Veterinary Microbiology 99 (2004) 6774

Identification of an alkaline ceramidase gene from

Dermatophilus congolensis
Alfredo Garca-Snchez b , Rosario Cerrato a , Jos Larrasa c , Nicholas C. Ambrose d,1 ,
Alberto Parra a , Juan M. Alonso a , Miguel Hermoso-de-Mendoza a ,
Joaqun M. Rey a , Javier Hermoso-de-Mendoza a,
a

Unidad de Patologa Infecciosa, Facultad de Veterinaria, Universidad de Extremadura, Campus UEX,

Av. Universidad s/n, 10071 Cceres, Spain
Bacteriology Division, Moredun Research Institute, Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, Scotland, UK
c Laboratorios Larrasa, Corredera Hernando de Soto 13-A, Jerez de los Caballeros, 06380 Badajoz, Spain
d Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh,
Easter Bush, Roslin, Midlothian EH25 9RG, Scotland, UK
Received 20 June 2003; received in revised form 15 October 2003; accepted 28 October 2003

Abstract
A random amplified polymorphic DNA (RAPD) procedure was used to identify a specific 0.6 kb DNA fragment unique to
Dermatophilus congolensis. This 0.6 kb fragment was evaluated as a specific DNA probe and used to design oligonucleotide
primers for polymerase chain reaction (PCR) amplification. The nucleotide sequences adjacent to this DNA fragment were
determined by inverse PCR allowing the identification of a 4.1 kb sequence. Analysis of this revealed a complete open reading
frame (ORF) with a high similarity to an alkaline ceramidase from Pseudomonas aeruginosa. The molecular weight of the
enzyme derived from the predicted amino acid sequence is 74,662 Da, its pI is 9.81. The predicted N-terminal sequence of the
enzyme contains a signal sequence indicating that the enzyme is exported by the bacterium. Since ceramides have important
protective and cell regulatory roles in the epidermis we suggest that this ceramidase may have a role in the pathogenesis of
dermatophilosis. It is the first completely sequenced gene described for D. congolensis.
2003 Elsevier B.V. All rights reserved.
Keywords: Alkaline ceramidase; RAPD; Inverse PCR; Dermatophilus congolensis; agc gene

1. Introduction
Corresponding author. Tel.: +34-927257129;
fax: +34-927257110.
E-mail address: jhermoso@unex.es (J. Hermoso-de-Mendoza).
1 Present address: Scottish Executive Environment and Rural
Affairs Department (SEERAD), The Scottish Executive, Room
440, Pentland House, 47 Robbs Loan, Edinburgh EH14 1TY,
Scotland.

Dermatophilus congolensis (Van Saceghem, 1915)

is a filamentous branching actinomycete that causes
dermatophilosis, an exudative dermatitis that occurs
throughout the world and affects a wide variety of
mammals and reptiles. The disease is economically
important in cattle in the tropical and sub-tropical re-

0378-1135/$ see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2003.10.028

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A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

gions, and in sheep in high rainfall areas, because it

causes deleterious effects on the production of meat,
milk, hides, skins and fibers (Lloyd, 1976). Currently
there is a lack of efficient management and immunological measures to control the disease. There is an urgent need for an improved understanding of the pathogenesis of the disease, to identify D. congolensis genes
that are involved and what might be targets for immunological or therapeutic interventions.
The clinical picture of the disease includes matting
of the hair or wool, scab and crust formation and sometimes generalized massive crust formation in chronic
cases which lead to loss of hair and even local loss
of the upper skin layers predisposing to secondary infections from Staphylococcus spp. (Devriese, 1983;
Lomax and Cole, 1983), or Pseudomonas aeruginosa
(Mathieson, 1983).
The pathogenesis of dermatophilosis is not well understood. It is known that D. congolensis is haemolytic
(Skalka and Pospsil, 1992), and produces phospholipases (Master et al., 1997) and proteolytic enzymes
(Gordon, 1964; Ambrose et al., 1997). A wide enzymatic activity with putative pathogenic role has been
also evidenced by means of commercial enzymatic activity test strips (API ZYM) (Hermoso de Mendoza
et al., 1993). Now there has been no detailed genetic characterization of these or other putative virulence factors. However the aforementioned studies
have demonstrated a high degree of phenotypic interstrain variation between D. congolensis strains.
Random amplified polymorphic DNA (RAPD)
products have been used with considerable success as
markers for strain identification. RAPD bands, often
originate from repetitive DNA sequences (Williams
et al., 1990). When they are analyzed, it is usual that
some of the DNA fragments appear in the fingerprints of all strains belonging to a given species. Once
demonstrated that one fragment is species specific,
it can be used to design specific polymerase chain
reaction (PCR) tests for taxonomical or diagnostic
research.
In the present work, we describe the use of an RAPD
technique to identify a specific DNA fragment that
could be used as a probe to identify D. congolensis. In
carrying out this work, sequencing of the fragment was
performed in order to design specific PCR primers by
comparison with published sequences of the EMBL
database. This process resulted in the recognition of

