Antigen for the indirect fluorescent antibody test (IFAT) was routinely prepared from infected erythrocytes from horses experimentally infected with Babesia equi and Babesia caballi. With the successful
establishment of in vitro cultures of B. equi and B. caballi, it is now possible to employ culture-
derived antigens in this test. In this study, in vitro-propagated B. equi- and B. caballi-infected
erythrocytes were used as antigen in the IFAT. Various modifications to an established protocol had
to be implemented to allow repeatable results. Cultures with 3-4% parasitized erythrocytes were
found to be most suitable. As cross-reactions of control sera on heterologous antigen were observed
at serum dilutions of up to 1/40, a reciprocal titre of 80 was considered to be positive. In positive
samples, specific fluorescence of Babesia parasites and/or erythrocyte membranes was observed.
Fifteen sera from Babesia-free horses from Japan all tested negative in the IFAT. One hundred and
ten field-horse sera from Central Mongolia were investigated in this study. The results indicate that
both B. equi and B. caballi are endemic in horses in Central Mongolia, with 88,2% and 84,5% of
horses being seropositive to B. equi and B. caballi, respectively.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Avarzed, A.

De Waal, D.T.

Igarashi, I.

Oyamada, T.

Saito, A.

Suzuki, N.

Toyoda, Y.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Antigen for the indirect fluorescent antibody test (IFAT) was routinely prepared from infected erythrocytes from horses experimentally infected with Babesia equi and Babesia caballi. With the successful
establishment of in vitro cultures of B. equi and B. caballi, it is now possible to employ culture-
derived antigens in this test. In this study, in vitro-propagated B. equi- and B. caballi-infected
erythrocytes were used as antigen in the IFAT. Various modifications to an established protocol had
to be implemented to allow repeatable results. Cultures with 3-4% parasitized erythrocytes were
found to be most suitable. As cross-reactions of control sera on heterologous antigen were observed
at serum dilutions of up to 1/40, a reciprocal titre of 80 was considered to be positive. In positive
samples, specific fluorescence of Babesia parasites and/or erythrocyte membranes was observed.
Fifteen sera from Babesia-free horses from Japan all tested negative in the IFAT. One hundred and
ten field-horse sera from Central Mongolia were investigated in this study. The results indicate that
both B. equi and B. caballi are endemic in horses in Central Mongolia, with 88,2% and 84,5% of
horses being seropositive to B. equi and B. caballi, respectively

