J O U R N A L O F V eterin ary S cien ce J. Vet. Sci . (2002), 3(2), 109-114 ABSTRA CT 8) Three do gs w ere ex perim en ta lly in fecte d w ith D ir ofila ri a im m i ti s. All d o gs w ere eu th a n ised a t 30, 36 and 37 weeks after inoculation of D. immitis for the recovery of adult w orms. Three cases accounted to 42.91 % recovery of inoculated w orm s. Serum samples from dogs experimentally inoculated w ith D. immitis w ere analyzed by ELISA and imm unoblotting methods. Antibody titers of dogs detected by ELISA peaked betw een 7 and 14 weeks then decreased betw een w eeks 15 to 24 follow ed by another increase during weeks 25 to 30 and persisted throughout the remainde of the experiment period. Analysis of adult D. immitis protein stained w ith Coomassie brilliant blue R-250 indicated separately more than 10 bands, and the major bands were 22, 40, 46, 56, 70, 72 and 89 kDa. Antigenic identification of extracts antigens of adults D. immitis by immunoblotting analysis revealed several bands from pooled sera of patent infection (30 w eeks after inoculation). The detected bands were 24, 70, 80 and 110 kDa, 22, 72 and 84 kDa, and 58 and 72 kDa in dogs 1, 2 and 3, respectively. Results of antibody titers re ached high levels on the 4th molting stage after inoculation of infective larva (L3), and reinforced previous findings that high molecular w eight regions are detected in young animals. Key w ords: Dirofilaria immit i s, experim ental in fection , ELISA, im m un oblot tin g ＊ Corresponding author: Tel: +82-42-821-6788, Fax: +82-42-821-4216 E-mail: kyuwoancho@cnu.ac.kr Introduction Dirofilaria im m itis, com m on ly ca lled ca n in e h ear twor m , is the ca u se of a seriou s pa rasitic disea se of dogs endem ic in tem pera te, subtr op ica l an d tropica l cou ntries [4]. In epizoot iologic su rveys of d irofila riasis in dogs, va riou s m et h ods for determ in ing in fect ion sta tus h ave been used, inclu din g microscopic exa m in ation of blood sm ear s, blood sa m ple concen tr a tion tech n iqu es, Kn ot t's test for detect in g circu la tin g microfila ria e, exa m in a tion for iden tifica tion of adu lt worms a t necropsy, a nd radiogra phic a nd a ngiogra phic evalu a tion s [2,23,26]. A m ajor pr oblem in m ak in g dia gn osis is the dirofilariasis without microfilaremia (occult dir ofila ria sis) wh ich has been sh own t o occu r in 10 % t o 67 % of dogs t ha t ha ve been in fected na tu ra lly. This h a s been at tribu ted t o sin gle-sex in fection , presen ce of im m ature a du lt s or im mu ne- mediated clearance of microfilariae [21]. These occult in fection s ar e very difficu lt to dia gn ose by m icr oscopic exa m in ation of blood, so t ha t, th e veterin a ry pr actition ers u sua lly m a ke t he dia gn osis ba sed on clin ica l signs, r a diograph ic evidence, a n d oth er in direct in dica tion of in fect ion [21]. The occu lt D . im m itis in fection s pose t h e m ost ser iou s dia gn ostic ch allen ge. An d, som etim es, even in pa tent infect ion s, obstacles cou ld be a voided in the dia gnosis of dirofila ria sis du e t o th e low n um bers of m icrofila ria e or th e difficu lt y in distin gu ish in g m icr ofilariae of D . im m itis from th ose of Dipen talon em a recon d itu m [5]. To overcom e these problem s, va riou s im m un ologica l tech n iqu es h ave recently been developed [3,5,6,8-12,14,15,18,19,28]. Am ong these meth ods, enzyme-lin ked im mun osorben t a ssay (E LIS A) a nd im m u noblott in g a na lysis h a ve been recogn ized a s sim p le, sen sit ive and p ar ticu la rly su it able for th e pa ra sitic disea ses like D. im m itis in fect ion [5,6,9,11]. Th e pr esent study wa s per for m ed to elu cida te the im m u n ological respon ses of dogs experim en ta lly in fected wit h infect ive la rvae of D . im m itis usin g ELISA a nd im m un oblot tin g. Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis Kun-Ho Song, Mineo Hayasaki1, Chusnul Choliq2, Kyu-Woan Cho*, Hong-Ryul Han3, Bung-Hyun Jeong4, Moo-Hyung Jeon5, Bae-Kun Park6 and Duck-Hwan Kim Department of Veterinary Internal Medicine, 5Department of Veterinary Microbiology and 6Department of Veterinary Parasitology, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea 1Veterinary Clinical Center, School of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan 2Veterinary Clinical Center, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor 16151, Indonesia 3Department of Veterinary Internal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea 4Department of Veterinary Internal Medicine, College of Veterinary Medicine, Kon-kuk University, Seoul 143-701, Korea Received F eb. 3, 2002 / Accepted May 7, 2002 110 Kun-Ho Song, Mineo Hayasaki, Chusnul Choliq, Kyu-Woan Cho, Hong-Ryul Han, Bung-Hyun Jeong, Moo-Hyung Jeon, Bae-Kun Park and Duck-Hwan Kim Materials and Methods Ex pe rim e n tal an im a ls Five h ea lthy m on gr el ju ven ile d ogs of appr oxim at ely th ree m on t hs of a ge wer e hou sed in mosqu ito-proof run s, an d t reat ed wit h pipera zine for in test in a l para sit es pr ior to experim en ta tion . Ex pe rim e n tal in fectio n Th ree dogs w er e experim en ta lly in fect ed wit h D. im m itis by t he procedu res descr ibed by H a ya saki [8]. Br iefly, m osquit oes, Aed es t ogoi, collect ed du r in g the la rval sta ge from th eir na tu r al spa wn in g ar ea s wer e rear ed u nd er labor a tory con dit ion s a n d in ocu la t ed by feedin g on a dog wit h a bou t 200 cir cu lating m icr ofilaria l (Mf) cou n ts per 20 ㎕ of blood (Table 1). Two dogs were used as the negative control. Infective larvae (L3) of D. immitis were recovered from the proboscis of the infected mosquitoes 10 to 14 days after blood feeding and suspended in saline during microscopic observation. Three dogs were subcutaneously injected in the inguinal region with each dog receiving 228 (dog No. 1), 278 (dog No. 2) and 248 (dog No. 3) infective larvae (L3), respectively. Experimental dogs were euthanised at 30, 36 and 37 after inoculation for the recovery of adult worms. Serum samples The blood was collected from the cephalic vein once a week, and serum separated and stored at -80 until analysis. Circulating Mf counts Dogs were screened for Mf by concentration method, and microfilarial density in 20 ㎕ of blood was measured by counting Mf on methylene blue stained smears [20]. Preparation of D. immitis antigen The crude extracts of D. immitis were prepared as previously described [7]. Briefly, the antigens used in this study were extracted from adult worms of D. immitis by phosphate buffered saline (PBS, pH7.2 , 0.1M). These worm s were h om ogenized a n d son ica ted by tissu e hom ogen izer (15m in, 4 ) a n d u lt ra son icator (50 watt , 15m in , 4 ), respect ively, and th en allowed to in cu ba te overnigh t at 4 . After cen trifuga tion at 18,000g, th e su pernatan t as a nt igen wa s collected and kept at -80 . Th e prot ein con cen tr at ion of th e an tigen wa s determ in ed usin g the m et hods of Low ry et a l.[17]. En zy m e -Lin ked Im m u noso rb en t Ass ay (ELIS A) E LISA wa s per form ed by m ethods of Grieve et a l.[6]. D . im m itis antigen wa s dilu ted to 10 ㎍ / ㎖ in PBS. 100 ㎕ of antigen was dispensed into each well and incubated for 30 minutes at room temperature. 