the sequence as a part of an alkaline ceramidase gene.

We therefore chose to carry out complete sequencing
of this gene, the first described from D. congolensis.

2. Materials and methods

2.1. Bacteria and culture condition
Fourteen isolates of D. congolensis were used to
develop the RAPD method (Table 1). Isolates were
grown in 20 ml of BHI-NSP (2% (w/v) brain heart
infusion, 1.7% (w/v) neutralized soya peptone, Oxoid) in glass McCartney bottles, at 37 C in air for
48 h.
2.2. DNA isolation, RAPD analysis and cloning
DNA was isolated using a modified version of the
protocol described by Wilson (1987) with an additional step of 5 h incubation at 37 C with lysozyme
(0.2 mg/ml) in TrisEDTA buffer, prior to a sodium
dodecyl sulfate (SDS) and a proteinase K treatment step, followed by standard phenolic extraction
(Larrasa et al., 2002). DNA extracted by a second
fast non-phenolic method based on that described by
Table 1
D. congolensis isolates
Strain

Host species

Origin

OA1 (NCTC 7915)

BE3 (ATCC 14637)
OG1
HK628
OMia3
B556
EEQ
EA2 (NCTC 5175)
O (AG)
OA8 (NM)
BE9
CH8
O124
D. chelonae

Sheep
Cattle
Sheep
Horse
Sheep
Cattle
Horse
Horse
Sheep
Sheep
Cattle
Deer
Sheep
Tortoise

UK
Zimbabwe
Spain
Germany
Spain
Spain
Spain
UK
Australia
Australia
Ghana
UK
Australia
Australia

They all, except D. chelonae, belong to twin collections at the

CTVM, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, and at the Veterinary Faculty, Universidad de
Extremadura. D chelonae strain belong to Animal Health Laboratories, Department of Agriculture of Western Australia, South
Perth, Australia.

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

Johansson et al. (1995) modified by Larrasa et al.

(2002) was also used.
Two RAPD primers, A (5 -CTTCACCTCGTTGTCCACCC-3 ) and B (5 -CCATGGTGAACGCGCTGCGGA-3 ), were tested using sequential optimization
of each component of the reaction mixture and amplification conditions (Williams et al., 1990; Larrasa
et al., 2002). Amplified products (10 !l) were resolved
by electrophoresis on a 2% (w/v) agaroseTBE gel
at 5 V cm1 for 2 h. A 1 kb DNA ladder was used
as a molecular weight marker. Each primer resulted
in RAPD fingerprints which contained several bands
present in all or almost all the D. congolensis strains.
Two bands of 0.50.6 kb were selected as they had an
easier-to-manage size.
The bands were cut and eluted from agarose gel,
and cloned into PCR-Script Amp ElectroporationCompetent Cell Cloning Kit (Stratagene). Randomly
selected white colonies were purified using UltraClean Mini Plasmid Prep Kit (Mobio) and the plasmid
DNA was sequenced using an ABI 377 autosequencer
with four fluorescent dyes. Computer-assisted analysis of the sequence data was performed using ABI
Prism sequencing analysis, Version 3.3 (Perkin-Elmer
Software). Further screening by comparison of these
sequences with published GenBank sequences allowed to select as probe the 0.6 kb band obtained with
primer A.
2.3. Southern blot analysis
The selected RAPD band was recovered from
agaroses gel by using UltraClean GelSpin Kit
(Mobio) and labeled with digoxigenin-11-dUTP
(DIG) using a PCR DIG probe synthesis kit
(BoehringerMannheim).
The PCR for preparation of DIG-labeled DNA
probe was performed in a thermal cycler (Mastercycler 5330 Plus, Eppendorf) programmed for a denaturation step at 94 C for 5 min and then 30 cycles
consisting of 15 s step at 94 C, 40 s step at 35 C,
and 30 s step at 72 C. Prehybridization, hybridization, and washing for Southern hybridization with
DIG-labeled probe was performed at 45 C, according
to the manufacturers protocol.
In order to test species specificity, genomic DNA
(2 !g) from isolates of D. congolensis (Table 1),
a Mycobacterium bovis field isolate, identified by