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Onderstepoort Journal of Veterinary Research, 64:141-145 ( 1997) Prevalence of equine piroplasmosis in Central Mongolia A. AVARZED1, D.T. DE WAAL1•2, I. IGARASHP, A. SAIT03, T. OYAMADA4 Y. TOYODA1 and N. SUZUKPA ABSTRACT AVARZED, A., DE WAAL, D.T., IGARASHI, 1., SAITO, A., OYAMADA, T., TOYODA, Y. & SUZUKI, N. 1997. Prevalence of equine piroplasmosis in Central Mongolia. Onderstepoort Journal of Veterinary Research, 64:141-145 Antigen for the indirect fluorescent antibody test (I FAT) was routinely prepared from infected eryth· rocytes from horses experimentally infected with Babesia equi and Babesia caba/li. With the suc-cessful establishment of in vitro cultures of B. equi and B. caba/li, it is now possible to employ cul-ture-derived antigens in this test. In this study, in vitro-propagated B. equi- and B. cabal/i-infected erythrocytes were used as antigen in the I FAT. Various modifications to an established protocol had to be implemented to allow repeatable results. Cultures with 3-4 % parasitized erythrocytes were found to be most suitable. As cross-reactions of control sera on heterologous antigen were observed at serum dilutions of up to 1/40, a reciprocal titre of 80 was considered to be positive. In positive samples, specific fluorescence of Babesia parasites and/or erythrocyte membranes was observed. Fifteen sera from Babesia-free horses from Japan all tested negative in the I FAT. One hundred and ten field-horse sera from Central Mongolia were investigated in this study. The results indicate that both B. equi and B. caballi are endemic in horses in Central Mongolia, with 88,2 % and 84,5 % of horses being seropositive to B. equi and B. caba/li, respectively. Keywords: Babesia caba/li, Babesia equi, equine piroplasmosis, prevalence INTRODUCTION Equine piroplasmosis is caused by Babesia equi and Babesia caballi and is endemic in equids throughout most of the tropical and subtropical regions of the world (Friedhoff, Tenter & Muller 1990). B. caballi, a typical large Babesia, infects and multiplies only in erythrocytes and is transmitted transovarially and transstadially by its tick vectors (Friedhoff 1988). In the case of B. equi, however, it has been reported that the parasite apparently first multiplies in lymphocytes (Schein, Rehbein, Voigt & Zweygarth 1981; Rehbein, Zweygarth, Voigt & Schein 1982) before invading erythrocytes in which merozoites can frequently be seen arranged in a typical Maltese-cross formation. It is transmitted transstadially only by its tick vectors. 1 The Research Centre for Protozoan Molecular Immunology, Obi-hiro University of Agriculture and Veterinary Medicine, Obihiro, 080 Hokkaido, Japan 2 Protozoology Division, Onderstepoort Veterinary Institute, Onder-stepoort, 011 0 South Africa 3 Department of Veterinary Physiology, Obihlro University of Agri-culture and Veterinary Medicine, Obihiro, 080 Hokkaido, Japan 4 Department of Veterinary Parasitology, Kitasato University, To-wada, 034 Aomori, Japan Accepted for publication 22 April 1997-Editor Babesia parasites are usually detected in blood smears only during the acute stage of the infection and animals that recover from the disease remain carriers of these parasites. It has been reported that horses can remain carriers of B. caba//iparasites for up to 4 years, while they probably remain life-long carriers after infection with B. equi (Friedhoff 1982). Identification of these carriers and previously ex-posed horses can most easily be accomplished by demonstrating specific antibodies in the serum with serological tests {Weiland & Reiter 1988). 141 Prevalence of equine piroplasmosis in Central Mongolia The indirect fluorescent antibody test (IFAT) is the most widely used serological technique to detect specific antibodies in protozoan infections. Ristic & Sibinovic (1964) first reported the use of the I FAT to detect B. caballi antibodies in horses. Madden & Holbrook (1968) successfully applied this test to dif-ferentiate between B. equi and B. caballi infection in horses and did not report any cross-reaction. Anti-bodies detectable by the IFAT start to rise shortly after the first parasites appear in blood smears, and persist for long periods (Donnelly, Joyner & Frank 1980; Weiland 1986; De Waal1995). In Mongolia, Dash (1957, 1959, cited by Dash 1967), using microscopic examination of thin blood smears, reported that both B. equi and B. caballi are wide-spread in horses. The tick vectors in this region are unknown, but Dermacentor nuttali (Oienev 1929, cited by