serodiagnosis of canine babesiosisclione.ne.jp/~sample/sentan/jsps/img/ilri-xuan.pdf ·...
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Serodiagnosis of Canine Babesiosis
Xuenan XUAN, DVM, PhDNational Research Center for Protozoan Diseases
Obihiro University of Agriculture and Veterinary MedicineObihiro, Hokkaido 080-8555, Japan
Tel.:+81-155-495648; Fax:+81-155-495643E-mail:[email protected]
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IntroductionTaxonomic classificationMorphologyLife cyclePathogenesisClinical signsHost defensesEpidemiologyDiagnosisTreatmentPrevention
Babesia spp infections in dogs
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IntroductionCanine babesiosis is tick-bone disease caused by two intraerythrocytic protozoan parasites, Babesia canis and Babesia gibsoni.
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Morphology
B.canis usually appears as paired pyriform organisms in canine red blood cells.
B.gibsoni usually appears as single ringform organisms in canine red blood cells.
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Phylum: ApicomplexaClass: SporozoasidaOrder: PiroplasmidaFamily: BabesiidaeGenus: Babesia
Taxonomic classification
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Life cycle
Following attachment of an infected tick, Babesia spp. trophozoites are released into the blood, infecting erythrocytes. Within the erythrocytes, the parasite multiplies by binary fission, an asexual form of schizogony. Naïve ticks attach to the dog and become infected with Babesia ssp. when they ingest a blood meal.
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PathogenesisThe transmission of parasites takes place after 2-3 days of attachment of the tick, at which time infective sporozoites migrate from the tick’s salivary glands into the host’s circulation. Babesia organisms are obligate intracellular parasites that invade, divide within, and rupture erythrocytes. An important pathogenic mechanism therefore is direct parasite-induced red cell damage resulting in an intravascular haemolysis, but the severity of this is usually not proportional to the low parasitemia that is typically observed. It is now recognised that other significant mechanisms are involved, including immun-mediated lysis and oxidative injury of the red cell membrane.
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Clinical signs
Acute infection: anemia with icterus, inappetence, thirst, pyrexia (>39℃).
Chronic infection: irregular temperature, capricious appetite, and loss of conditions.
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Host defenses
Humoral responsesCell-mediated responsesNonspecific responses
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EpidemiologyB. canis canis: Europe, AsiaB. canis rossi: South AfricaB. canis vogeli: USA, tropical and subtropical areasB. gibsoni Asian type: AsiaB. gibsoni Spanish type: EuropeB. gibsoni California type: USA
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Diagnosis
Microscopic examinationSerodiagnosisMolecular diagnosis
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Microscopic examination
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Enzyme-linked immunosorbent assay (ELISA)
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Immunofluorescent-antibody test (IFAT)
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Immunochromatographic test (ICT)
Examples of the ICT strips pre- (1) and post- (2 and 3) tests. +, the positive result; -, the negative result.
Control lineTest line
Absorbent pad
Conjugate pad
Sample pad
+ –
1 2 3
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Polymerase chain reaction (PCR)
M 1 2 3 4 5 6 7
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Loop-mediated isothermal amplification (LAMP)
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Treatment Chemotherapy against canine babesiosis at the early phase of infection is very important to reduce the severity of disease and mortality, although it cannot completely eliminate the parasites. Diminazene acceturate, phenamicine isethionate, and pentamicine isethionate have been demonstrated to be effective against canine babesiosis. Supportive therapy, such as intravenous fluids and transfusions, is recommended for canine babesiosis, particularly for dogs with severe anemia.
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Prevention For the control of canine babesiosis, vaccination is generally considered to be the most effective means. It is known that the inactivated whole parasite antigen or soluble parasite antigen that is derived from a supernatant of an in vitro culture of Babesia parasites is useful antigen for vaccination and induces partial protection against canine babesiosis. On the other hand, tick control is considered the most important means for prevention of canine babesiosis, since treatment is not always successful.