69

biochemical methods and multiplex PCR amplifying the MPB20 sequence (Cousins et al., 1991) and
RNAr16S (Woese, 1987), a Corynebacterium propinquum field isolate, identified by API-CoryneTM ,
and a Dermatophilus chelonae type strain (ATCC
51576) were digested with the restriction enzyme
PstI, separated by electrophoresis on 0.8% agarose
gel and transferred onto Hybond N + membrane
(BoehringerMannheim).
2.4. PCR conditions
For specific amplification of D. congolensis, two
oligonucleotide primers, ESP1 and ESP2, were designed from the nucleotide sequence of the 0.6 kbRAPD fragment cloned from D. congolensis (NCTC
5175). ESP1 (5 -CTTCAGCAGAAAATTCACCA-3 )
and ESP2 (5 -CGTACATTCCCGGAATCTTC-3 ),
delineating a 438 bp fragment. DNA was amplified in
100 !l of reaction mixture which contained 100 ng of
genomic DNA, 1.5 mM MgCl2 , 1 !M of each primers,
250 !M (each) deoxynucleoside triphosphate (dNTP),
1.5 units of Taq DNA polymerase (BioTaqTM , Bioline), 10 mM TrisHCl, pH 8, 50 mM KCl and 0.1%
Triton 100. PCR conditions were programmed for denaturation at 94 C for 5 min and then for 35 cycles of
1 min at 94 C, 1 min at 55 C, and 1.5 min at 72 C.
To clone the 5 - and 3 -flanking regions of the RAPD
product, amplification by inverse PCR (Moreau et al.,
1997) was performed. Chromosomal DNA from the
type culture strain (NCTC 5175) was digested with
different restriction enzymes PstI, AgeI, EcoRI, BglII
and SmaI (Amersham Biosciences), the restriction endonucleases were then thermally inactivated (80 C,
20 min), and aliquots were incubated with T4 DNA
ligase (Amersham Biosciences) overnight at 16 C.
The resulting self-ligated DNA was precipitated with
ethanol and resuspended in sterile distilled water to
give a final concentration of 10 ng !l1 . Then, it was
used as the template in further PCR reactions with
primers based on the RAPD band sequence.
PCR reactions (100 !l) contained 5 or 10 !l
self-ligated chromosomal DNA, 0.5 mM of each
primer, 0.2 mM concentrations of each deoxynucleotide triphosphate, 1.5 units of Taq DNA polymerase (BioTaq), 1.5 mM MgCl2 , 10 mM TrisHCl,
pH 8, 50 mM KCl and 0.1% Triton 100. The thermocycler was programmed as follows: 5 min warm-up/hold

70

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

at 94 C, followed by 30 cycles of: 1 min at 94 C,

1 min at annealing temperature (55 C) and 2 min at
72 C, 1, followed by a 15 min step holding at 72 C.
PCR products were cloned into a pPCR-Script Amp
SK(+) vector, following the manufacturers protocol
(Stratagene).
2.5. Computer sequence analysis
Nucleotide sequence data were compiled using the
DNA Strider 1.0 software (Marck, 1988). Database
searches were performed using the BlastX (http://
www.ncbi.nlm.nih.gov/BLAST) (Altschul et al.,
1990). ORFs were identified using ORF Finder
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Signal
sequence prediction was performed using SignalP
(http:// www.cbs.dtu.dk/services/SignalP) (Henrik
et al., 1997). Multiple sequence alignments were performed using PILEUP (GCG software, Version 9.0,
Devereux et al., 1984). Protein masses and isoelectric points were determined online, using ProtParam
tools (http://us.expasy.org/tools/protparam.html). Proteins were examined for conserved motifs using Pfam
(http://pfam.wustl.edu/hmmsearch.shtml). The sequence described in this work has been deposited in
GenBank (AJ496026).

