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Babesial antibody dynamics after cattle immunisation with live vaccines, measured with an indirect immunofluorescence test
veterinary parasitok~y ELSEVIER
Veterinary Parasitology70 (1997) 33-39
Babesial antibody dynamics after cattle immunisation with live vaccines, measured with an indirect immunofluorescence test A.A. Guglielmone *, C.I. Lugaresi, M.M. Volpogni, O.S. Anziani, V.R. Vanzini lnstituto Nacional de Tecnologla Agropecuaria, Estaci6n Experimental Agropecuaria Rafaela, CC 22, CP 2300 Rafaela (Santa Fe), Argentina Received 5 September 1996; accepted 19 November 1996
Abstract
The efficacy of vaccination of Argentinean cattle against babesiosis and anaplasmosis using live immunogens was tested to detect specific antibodies in samples obtained about 60 days after vaccination. Under these conditions a higher than expected proportion of cattle failed to show antibodies against Babesia bigemina. Therefore, a study was designed to evaluate if this failure was due to insensitivity of the routine test to detect antibodies to B. bigemina or to lack of infectivity of the live vaccine. Four groups (G) of cattle were each inoculated subcutaneously with 10 million Babesia boris (vaccinal strain R1A), l0 million B. bigemina (vaccinal strain SIA) and 10 million Anaplasma centrale (strain M1). G1 and G2 consisted of ten Angus bulls 20-24 months' old and ten Angus bulls 15-18 months old, respectively; G3 and G4 consisted of ten and 16 Holstein 1-month-old male calves, respectively. Blood samples were obtained on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed with an indirect immunofluorescent (IFA) test to detect antibodies to B. boris (baseline dilution for a positive result 1:60) and B. bigemina (baseline dilution l:120). Positive IFA titres were considered as evidence of the infectivity of the Babesia vaccinal strains contained in the vaccine. All Angus bulls were found positive to antibodies against both Babesia species, by day 20 (B. boris) and day 30 (B. bigemina), whereas 10-25% of Holstein calves were negative throughout.
*
Corresponding author.
0304-4017/97/$17.00 Copyright © 1997 Elsevier ScienceB.V. All rights reserved. Pll S0304-4017(96)01143-0
34
A.A. Guglielmoneet al. / VeterinaryParasitology 70 (1997) 33-39
The partial lack of vaccine infectivity in the calves was considered to be a consequence of innate resistance of young calves to Babesia. Antibody titres to B. boris and B. bigemina declined by day 60 after vaccination. However, all cattle that were positive to B. boris antibodies on day 50 were still positive to the IFA test 10 days later while 10%, 30% and 12% of cattle of G1, G2 and G3 that were positive to B. bigemina antibodies on day 50 after vaccination were found negative to the IFA test on day 60. In future, samples taken on days 40-50 will be used for detection of B. bigemina antibodies in vaccinated cattle, on day 60 for A. centrale and on either occasion for B. boris. The reaction to the inoculation of B. bigemina SIA strain appears to lag behind the reaction to B. boris R1A strain. It is not certain if this is a normal reaction to this B. bigemina strain or the result of interaction with the B. boris strain. Keywords: Babesiabigemina; Babesia boris; Vaccines;Immunisation-Protozoa;Antibodydynamics; IFA test
1. Introduction Argentinean cattle at risk to babesiosis (Babesia bovis, Babesia bigemina) and anaplasmosis (Anaplasma marginale) are usually immunised simultaneously with live vaccines containing attenuated B. bovis and B. bigemina strains and Anaplasma centrale (Guglielmone, 1994). Unlike B. boris and B. bigemina, A. centrale has a long pre-patent period (Mellors et al., 1982; Potgieter and Van Rensburg, 1983; Mangold et al., 1990), and antibodies against this rickettsia are not detected in cattle blood until 60 days after vaccination using the card agglutination (CA) test, which is routinely utilised in our laboratory (De Echaide et al., 1989). There is an increasing demand for highly valued cattle from the temperate region of Argentina in tropical and sub-tropical areas within Argentina and in neighbouring countries. Such cattle are vaccinated against babesiosis and anaplasmosis before sale and veterinary practitioners demand positive antibody diagnosis to confirm the efficacy of vaccination against these diseases. To reduce sampling costs antibody evaluation is made at about 60 days after vaccination. Under these conditions, B. bovis and A. centrale antibodies are found in more than 95% of cattle tested, whereas B. bigemina antibody detection are often found in less than 80% of vaccinated cattle. The failure to detect B. bigemina antibodies may be due to a lack of infectivity of the vaccinal strain or to insensitivity of the immunological test. We routinely use an indirect immunofluorescence antibody (IFA) test for both species of Babesia. Lack of specificity has been mentioned as a problem for the 1FA test applied to B. bigemina, and this has not been improved by using the indirect haemagglutination test (Goodger, 1973) or complement fixation test (Mahoney, 1967). Use of the IFA test for Babesia bigemina in laboratory (Aguirre et al., 1990) or Argentinean field conditions (De R~os et al., 1988, 1989) demonstrated a direct relationship between IFA titres and cattle parasitaemias. These results were obtained using a baseline dilution double that used for B. boris antibodies (De Rfos et al., 1988) to avoid cross-reactions of B. bigemina antibodies with B. bovis antigens (Goldman et al., 1972; Fujinaga et al., 1980). In this paper we describe the course of B. boris and B. bigemina antibody titres measured with the IFA test. Cattle of different ages and breeds vaccinated with live
A.A. Guglielmoneet al. / VeterinaryParasitology70 (1997) 33-39
35
attenuated strains of both protozoan were used to respond to the important question rised by veterinary practitioners.