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Cloning of a novel gene encoding a major surface antigen P50 of Babesia gibsoni
and development of serodiagnosticmethods using recombinant P50
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Babesia-infected RBC
cDNA library
mRNA
Immunoscreening
Novel genes (P50)
DNA vaccineVector vaccineSubunit vaccine
ELISAICTPCR
Gene hunting
Vaccine development Diagnostic methods
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AtgaatgtcgttcgttcattcctgtttttcccaatcgccttctccctggtaagggcaaatggtgaggggaagacggcagaggccacccctgcaggaacatcgacaMetAsnValValArgSerPheLeuPhePheProIleAlaPheSerLeuValArgAlaAsnGlyGluGlyLysThrAlaGluAlaThrProAlaGlyThrSerThr
CccactgaacctaaggcagctgaggctgctcccaaagcagtagacgcagctgctgttacctttaaacagtatctggactttgcaatgaagttaaacgaggccgtgProThrGluProLysAlaAlaGluAlaAlaProLysAlaValAspAlaAlaAlaValThrPheLysGlnTyrLeuAspPheAlaMetLysLeuAsnGluAlaVal
AcactccgtgaggaagacactaggaaaaagcttttggttaacttccctcttttcggagctcccccgttcgatggtgcatggggggatttgaaagacttattgaagThrLeuArgGluGluAspThrArgLysLysLeuLeuValAsnPheProLeuPheGlyAlaProProPheAspGlyAlaTrpGlyAspLeuLysAspLeuLeuLys
AaagttactgagcttcgggcacttctacttaagggtcacacattcggtttaccagcggcaaccacaacagacaaacagcaacaggatgctaaccaaactgtcggtLysValThrGluLeuArgAlaLeuLeuLeuLysGlyHisThrPheGlyLeuProAlaAlaThrThrThrAspLysGlnGlnGlnAspAlaAsnGlnThrValGly
GctttatttgatttcattgtcggagtagcaactgatgcagtcaccgttgctgataaggctactagggctgttactggaatggaccccgataaagccgtgggattcAlaLeuPheAspPheIleValGlyValAlaThrAspAlaValThrValAlaAspLysAlaThrArgAlaValThrGlyMetAspProAspLysAlaValGlyPhe
CacgtcacaccagcaacggctgatgccctatttgagtttgttccagatctctatgaaaagttgaaggatttgcatagtaaggttggagagtgggttgaaattaagHisValThrProAlaThrAlaAspAlaLeuPheGluPheValProAspLeuTyrGluLysLeuLysAspLeuHisSerLysValGlyGluTrpValGluIleLys
TccacctttgatgacacgaaattggtaacccaagctggtgatcacaggccgaaacactggttaaggcagggtgggtttactgaccaggaggttaaaggtgataccSerThrPheAspAspThrLysLeuValThrGlnAlaGlyAspHisArgProLysHisTrpLeuArgGlnGlyGlyPheThrAspGlnGluValLysGlyAspThr
AccttggaaactttgaaaactaaactgggtgagctcgttggtcctactaagccttgtgagaaggttttgtgtacccttgcttcatatgcgcttatgaagacccctThrLeuGluThrLeuLysThrLysLeuGlyGluLeuValGlyProThrLysProCysGluLysValLeuCysThrLeuAlaSerTyrAlaLeuMetLysThrPro
CaagatgcagctggcaagcaggcatggatctttttattggcaagtgcaatgaataacaatgctatgaaagctaagcttgaggtagcagtaaacgcggttactcccGlnAspAlaAlaGlyLysGlnAlaTrpIlePheLeuLeuAlaSerAlaMetAsnAsnAsnAlaMetLysAlaLysLeuGluValAlaValAsnAlaValThrPro
GgtaagggagaaacctttgtcaaccaactaaaggaggttggcaaatcactccagcttcccaaggaacaagttcctaagcaatatcgtttccctggtgtctatgcaGlyLysGlyGluThrPheValAsnGlnLeuLysGluValGlyLysSerLeuGlnLeuProLysGluGlnValProLysGlnTyrArgPheProGlyValTyrAla
AacctcgatgtgcaacacttttggactgtgttaaccggcgtctttggcactatactgactgaccttgaggttgacgaaaaggatgctcagggtaaagcaggacagAsnLeuAspValGlnHisPheTrpThrValLeuThrGlyValPheGlyThrIleLeuThrAspLeuGluValAspGluLysAspAlaGlnGlyLysAlaGlyGln
GttgctactagagttgcggagcttgtcaaggtggaaggtccacttcacagcctcactgtgcaagtagctgagatgactaaggctggagcgggtgctggtggtgaaValAlaThrArgValAlaGluLeuValLysValGluGlyProLeuHisSerLeuThrValGlnValAlaGluMetThrLysAlaGlyAlaGlyAlaGlyGlyGlu
GctcctgctcaggcagctgctgggacagcaggagcacgtgcagaagctccagccaaggaaggccaaggtgaggatggtgcgcacttttgtggcatcggaatgacaAlaProAlaGlnAlaAlaAlaGlyThrAlaGlyAlaArgAlaGluAlaProAlaLysGluGlyGlnGlyGluAspGlyAlaHisPheCysGlyIleGlyMetThr
gttttctttgtttctGtggttatcgctgtcttttaa 1401ValPhePheValSerValValIleAlaValPhe*** 466