3. Results
The optimal RAPD mixture and amplification conditions with primer A were as follows: PCR reactions
(100 !l) contained 100 ng of genomic DNA, 1 !M of
each primer, 300 !M dNTP, 2.5 units of Taq DNA
polymerase (BioTaq), 2 mM MgCl2 , 10 mM TrisHCl,
pH 8, 50 mM KCl and 0.1% Triton 100. The thermocycler was programmed as follows: 5 min warm-up/hold
at 94 C, followed by 30 cycles of: 15 s at 94 C, 40 s
at annealing temperature (35 C) and 30 s at 72 C,
1, followed by a 15 min step holding at 72 C. With
primer B, the conditions were as follows: PCR reactions (100 !l) contained 100 ng of genomic DNA,
1 !M of each primer, 250 !M dNTP, 1.5 units of Taq
DNA polymerase (BioTaq), 1.5 mM MgCl2 , 10 mM
TrisHCl, pH 8, 50 mM KCl and 0.1% Triton 100.
The thermocycler was programmed as follows: 5 min
warm-up/hold at 94 C, followed by 30 cycles of:
1 min at 94 C, 1 min at annealing temperature (45 C)

Fig. 1. The RAPD patterns of 13 D. congolensis isolates. Lane

M, marker lane (ladder 100 pb, Amersham Biosciences).

and 90 s at 72 C, 1, followed by a 15 min step holding at 72 C. The RAPD amplification patterns obtained with these primers on the DNA extracts of D.
congolensis were reproducible and revealed clearly
marked products of about 0.6 kb (Fig. 1).
The selected DNA fragments were gel purified and
cloned in plasmid vector and the insert were sequenced
from both directions. The DNA sequenced fragments
were searched for homologies with EMBL and GenBank and the band from primer A showed a high degree of homology with a portion of the P. aeruginosa
alkaline ceramidase gene (Okino et al., 1999), whereas
the band from B was nor significantly homologous
with previously characterized gene sequences.
The 0.6 kb DNA fragment was eluted from agarose
gel, labeled with DIG by PCR with the primer A, and
used as a probe against PstI digested total DNAs of
D. congolensis and one isolate of M. bovis, one of
D. chelonae and other of C. propinquum. The results
showed that the 0.6 kb DNA fragment hybridized to
an approximately 3.0 kb PstI fragment in the genomic
DNA of D. congolensis, but not with those of any
other species tested.
Based on this DNA sequence, two oligonucleotides
(ESP1 and ESP2) were used for subsequent PCR amplification tests. To determine the specificity of these
primers, PCRs were carried out with DNA of all of the
strains listed in Table 1, DNA from a M. bovis field isolate and from a C. propinquum field isolate. The PCR

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

71

Fig. 2. The PCR amplification using ESP1 and ESP2 primers generated an approximately 0.4 kb size amplified DNA fragment in all D.
congolensis strains tested (lanes 415), in addition a minor second band was also observed from isolates of ovine origin. No amplifications
were observed with the M. bovis, C. propinquum and D. chelonae strains tested (lanes 13) (ladder 0.5 kb, Amersham Biosciences).

amplification using ESP1 and ESP2 primers generated

an approximately 0.4 kb size amplified DNA fragment
in all D. congolensis strains tested. No amplification
was observed with the M. bovis, C. propinquum and
D. chelonae isolates tested (Fig. 2).
The inverse PCR performed with the primers Inv1
(5 -GAAGATTCCGGGAATGTACG-3 ) and Inv2
(5 -TGGTGAATTTTCTGCTGAAG-3 ) which were
oriented in inverted tail-to-tail direction, generated
an approximately 1.5 kb DNA fragment on genomic
DNA from D. congolensis strain (NCTC 5175), previously digested with the restriction endonuclease AgeI
and circularized with T4 DNA ligase. The positions
of restriction endonuclease sites and DNA sequence
of the 1.5 kb AgeI fragment were determined. This
sequence also revealed extensive homology with the
P. aeruginosa alkaline ceramidase gene (Okino et al.,
1999) in the EMBL/GenBank Data Library.
The inverse PCR assay was repeated with different pairs of primers on self-ligated circular DNA
derived from PstI-digested (NCTC 5175) genomic
DNA. We choose a specific pair of primers Inv3
(5 -GTTTCTGCGGCAGCACCGGT-3 ) and Inv4
(5 -TGTTCCCCTCGATCACCTCTG-3 ) designed
from upstream of the unique PstI site on the known
sequence and other pair from downstream Inv2
(5 -TGGTGAATTTTCTGCTGAAG-3 ) and Inv5
(5 -GTTTGACAGTAAGCCAGCAG-3 ). A DNA
fragment of approximately 1 kb was obtained with
primers Inv3 and Inv4, and another DNA fragment of
approximately 2.7 kb was obtained with primers Inv2
and Inv5. Both fragments were cloned and sequenced.
Sequences were aligned and assembled to obtain
a final sequence 4183 bp long. Analysis revealed one
complete open reading frame (ORF), which we term

