is'i!!liA'!itt Taiwan Vet J 32 (2): 76-87, 2006

Identification of the Causative Agents of Ehrlichia canis and Anaplasma phagocytophilum in Dogs in Taiwan by Nested PCR, Indirect Immunofluorescent-Antibody Assay, and Sequence Analysis of the 16S rRNA Gene *1Hung J. L1U , 2Chia C. YIN, 2Yao C. HSI EH , 3Yu C. CH IANG, 'Ching D. CHANG , 1Ming H. L1AO , 'Chiang H. CHIANG , 'Yeong H. WU , 'Suen C. LIN "Dep artmen t of Veterinary Medicine, National Pingtuag University. NO. I, Syuefu Rd., Neipu, Pintung 9 12, Taiwan, R. O. C. 'Deper tment of Life Science, National Pingtung University of Science and Technology, Pingtung 912,

Taiwan, R.O.C. 2

Taiwan Hsien Livestock Disease Control Center, Hsin- Ying, Tainan 730, Taiwan, R.O.C.

(Received: July 28, 2005. Accepted: December 27, 2005. )

ABSTRACT To determine th e prevalence of Ehrlichia canis and Anaplasma phagocytophilum (formerly know n as E. equi) in dogs in Taiwan, we collected canine blood samples randomly f rom clinical cases at Department of Teaching Animal Hospital, National Pingtung University of Science and Technology from June, 1999 to March 2000. These samples we re examined by blood smear examination, indirect immunof luorescent-ant ibody ( IFA ) assay, and nested polymerase chain reaction ( PCR ) were used to examine one hundred-and-ten blood samples from dogs. Two sets of t he nested PCR we re developed for detecting Ehrlichia species in dogs, and predicted nested PCR products from E. canis and Anaplasma phago cytophilum were 520 bp and 925 bp, respectively. Tw enty two of 110 dogs ( 20% ) showed positive results of E. canis in nested PCR, while only two of 110 dogs ( 1.82% ) showed positive results of Anaplasma phagocytophilum in nested PCR. The PCR products from E. canis and Anaplasma phago cytophilum were sequenced and compared with t he previously published sequences. The sequence similarities of the 16S rRNA gene among them were above 97% . Phylogeneti c tree were inferred from nucleot ide sequences of the 16S rRNA gene, showing that the Taiwanese species were closely related to other Ehrlichia species. By IFA test, 24 of 60 ( 40% ) dogs we re seroreactive against E. canis antigens wh ile only 17 of 60 ( 28.3% ) dogs were nested PCR positive . The discordant IFA and nested PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E. canis . Ehrlichial morulae in peripheral lymphocytes were found in 5 of 22 cases of PCR positive dogs. The peripheral lymphocytes containing elementary bodies ( EB ) and/or initial bodies ( IB ) less t han 20% was found in 93.94% (62/66) of PCR negative dogs, and t he absolute number of simi lar lymphocytes less than 600/IJL was found in 95.52% (64/66) of PCR negative dogs. Alt hough the positive rate was highest in IFA test , the nested PCR exhibited the higher specificity than IFA test in the detection of canine ehrlichiosis using blood samples. The results suggested that nested PCR in combinat ion with IFA test is the best suited for the detection of canine ehrlichiosis while acute or chronic phase serum samples were tested . I" L iu HJ, Yi n CC, Hsieh V C, Chiang V C, Chang CD, L iao MH, Chiang CH, Wu VH, Lin Sc. Identifi cation of the Causative A gents of Ehrlichia Canis and Anapla sma Phagocytophilum in Dogs in Taiwa n by N ested PCR, Indirect Immunofluorescent- Ant ibody Assay, and Sequence An alysis of the 16S rRN A Gene. Taiw an V et J 32(2): 76-87, 2006. * Corresponding author TEL : 886-8-774 0319, FAX: 886-8-7700447, E-mail : [email protected].]

Key words: Ehrlichiosis, phylogenetic analysis, indirect immunofluorescent-antibody assay, nested peR

Hung J. Liu et al

77

INTRODUCTION Canine ehrlichiosis, a Tick-brone disease of dogs caused by the rickettsia Ehrl ichia canis , was first recognized as a disease entity in Algeria ( 5 J. Since then it has been acknowledged as a significant canine disease ( I J and particularly frequent in tropical and subtropical regions. It exhibits acute, subclinical, and chronic consecutive stages ( 1,3,24 J. In the acute phase, clinical signs such as fever, dyspnea, depression, anorexia, and lymphadenopathy are observed. The subclinical phase of infection follows the acute period and is associated with persistent E. canis infection. Thrombocytop enia, variable leukopenia and, anemia persist in the absence of clinical signs ( 10 J. The indirect immunofluorescent-antibody ( IFA ) assay with E. canis antigen has been widely used for the diagnosis of E. canis infection of dogs. Although the IFA test is very sensitive in detecting the prevalence of exposure to E. canis, it is not useful for assessing clearance of E. canis after antibiotic treatment or determining current infection status, since dogs and cats remain IFA positive for a long period of time after clearance of the organism ( 13 J. Ehrlichia canis infection in cats in Taiwan was reported previously (30 J. The purpose of the present study was to identify Ehrlichia and Anap lasma species infecting dogs in Taiwan by using nested PCR, IFA and sequence analysis of the 16S rRNA gene of E. canis and A. phagocytopliilum.