Nucleotide and amino acid sequences of P50 gene
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-2.0
-1.0
0.0
1.0
2.0
3.0
50 100 150 200 250 300 350 400 450
P50f
P50t
Hyd
roph
ilici
ty
nt 1 1398
nt 58 1341
466aa 1
aa 20 446
Amino acid number
Truncated P50 gene without SP and TM
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9764
43
30
20
kDa M 1 2 3
Expression of rP50 in E. coli
4
1, rP50f-soluble2, rP50f-insoluble3, rP50t-soluble4, rP50t-insoluble
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9764
43
30
20
kDa M 1 2 3
Purification of rP50 expressed in E. coli
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GST-P50t
(A) (B) ( C )
GST
1 2 1 2 1 2
Antigenicity of rP50 expressed in E. coli
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A B
Anti-P50 DIC
IFAT
94
67
43
30
kDa B. gibs
oni
nRBC
P50
Western blotting
Identification of native P50 on parasites
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The principle of ELISA:(1) Coat antigen; (2) Wash un-coated antigen; (3) Add serum samples; (4) Wash non-bound antibodies; (5) Add HRP-condugated anti-dog IgG antibody; (6) Wash un-bound 2nd antibody; (7) Add substrate (ABTS); (8) Positive result appears as blue color.
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Add subatrate solution (100µl/well) ↓
RT, dark place, 1 h
↓
Read the absorbance at 415 nm
↓
Judge4)
1) Add the diluted GST-rP50 (2 µg/ml) to lanes 2, 4, 6, 8, and 10; add the dilutedcontrol GST (2 µg/ml) to lanes 3, 5, 7, 9, and 11.
2) Add each sample to duplicate wells.
3) To check the optimal secondary antibody titers in advance.
4) The result is judged as positive when the OD value (substrate the average of
2 wells with GST from the average of 2 wells with GST-rP50) equal to or greater
than 0.1.
Samples
Positive control
Negative control
Blank
Materials
GST-NcSAG1 (purified)
GST(purified)
Antigen coating buffer(50 mM carbonate-bicarbonate 、pH9.6)
Antibody diluting buffer(PBS containing 3% skim milk)
Substrate buffer(0.1 M citric-0.2 M sodium phosphate, pH4.0)
Washing buffer(PBS containing 0.05% Tween 20)
ABTS(2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid))
H2O2(30%)
Substrate solution(substrate buffer containing 0.05% ABTS and 0.003% H2O2)
HRP-conjugated goat anti-dog IgG(ICN Biochemicals 、USA)
ELISA plate(NUNC, Denmark)
Methods
Coat antigens (50µl/well)1)
↓
4℃, overnight
↓
Wash once
↓
Add antibody diluting buffer (100µl/well) ↓
37℃, 1 h
↓
Wash once
↓
Add canine sera (1:100, 50µl/well)2)
↓
37℃, 1 h
↓
Wash six times
↓
Add HRP-conjugated goat anti-dog IgG (1:4000, 50µl/well)3)
↓
37℃, 1 h
↓
Wash six times
↓
Procedure of ELISA with rP50
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0
2.5
5
7.5
10
1E+00
1E+01
1E+02
1E+03
1E+04
1E+05
1E+06
0 100 200 300 400
Parasitemia
ELISA titer
Ant
ibod
y tit
erParasitem
ia (%)
Days-post infection
Detection of antibody to P50 in a dog experimentally infected with B. gibsoni
by the ELISA
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
0 1 2 3 4 5 6 7
OD
415
nm
Sensitivity and specificity of the ELISA
1. B. gibsoni-infected dog sera (n=22)
2. SPF dog sera (n=30)3. B. canis canis-infected
dog sera (n=2)4. B. canis canis-infected
dog sera (n=2)5. B. canis vogeli-infected
dog sera (n=2)6. B. canis rossi-infected dog
sera (n=2)7. L. infantum-infected dog
sera (n=2)