200 ㎕ of 1% bovine serum albumin(BSA) solution in PBS was added and the plates were incubated at 4 overnight. The wells were washed three times with 200 ㎕ of 0.1% Tween 20 in PBS (TPBS) in 3 minutes for each wash. 100 ㎕ of the serum from the test subject was diluted to 1:100 in PBS and added to each well. The microplates were incubated at 37 for 1 hour, washed with TPBS, and 100 ㎕ of peroxidase-conjugated goat anti-dog IgG (ICN Pharmaceuticals, Inc., USA) diluted to 1:200 in PBS, was added to each well and incubated at 37 for 1 hour, then washed with TPBS three times (3 minutes for each wash). A fresh preparation of substrate working solution was made from 1% o-phenylenediamine in methanol, 0.1M citrate buffer (pH4) and 3% hydrogen peroxide, and 100 ㎕ of the solution was added to each well and left at room temperature for 30 minutes. The enzyme reaction was stopped by adding 50 ㎕ 4 N sulfuric acid and values of optical density (OD) were read at 498 ㎚ (Sanko Junyaku co. Japan). The specificity of the assay was evaluated by cross-checking with the DiroCHEK kit (Synbiotics co., San Diego, USA) using five of the known positive and ten known negative serum samples. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) A minislab gel consisting of 12.5% acrylamide and 0.1% SDS was used as described by Laemmli [16]. The serum samples were diluted to 500 ㎍ protein/ ㎖ in the sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.00125% bromophenol blue) and heated for 5 minutes in boiling water. 10 ㎎ of protein was Table 1 . Wor m bur den of dogs experim en tally in ocu lated w ith D. im m itis D og N o. No . o f L3 inje cted Du ra tion o f in fe ctio n (w ee ks) N o. o f a du lts w o rm reco ve red (F /M) Re co v ery ra te 1 2 3 248 278 228 37 31 38 151(95/96) 31(16/15) 127(66/61) 60.88 11.15 56.70 Mea n(%) 42.91 L3: In fect ive la rvae F /M: Fem ale/Ma le Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis 111 th en loa ded in ea ch well, followed by elect roph or esis on 20 ㎃ currents at 4 for 120 minutes. The gels were subsequently stained with Coomassie brilliant blue R-250 (Katayama chemical, Japan). Approximate molecular weight of sepa ra ted bands w er e estim ated u sin g m olecu la r weigh t m a rkers (BIO-RAD, U SA). Im m u n ob lottin g Follow in g SDS-P AG E, th e pr ot ein bands in t he polya cryla m ide gel w er e tra nsfer red elect roph oretica lly to a nit rocellu lose m em bran e sh eet s (pore size 0.45 ㎛ , BIO-RAD, USA) at 80 volts at 4 for 120 minutes, with a Tris-glycine electro-transfer buffer (0.025 M Tris, 0.192 M glycine, 20% methanol and 0.1% SDS). The nitrocellulose sheets were blocked for overnight in Tris-buffered saline (TBS) (0.02M Tris-HCl, pH 7.5, 0.5M NaCl) containing 3% gelatin (BIO-RAD, USA). The nitrocellulose sheets were cut into strips and placed into p l a st ic tra ys. St rip s were separ at ely rea ct ed to test sa m ples tha t wer e dilu ted 1:500 in TBS con ta inin g 1% gela tin , a t r oom tempera ture for 2 h rs, peroxidase-conjugated goat anti dog IgG (ICN Ph a rm a ceu tica ls In c, U SA) ser um dilu ted t o 1:500 in TBS con ta inin g 1% gela tin . Str ips were t hen wa sh ed wit h TBS. Substra te solu tion con sistin g of 15 ㎎ of 4-chloro-1-naphtol dissolved in 5 ㎖ methanol, 15 ㎕ hydrogen peroxide, was added to each well and allowed to develop at room temperature for 20 minutes. The reactions were stopped by replacing the substrate with TBS. Results Recovery of w orms at necropsy Dogs were euthanised on week 30, 36 and 37 post inoculation for the recovery of adult worms. At necropsy, 151 adult worms (95 females and 56 males) in Dog No.1, 31 adult worms (16 females and 15 males) in Dog No.2 and 127 adult worms (66 females and 61 males) in Dog No.3. were recovered from the right ventricle and pulmonary arteries. These represent