agc gene, and one incomplete ORF. The first ORF

(agc) starts at nucleotide 738 with an ATG start codon
and ends at position 2855 with a TAA stop codon, encoding a putative alkaline ceramidase with 705 amino
acids (Mr = 74,662). The next ORF begins at nucleotide 3276 with a CTG start codon, however a stop
codon was not found after 917 bp of sequence. However, based on this the second partial gene encodes a
putative pyruvate-formate lyase with a conserved domain.

4. Discussion
In this study, the RAPD procedure was used to find
specific DNA fragments unique to D. congolensis.
A 0.6 kb fragment was evaluated as a specific DNA
probe and used to design oligonucleotide primers for
PCR amplification. The primers amplified a conspicuous 0.43 kb PCR product, from all D. congolensis
isolates tested, the product could be easily visualized
on a standard 0.81% agarose gel. The primers did
not amplify DNA from a selection of related other
pathogenic species, nor from the closely related D.
chelonae. This study demonstrates that the selection
and analysis of taxon-specific bands within patterns of
RAPD offers an effective method of identifying nucleic acid sequences on which to base specific diagnostic primers for conventional high-stringency PCR.
Using the data generated in this study it would now
be straightforward to develop and apply a PCR for diagnosis or detection of D. congolensis.
The nucleotide sequences adjacent to this 0.6 kb
DNA fragment were determined by inverse PCR allowing the identification of the agc gene. Interpretation

72

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

of the deduced amino acid sequence revealed a conserved alkaline ceramidase domain with around 35%
similarity with the P. aeruginosa and M. tuberculosis
ceramidases (Fig. 3) and a theoretical isoelectric point
of 9.81. Interestingly, the D. congolensis ceramidase
showed a putative signal peptide sequence suggesting

CER2
CER3
CER1

the enzyme is secreted by the bacterium. The logical

target for this secreted enzyme would be the host epidermis.
Ceramide is a major end-product of differentiation and the keratinization process in the mammalian
epidermis. It is secreted into extracellular spaces to

