Diagnosis of Bovine Babesiosis by the Capillary-Tube Agglutination Test

Transcription

1 Diagnosis of Bovine Babesiosis by the Capiary-Tube Aggutination Test By TETSURO MINAMI Kyushu Branch, Nationa Institute of Anima Heath Three species of Ba1esia, parasitic to catte in Japan, have been so far reported" ' ". Two of them, Babesia bigemina and B. argentina, with high pathogenicity are distributed in Okinawa district as a main cause of piropasmosis in that area. The third one (referred to the Japanese Babesia species), which is widey distributed in Japan except Okinawa Prefecture, is Jess pathogenic than the former two species, but it often shows the mixedinfection with Theieria sergenti, Anapasma etc., giving the compicated symptom of piropasmosis of grazing catte. The decisive diagnosis of babesiosis is to detect the pathogenic protozoa. The microscopic examination of stained bood-smear preparations is most frequenty empoyed for detecting the p1 otozoa. In catte infected with T. serg&nti, the protozoa can easiy be detected in bood even time eapsed after infection. However, with Babesia, athough it can be detected easiy at the stage immediatey after the initia infection, the microscopic detection becomes very difficut after time eapsed because the number of the protozoa in the circuating bood decreases rapidy. Therefore, with the toerant, catte, it is not possibe to carry out microscopic examinations of protozoa and the diagnosis of pathogenicity, uness the protozoa is mutipied by inocuating the bood sampe to the heathy spenectomized catte. With regard to the seroogica methods of diagnosis, it was reported that the compementfixation test, fuorescent antibody technique, ge-precipitation test, etc. have aready been appied and proved to be highy specific >. However, these methods are generay compicated in procedures requiring skifuness, and sti have probems reated to the positive imit or their popuarization. As to the aggutination test, various methods of test have been deveoped. As the indirect aggutination test, the passive haemaggutination test' '> or the rapid side aggutination test» by using atex partices is being empoyed for diagnosis of B. argentina and B. bigemina. As the direct aggutination test, the capiary-tube aggutination test >, side aggutination test">, and card aggutination test"> are used for B. bige- 1nina, and the parasitized erythrocyte aggutination test'> for B. argentina. Procedures of these aggutination tests are generay simpe, and particua'iy the capiary-tube aggutination (CA) test is considered as a seroogica method of diagnosis with high practicabiity. Therefore, the author attempted to utiize the CA test in diagnosing the Japanese Babesia species as an initia step in the effort to simpify the seroogica diagnosis of bovine babesiosis in Japan. Preparation of As Babesia strains, the Miyake strain of the Japanese Babesia species and the Kochinda strain of B. bige mina, both stored at the tempearture beow - 76 C by the rapid-freezing method' 0 > were used. As experimenta animas, the Hostein caves spenectomized of 3-5 months of age were used. Bood showing more than 15% in the ratio of parasitized erythrocytes to the tota number of erythrocytes was used for the preparation