approximately 42.91% recovery of inoculated worms. ELISA of sera from experimentally infected dogs In the Dog No.1, No.2 and No.3, antibody titers in the sera of inoculated dogs were significantly detected 7 weeks post inoculation and persisted until 14 weeks. Antibody titers of dogs detected by ELISA peaked between 7 and 14 weeks then decreased between weeks 15 to 24 followed by another increase during weeks 25 to 30 and persisted throughout the remainde of the experiment period. Analysis of adult D. immitis protein by Coomassie brilliant blue R-250 staining D. immitis proteins were separated into more than 10 bands stained with Coomassie brilliant blue R-250 (CBB) staining, the major bands were 22, 40, 46, 56, 70, 72 and 89 kDa. Fig 1. Antibody responses of dogs to experimental D. immitis infection by ELISA using D. immitis antigens. *The range of optical density of control group. The arrow indicates the day of inoculation. ＊ 112 Kun-Ho Song, Mineo Hayasaki, Chusnul Choliq, Kyu-Woan Cho, Hong-Ryul Han, Bung-Hyun Jeong, Moo-Hyung Jeon, Bae-Kun Park and Duck-Hwan Kim Antigenic identification of crude extract antigens of adult D. immitis by im munoblotting Pooled sera of patent infection (30 weeks after inoculation) from Dog No.1 revealed four antigenic bands with 24, 70, 80 and 110 kDa. Dog No.2 showed three antigenic bands of 22, 72 and 84 kDa, while two antigenic bands were detected in Dog No. 3 with 58 and 72 kDa. Discussion Experimental D. immitis infections in dogs have been performed by various investigators [6, 8, 27, 29]. The recovery rate of adult worms from dogs experimentally inoculated with the infective larvae of D. immitis were 45%, Hayasaki [8], 66%, Thrall et al.[27] and 50%, Wong et al.[29], respectively. In this study, three cases accounted to 42.91 % recovery of inoculated worms. Although this study did not confirm the correlation between antibody titer and the number of adult worm, Grieve et al.[6] reported that there was no relationship between them. The ELISA used in conjunction with a Knott's test, exposure history, clinical signs, laboratory results, and radiographic changes are useful for studying seroepizootiologic pattern and risk factors of heartworm infection. ELISA testing has been shown to be capable of identifying prepatent infections [6]. Diagnosis of prepatent infection or asymptomatic occult infection is important to the practitioner who may be preparing to initiate a heartworm preventive program based on negative results of a Knott`s test. In addition, experimental studies of beagles have shown abnormalities of pulmonary arteriograms 6 months after inoculation with infective D. immitis larvae, at which time the dogs were still amicrofilaremic and results of indirect fluorescent antibody testing for antimicrofilarial antibodies were negative [22]. ELISA testing for the detection of D. immitis provide a useful and improved assay for serological characterization and detection of infection. Grieve et al.[6] reported that ELISA titers were not significantly increased until 11 or 16 weeks, and remained at maximum levels for the duration of the observation period. However, the present study revealed that antibody levels were detected as early as 7 weeks post inoculation, and developed continuously to 14 weeks, and then diminished to 15 to 24 weeks, then climbed again to 25 to 30 weeks, and high titer levels persisted throughout later experiment time. One explanation of these different results may be the different responses of individual dogs to the variable extent and duration of parasitic exposure [13]. Another explanation may be that different antibodies are being detected by different protocols used [11]. Konno et al.