Embed Size (px)
Citation preview
635
Serological Evidence of Exposure to
Ehrlichia
Species in Dogs in Spain
ANGEL SAINZ,
a
CHANG-HYUN KIM,
b
MIGUEL A. TESOURO,
c
RICHARD HANSEN,
b
INMACULADA AMUSATEGUI,
a
HYUN-YOUNG KOO,
b
AND IBULAIMU KAKOMA
b,d
a
Department of Animal Pathology II, College of Veterinary Medicine, University Complutense, Madrid, Spain
b
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA
c
Department of Veterinary Medicine, College of Veterinary Medicine, University of Leon, Spain.
INTRODUCTION
To date, only three
Ehrlichia
spp. have been diagnosed in Spain:
E. canis
1
and
E. platys
2
in dogs and
E. phagocytophila
in cattle.
3
However, a recent report of dif-ferent
Ehrlichia
spp. in other European countries
4,5
and the presence of vectors forthese species and a similar environment in some areas of Spain, suggests the exist-ence of yet unidentified
Ehrlichia
spp. in Spain. The purpose of the investigationreported here was to delineate the situation of ehrlichiosis in Madrid, Spain as amodel for studying the disease in the whole country.
MATERIALS AND METHODS
Clinical Cases
Thirteen clinical cases (dogs of different age, sex, and breed) were attended inTorron Veterinary Clinic (Vigo, Galicia, Northwestern Spain) with clinical signscompatible with babesiosis (apathy, anorexia, anemic membrane mucoses, bilirubi-nuria, etc). Canine babesiosis is a very common disease in this area of Spain. Diag-nosis was confirmed in 12 of 13 dogs by detection of intraerythrocytic
Babesia
incapillary blood.
Ehrlichia
-like
5,6
inclusion bodies were detected in lymphocytes andmonocytes of the 13 dogs. Serum samples were collected the day when babesiosiswas diagnosed and 2–3 weeks later. Similarly, 66 random samples were collectedfrom dogs admitted to the Veterinary Hospital, College of Veterinary Medicine ofMadrid.
The immunofluorescent antibody test
7
and Western blot
8,9
analysis were per-formed as previously described.
d
Corresponding author. Voice: 217-333-1859; fax: [email protected]
636 ANNALS NEW YORK ACADEMY OF SCIENCES
RESULTS
Case #1:
• Five year old female mastiff: anorexia, lethargy, weight loss, jaundice.
• Both
Babesia
spp. and
Ehrlichia
spp observed.
• Major hematologic abnormalities included thrombocytopenia.
• Good initial response to Imizol and tetracycline.
• Condition subsequently deteriorated, euthanatized.
Case #2:
• Eight year old female Schnauzer: fever, anorexia, thrombocytopenia.
•
Babesia
spp and
Ehrlichia
spp. observed.
• Thrombocytopenia persisted for one month.
• Initial response to treatment with Imizol was good.
• Ehrlichi inclusions still present after one month.
Parasitological Observations
F
IGURES
1–6 show the various morphologic forms observed according to estab-lished criteria.
5,6
In most cases concurrent infection with
Babesia
species was alsonoted.
Serologic Data
Immunofluorescent antibody test and polymerase chain reaction assays for
E.canis
,
E. risticii,
and
E. ewingii
were negative on two samples that were clinicallydiagnosed to be ehrlichiosis, as described above. No definitive parasitologic or sero-logic diagnosis was tenable.
Analysis of Clinical Samples from Madrid
Nine of the 66 samples were positive for
E. canis,
six of 66 were positive for
E.risiticii
by Western blot, as shown in F
IGURE
7.
Overall Incidence of Canine Ehrlichiosis in Spain
A summary of the incidence of canine ehrlichiosis in Spain is shown in F
IGURE
8.This distribution strongly reflects the distribution of the vectors.
DISCUSSION
The main objective of these investigations was to use data from the Madrid regionof Spain and extend the findings nationwide in Spain. We have now established the
637SAINZ
et al.
: CANINE EHRLICHIOSIS IN SPAIN
FIGURE 1. Additional morula-like inclusions in mononuclear cells.
FIGURE 2. A case of erythrophagocytosis and a morula compatible with Ehrlichia species.
638 ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 3. Multiple inclusions in several infected cells.
FIGURE 4. Piroplasms in the erythrocytes in association with Ehrlichia-like inclu-sions in mononuclear cells.
639SAINZ
et al.
: CANINE EHRLICHIOSIS IN SPAIN
FIGURE 5. Multiple inclusions in a mononuclear cell.
FIGURE 6. Western Immunoblot analysis for E. risticii on some samples from Spain.