2. Materials and methods Forty-eight cattle born and raised in the area free of the only vector (Boophilus microplus ticks) of B. boris and B. bigemina in Argentina were allocated into four groups (G) as follows: GI, ten Angus bulls 20-24 months old; G2, ten Angus bulls 15-18 months old; G3, ten Holstein male calves 1 month old; G4, 16 Holstein male calves 1 month old. Individuals of G1, G2 and G3 were inoculated (subcutaneously) with a vaccine containing 10 million B. boris attenuated strain R1A, 10 million B. bigemina attenuated strain S1A and 10 million A. centrale M1 strain from the commercial batch No. 36 containing 5000 doses and made on 22 August 1995. Calves of G4 were vaccinated with the same doses and strains of the commercial batch No. 42 (6000 doses) produced on 8 April 1996. Both Babesia strains were grown in vitro as previously described (Mangold et al., 1996). Blood samples were obtained from all cattle on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed for B. boris and B. bigemina antibodies using the IFA test according to De Rios et al. (1988) and De Echaide et al. (1995) with antigens derived from in vitro cultures of both Babesia species and locally made fluorescein-labelled rabbit antibovine IgG. Positive and negative reference sera were from a freeze-dried batch of patron sera produced in 1992 (Guglielmone et al., 1997). The baseline dilution for a positive result was 1:60 for B. boris and 1:120 for B. bigemina (De Rios et al., 1988). Positive sera were double diluted to the final titre. Additionally, B. boris and B. bigemina parasitaemia was evaluated in bulls of G1 and G2 by inspection of thin and thick peripheral blood smears, Giemsa stained, obtained every 2 - 3 days from day 5 to 18 after vaccination. Positive results to the IFA test were used as evidence of infection. Antibody titres were log transformed for analysis of variance to detect significant differences between sampling days within each group. In both cases the analysis started on samples obtained on day 20 when the majority of cattle became positive to the IFA test, with the exception of calves from G3 that were still negative to B. bigemina antibodies on that day. Negative results were not included in the statistical analysis (Thrusfield, 1986).
3. Results
B. boris and B. bigemina were found in the blood smears of all cattle from G1 and G2 and all of them were found positive to the IFA test. Ten percent and 25% of calves of G3 and G4, respectively, remained negative to B. boris antibodies and 20% of calves from G3 and 13% of those calves from G4 remained negative to B. bigemina antibodies. Fourteen of the 41 cattle that were detected positive to the IFA test for B. boris antibodies showed their initial titres on day 10 post-vaccination and the remainder were
36
A.A. Guglielmone et al. / Veterinar3, Parasitology 70 (1997) 33-39
Table 1 Percentage of positive titres to Babesia boris and Babesia bigemina antibodies (IFA test) from day 0 to day 60 after the simultaneous inoculation of vaccinal strains in different groups (G) of cattle Days after vaccination
0 10 20 30 40 50 60
B. boris
B. bigemina
GI
G2
G3
G4
G1
G2
G3
G4
0 50 100 100 100 100 100
0 40 100 100 100 100 100
0 0 90 90 90 90 90
0 3l 75 75 75 75 75
0 0 90 100 100 100 90
0 10 90 100 100 100 70
0 0 0 50 80 80 70
0 25 50 87 87 87 87
G1, ten Angus bulls 20-24 months old; G2, ten Angus bulls 15-18 months old; G3, ten Holstein male calves 1 month old; G4, 16 Holstein male calves 1 month old.
first d e t e c t e d p o s i t i v e o n day 20 after v a c c i n a t i o n . T h i s p a t t e r n was d i f f e r e n t for B. bigemina a n t i b o d i e s : 5, 21, 13 a n d 3 o f the 4 2 cattle p o s i t i v e to I F A test s h o w e d t h e i r initial titres o n d a y s 10, 20, 30 a n d 4 0 a f t e r v a c c i n a t i o n . T e n p e r c e n t , 3 0 % a n d 12% o f i n d i v i d u a l s o f G1, G 2 a n d G3, respectively, that w e r e p o s i t i v e to I F A test for B. bigemina a n t i b o d i e s o n day 5 0 b e c o m e n e g a t i v e on d a y 60. T h e p e r c e n t a g e o f i n d i v i d u a l s o f all g r o u p s p o s i t i v e to I F A test for B. boris a n d B. bigemina a n t i b o d i e s f r o m day 0 to 60 after v a c c i n a t i o n are s h o w n in T a b l e 1. T h e e v o l u t i o n o f m e a n a n t i b o d y titres to B. boris a n d B. bigemina m e a s u r e d b y I F A test in the i n d i v i d u a l s o f G1, G2, G 3 a n d G4, a l o n g w i t h the results o f the statistical a n a l y s i s are p r e s e n t e d in T a b l e 2. Titres o f B. boris w e r e c o n s i s t e n t l y h i g h e r t h a n the c o r r e s p o n d i n g titre to B. bigemina o n all s a m p l i n g days. T h e h i g h e s t a n t i b o d y titres to b o t h p r o t o z o a were f o u n d o n d a y s 4 0 ( G 4 ) a n d 50 p o s t - v a c c i n a t i o n (G1, G 2 a n d G3), b u t with the e x c e p t i o n o f B. boris in G 2 a n d B. bigemina in G 4 the a n t i b o d y p e a k s
Table 2 Mean antibody IFA titer (log 10) to Babesia bocis and Babesia bigemina and statistical significance from day 20 to 60 after the simultaneous inoculation of vaccinal strains in different groups of cattle. Negative samples were not included Daysafiervaccination 20 30 40 50 60
B. bocis
B. bigemina
GI ~
G2
G3
G4
GI
G2
G3
G4
3.20a 3.07a 3.16a 3.22a 2.95a
3.25a 2.80b 3.04ab 3.3 la 2.86b
2.71b 3.15a 3.08a 3.31 a 3.15a
1.80c 2.98a 3.12a 2.78ab 2.46bc
2.58a 2.62a 2.53a 2.77a 2.64a
2.68a 2.47a 2.44a 2.77a 2.58a
_ b 2.14b 2.46ab 2.64a 2.34ab
2.08d 2.55bc 2.83a 2.77ab 2.36cd
a See Table 1 or text for group characteristics. b All individuals in the group were negative to the IFA test. Means in a column followed by different letters are significantly different ( P < 0.01).