MATERIALS AND METHODS Blood spe cimens and DNA extraction A small volume of blood ( 5 mL with heparin and 5 ml, drawn with EDTA ) was collected randomly from 110 dogs of clinical cases at Department of Teaching Animal Hospital, National Pingtung University of Science and Technology from June, 1999 to March, 2000. The EDTA-blood samples were used for haematological examination. Haematological assays were performed on the same day and the following parameters were determined: packed cell volume, haemoglobin concentration, total erythrocyte count, mean corpuscular haemoglobin concentration, total and differential white blood count, total platelet count, and mean

platelet volume. The blood samples, in heparin anticoagulant tubes, were used for indirect immunofluorecent-antibody ( IFA ) assay and nested PCR assay. Each blood sample was centrifuged at 2,500 xg for 5 min. Plasma was saved for the IFA test, and 200 ul. of the buffy coat layer was harvested for polymerase chain reaction ( PCR ) . The buffy coat layer was dissolved in lysis buffer ( 10 mM Tris-HCI, 0.5M EDTA, 0.1% SDS, 0.5 mg/ mL proteinase K ) . After incubation for 14 h at 37°C, total DNA was isolated by two consecutiv e phenol: chloroform: isoamyl alcohol ( 25:24:I ) extractions and recovered by precipitation with ethanol containing 0.4 M LiCI ( 15,16 J. The purified DNA was stored at 4°C until it was used as the template for PCR amplification .

Nested Pf.R amplification of the 16S rRNA gene of E. canis and A. phagocytophilum Primers for detection of E. canis were designed from a variable region of the 16S rRNA gene sequence s of E. canis, which is available in GenBank ( accession number M73221 ) . Primary reactions used 5 ul, of purified DNA as the template in a total volume of 50 ul.. Amplification contained 20 ulvl of each dedoxyribonucl eoside triphosphate ( dATP, dCTP, dGTP, a nd dTTP ), 1.25 U of Pfu Taq po lymeras e, and 0.5 ~lM each primer. Primers for detection of E. canis were ECI ( 5'-GTTTGATCATG GCTCAG-3'; identical to nucleotides 3- 19) and EC2 (5'-TTAAAGCCGCTCCA AA-3'; complement ary to nucleotide s 6 15-60I ) . The primers for detection of A. phagocytophilum were as follows: EEl: 5'-TCCTGGCTCAGAACGAACGCTG GCGGC-3' ( identical to nucleotides 9-36 ) and EE2: 5'-CAGCCATITAAGG TGG TCAGTGACT-3' ( complementary to nucleotides 1440-1413 ) . The predicted PCR products from E. canis and A. phagocyt opliilum were 6 13 bp and 1432 bp, respectively. PCR reactions were subjected to 35 cycles consisting of denaturation at 94°C for I minute, annealing at 55"C for I minute, and extension at n oc for 2 minutes and one final extension cycle at n oc for 7 minutes. Nested amplifica tion used I ul, of the primary PCR product as the temp late in a total volu me of 50 ul. . Each nested amplification containied 20 ~lM of each dedoxynuc leoside triphosphate (dATP, dCTP,

Identification of the Causative Agents of Ehrlicliie Canis and Anaplasma Plisgocy tophilum in Dogs in Taiwan

78

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Fig. 1A-B. Nested PCR for detection of the 168 rRNA gene of E. canis ( 520 bp ) in dog blood samples. (A) Lane M: DNA molecular marker; lanes 1-11 : TW16611, TW16659, TW16913, TW16924, TW16927, TW16933, TW16934, TW16945 , TW16949, TW16977 , and TW16980, respectively; lane N: a negative control. ( B ) Lane M: DNA molecular marker; lanes 1-11 : TW16983, TW17122 , TW1712 3, TW17124 , TW17128 , TW171 34, TW17146, TW17196, TW17209 , TW1216 , and TW16113, respectively ; lane N: a negative control. The numbers on the right indicate molecular size in base pairs.

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dGTP, and dTTP ), 1.25 U of Pfu Taq polymerase, and 0.5 ul. M each prim er. Prim ers for E. canis were EC3

( 5'-TTATAGC CTCTGGCTATAGG-3'; identical to nucl eotid es 52-67 ) and EC4 ( 5'-CACTTTAACT ACTAGTCCA-3'; complementary to nucleotides 581-569 ) . The primers for detection of A. phagocytophifum were as follows: EE3: 5'-GTCGAACGGATTATTC TTTATAGC TT GC-3' ( identical to nucleotides 52-80) and EE4 :5'-GGAGATTAGAT CCTTCT TAACGGAAGGG-3' (compleme ntary to nucleot ides 976-949 ) . The predi cted PCR produ cts from E. canis and A. phagocytophilum we re 520 bp and 925 bp, respectively. Nested cycl ing conditions were as described for the primary amplification. Reaction products we re subsequently maintain ed at 4°C until they were analyzed by agarose gel electrophoresis or puri fied for DNA cloning.

79

Hung J. Liu et al

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