1
50
~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~ML
~~~~~~~~~~ ~~~~~~~~~~ ~MSRSAFTAL LLSCVLLALS MPARADDLPY
MTRRTRKITL SKIMAPAVVV ALALPAGTAF AAQPHVVHTS AVKTDGSGAY

CER2
CER3
CER1

51
SVGRGIADIT
RFGLGKADIT
LVGSGMYDIT
--*-*--***

GEAADCGMLG
GEAAEVGMMG
GAAAETGMLG
*-**--**-*

YGKSDQRTAG
YSSLEQKTAG
YAAS.QEVDG
*----*---*

IHQRLRSRAF
IHMRQWARAF
LHMRLYSRAF
-*-*---***

100
VFRDDSQDGD
VI.EEAASG.
VVAD..QKSG
*---------

CER2
CER3
CER1

101
ARLLLIVAEL
RRLVYVNTDL
KRVAMVTTDM
-*--------

PLPMQNVNEE
GMTFQAVHLK
GAMFPSITSA
----------

VLRRLADLYG
VLARLKAKYP
VVAKLQQKFG
*---*-----

DTYSEQNTLI
GVYDENNVML
DKYTPKNVLI
--*---*---

150
TATHTHAGPG
AATHTHSGPG
AATHTHVGNS
-*****-*--

CER2
CER3
CER1

151
GYCGYLLYNL
GFSHYAMYNL
GMSGDRLYQV
*------*--

.....TTSGF
.....SVLGF
AGADSTSAGY
--------*-

RPATFAAIVD
QEKTFNAIVD
DKKNFGTVVN
----*---*-

GIVESVEHAH
GIVRSIERAQ
GIVESISRAH
***-*---*-

200
ADVAPAEVSL
ARLQPGRLFY
TSLAPGTVQR
----*-----

CER2
CER3
CER1

201
SHGELYGASI
GSGELRNASR
SEGELKGATR
--***--*--

NRSPSAF..D
NRSLLSH..L
NRSLPAHRAN
***-------

RNPPADKAFF
KNP..DIAGY
KNPGSE....
-**-------

PKRVDPHTTL
EDGIDPQMSV
...VDSSMTQ
----*-----

250
VR.IDRGEAT
LSFVDANGEL
LEFRRSNGQA
----------

CER2
CER3
CER1

251
VGVIHFFATH
AGAISWFPVH
VGVLNWFAIH
-*----*--*

GTSMTNRNHL
STSMTNANHL
PTSFSRKFTK
-**-------

ISGDNKGFAA
ISPDNKGYAS
LSGDNKGYAS
-*-****-*-

YHWERTVGGA
YHWEHDV...
YMFEKQMGGD
*--*------

300
DYLAGQPDFI
...SRKSGFV
PDKAG..SFV
--------*-

CER2
CER3
CER1

301
AAFAQTNPGD
AAFAQTNAGN
AAFANSAVGD
****----*-

MSP.NVDGPL
LSP.NLN..L
VVPAQGNAHS
--*-------

SPEAPP.DRE
KPGSGPFDNE
APGYGGSSDE
-*-------*

FDNTRRTGLC
FDNTREIGLR
YHNTHVAGEA
--**---*--

350
QFEDAFTQLS
QFAKAY.EIA
QLGKA.RQLW
*---*-----

CER2
CER3
CER1

351
GAT..PIGAG
GQAQEEVLGE
AAQGQAQGGP
----------

IDARFTYVDL
LDSRFRFVDF
VDFRSRHIDL
-*-*----*-

GSVLVRGEYT
TRLPIRPEFT
RNYMVDAKYA
----------

PDGEERRTGR
.DGQPRQLCT
.GGKAVQLCK
--*-------

400
PMFGAGAMAG
AAIGTSLAAG
AARGFSFASG
---*-----*

CER2
CER3
CER1

401
.TDEGPGFH.
STEDGPGPL.
G.ENGPSKIP
----**----

GFRQG..RNP
GLEEG..NNP
GMYEGMTRDS
*---*-----

FW........
FL........
FSISDKIKKV
*---------

..DRLSR...
..SALGG...
IPSALGGLTR
----*-----

450
.AMYRL.ARP
.LLTGVPPQE
LAFAGVSALH
----------

CER2
CER3
CER1

451
TAAAQAPKGI
LVQCQAEKTI
QDKCHAEKPI
-----*-*-*

VMPARLPNRI
LADTG.NKKP
PLPTGA....
----------

HPFVQEIVPV
YPWTPTVLPI
WKMVSSVVQV
----------

QLVRIGRLYL
QMFRIGQLEL
QLVRVGNTAV
*--*-*----

500
IGIPGEPTIV
LGAPAEFTVM
LALPVEPTTV
---*-*-*--

Fig. 3. Alignment of Dermatophilus congolensis CDase (CER1), Mycobacterium CDase (CER2) and Pseudomonas CDase (CER3). Pileup
algorithm (Devereux et al., 1984) was used. Identical amino acid are indicated by a consensus symbol (asterisk). Gaps inserted into the
sequences are indicated by dots.

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

CER2
CER3
CER1

501
AGLRLRRMV.
AGVRIRRAVQ
ASRRLQERVA
*--*----*-

ASIVGADLAD
AASEAAGIRH
AELAGTGVNR
*---------

VLCVGYTNAY
VVFNGYANAY
VVIAGVANGY
*---*--*-*

IHYVTTPEEY
ASYVTTREEY
NGYLATREEY
--*--*-***

550
LEQRYEGGST
AAQEYEGGST
AAQHYEGAST
--*-***-**

CER2
CER3
CER1

551
LFGRWELCAL
LYGPWTQAAY
EFGPFEFAAF
--*-----*-

MQTVAELAEA
QQLFVDMAVA
EQEAAGLASA
-*-----*-*

MRDGRPV.TL
LRERLPVETS
MKKGAAVSDA
------*---

GRRP.RPTRE
AIAPDLSCCQ
ASPAGSFTAK
----------

600
LSWVRGAPAD
MNFQTGVVAD
TPARPGVLFD
-----*---*

CER2
CER3
CER1

601
....AGSFGA
DPYIGKSFGD
SKPAGQQFGQ
-------**-

VIAEPSATYR
VLQQPRESYR
VLGQPSQSYS
*---*---*-

PGQAVEAVFV
IGDKVTVAFV
AGQVASAVFR
-*------*-

SALPNNDLRR
TGHPKNDLRT
