,
we investigated the serological reactivity patterns of test sera with the 23
treponemal antigens. The results indicated the existence of at least three
serogroups / -types, consistent with the phylogenetic divisions. The ELISA
results for representatives of each group showed marked similarity. The T.
phagedenis-like isolates, which were most commonly identified on the farms
of origin, solicited the highest response, regardless of clinical status. These
antigens were related to treponemes that Moter et al. (1998) demonstrated using
an in situ hybridization technique to penetrate most deeply into the lesions.
It is therefore possible that this group has the highest pathogenic 160
significance.
There was, however, substantial cross-reactivity. This
could be a consequence of a lack of antigenic specificity of the
IgG2 antibodies. Alternatively, it could be due to mixed infections
with treponemes from multiple groups – which is plausible considering the
mixed treponemal populations identified on farm level. We were unable to
clarify this, which impeded interpretation of the serology; our results
indicate that the existence of serogroups is likely, but we are currently
unable to distinguish between these. Serological diversity is well documented
for Brachyspira hyodysenteriae, to which the treponemes are related.
For the application of a ‘general purpose’ ELISA, we
combined five antigens, including representatives of each of the three groups,
into a ‘cocktail’. The performance and repeatability of this ELISA
was assessed. The variability of the test was small and it was found to be
precise and hence consistent (Dohoo et al., 2003; Thrusfield, 1997). It was
therefore considered to be a suitable procedure for routine analysis, and an
improvement on the ELISA described by Demirkan et al. (1999); it was applied in
subsequent observational studies. A disadvantage of this ELISA was that it
could not identify potential predominance of certain antigenic strains over
others on the farm level.
Acknowledgement. The
greatest part of the microbiological work was carried out within the project
framework by colleagues at the Connective Tissue Research group of the Faculty
of Veterinary Science at the University of Liverpool, namely by Dr. Nick Evans,
Ms. Jenni Brown and Dr. Ibrahim Demirkan. |
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©
Willem Daniel Vink 2006 |
