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Humoral immune response and hematologic evaluation of pregnant Jersey cows after vaccination with Anaplasma centrale
Veterinary Microbiology 94 (2003) 335–339
Humoral immune response and hematologic evaluation of pregnant Jersey cows after vaccination with Anaplasma centrale R.D. Meléndez a,∗ , M. Toro Ben´ıtez b , G. Niccita c , J. Moreno b , S. Puzzar a , J. Morales c a
c
Decanato de Ciencias Veterinarias, Area de Parasitolog´ıa, Universidad Centroccidental “Lisandro Alvarado” (UCLA), Apartado Postal 665, Barquisimeto, Lara, Venezuela b ANACENT Co., Naguanagua, Carabobo, Venezuela Centro de Investigaciones Médicas y Biotecnológicas (CIMBUC), Universidad de Carabobo (UC), Naguanagua, Carabobo, Venezuela Received 9 August 2002; received in revised form 7 April 2003; accepted 8 April 2003
Abstract The main objective of this work was to evaluate the safety of an Anaplasma centrale vaccine in pregnant pure bred Jersey cows selected from a herd located at Miranda State, Venezuela. Ten cows of 3–5 months of gestation were chosen and previous vaccination all cows were tested for Anaplasma antibodies by the indirect immunofluorescence assay (IFA), so only seronegative cows were included in the group, and for blood parameters, rectal temperature, and pregnancy. Selected cows were vaccinated intramuscularly with 1 ml of an A. centrale live vaccine which had 108 A. centrale per ml. Over the next 2 months cows were checked weekly for hematological parameters and Anaplasma antibodies, and then for the next 2 months these evaluations were performed monthly. Among the values monitored were: A. centrale parasitemia, hematocrit, hemoglobin, and white blood cells (WBCs) (neutrophil, lymphocyte and eosinophil counts). Levels of Anaplasma antibodies were measured by IFA. Anaplasma were observed for the first time in blood films of two vaccinated cows at 14 days post-vaccination (PV), 6 out of 10 cows were A. centrale positive at 30 days PV, and all cows were A. centrale positive at 42 days PV. A. centrale often showed low parasitemia, 1–3%. Anaplasma antibodies were detected at day 14 PV in all vaccinated cows with a mean group titre of 360 (range: 80–1280). All vaccinated cows showed few changes in their hematologic parameters or in rectal temperature, and all gave birth to healthy calves. In conclusion, adult pregnant cows were
∗
Corresponding author. Tel./fax: +58-251-259-2440. E-mail address: [email protected] (R.D. Mel´endez). 0378-1135/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1135(03)00128-7
336
R.D. Mel´endez et al. / Veterinary Microbiology 94 (2003) 335–339
safely vaccinated with this live A. centrale vaccine, which may help to develop a cross-protective immunity against field strains of A. marginale. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Anaplasma centrale; Vaccination; Cattle; Rickettsia
1. Introduction Bovine anaplasmosis is an arthropod-borne hemoparasitic disease caused by the intraerythrocytic rickettsia Anaplasma marginale. This hemoparasite causes severe anaemia, fever, high levels of rickettsemia and even death, particularly in animals aged over 2 years (Ristic, 1977). Cattle that survive to acute anaplasmosis develop a chronic carrier state characterized by persistent low level infection, which generally is asymptomatic (“premunition stage”). Anaplasma centrale is another hemoparasite of cattle which was also discovered in South Africa by Theiler (1911). Its current taxonomic classification is A. marginale ss. centrale, which is considered less pathogenic than A. marginale, since it causes only mild anaplasmosis. In addition, A. centrale shows a central location in the erythrocytes on Giemsa-stained blood smears (Theiler, 1911; Ristic, 1977). After Theiler isolated A. centrale in the early 1900s he took advantage of the biological properties of this subspecies to develop a live A. centrale vaccine, which is the most widely used live vaccine strain for control of bovine anaplasmosis (Theiler, 1911), since cattle infected or vaccinated with A. centrale develop protective immunity against A. marginale infections. The A. centrale strain continues to be used for vaccine production in several regions of the world including Africa, Australia, Israel and some Latin America countries, i.e., Argentina, Brazil, Uruguay, and Venezuela (Pipano et al., 1985; Abdala et al., 1990). Both A. centrale and A. marginale share immunodominant epitopes that may play a role in the protection induced by A. centrale (Nakamura et al., 1991; Shkap et al., 1991). Recent studies have demonstrated that antigenic variation of major surface protein 2 (MSP2) occurs during persistent A. centrale infections in a manner similar to that described for A. marginale (Palmer et al., 1999). In addition, CD4+ T-cell epitopes were conserved between the two species, which may contribute to the cross-protection afforded by the A. centrale live vaccine (Shkap et al., 2002). Recent studies suggest that cattle can become infected with one genotype of A. marginale since only one was found per animal in an area where many genotypes were diagnosed (Palmer et al., 2001). Field and laboratory failures of A. centrale vaccines have been reported and are not uncommon (Brizuela et al., 1998; Guglielmone and Vanzini, 1999). In addition, the vaccine strain has been reported to cause severe anaplasmosis in splenectomized adult cattle (Kuttler, 1966; Pipano et al., 1985); by contrast, the vaccine has been most successfully used in young cattle (Pipano et al., 1985). In a recent study carried out in Australia (Bock and de Vos, 2001) use of the live A. centrale vaccine appeared to be justified because, although the bovine response was variable, protection against challenge-exposure was sufficient in most cases to prevent disease. In spite of these facts, A. centrale vaccines have been used for more than 90 years, and it appears that cattlemen and veterinarians promote the use of this live vaccine for prevention