[15] reported that protein bands in extracted fig. 2. Protein fractions of adult D. immitis were separated by SDS- P AGE sta ined w ith Coom a ssie brillia nt blu e R-250(A), a n d D. im m itis an tibody respon ses with D. im m itis a ntigen by im m un oblot tin g a n alysis(B). La ne 1:sta n da rd mark er, la n e 2-4: n ega tive con t rol, la n e 5-7: pa tent in fection (la n e 2,5: N o.1, lane 3,6: N o.2, la ne 4,7: No.3). Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis 113 an tigen of D. im m itis w as det ect ed by Coom assie brillia n t blu e R-250 st ain in g were 44 ba n ds (14 to 230 k Da ). Th e present study revea led tha t D. im m itis protein was separ a ted int o m ore th a n 10 ba nds (22 t o 89 kDa ) by Coom a ssie br illia n t blu e R-250 stain ing, t he m ajor ba nd s detect ed were 22, 40, 46, 56, 70, 72 an d 89 kD a. Alt hough the n um ber of prot ein ba nds wa s less th an th o s e of Konno et al.[15], similar protein bands were detected by Coomassie brilliant blue R-250 staining. Immunoblotting in th e D. im m itis infection helps to clarify the relation of antigen- antibody in immune response, a more effective application of immunologic diagnosis [9,12,24]. Tamashiro et al.[24] and Boto et al.[1] reported that low molecular weight reactivity as more prevalent in older and /or more heavily infected dogs in experimental D. i m mitis infectio n by means of immunoblotting. In na t ur al D. im m i t is infection, Tanaka et al.[25] reported the trends in the distribution of immunoblotting patterns with either young animals and/or mild infection tended to reveal antibodies with reactivity to antigen in the higher molecular weight regions of immunoblotting (more than 80 kDa), he also reported that the majority of antibodies with reactivity to antigens in the low molecular weight regions (less than 37 kDa), occurred more frequently in older dogs and/or heavier infections. The results of Tamashiro et al.[24], Tanaka et al.[25] and Boto et al.[1] suggested that immunoblotting will produce similar results in both experimental and natural infections. In the present s tu dy, an tigenic iden tifica tion of crude extr act a n tigens of a du lts D. im m itis by im m un oblot ting an alysis revea led severa l ba n ds from th e pooled ser a of pa tent in fection (30 weeks a ft er in ocu la tion). The detect ed ba nds w er e 24, 70, 80 and 110 kD a in Dog N o.1, 22, 72 a n d 84 kDa in Dog N o.2, a nd 58 a nd 72 kDa in Dog No.3. The resu lt s pr esen ted here are sim ila r to th ose of Tam ash iro et al.[24] an d Bot o et a l.[1], in w hich th e h igher molecu la r weight region s were detect ed by im munoblot tin g in you n g an im a ls (m or e t ha n 70 kDa ). In sum m a ry, an tibody titers by E LISA reach high levels at th e 4th m olt in g st age a fter in ocu la tion of L3, a nd th e high er m olecula r weigh t region s ar e m a in ly det ect ed by im m u n oblot tin g in you n g anim a ls. F u rth er stu dies by oth er tech n iqu es are need ed to elucidat e im m un ologica l resp on ses in D . im m itis-in fected dogs. Acknow ledgem ent Th is stu dy wa s fin a ncia lly su pported by 2001 of Aca dem ic Resear ch & Schola rsh ip F ou n dation, Ch u ngn am N at ion a l U niver sit y. References 1. B oto , W. M., P o w er, K. G. a n d Le v y, D . A. 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Immunological Responses of Dogs Experimentally Infected with Dirofilaria immitis 113 an tigen of D. im m itis w as det ect ed by Coom assie brillia n. antibody production in dogs infected with Dirofilaria immitis. Jpn. J. Vet. Sci. 1982, 44 , 63-70. 9. Hayasaki, M., Nakamura, F. and Katsuhiko, K. Immunoblotting analysis of somatic components of Dirofilaria