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:gen@obihiro.ac.jp

IntroductionTaxonomic classificationMorphologyLife cyclePathogenesisClinical signsHost defensesEpidemiologyDiagnosisTreatmentPrevention

Babesia spp infections in dogs

IntroductionCanine babesiosis is tick-bone disease caused by two intraerythrocytic protozoan parasites, Babesia canis and Babesia gibsoni.

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.

Phylum: ApicomplexaClass: SporozoasidaOrder: PiroplasmidaFamily: BabesiidaeGenus: Babesia

Taxonomic classification

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.

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.

Clinical signs

Acute infection: anemia with icterus, inappetence, thirst, pyrexia (>39℃).

Chronic infection: irregular temperature, capricious appetite, and loss of conditions.

Host defenses

Humoral responsesCell-mediated responsesNonspecific responses

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

Diagnosis

Microscopic examinationSerodiagnosisMolecular diagnosis

Microscopic examination

Enzyme-linked immunosorbent assay (ELISA)

Immunofluorescent-antibody test (IFAT)

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

Polymerase chain reaction (PCR)

M 1 2 3 4 5 6 7

Loop-mediated isothermal amplification (LAMP)

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.

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.

Cloning of a novel gene encoding a major surface antigen P50 of Babesia gibsoni

and development of serodiagnosticmethods using recombinant P50

Babesia-infected RBC

cDNA library

mRNA

Immunoscreening

Novel genes (P50)

DNA vaccineVector vaccineSubunit vaccine

ELISAICTPCR

Gene hunting

Vaccine development Diagnostic methods

AtgaatgtcgttcgttcattcctgtttttcccaatcgccttctccctggtaagggcaaatggtgaggggaagacggcagaggccacccctgcaggaacatcgacaMetAsnValValArgSerPheLeuPhePheProIleAlaPheSerLeuValArgAlaAsnGlyGluGlyLysThrAlaGluAlaThrProAlaGlyThrSerThr

CccactgaacctaaggcagctgaggctgctcccaaagcagtagacgcagctgctgttacctttaaacagtatctggactttgcaatgaagttaaacgaggccgtgProThrGluProLysAlaAlaGluAlaAlaProLysAlaValAspAlaAlaAlaValThrPheLysGlnTyrLeuAspPheAlaMetLysLeuAsnGluAlaVal

AcactccgtgaggaagacactaggaaaaagcttttggttaacttccctcttttcggagctcccccgttcgatggtgcatggggggatttgaaagacttattgaagThrLeuArgGluGluAspThrArgLysLysLeuLeuValAsnPheProLeuPheGlyAlaProProPheAspGlyAlaTrpGlyAspLeuLysAspLeuLeuLys

AaagttactgagcttcgggcacttctacttaagggtcacacattcggtttaccagcggcaaccacaacagacaaacagcaacaggatgctaaccaaactgtcggtLysValThrGluLeuArgAlaLeuLeuLeuLysGlyHisThrPheGlyLeuProAlaAlaThrThrThrAspLysGlnGlnGlnAspAlaAsnGlnThrValGly

GctttatttgatttcattgtcggagtagcaactgatgcagtcaccgttgctgataaggctactagggctgttactggaatggaccccgataaagccgtgggattcAlaLeuPheAspPheIleValGlyValAlaThrAspAlaValThrValAlaAspLysAlaThrArgAlaValThrGlyMetAspProAspLysAlaValGlyPhe

CacgtcacaccagcaacggctgatgccctatttgagtttgttccagatctctatgaaaagttgaaggatttgcatagtaaggttggagagtgggttgaaattaagHisValThrProAlaThrAlaAspAlaLeuPheGluPheValProAspLeuTyrGluLysLeuLysAspLeuHisSerLysValGlyGluTrpValGluIleLys

TccacctttgatgacacgaaattggtaacccaagctggtgatcacaggccgaaacactggttaaggcagggtgggtttactgaccaggaggttaaaggtgataccSerThrPheAspAspThrLysLeuValThrGlnAlaGlyAspHisArgProLysHisTrpLeuArgGlnGlyGlyPheThrAspGlnGluValLysGlyAspThr

AccttggaaactttgaaaactaaactgggtgagctcgttggtcctactaagccttgtgagaaggttttgtgtacccttgcttcatatgcgcttatgaagacccctThrLeuGluThrLeuLysThrLysLeuGlyGluLeuValGlyProThrLysProCysGluLysValLeuCysThrLeuAlaSerTyrAlaLeuMetLysThrPro

CaagatgcagctggcaagcaggcatggatctttttattggcaagtgcaatgaataacaatgctatgaaagctaagcttgaggtagcagtaaacgcggttactcccGlnAspAlaAlaGlyLysGlnAlaTrpIlePheLeuLeuAlaSerAlaMetAsnAsnAsnAlaMetLysAlaLysLeuGluValAlaValAsnAlaValThrPro

GgtaagggagaaacctttgtcaaccaactaaaggaggttggcaaatcactccagcttcccaaggaacaagttcctaagcaatatcgtttccctggtgtctatgcaGlyLysGlyGluThrPheValAsnGlnLeuLysGluValGlyLysSerLeuGlnLeuProLysGluGlnValProLysGlnTyrArgPheProGlyValTyrAla

AacctcgatgtgcaacacttttggactgtgttaaccggcgtctttggcactatactgactgaccttgaggttgacgaaaaggatgctcagggtaaagcaggacagAsnLeuAspValGlnHisPheTrpThrValLeuThrGlyValPheGlyThrIleLeuThrAspLeuGluValAspGluLysAspAlaGlnGlyLysAlaGlyGln

GttgctactagagttgcggagcttgtcaaggtggaaggtccacttcacagcctcactgtgcaagtagctgagatgactaaggctggagcgggtgctggtggtgaaValAlaThrArgValAlaGluLeuValLysValGluGlyProLeuHisSerLeuThrValGlnValAlaGluMetThrLysAlaGlyAlaGlyAlaGlyGlyGlu

GctcctgctcaggcagctgctgggacagcaggagcacgtgcagaagctccagccaaggaaggccaaggtgaggatggtgcgcacttttgtggcatcggaatgacaAlaProAlaGlnAlaAlaAlaGlyThrAlaGlyAlaArgAlaGluAlaProAlaLysGluGlyGlnGlyGluAspGlyAlaHisPheCysGlyIleGlyMetThr

gttttctttgtttctGtggttatcgctgtcttttaa 1401ValPhePheValSerValValIleAlaValPhe*** 466

Nucleotide and amino acid sequences of P50 gene

-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

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

9764

43

30

20

kDa M 1 2 3

Purification of rP50 expressed in E. coli

GST-P50t

(A) (B) ( C )

GST

1 2 1 2 1 2

Antigenicity of rP50 expressed in E. coli

A B

Anti-P50 DIC

IFAT

94

67

43

30

kDa B. gibs

oni

nRBC

P50

Western blotting

Identification of native P50 on parasites

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.

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

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

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)

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)

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

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

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

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

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

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

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.

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)

Other ELISAsB. equi ELISA-BeEMA2B. caballi ELISA-BcP48B. bovis ELISA-BboRAP1B. bigemina ELISA-BbiRAP1T. gondii ELISA-TgSAG2N. caninum ELISA-NcSAG1C. parvum ELISA-CpP23

Other ICTsB. equi ICT-BeEMA2B. caballi ICT-BcP48B. bovis ICT-BboRAP1B. bigemina ICT-BbiRAP1T. gondii ICT-TgSAG2N. caninum ICT-NcSAG1