2 235 of the. The was prepared by the Lohr & Ross method (origina method) and the author's modified method. The CA by the origina method (refened to origina ) was prepared by treating 50% suspension of washed infected erythrocytes with an utrasonic disintegrator (MSE, 150W) at its maximum output for 5 min, and repeating washing by utra-centrifugation at 30,000 rpm for 30 min and homogenizing. The CA by the modified method (referred to modified ) was prepared as foows: washed infected erythrocytes were hemoyzed with 0.35% NaC soution. After removing eucocytes and unhemoyzed erythrocytes, the ysate was centrifuged at 10,000 rpm for 15 min. The sediment, washed, was subjected to the sonic treatment at the minimum output for sec, and then washed and homogenized repeatedy (Fig. 1). In the origina, a the protozoa were crushed, whereas the modified differs from the origina in that the origina form of the protozoa was mosty kept unchanged. The CA s of the Japanese Bcibesia species, prepared by both methods were compared by box-titration. The modified showed higher and antibody titer vaues as we as more cear aggutination figures than the origina. In addition, the microscopic examination of Giemsa stained smear preparations of the modified and its aggutination figures after reaction detected the protozoa in the, and it was considered that the aggutination in the CA test is mosty due to the aggutination of p'otozoa. With the origina, no protozoa was observed. Therefore, it was decided to use the modified, standardized by box-titration, in a future works. Reaction procedures The CA test was carried out foowing the Lohr & Ross method"'. Bovine serum was taken after the experimenta infection of Babesia, and heated at 56 C for 30 min to inactivate. Bood infected with Babesia! Washing of bood ce with physioogica saine nt 2,500 rpm, 4 C, for 15 min, 3times. Hemoysis of bood ce with JO voumes of 0. 35% NaC soution at 4 C for min. Centrifugation! at 6,000 rpm ror 30 min. Washing of sediment (upper ayer) with physioogica saine at JO, 000 rpm, 4 c. for 15 min. 3 times, Suspension of sediment! in verona buffered s aine (VBS) or 5 voumes. Sonication at the minimum output of utrasonic disintcgrator (20 J<cs) for I0 30 sec. Washing of sediment with VBS at JO, 000 rpm, 4 C, for 15 min. 2 times and homogenizing by gass gringer for I min. Suspension of sediment! in VOS of 2-4 voumes. Homogenizing! by gass grinder for 1 min. sighty. Low speed centrifugation! at I, 000 ri>m for 1 min. Supernatant fuid! add formain to the rate of 0. 2% and storage at 4 c. CA Fig. I. Modified preparation method of Babesia CA Origina serum or that made to twofod se'ia diutions with verona buffered saine (VBS) were used. Procedure of the CA test is as foows: Firsty, the CA is we mixed, sucked into a gass capiary tube 90 mm in ength and 0.5 mm of inside diameter to about 10 mm high, and the cinging outside the capijary tube was wiped off. Secondy, the inactivated serum was sucked into the capiary tube from the same opening used for the to the height of 60 mm, and outerwa of the tube was ceaned again. Finay, the tube was paced verticay with that opening upwa1 ds. The reaction was run at room temperature (22-25 C). When strong reaction occurred, the rating was made after 1-2 hrs. When the reaction was weak or doubtfu, observation of aggutination figures in the capiary tube was made after aowing to stand sti overnight.

3 236 The rating made by naked eyes based on the time required for the occurrence of aggutination reaction and on the extent of fo 1 ming aggutination figures was as foows (Pate 1). fh- tt- + Pate 1. Rating criteria in CA reaction +++ : Apparent aggutiantion cumps were 'ecognized in more than 1/ 2 of the reaction mixture after 1-2 hrs. ++ : Apparent cumps were recognized in ess than 1 /2 of the reaction mixture after 1-2 hrs. + : Aggutination reaction did not appear or was doubtfu after 1-2 hrs, but apparent cumps or a arge number of fine cumps were recognized af ter keeping the reaction mixture stand sti overnight. ± : A sma number of fine cumps were observed. No cumps were observed at a. Serum which shows aggutination figures higher than + was referred as CA test positive, and that ower than ± as negative. Antibody titer was expressed by the maximum J ARQ Vo. 11, No. 4, 1977 diution times of serum showing the reaction higher than +. Specificity of the CA test To examine the specificity of CA of the Japanese Babesia species, CA s were prepared by the modified method from bood infected with B. bigemina and norma bovine bood, and the cross CA test was carried out using sera coected from heathy catte and the animas experimentay infected with the Japanese Babesia species, B. bigemina, B. argentina, T. sergenti, Anapasrna rnargina!e, A. centrae, and Eperythrozoon wenyonii. Resuts indicated that the CA of the Japanese Babesia species did not react with any other serum than Babesia-infected serum, and that the norma did not react with any serum. Among the species of Babesia, a cross-reaction was observed, ike other seroogica reactions. However, the degree of reaction was apparenty strong for a same species, with titers of 4-1,024 times. On the contrary between different species, no reaction or weak reaction with titers of 2-16 times, even when cross-reactions occurred, were observed (Tabe 1). The CA antibody of the Japanese Babesia species coud be detected within about 5 days after the appearance of the protozoa, and its titer vaue reached the maximum, 128-1,024 times, after 3-10 days. Athough the parasitic protozoa disappeared from bood generay 3-21 days after infection, the CA antibody coud be detected over a period more than 1 year. These resuts were considered amost the same as the case of B. bigemina reported by Lohr & Ross > (Fig. 2). The CA, stored for days at 4 C after formain was added, was examined. It was demonstrated that even after 7 months of storage, the maintained its activity without any oss, showing a strong reaction to the antibody of the same species. In concusion, the CA test can be a rapid and simpe method of seroogica diagnosis of the Japanese Babesia species, which is widey