A.A. Guglielmone et al. / Veterinary Parasitology 70 (1997) 33-39
a'S l
o o
,:, I-
37
B. bovis
2.5
B. bigemina 2 1,5
z :E 0.5
o~w~f 0
-
10
20
30
40
50
60
DAYS AFTER VACCINATION
Fig. 1. Evolution of antibody IFA titres to Babesia boris and Babesia bigemina in Angus bulls, 20-24 months of age, vaccinated simultaneously with vaccinal strains of both protozoans.
were not statistically higher than the titres found on day 60. An example of the evolution of antibody titres is shown in Fig. 1 using the results obtained from cattle of G1.
4. Discussion The partial lack of infectivity in 1-month-old Holstein calves of G3 and G4 may not be due to a vaccine defect or a cattle breed effect. Vaccination of Holstein cattle older than five months with the strains used in this study (Mangold et al., 1990, 1996; Echaide et al., 1993) or other Babesia strains (Latif et al., 1979; Pipano et al., 1987) resulted in the infection of all inoculated cattle. Therefore, the results found in G3 and G4 may be related to the innate resistance of young calves to Babesia (Levy et al., 1982). The decrease of B. boris antibody titre on day 60 did not alter the percentage of cattle found positive to the IFA test (Table 1), and the titre may remain above the baseline dilution for long periods of time (Mahoney et al., 1979; Kung'u and Goodger, 1990). However, the decrease in antibody titres to B. bigemina resulted in up to 30% of cattle being negative to the IFA test on day 60 after vaccination in comparison with day 50. This experiment seems to show that B. bigemina antibody detection by using IFA test becomes increasingly less reliable from day 50 post-infection together with the risk of underestimating the proportion of cattle properly vaccinated with the B. bigemina S 1A strain. Schunter and Wright (1989) found that a radioimmunoassay detected antibodies to the pathogenic Australian Lismore strain of B. bigemina in all 13 cattle with induced infections. Highest titres were found from day 14 to 47 after inoculation but the authors mentioned that data extrapolation suggested that 31% of them could become negative to this test 70-90 days after infection. The marginal improvement obtained with the radioimmunoassay does not warrant a change of a simple routine technique to another
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A.A. Guglielmone et al. / Veterinary Parasitology 70 (1997) 33-39
involving the manipulation of radioctive material. We have had no opportunity to evaluate any ELISA for B. bigemina antibodies. Therefore, until better techniques are available, the IFA test will continue to be our routine method of diagnosis to B. bigemina antibodies but in future samples obtained on day 40-50 will be used to detect B. bigemina antibodies. An additional sample to detect antibodies to A. centrale will be taken at about 60 days from vaccination. Diagnosis of vaccinal B. bovis antibodies can be done with samples obtained in any of the given post-vaccination period. Higher antibody titres to B. boris than to B. bigemina are usually found in cattle inoculated simultaneously with the Argentinean vaccinal strains used in the present study (Guglielmone et al., 1989; Aguirre et al., 1990). The opposite was found by Bessenger and Schoeman (1983) using South African vaccinal strains of B. bovis and B. bigemina, and Pipano et al. (1987) detected a similar range of IFA titres to B. boris and B. bigemina in cattle inoculated simultaneously with Israel vaccinal strains of both protozoan. The disparity of the results may be a consequence of difference amongst vaccinal strains used. Further differences are suggested by the fact that the Israeli strains induced the highest antibody titres before day 36 after inoculation whereas the South African and Argentinean strains reached their highest antibody titres after that period. The initial detection of B. bigemina and B. bovis antibodies using IFA test after monospecific cattle infections ranged from 5 - 7 days (Dwived and Gautam, 1979; Latif et al., 1979; Fujinaga et al., 1980; Kuttler et al., 1983) to 21-24 days (Latif et al., 1979; Bessenger and Schoeman, 1983). However, Bessenger and Schoeman (1983) inoculated four calves simultaneously with B. bovis and B. bigemina and all of them were first found positive to the IFA test for B. bigemina on day 14 after inoculation and to B. boris 7-21 days post-inoculation. This appears to show that the results obtained in this study agreed with the literature on B. boris but to a lesser extent on B. bigemina. Fig. l and Tables 1 and 2 seem to demonstrate that the reaction to B. bigemina SlA strain lagged behind the reaction to B. bovis RIA. It is not certain if this is a normal effect of B. bigemina S1A strain or the result of competition with the B. bovis R1A strain, which will be the subject of a future study.