Dash 1967), Dermacentor salvarum (Shulth 1933, cited by Dash 1967) and Hyalomma dromedari (Svirskaya 1961, cited by Dash 1967) frequently in-fest horses and all have been reported to be vectors of B. caballi elsewhere. However, no serological sur-vey has been conducted to determine the prevalence of these two parasites in horses. In this study in vitro-propagated parasites were used as antigen in the I FAT to determine the prevalence of B. equi and B. caballi antibodies in field horses from Central Mon-golia. MATERIALS AND METHODS Strains of Babesia species B. equi and B. caballi, both USDA strains, were used in this study and were obtained from experimentally infected horses at Tochigi Research Station of the Equine Research Institute, Japan Racing Associa-tion. Serum samples Serum samples from horses experimentally infected with B. equi, OP isolate (horse no. 460-9) and B. caballi, SWA isolate (horse no. 042-9), were obtained from Onderstepoort Veterinary Institute (OVI), South Africa and used as positive control sera. Negative serum was obtained from a horse raised under tick-TABLE 1 Summary of IFAT results of Mongolian horse sera Number of horses Antigen Seronegativea Babesia equi 13 (11,9%) Babesia cabal/i 17 (15,5%) a Reciprocal titre < 80 b Reciprocal titre ~ 80 142 Seropositiveb 97 (88,2%) 93 (84,5%) free conditions at OVI. Blood samples were collected from 15 Babesia-free horses in Japan and from 11 0 horses of the Mongolian native breed, originating from two different herds in the Central Province of Mongolia. These were allowed to clot at room tem-perature overnight before serum was removed and stored at -20°C. At the time of blood collection, thin blood smears were also prepared and later stained with Giemsa solution. Antigen preparation for IFAT Antigen was prepared from in vitro-propagated B. equi- or B. caba//i-infected erythrocytes (Avarzed, Igarashi, Kanemaru, Ornata, Saito, Nagasawa, Toy-ada & Suzuki 1995). In vitro-propagated Babesia parasites were transferred from one well of a 6-well culture plate to a 15-mQ centrifuge tube and centri-fuged for 15 min at 700 gat 4 oc. After the first centri-fugation, the erythrocyte pellet was washed five times by centrifugation for 5 min at 350 g and after each wash, reconstituted with 1 0 mQ of PBS(-)[147 mM NaCI, 5 mM NaH2P04 ,5 mM Na2HP04 , pH 7,2].Any visible white-blood-cell layer was removed after each wash. After the fifth wash, one part packed red-blood cells was reconstituted with two parts of 4% bovine serum albumin in PBS(-) (1 :2, v/v). The blood mix-ture was dispensed onto previously cleaned glass slides by means of a 1-mQ syringe and 27 -G needle. Fifteen antigen spots, 5 mm in diameter (three rows of five spots), were prepared per slide. The slides were dried for 30 min at 45 oc in a drying oven (forced-convection oven FC-61 0, Toyo Seisaku, Co., Ltd) and after that for a further 30 min at room temperature. The slides were then wrapped in tissue paper, cov-ered with aluminum foil and stored at -20 oc. I FAT technique Antigen slides were removed from storage at-20°C and placed in a drying oven at 45°C for 1 h. The an-tigen spots on the slides were encircled with a pap pen (Kantoukagaku, Japan) and fixed in a large vol-ume of cold acetone (-20 oc) for 5 min. Two-fold serum dilutions were prepared in PBS(-) starting at 1/1 o-1 /5120 dilution. Every test included B. equi and TABLE 2 Prevalence of Babesia equi and B. caballi in different age groups of Mongolian horses Age Seronegative Seropositive (years) B. equi B. caballi B. equi B. caballi 1 0 1 16 15 2 1 3 21 19 3 2 3 12 11 4 0 3 13 10 5 0 0 11 11 6-10 6 3 23 26 11-19 4 4 1 1 B. caballi positive and negative control sera as well as a PBS control. Ten microliters of each serum di-lution were then placed on the antigen spots. Slides were incubated in a humid chamber at 37 oc for 30 min. The slides were then rinsed in fresh PBS(-) and washed once for 10 min in PBS(-), followed by a further washing with deionized-distilled water for 5 min. Goat anti-horse lgG FITC conjugate (Bethyl Laboratories, Inc., USA.) was diluted 1/80 in 0,01% Evans blue-PBS(-) (to reduce non-specific fluores-cence) and 1 0 m~ of diluted conjugate was pipetted onto each spot. The slides were incubated, rinsed and washed as described above. After the last wash, slides were allowed to air dry slightly before being overlaid with 50% glycerin-PBS(-) (1 :1) and covered with a 24 x 60 mm cover slip. The slides were ex-amined under a fluorescent microscope with the use of a 40x objective lens (Microphot