4 237 Serum Tabe Specificity of CA CA antibody titer Species Catte The Japanese Babesia species B. bigemina number The Japanese Babesia species I, B. bige111i11a B. arge11ti11a ! 12 T. sergenti { A. marginae { A. centrae { E. we11yo11ii { Norma serum { * CA prepared from norma bovine bood. Norma* 2, e i :3 10' '8 >, 10',D.. a 10' 10 C. 0 6 <I Caf NO L/ 512 Caf NO }:.. 32 :; >, 'O :; 10'.& :; e 10' ;i 2 j 10 C uo Fig. 2. Days o-o CA titer after infection -- Parasitcmia Deveopment of CA antibody in caves infected with the Japanese Babesia species

5 238 dist'ibuted in Japan, with high specificity and appicabiity for the diagnosis at the ch'onic stage of babesiosis. Resuts of the c"oss-tests showed that the discrimination from B. bigem.ina and B. a1 gentina was amost possibe, but further detaied examination using serum coected at given inte'vas of time wi be needed. References 1) Curnow, J. A. & Curnow, B. A.: An indirect haemaggutination test for the diagnosis of Bcibesic, a1 gentina infection in catte. Austra. Vet. J., 43, (1967). 2) Curnow, J. A.: In vitro aggutination of bovine erythrocytes infected Babesia cirgcn,,.. tina. Natiwe, 217, (1968). 3) Curnow, J. A.: The use of a side aggutination test to demonstrate ic differences between Babesia bige-mina purasites. Aiistm. Vet. J., 49, (1973). 4) Goodg er, B. V.: Preparation and preiminary assessment of pu'ified s in the passive haemaggutination test for bovine babesiosis. Ai1st1 a. Vet. J., 47, (1971}. 5) Goodge1 1 B. V. & Mahoney, D. F.: A rapid JARQ Vo. 11, No. 4, 1977 side aggutination test for the herd diagnosis of Babesia argentina infection. Ai1st1 a. Vet. J., 50, (1974). 6) Hamakawa, M. et a.: Bovine Piropas1nci in Ryukyu Isands. Preiminary report. Abstract of 78th meeting of the Japanese Society of Veterinary Science (1972). 7) Ishihara, T.: Bovine piropasmosis in Japan. JARQ, 3, (1968). 8) Ishihara, T.: Bovine babesiasis and theieriasis in Japan. Bu. Nat. Inst. Ani1n. Hth, No. 62, (1971) [In Japanese]. 9) Liihr, K. F. & Ross, J. P. J.: Ein Kapiarr6hrchen-Aggutinationstest zum Nachweis von Babesia bige1nin<i-antikorpern. z. T1'0pen11icc. Parasito., 20, (1969). 10) Minami, T. 1 Ishiha1 a, T. & Yamabe, K.: Cryopreservation of T1 ypanosoma, 1'heieric,, Babesia, and Anapctsma after rapidfreezing. Bu. Nat. Inst. Ani111.. Hth, No. 73, (1976). [In Japanese with Engish summary), Nat. Inst. Anim. Hth Qua1 t. 1 16, (1976). 11) Ristic, M.: Babesiosis and Theieriosis. Immunity to Parasitic Animas Vo. 2, , Appeton-Century-Crofts, New York (1970). 12) Todorovic, R. A. & Kutter, K. L.: A babesiasis card aggutination test. Amer..J. Vet. Res., 35, (1974).