References Aguirre, D.H., Mangold, A.J., de R~os, L.G. and Guglielmone, A.A., 1990. Respuesta cl~nica y evoluci6n del peso corporal en terneras (Bos taurus) vacunadas simult~neamente contra babesiosis y anaplasmosis con inmun6genos vivos. Med. Vet. (Barcelona), 8: 95-101. Bessenger, R. and Schoeman, J.H., 1983. Serological response of cattle to infection with Babesia bigemina and Babesia boris in southern Africa. Onderstepoort J. Vet. Res., 50:115-117. De Echaide, S.T., de Rios, L.G. and Aguirre, D.H., 1989. Evaluaci6n de un ant[geno de Anaplasma marginale para la prueba de aglutinaci6n en placa. Mem. 5ta. Reun. Anu. Asoc. Arg. Vet. Lab. Diag., Rio Cuarto, Crrdoba, Argentina, 8 - 9 dic. 1989, pp. 143-152. De Echaide, S.T., Echaide, I.E., Gaido, A.B., Mangold, A.J., Lugaresi, C.I., Vanzini, V.R. and Guglielmone, A.A., 1995. Evaluation of an enzyme linked immunosorbent assay-kit to detect Babesia boris antibodies in cattle. Prev. Vet. Med., 24: 277-283. De Rfos, L.G., Aguirre, D.H. and Gaido, A.B., 1988. Evaluaci6n de la din~imica de la infecci6n por Babesia boris y Babesia bigemina en terneros. Diagn6stico por microscopla directa y prueba de inmunofluorescencia indirecta. Rev. Med. Vet. (Buenos Aires), 69: 254-260.
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De RJos, L.G., Aguirre, D.H. and Gaido, A.B., 1989. Infecci6n natural por Babesia boris y Babesia bigemina en dos rodeos bovinos con diferentes niveles de infestaci6n por Boophilus microplus. Rev. Latinoam. Microbiol., 31: 39-43. Dwived, S.K. and Gautam, O.P., 1979. A note on indirect fluorescent antibody test for the diagnosis of experimental babesiosis in calves. Indian J. Anim. Sci., 49: 664-666. Echaide, I.E., de Echaide, S.T. and Guglielmone, A.A., 1993. Live and soluble antigens for cattle protection to Babesia bigemina. Vet. Parasitol., 51: 35-40. Fujinaga, T., Minami, T. and Ishihara, T., 1980. Serological relationship between a large Babesia found in Japanese cattle and Babesia major, B. bigemina and B. major. Res. Vet. Sci., 29: 230-234. Goldman, M., Pipano, E. and Rosenberg, A.S., 1972. Fluorescent antibody tests to Babesia bigemina and B. berbera. Res. Vet. Sci., 13: 77-81. Goodger, V.B., 1973. Further studies of haemagglutinating antigens of Babesia bigemina. Aust. Vet. J., 49: 81-84. Guglielmone, A.A., 1994. Epidemiologla y prevenci6n de los hemoparfisitos (Babesia y Anaplasma) en la Argentina. In: A. Nari and C. Fiel (Editors), Enfermedades Parasitarias de Importancia Econ6mica en Bovinos. Bases Epidemiol6gicas para su Prevenci6n y Control. Editorial Hemisferio Sur, Montevideo, pp. 461-479. Guglielmone, A.A., Mangold, A.J., Aguirre, D.H., de R[os, L.G. and Olsen, A.A., 1989. Vacunaci6n simultalnea con Babesia boris y Babesia bigemina atenuadas, congeladas en nitr6geno lfquido con DMSO o glicerol como crioprotectores. Rev. Med. Vet. (Buenos Aires), 70: 176-181. Guglielmone, A.A., de Echaide, S.T., Mangold, A.J., Echaide, I.E., Abdala, A.A., Anziani, O.S., Aguirre, D.H., Montenegro-James, S., Patarroyo, J., Alonso, M., Camus, E., Cordov6s, C., Kessler, R.H., Martins, J.R., Nari, A., Solari, M.A., Toro, M., Vega y Murgula, C. and Arellano Sota, C., 1997. Producci6n de sueros patrones anti-Babesia bovis, anti-Babesia bigemina, anti-Anaplasma marginale y negativos. Ser. Gan., in press. Kung'u, M.W. and Goodger, B.V., 1990. A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia boris infections and for the screening of Babesia-specific monoclonal antibodies. Int. J. Parasitol., 20: 341-345. Kuttler, K.L., Levy, M.G. and Ristic, M., 1983. Cell cultured-derived Babesia boris vaccine: sequential challenge exposure of protective immunity during a 6-month postvaccination period. Am. J. Vet. Res., 44: 1456-1459. Latif, B.M.A., Said, M.S. and All, S.R., 1979. Effect of age on the immune response of cattle experimentally infected with Babesia bigemina. Vet. Parasitol., 5: 307-314. Levy, M.G., Clabaugh, G. and Ristic, M., 1982. Age resistance in bovine babesiosis: rote of blood factors in resistance to Babesia boris. Inf. Immun., 37:1127-1131. Mahoney, D.F., 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Exp. Parasitol., 20: 223-241. Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1979. Immunity in cattle to Babesia bovis after single infections with parasites of various origins. Aust. Vet. J., 55: 10-12. Mangold, A.J., Aguirre, D.H. and Guglielmone, A.A., 1990. Post-thawing viability of vaccines for bovine babesiosis and anaplasmosis eryopreserved with glycerol. Vet. Parasitol., 37:311-306. Marigold, A.J., Vanzini, V.R., Echaide, I.E., de Echaide, S.T., Volpogni, M.M. and Guglielmone, A.A., 1996. Viability after thawing and dilution of simultaneously cryopreserved vaccinal Babesia bovis and Babesia bigemina strains cultured in vitro. Vet. Parasitol., 61: 345-348. MeUors, R.T., Daigliesh, R.J., Timms, P., Rodwell, B.J. and Callow, L.L., 1982. Preparation and laboratory testing of a frozen vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale. Res. Vet. Sci., 32: 194-197. Pipano, E., Markovics, A., Kriegel, Y., Frank, M. and Fish, L., 1987. Use of long-acting oxytetracycline in the immunisation of cattle against Babesia bovis and B. bigemina. Res. Vet. Sci., 43: 64-66. Potgieter, F.T. and van Rensburg, L., 1983. Infectivity virulence and immunogenicity of Anaplasma centrale live blood vaccine. Onderstepoort J. Vet. Sci., 50: 29-31. Schunter, C.A. and Wright, I.G., 1989. Detection of Babesia bigemina in cattle by a radioimmunoassay incorporating specifically depleted antigen. Vet. Parasitol., 31: 229-241. Thrusfield, M., 1986. Veterinary Epidemiology. Butterworth, London, 280 pp.