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Conjugate pad
Nitrocellulose membrane
Test line
Absorbent pad
Colloidal gold particle-conjugated antigen
AntigenColloidal gold particle
The composition of ICT
Easy performanceQuick results (~15 min)Sensitive and specific as ELISA
Immunochromatographic test (ICT)
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Principle of ICT for B. gibsoni infection1
3
2
rP50 conjugated with gold colloids
rP50Rabbit anti-rP50 IgG
Antibodies in serum
-
+
4
+ –
1 2 3
1) Pre-test 2) Positive serum3) Negative serum
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Absorbent pad
Control lineTest line
Conjugate pad
Sample pad
1 2 3 4 5 6 7 84 5 6 7 8
The sensitivity and specificity of the ICT
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0
Days post infection2482 6 28 1444116 11610 52
0
0.5
1
1.5
2
2.5
3
0 20 40 60 80 100 120 140
Days post infection
Abs
orba
nce
OD
415
nm0
2.5
5
7.5
10
Para
site
mia
(%)
ELISA
Parasitemia
Detection of antibodies to B. gibsoni in an experimentally infected dog by ELISA and ICT
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0
1
2
3
4
5
6
0 2 4 6 8 10 12 14
β-gal
NRS
P50
Days-post challenge
Para
site
mia
(%)
( n = 6, * P < 0.05 )
* ** * ***
Anti-rP50 antibodies significantly inhibited the growth of B. gibsoni in SCID mouse model
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0
0.5
1
1.5
2
2.5
3
-56 -42 -28 -14 0 14 28 42
Con
β-gal
rP50t
Immunization Challenge ( 2 x 108 parasites )
Days-post challenge
Abs
orba
nce
( OD
at 4
15nm
) ( n = 3, * P < 0.05 )
*
*** ***
Recombinant P50 could induce strong humoral immunity in dogs
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0
2
4
6
8
10
12
0 10 20 30 40
Con
β-gal
rP50t
Para
site
mia
(%)
Days-post challenge
( n = 3, * P < 0.05 )
* *
Recombinant P50 could induce protective immunity in dogs
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ConclusionsA gene encoding major surface protein P50 of B. gibsoni was cloned.P50 was identified as an immunodominant antigen in both acute and chronic B. gibsoniinfections in dogs. Recombinant P50 expressed in E. coli is a promising diagnostic antigen for detection of specific antibodies to B. gibsoni in dogs.Recombinant P50 is a promising vaccine candidate to control B. gibsoni infection in dogs.
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Publications about P50 of B. gibsoni1. Fukumoto et al., J. Clin. Microbiol. 39: 2603-2609,
2001.2. Fukumoto et al., Clin. Diagn. Lab. Immunol. 10:
596-601, 2003.3. Fukumoto et al., Infect. Immun. 72: 1795-1798, 2004.4. Fukumoto et al., J. Parasitol. 90: 387-391, 2004.5. Verdida et al., J. Vet. Med. Sci. 66: 1517-1521, 2004.6. Verdida et al., Parasitology 131: 769-774. 2005.7. Fukumoto et al., Clin. Diagn. Lab. Immunol. 12:
557-559, 2005.8. Fukumoto et al., Vaccine (in press)
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Other ELISAsB. equi ELISA-BeEMA2B. caballi ELISA-BcP48B. bovis ELISA-BboRAP1B. bigemina ELISA-BbiRAP1T. gondii ELISA-TgSAG2N. caninum ELISA-NcSAG1C. parvum ELISA-CpP23
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Other ICTsB. equi ICT-BeEMA2B. caballi ICT-BcP48B. bovis ICT-BboRAP1B. bigemina ICT-BbiRAP1T. gondii ICT-TgSAG2N. caninum ICT-NcSAG1
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