EPI-FL, Nikon, Japan). Fluorescence was interpreted as positive ( + ), trace(±) or negative (-). The highest serum dilution showing positive fluorescence was taken as the ti-tre of the sample. RESULTS Validation of IFA with parasites from in vitro culture In vitro-propagated B. equiand B. caballiwere inves-tigated as antigen source in the I FAT. When the test was performed with B. equi or B. caballi antigen and homologous positive control sera, strong specific fluorescence of the parasites within the erythrocytes was observed (Fig. 1 }. Some cross-reactions were observed between the positive-reference serum and the reciprocal antigen at low serum dilutions$ 1/40 (data not shown) but no specific fluorescence was observed at 1/80 or higher dilutions. Sera from blood samples collected from the 15 horses born and reared in Japan and regarded as Babesia-free, a • . ., " • "" • • • J: \ • •• • :,. •• • • • • •• • ; .... • • ' # 4 f.. \ • .. A. AVARZED eta/. showed some non-specific fluorescence at low se-rum dilutions ($ 1 /40) on both B. equi and B. caballi antigen (Fig. 2). Therefore, a reciprocal titre of 80 with antigens cultured in vitro was considered as being positive for the infections. Screening of Mongolian sera with IFA Although no blood parasites were detected in the Giemsa-stained blood smears from any of the 11 0 horses from Central Mongolia, a wide range (1 /2Q-1 I 2560) of IFA end titres to both B. equi and B. cabali was found in them (Fig. 3). Based on the criterion for positivity as described above, the prevalence of B. equi and B. caballi infection in Mongolia was 88,2 % and 84,5%, respectively (Table 1 ). The ages of the Mongolian horses varied from 1-1 9 years and B. equi and B. caballi antibodies were detected in all age groups, with 90 horses (81 ,8%) being seropositive to both infections (Table 2). DISCUSSION Until recently, most researchers have performed the I FAT for equine piroplasmosis on infected erythrocyte antigen prepared from experimentally infected horses (Ristic & Sibinovic 1964; Madden & Holbrook 1968; Tenter & Friedhoff 1986; Weiland 1986; De Waal1995). With the successful establishment of in vitro cultures of Babesia, it is now possible to em-ploy culture-derived antigens in the I FAT and to avoid the use of experimentally infected animals as anti-gen sources. Although Bose (1 994) reported the use of micro-aerophilous stationary-phase (MASP) cul-tures as an antigen source for B. caballi I FAT, he did not comment on the antigen-preparation procedure. Many difficulties were in~tially experienced in estab-lishing the IFAT procedure when MASP cultures of B. equi or B. caballi were used as antigens for the IFAT in comparison with the antigens obtained from · b •• • •' •• • • • ' • • • • ' • • •• . ... •• ~:• • • ,. ..... • • • • .. .. • .. .. , ... .. • • • • • .. • • .. " • ••• . •. • :s • • • FIG. 1 Immunofluorescent reactions; (a) Babesia caballi antigen with B. cabal/i positive serum, (b) Babesia equi antigen with B. equi positive serum 143 Prevalence of equine piroplasmosis in Central Mongolia 7 6 Ill Q) 5 Ill .. 0 ~ 4 0 .. 3 Q) .a E 2 :::J z 0 <1 0 10 20 40 80 Reciprocal titre • B. equi • B. caballi FIG. 2 Distribution of I FAT end titres for Babesia equi and Babe-sia caballi in Babesia-free horses from Japan 30 Ill 25 Q) Ill .. 20 0 ~ 0 15 .. Q) .a 10 E :::J z 5 0 <20 20 40 80 160 320 640 1280 2560 5120 Reciprocal titre • B. equi • B.caballi FIG. 3 Distribution of I FAT end titres for Babesia equi and Babe-sia caballi in Mongolian horses infected horses (unpublished observations). Conse-quently, various modifications had to be implemented to allow repeatable and consistent results. In this study, cultures with 3-4% parasitized erythrocytes were found to be most suitable. Cross-reactions of con-trol sera with heterologous antigen were observed at serum dilutions of up to 1/40. Similar results were reported by other workers using antigen from experi-mentally infected horses (Ristic & Sibinovic 1964; Madden & Holbrook 1968; Weiland 1986; Tenter & Friedhoff 1986; De Waal 1995). Although we were unable to completely eliminate the non-specific bind-ing of conjugate to B. caballi antigen, it was distinctly different from the positive control and did not inter-fere with the interpretation of the test. The Mongolian horse, famous for its hacking quali-ties, is also used for racing, draught, and milk and meat production. Horse husbandry in Mongolia has 144 remained a pastoral nomadic system, which includes seasonal migrations and rotations of migration routes. This seems to be a