Veterinary Parasitology70 (1997) 33-39
Babesial antibody dynamics after cattle immunisation with live vaccines, measured with an indirect immunofluorescence test A.A. Guglielmone *, C.I. Lugaresi, M.M. Volpogni, O.S. Anziani, V.R. Vanzini lnstituto Nacional de Tecnologla Agropecuaria, Estaci6n Experimental Agropecuaria Rafaela, CC 22, CP 2300 Rafaela (Santa Fe), Argentina Received 5 September 1996; accepted 19 November 1996
Abstract
The efficacy of vaccination of Argentinean cattle against babesiosis and anaplasmosis using live immunogens was tested to detect specific antibodies in samples obtained about 60 days after vaccination. Under these conditions a higher than expected proportion of cattle failed to show antibodies against Babesia bigemina. Therefore, a study was designed to evaluate if this failure was due to insensitivity of the routine test to detect antibodies to B. bigemina or to lack of infectivity of the live vaccine. Four groups (G) of cattle were each inoculated subcutaneously with 10 million Babesia boris (vaccinal strain R1A), l0 million B. bigemina (vaccinal strain SIA) and 10 million Anaplasma centrale (strain M1). G1 and G2 consisted of ten Angus bulls 20-24 months' old and ten Angus bulls 15-18 months old, respectively; G3 and G4 consisted of ten and 16 Holstein 1-month-old male calves, respectively. Blood samples were obtained on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed with an indirect immunofluorescent (IFA) test to detect antibodies to B. boris (baseline dilution for a positive result 1:60) and B. bigemina (baseline dilution l:120). Positive IFA titres were considered as evidence of the infectivity of the Babesia vaccinal strains contained in the vaccine. All Angus bulls were found positive to antibodies against both Babesia species, by day 20 (B. boris) and day 30 (B. bigemina), whereas 10-25% of Holstein calves were negative throughout.
*
Corresponding author.
0304-4017/97/$17.00 Copyright © 1997 Elsevier ScienceB.V. All rights reserved. Pll S0304-4017(96)01143-0
34
A.A. Guglielmoneet al. / VeterinaryParasitology 70 (1997) 33-39
The partial lack of vaccine infectivity in the calves was considered to be a consequence of innate resistance of young calves to Babesia. Antibody titres to B. boris and B. bigemina declined by day 60 after vaccination. However, all cattle that were positive to B. boris antibodies on day 50 were still positive to the IFA test 10 days later while 10%, 30% and 12% of cattle of G1, G2 and G3 that were positive to B. bigemina antibodies on day 50 after vaccination were found negative to the IFA test on day 60. In future, samples taken on days 40-50 will be used for detection of B. bigemina antibodies in vaccinated cattle, on day 60 for A. centrale and on either occasion for B. boris. The reaction to the inoculation of B. bigemina SIA strain appears to lag behind the reaction to B. boris R1A strain. It is not certain if this is a normal reaction to this B. bigemina strain or the result of interaction with the B. boris strain. Keywords: Babesiabigemina; Babesia boris; Vaccines;Immunisation-Protozoa;Antibodydynamics; IFA test
1. Introduction Argentinean cattle at risk to babesiosis (Babesia bovis, Babesia bigemina) and anaplasmosis (Anaplasma marginale) are usually immunised simultaneously with live vaccines containing attenuated B. bovis and B. bigemina strains and Anaplasma centrale (Guglielmone, 1994). Unlike B. boris and B. bigemina, A. centrale has a long pre-patent period (Mellors et al., 1982; Potgieter and Van Rensburg, 1983; Mangold et al., 1990), and antibodies against this rickettsia are not detected in cattle blood until 60 days after vaccination using the card agglutination (CA) test, which is routinely utilised in our laboratory (De Echaide et al., 1989). There is an increasing demand for highly valued cattle from the temperate region of Argentina in tropical and sub-tropical areas within Argentina and in neighbouring countries. Such cattle are vaccinated against babesiosis and anaplasmosis before sale and veterinary practitioners demand positive antibody diagnosis to confirm the efficacy of vaccination against these diseases. To reduce sampling costs antibody evaluation is made at about 60 days after vaccination. Under these conditions, B. bovis and A. centrale antibodies are found in more than 95% of cattle tested, whereas B. bigemina antibody detection are often found in less than 80% of vaccinated cattle. The failure to detect B. bigemina antibodies may be due to a lack of infectivity of the vaccinal strain or to insensitivity of the immunological test. We routinely use an indirect immunofluorescence antibody (IFA) test for both species of Babesia. Lack of specificity has been mentioned as a problem for the 1FA test applied to B. bigemina, and this has not been improved by using the indirect haemagglutination test (Goodger, 1973) or complement fixation test (Mahoney, 1967). Use of the IFA test for Babesia bigemina in laboratory (Aguirre et al., 1990) or Argentinean field conditions (De R~os et al., 1988, 1989) demonstrated a direct relationship between IFA titres and cattle parasitaemias. These results were obtained using a baseline dilution double that used for B. boris antibodies (De Rfos et al., 1988) to avoid cross-reactions of B. bigemina antibodies with B. bovis antigens (Goldman et al., 1972; Fujinaga et al., 1980). In this paper we describe the course of B. boris and B. bigemina antibody titres measured with the IFA test. Cattle of different ages and breeds vaccinated with live
A.A. Guglielmoneet al. / VeterinaryParasitology70 (1997) 33-39
35
attenuated strains of both protozoan were used to respond to the important question rised by veterinary practitioners.