640 ANNALS NEW YORK ACADEMY OF SCIENCES
basic distribution profile of canine ehrlichiosis in the country, thus confirming theoriginal data of Sainz
et al.
2
More recently,
Ehrlichia
-like inclusion bodies have been detected in monocytesand lymphocytes from some dogs in Spain (Sainz, unpublished data). Serum sam-ples from two of these clinically diagnosed cases were negative for the major ehrli-chial species using the standard indirect immunofluorescent antibody technique.These findings suggest the presence of yet unidentified species of
Ehrlichia
in Spain.We have observed inclusions associated with erythrophagocytosis (especially whenthere is a coinfection with
Babesia
species). In many cases inclusions compatiblewith
Ehrlichia
infection
1,6
were also detected.Using Western blot, the reactivity pattern observed among Spanish samples was
strikingly similar to that seen in
E. risticii
infected dogs in USA.
10
To our knowl-edge, this is the first evidence of exposure to
Ehrlichia risticii
or a related species of
FIGURE 7
641SAINZ
et al.
: CANINE EHRLICHIOSIS IN SPAIN
Ehrlichia
in Spain. These data are consistent with reports that this organism cancause disease in non-equine species.
10,11
Further studies are necessary to isolate andcharacterize this putative
Ehrlichia
species and to determine its pathogenicity fordogs in Spain and other geographic areas.
REFERENCES
1. F
ONT
, J., J. C
AIRÓ
& A. C
ALLÉS
. 1988. Ehrlichiosis canina. Revista de AVEPA
8:
141–148.
2. S
AINZ
, A.R. 1996. Aspectos Clinicos y epizootiologicos de la ehrlichiosis canina.Estudio cmparado de la eficacia terapeutica de la doxiclina y el dipropionato de imi-docarb. Ph.D. Thesis, Universidad Computense de Madrid, Spain.
3. J
USTE
, R.A., G.R. S
COTT
, E.A. P
AXTON
, J.L. G
ELABERT
& S. J
IMÉNEZ
. 1989. Presenceof
Cytoectes phagocytophilia
in an atypical disease of cattle in Spain. Vet. Rec.
124:
636.4. J
OHANSSON
, K.E., B. P
ETTERSSON
, M. U
HLÉN
, A. G
UNNARSSON
, M. M
ALMQVIST
& E.O
LSSON
. 1995. Identification of the causative agent of granulocytic ehrlichiosis inSwedish dog and horses by direct solid phase sequencing of PCR products from the16S rRNA gene. Res. Vet. Sci.
58:
109–112.5. E
LIAS
, E. 1991. Diagnosis of ehrlichiosis from the presence of inclusion bodies ormorulae of
E. canis
. J. Small An. Pract.
33:
540–543.6. R
ISTIC
, M. & D.L. H
UXSOLL
. 1984. Ehrlichieae.
In
Bergey’s Manual of SystematicBacteriology. N.R. Kreig & J.G. Holt, Eds.: 704–709. Baltimore, Maryland.
7. R
ISTIC
, M., D.L. H
UXSOLL
, R.M. W
EISIGER
, P.K. H
ILDEBRANT
& M.B.A. N
YINDO
.1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluo-rescent. Infect. Immun.
6:
226–231.8. N
YINDO
, M.B.A., I. K
AKOMA
& R.D. H
ANSEN
. 1991. Antigenic analysis of four spe-cies of the genus
Ehrlichia
by use of protein immunoblot. Am. J. Vet. Res.
52:
1225–1230.
9. R
IKIHISA
, Y., S.A. E
WING
& J.C. C
OX
. 1994. Western immunoblot analysis of
Ehrli-chia chaffeensis, Ehrlichia canis,
or
Ehrlichia ewingii
infections in dogs andhumans. J. Clin. Microbiol.
32:
2107–2112.
FIGURE 8. Map showing the distribution of canine ehrlichiosis in Spain.
642 ANNALS NEW YORK ACADEMY OF SCIENCES
10. K
AKOMA
, I., R.D. H
ANSEN
, B.E. ANDERSON, T.A. HANLEY, K.G. SIMS, L. LIU,C. BELLAMY, M.T. LONG & B.K. BAEK. 1994. Cultural, molecular and immunologi-cal characterization of the etiologic agent for atypical canine ehrlichiosis. J. Clin.Microbiol. 32(1): 170–175.
11. PEAVY, G.M., C.J. HOLLAND, S.K. DUTTA, G. SMITH, A. MOORE, L.J. RICH, M.R.LAPPIN & K. RICHTER. 1996. Suspected ehrlichial infection in five cats from ahousehold. J. Am. Vet. Med. Assoc. 210: 231–234.