AGHPKNDYRT
---*-**-*-

650
GGTYLEVVR.
EKTFLEVVNI
MGSFLQVQR.
----*-*---

CER2
CER3
CER1

651
.REGASW.VR
GKDGKQTPVT
.QEGGQWK.T
---*------

IADDGDWATS
VATDNDWDTQ
VRTDRDWDTT
---*-**-*-

FRWQRQGRAG
YRWERVGISA
YAWKREGVAF
--*-*-*---

SHVSIRWDVP
SKATISWSIP
SRATVQWRIP
*-----*--*

700
GDTTPGQYRI
PGTEPGHYYI
KGTPAGTYRL
--*--*-*--

CER2
CER3
CER1

701
VHHGTARD.R
RHYGNAKNFW
VQTGDWKNAR
---*------

NGMLTAFSAT
TQKISEIGGS
GGKVSPYVGM
----------

731
TREFTVV~~~ ~
TRSFEVLGTT P
SRSFTVRZ~~ ~
-*-*-*-*** *

73

Fig. 3. (Continued ).

form a mantle that surrounds horny (keratinized) cells

(Lampe et al., 1983; Wertz et al., 1987). Extracellular
ceramide, which is arranged in a lamellar structure
(Hamanaka et al., 1989), is a major component of
the permeability barrier and the water reservoir of
the skin (Imokawa et al., 1986). In addition ceramide
modulates cell kinetics in the process of epidermal
proliferation, differentiation and apoptosis (Wakita
et al., 1994).
In normal epidermis, ceramidase catalyzes the
cleavage of ceramide to fatty acids and sphingosine
(Ohnishi et al., 1999). Imbalances in levels of both
products may modify the features of epidermis, including its permeability, immune functions, and other
physical characteristics determined by the normal
proliferation and differentiation of epidermal keratinocytes. Further studies are required to establish the
role of this enzyme in the life cycle of D. congolensis,
However we suggest that the secretion of ceramidase
in the infective phase would favor the spread of D. congolensis in the epidermis where ceramide is present.
Alkaline ceramidases from pathogenic bacteria, such
as P. aeruginosa, use mammalian ceramides as a
substrate (Okino et al., 1998), causing fissures in the
protective barrier that increase levels of allergens in
skin of patients with atopic dermatitis. This situation
aggravates the allergic picture and allows the recur-

rent eczematous lesion typical of this condition (Bos

et al., 1994) which favors the survival the bacterium
(Ohnishi et al., 1999). A similar process might occur
with a D. congolensis ceramidase having direct and
indirect effects to assist Dermatophilus penetration of
the epidermis, perhaps in parallel to the reported exoprotease activity (Hnel et al., 1991; Ambrose et al.,
1998). Interestingly D. congolensis has shown the
greatest proteolytic activity in neutral to alkaline pH
(Ambrose et al., 1998). This may be because the pH
of the skin surface will become alkaline as a result of
leakage of plasma during inflammation, a change that
also favors the activity of our alkaline ceramidase.

Acknowledgements
This work was supported by a Grant from the European Union Programme INCO-DC Genetic and
Immunological Control on Dermatophilosis contract
no. IC18-CT98-0334 (DGXII-SNRD). Special thanks
to Dr. Alberto Quesada, Department of Biochemistry,
University of Extremadura, for his suggestions for the
characterization of the gene, to Dr. Anne Masters, Department of Agriculture of Western Australia, Animal
Health Laboratories, South Perth, Australia, for the
D. chelonae reference strain and for Australian ovine

74

A. Garca-Sanchez et al. / Veterinary Microbiology 99 (2004) 6774

D. congolensis strains, and to Dr. K.H. Bhm, Institut fur Mikrobiologie und Tierseuchen, Tierarztlichen
Hochschule, Hannover, Germany, for providing German horse isolates of D. congolensis.

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