2. Materials and methods Forty-eight cattle born and raised in the area free of the only vector (Boophilus microplus ticks) of B. boris and B. bigemina in Argentina were allocated into four groups (G) as follows: GI, ten Angus bulls 20-24 months old; G2, ten Angus bulls 15-18 months old; G3, ten Holstein male calves 1 month old; G4, 16 Holstein male calves 1 month old. Individuals of G1, G2 and G3 were inoculated (subcutaneously) with a vaccine containing 10 million B. boris attenuated strain R1A, 10 million B. bigemina attenuated strain S1A and 10 million A. centrale M1 strain from the commercial batch No. 36 containing 5000 doses and made on 22 August 1995. Calves of G4 were vaccinated with the same doses and strains of the commercial batch No. 42 (6000 doses) produced on 8 April 1996. Both Babesia strains were grown in vitro as previously described (Mangold et al., 1996). Blood samples were obtained from all cattle on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed for B. boris and B. bigemina antibodies using the IFA test according to De Rios et al. (1988) and De Echaide et al. (1995) with antigens derived from in vitro cultures of both Babesia species and locally made fluorescein-labelled rabbit antibovine IgG. Positive and negative reference sera were from a freeze-dried batch of patron sera produced in 1992 (Guglielmone et al., 1997). The baseline dilution for a positive result was 1:60 for B. boris and 1:120 for B. bigemina (De Rios et al., 1988). Positive sera were double diluted to the final titre. Additionally, B. boris and B. bigemina parasitaemia was evaluated in bulls of G1 and G2 by inspection of thin and thick peripheral blood smears, Giemsa stained, obtained every 2 - 3 days from day 5 to 18 after vaccination. Positive results to the IFA test were used as evidence of infection. Antibody titres were log transformed for analysis of variance to detect significant differences between sampling days within each group. In both cases the analysis started on samples obtained on day 20 when the majority of cattle became positive to the IFA test, with the exception of calves from G3 that were still negative to B. bigemina antibodies on that day. Negative results were not included in the statistical analysis (Thrusfield, 1986).
3. Results
B. boris and B. bigemina were found in the blood smears of all cattle from G1 and G2 and all of them were found positive to the IFA test. Ten percent and 25% of calves of G3 and G4, respectively, remained negative to B. boris antibodies and 20% of calves from G3 and 13% of those calves from G4 remained negative to B. bigemina antibodies. Fourteen of the 41 cattle that were detected positive to the IFA test for B. boris antibodies showed their initial titres on day 10 post-vaccination and the remainder were
36
A.A. Guglielmone et al. / Veterinar3, Parasitology 70 (1997) 33-39
Table 1 Percentage of positive titres to Babesia boris and Babesia bigemina antibodies (IFA test) from day 0 to day 60 after the simultaneous inoculation of vaccinal strains in different groups (G) of cattle Days after vaccination
0 10 20 30 40 50 60
B. boris
B. bigemina
GI
G2
G3
G4
G1
G2
G3
G4
0 50 100 100 100 100 100
0 40 100 100 100 100 100
0 0 90 90 90 90 90
0 3l 75 75 75 75 75
0 0 90 100 100 100 90
0 10 90 100 100 100 70
0 0 0 50 80 80 70
0 25 50 87 87 87 87
G1, ten Angus bulls 20-24 months old; G2, ten Angus bulls 15-18 months old; G3, ten Holstein male calves 1 month old; G4, 16 Holstein male calves 1 month old.