possible reason for re-and mixed infections of horses with Babesia para-sites. The present study confirmed that both babesia! infections occur in horses in Central Mongolia. As shown in Fig. 3, 90 horses had high titres (1/160-1/ 2560) for B. equi and 82 horses for B. caballi. The results from this limited survey suggest that equine piroplasmosis is probably endemic in Central Mon-golia. Very few clinical cases of equine piroplasmo-sis are reported in horses in Central Mongolia, prob-ably owing to the fact that most horses in this survey were infected early in life (93, 75% of 1-year-old horses being sera positive to both parasites) when they were protected by colostral immunity and other non-specific factors, and then became carriers (De Waal1995). However, surprisingly few horses in the 11-19-year age group were seropositive, which is dif-ficult to explain in the light of the high prevalence in the other age groups. The tick vectors of these para-sites in Mongolia are still unknown, but D. nutalli (Oiesnev 1929, cited by Dash 1967), D. salvarum (Shulth 1933, cited by Dash 1967) and H. dromedari (Svirskaya 1961, cited by Dash 1967) frequently in-fest horses and all have been reported to be vectors of B. caballi in Europe and Russia (Friedhoff 1988). Further surveys and studies on the vectors of equine piroplasmosis are necessary to evaluate the epide-miological status of this disease in Mongolia. ACKNOWLEDGEMENT The study was supported by Grant-in-Aid for Scien-tific Research (C), The Ministry of Education, Sci-ence, Sports and Culture, Japan. REFERENCES AVARZED,A., IGARASHI, 1., KANEMARU, T., OMATA, Y., SAITO, A., NAGASAWA, H. , TOYODA, Y. & SUZUKI, N. 1995. Method of continuous in vitro cultivation of horse Babesia parasites. Proceedings of the 120th Conference of Japanese Society of Veterinary Science, Tottori, Japan, 1995: 89 (in Japanese). BOSE, R. 1994. Polyclonal antibody characterization of Babe-sia cabal/i antigens. International Journal for Parasitology, 24:511-517. DASH, M. 1967. Study on diagnosis and treatment of horse piroplasmosis. Proceedings of Scientific Works of The Re-search Institute of Animal Husbandry, Ulanbotar, 1967, 16:47-61 (in Mongolian). DE WAAL, D.T. 1995. Distribution, transmission and serodiag-nosis of Babesia equi and Babesia caballi in South Africa. Ph.D. thesis, University of Pretoria. DONNELLY, J., JOYNER, L.P. & FRANK, C. 1980. Quantitative epidemiological studies on the prevalence of babesiosis in horses in Kuwait. Tropi~al Animal Health and Production, 12:253-258. FRIEDHOFF, K.T. 1982. Die Piroplasmen der Equiden- Be-deutung fOr der lnternationalen Pferdeverkehr. Berlin Man-chen Tierartzliche Wochenschrift, 95:368-374. FRIEDHOFF, K.T. 1988. Transmission of Babesia, in Babesio-sis of domestic animals and man, edited by M. Ristic. Boca Raton, Florida: CRC Press: 23-52. FRIEDHOFF, K.T., TENTER, A.M. & MULLER, I. 1990. Haemo-parasites of equines: impact of international trade of horses. Revue Scientifique Office International des Epizooties, 9: 1187-1194. MADDEN, P. A. & HOLBROOK, A.A. 1968. Equine piroplasmo-sis : Indirect fluorescent antibody test for Babesia caba/li. American Journal of Veterinary Research, 29:117-123. REHBEIN, G. , ZWEYGARTH, E., VOIGT, W.P. & SCHEIN, E. 1982. Establishment of Babesia equi-infected lymphoblastoid cell lines. Zeitschrift fOr Parasitenkunde, 67:125--127. RISTIC, M. & SIBINOVIC, S. 1964. Equine piroplasmosis-a mixed strain of Piroplasma caba/li and Piroplasma equi iso-A. AVARZED eta/. lated in Florida and studied by the fluorescent-antibody tech-nique. American Journal of Veterinary Research, 1 04:15--23. SCHEIN, E., REHBEIN, G., VOIGT, W.P. & ZWEYGARTH, E. 1981. Babesia equi (Lave ran 1901 ). I. Development in horses and in lymphocyte culture. Tropenmedizin und Parasitologie, 32:223-227. TENTER, A.M. & FRIEDHOFF, K.T. 1986. Serodiagnosis of ex-perimental and natural Babesia equi and B. caballi infections. Veterinary Parasitology, 20:49-61. WEILAND, G. 1986. Species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test, immunofluorescence and enzyme-linked immunosorbent assay. Veterinary Parasitology, 20:43-48. WEILAND, G. & REITER, I. 1988. Methods for the measurement of serological response to Babesia, in Babesiosis of Domes-tic Animals and Man, edited by M. Ristic. Boca Raton, Florida: CRC Press: 143-162. 145 

Prevalence of equine piroplasmosis in Central Mongolia

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Prevalence of equine piroplasmosis in Central Mongolia

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