first d e t e c t e d p o s i t i v e o n day 20 after v a c c i n a t i o n . T h i s p a t t e r n was d i f f e r e n t for B. bigemina a n t i b o d i e s : 5, 21, 13 a n d 3 o f the 4 2 cattle p o s i t i v e to I F A test s h o w e d t h e i r initial titres o n d a y s 10, 20, 30 a n d 4 0 a f t e r v a c c i n a t i o n . T e n p e r c e n t , 3 0 % a n d 12% o f i n d i v i d u a l s o f G1, G 2 a n d G3, respectively, that w e r e p o s i t i v e to I F A test for B. bigemina a n t i b o d i e s o n day 5 0 b e c o m e n e g a t i v e on d a y 60. T h e p e r c e n t a g e o f i n d i v i d u a l s o f all g r o u p s p o s i t i v e to I F A test for B. boris a n d B. bigemina a n t i b o d i e s f r o m day 0 to 60 after v a c c i n a t i o n are s h o w n in T a b l e 1. T h e e v o l u t i o n o f m e a n a n t i b o d y titres to B. boris a n d B. bigemina m e a s u r e d b y I F A test in the i n d i v i d u a l s o f G1, G2, G 3 a n d G4, a l o n g w i t h the results o f the statistical a n a l y s i s are p r e s e n t e d in T a b l e 2. Titres o f B. boris w e r e c o n s i s t e n t l y h i g h e r t h a n the c o r r e s p o n d i n g titre to B. bigemina o n all s a m p l i n g days. T h e h i g h e s t a n t i b o d y titres to b o t h p r o t o z o a were f o u n d o n d a y s 4 0 ( G 4 ) a n d 50 p o s t - v a c c i n a t i o n (G1, G 2 a n d G3), b u t with the e x c e p t i o n o f B. boris in G 2 a n d B. bigemina in G 4 the a n t i b o d y p e a k s
Table 2 Mean antibody IFA titer (log 10) to Babesia bocis and Babesia bigemina and statistical significance from day 20 to 60 after the simultaneous inoculation of vaccinal strains in different groups of cattle. Negative samples were not included Daysafiervaccination 20 30 40 50 60
B. bocis
B. bigemina
GI ~
G2
G3
G4
GI
G2
G3
G4
3.20a 3.07a 3.16a 3.22a 2.95a
3.25a 2.80b 3.04ab 3.3 la 2.86b
2.71b 3.15a 3.08a 3.31 a 3.15a
1.80c 2.98a 3.12a 2.78ab 2.46bc
2.58a 2.62a 2.53a 2.77a 2.64a
2.68a 2.47a 2.44a 2.77a 2.58a
_ b 2.14b 2.46ab 2.64a 2.34ab
2.08d 2.55bc 2.83a 2.77ab 2.36cd
a See Table 1 or text for group characteristics. b All individuals in the group were negative to the IFA test. Means in a column followed by different letters are significantly different ( P < 0.01).
A.A. Guglielmone et al. / Veterinary Parasitology 70 (1997) 33-39
a'S l
o o
,:, I-
37
B. bovis
2.5
B. bigemina 2 1,5
z :E 0.5
o~w~f 0
-
10
20
30
40
50
60
DAYS AFTER VACCINATION
Fig. 1. Evolution of antibody IFA titres to Babesia boris and Babesia bigemina in Angus bulls, 20-24 months of age, vaccinated simultaneously with vaccinal strains of both protozoans.
were not statistically higher than the titres found on day 60. An example of the evolution of antibody titres is shown in Fig. 1 using the results obtained from cattle of G1.
4. Discussion The partial lack of infectivity in 1-month-old Holstein calves of G3 and G4 may not be due to a vaccine defect or a cattle breed effect. Vaccination of Holstein cattle older than five months with the strains used in this study (Mangold et al., 1990, 1996; Echaide et al., 1993) or other Babesia strains (Latif et al., 1979; Pipano et al., 1987) resulted in the infection of all inoculated cattle. Therefore, the results found in G3 and G4 may be related to the innate resistance of young calves to Babesia (Levy et al., 1982). The decrease of B. boris antibody titre on day 60 did not alter the percentage of cattle found positive to the IFA test (Table 1), and the titre may remain above the baseline dilution for long periods of time (Mahoney et al., 1979; Kung'u and Goodger, 1990). However, the decrease in antibody titres to B. bigemina resulted in up to 30% of cattle being negative to the IFA test on day 60 after vaccination in comparison with day 50. This experiment seems to show that B. bigemina antibody detection by using IFA test becomes increasingly less reliable from day 50 post-infection together with the risk of underestimating the proportion of cattle properly vaccinated with the B. bigemina S 1A strain. Schunter and Wright (1989) found that a radioimmunoassay detected antibodies to the pathogenic Australian Lismore strain of B. bigemina in all 13 cattle with induced infections. Highest titres were found from day 14 to 47 after inoculation but the authors mentioned that data extrapolation suggested that 31% of them could become negative to this test 70-90 days after infection. The marginal improvement obtained with the radioimmunoassay does not warrant a change of a simple routine technique to another
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A.A. Guglielmone et al. / Veterinary Parasitology 70 (1997) 33-39
involving the manipulation of radioctive material. We have had no opportunity to evaluate any ELISA for B. bigemina antibodies. Therefore, until better techniques are available, the IFA test will continue to be our routine method of diagnosis to B. bigemina antibodies but in future samples obtained on day 40-50 will be used to detect B. bigemina antibodies. An additional sample to detect antibodies to A. centrale will be taken at about 60 days from vaccination. Diagnosis of vaccinal B. bovis antibodies can be done with samples obtained in any of the given post-vaccination period. Higher antibody titres to B. boris than to B. bigemina are usually found in cattle inoculated simultaneously with the Argentinean vaccinal strains used in the present study (Guglielmone et al., 1989; Aguirre et al., 1990). The opposite was found by Bessenger and Schoeman (1983) using South African vaccinal strains of B. bovis and B. bigemina, and Pipano et al. (1987) detected a similar range of IFA titres to B. boris and B. bigemina in cattle inoculated simultaneously with Israel vaccinal strains of both protozoan. The disparity of the results may be a consequence of difference amongst vaccinal strains used. Further differences are suggested by the fact that the Israeli strains induced the highest antibody titres before day 36 after inoculation whereas the South African and Argentinean strains reached their highest antibody titres after that period. The initial detection of B. bigemina and B. bovis antibodies using IFA test after monospecific cattle infections ranged from 5 - 7 days (Dwived and Gautam, 1979; Latif et al., 1979; Fujinaga et al., 1980; Kuttler et al., 1983) to 21-24 days (Latif et al., 1979; Bessenger and Schoeman, 1983). However, Bessenger and Schoeman (1983) inoculated four calves simultaneously with B. bovis and B. bigemina and all of them were first found positive to the IFA test for B. bigemina on day 14 after inoculation and to B. boris 7-21 days post-inoculation. This appears to show that the results obtained in this study agreed with the literature on B. boris but to a lesser extent on B. bigemina. Fig. l and Tables 1 and 2 seem to demonstrate that the reaction to B. bigemina SlA strain lagged behind the reaction to B. bovis RIA. It is not certain if this is a normal effect of B. bigemina S1A strain or the result of competition with the B. bovis R1A strain, which will be the subject of a future study.
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De RJos, L.G., Aguirre, D.H. and Gaido, A.B., 1989. Infecci6n natural por Babesia boris y Babesia bigemina en dos rodeos bovinos con diferentes niveles de infestaci6n por Boophilus microplus. Rev. Latinoam. Microbiol., 31: 39-43. Dwived, S.K. and Gautam, O.P., 1979. A note on indirect fluorescent antibody test for the diagnosis of experimental babesiosis in calves. Indian J. Anim. Sci., 49: 664-666. Echaide, I.E., de Echaide, S.T. and Guglielmone, A.A., 1993. Live and soluble antigens for cattle protection to Babesia bigemina. Vet. Parasitol., 51: 35-40. Fujinaga, T., Minami, T. and Ishihara, T., 1980. Serological relationship between a large Babesia found in Japanese cattle and Babesia major, B. bigemina and B. major. Res. Vet. Sci., 29: 230-234. Goldman, M., Pipano, E. and Rosenberg, A.S., 1972. Fluorescent antibody tests to Babesia bigemina and B. berbera. Res. Vet. Sci., 13: 77-81. Goodger, V.B., 1973. Further studies of haemagglutinating antigens of Babesia bigemina. Aust. Vet. J., 49: 81-84. Guglielmone, A.A., 1994. Epidemiologla y prevenci6n de los hemoparfisitos (Babesia y Anaplasma) en la Argentina. In: A. Nari and C. Fiel (Editors), Enfermedades Parasitarias de Importancia Econ6mica en Bovinos. Bases Epidemiol6gicas para su Prevenci6n y Control. Editorial Hemisferio Sur, Montevideo, pp. 461-479. Guglielmone, A.A., Mangold, A.J., Aguirre, D.H., de R[os, L.G. and Olsen, A.A., 1989. Vacunaci6n simultalnea con Babesia boris y Babesia bigemina atenuadas, congeladas en nitr6geno lfquido con DMSO o glicerol como crioprotectores. Rev. Med. Vet. (Buenos Aires), 70: 176-181. Guglielmone, A.A., de Echaide, S.T., Mangold, A.J., Echaide, I.E., Abdala, A.A., Anziani, O.S., Aguirre, D.H., Montenegro-James, S., Patarroyo, J., Alonso, M., Camus, E., Cordov6s, C., Kessler, R.H., Martins, J.R., Nari, A., Solari, M.A., Toro, M., Vega y Murgula, C. and Arellano Sota, C., 1997. Producci6n de sueros patrones anti-Babesia bovis, anti-Babesia bigemina, anti-Anaplasma marginale y negativos. Ser. Gan., in press. Kung'u, M.W. and Goodger, B.V., 1990. A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia boris infections and for the screening of Babesia-specific monoclonal antibodies. Int. J. Parasitol., 20: 341-345. Kuttler, K.L., Levy, M.G. and Ristic, M., 1983. Cell cultured-derived Babesia boris vaccine: sequential challenge exposure of protective immunity during a 6-month postvaccination period. Am. J. Vet. Res., 44: 1456-1459. Latif, B.M.A., Said, M.S. and All, S.R., 1979. Effect of age on the immune response of cattle experimentally infected with Babesia bigemina. Vet. Parasitol., 5: 307-314. Levy, M.G., Clabaugh, G. and Ristic, M., 1982. Age resistance in bovine babesiosis: rote of blood factors in resistance to Babesia boris. Inf. Immun., 37:1127-1131. Mahoney, D.F., 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Exp. Parasitol., 20: 223-241. Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1979. Immunity in cattle to Babesia bovis after single infections with parasites of various origins. Aust. Vet. J., 55: 10-12. Mangold, A.J., Aguirre, D.H. and Guglielmone, A.A., 1990. Post-thawing viability of vaccines for bovine babesiosis and anaplasmosis eryopreserved with glycerol. Vet. Parasitol., 37:311-306. Marigold, A.J., Vanzini, V.R., Echaide, I.E., de Echaide, S.T., Volpogni, M.M. and Guglielmone, A.A., 1996. Viability after thawing and dilution of simultaneously cryopreserved vaccinal Babesia bovis and Babesia bigemina strains cultured in vitro. Vet. Parasitol., 61: 345-348. MeUors, R.T., Daigliesh, R.J., Timms, P., Rodwell, B.J. and Callow, L.L., 1982. Preparation and laboratory testing of a frozen vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale. Res. Vet. Sci., 32: 194-197. Pipano, E., Markovics, A., Kriegel, Y., Frank, M. and Fish, L., 1987. Use of long-acting oxytetracycline in the immunisation of cattle against Babesia bovis and B. bigemina. Res. Vet. Sci., 43: 64-66. Potgieter, F.T. and van Rensburg, L., 1983. Infectivity virulence and immunogenicity of Anaplasma centrale live blood vaccine. Onderstepoort J. Vet. Sci., 50: 29-31. Schunter, C.A. and Wright, I.G., 1989. Detection of Babesia bigemina in cattle by a radioimmunoassay incorporating specifically depleted antigen. Vet. Parasitol., 31: 229-241. Thrusfield, M., 1986. Veterinary Epidemiology. Butterworth, London, 280 pp.