R.D. Mel´endez et al. / Veterinary Microbiology 94 (2003) 335–339
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of anaplasmosis outbreaks in the field. This is a reality in countries like Argentina, Australia, Brazil, South Africa, Uruguay and Venezuela, where several thousands doses are sold yearly, and use of the vaccine will most likely continue until more effective Anaplasma vaccines become available. In Venezuela, a live A. centrale vaccine was introduced from Australia in 1997 and it has been annually produced through a joint agreement between University of Carabobo and Anacent Co. So far, more than 200,000 doses have been sold and applied both in dairy and beef cattle. In order to officially register this live vaccine in Venezuela several research projects were undertaken. In this case, the main objective was to evaluate the safety of the live A. centrale vaccine in pregnant pure bred Jersey cows.
2. Materials and methods 2.1. Animals The assay was carried out at a farm in Miranda State, Venezuela, located 110 km south of Caracas, on a herd of pregnant pure bred Jersey cows. Ten cows, 3–5 months of gestation were chosen to form the vaccinated group. Previously, all cows had been tested for Anaplasma antibodies by the immunofluorescence assay (IFA) in order to include only seronegative cows in the experimental group. Simultaneously, these selected cows were also checked for blood parameters, rectal temperature, and pregnancy. 2.2. Hematological samples The samples were collected as follows: (1) 1 day before vaccination; (2) weekly postvaccination (PV) for the next 2 months; (3) monthly for the final 2 months. Among the samples collected were: capillary blood for thin blood smears to monitor A. centrale parasitemia, hematocrit, hemoglobin (Hb), and white blood cells (WBCs) (neutrophil, lymphocyte, and eosinophil counts). Anaplasma antibodies were measured on sera by a modified IFA (Montenegro-James et al., 1985). 2.3. Pregnancy and clinical conditions Pregnancy was diagnosed through rectal palpation by a veterinarian expert in bovine reproduction, whereas clinical conditions and rectal temperature were evaluated with the same frequency established for blood samples. 2.4. Vaccination All pregnant Jersey cows in the experimental group were vaccinated intramuscularly (IM) with 1 ml of a live A. centrale vaccine, which had 108 A. centrale per ml. The vaccine is kept frozen in nitrogen tanks in vials with 1 ml of A. centrale infected blood. Before vaccination vials were thawed, infected blood was resuspended in 99 ml of medium 199, and 1 ml of this mixture was injected IM to each cow.
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3. Results A. centrale infected erythrocytes were observed for the first time at 14 days PV in thin blood films from two vaccinated pregnant cows. In addition, 6 out of 10 cows were A. centrale positive at 30 days PV, and in general all cows showed low parasitemia to A. centrale (1–3%); all experimental cows were A. centrale positive at 42 days PV. After 2 months PV all cows in the vaccinated group showed low parasitemia to A. centrale. The mean Hb and the mean hematocrit for cows of the vaccinated group during the whole assay were 9.11 g/dl and 26.94%, whereas the mean values for the WBC counts were on normal levels. Fever was not diagnosed in the group of cows PV. These data show that the A. centrale vaccine only caused a mild infection in vaccinated pregnant cows, and that their hematological parameters were not altered PV. Anaplasma antibodies were detected for the first time by IFA at day 14 PV in all pregnant cows of the vaccinated group which had a mean group titre of 360 (range: 80–1280) for the whole assay. It is important to stress that in this experiment all pregnant cows in the vaccinated group gave birth to healthy calves.
4. Discussion All vaccinated cows showed a low parasitemia with A. centrale, that lasted for a short time, and these cows stayed free of symptoms of anaplasmosis. All these observations are consistent with the less pathogenic condition of A. centrale when compared with A. marginale infections. The fact that the hematological values evaluated during this assay were little altered in pregnant cows of the vaccinated group is also in agreement with the low virulence of A. centrale infections, and also confirms previous results (Potgieter and Van Rensburg, 1983; Anziani et al., 1987). The mean group titre of Anaplasma antibodies in the vaccinated group was only 360 which seems a low mean titre. Nonetheless, it is appropriate to mention that the antigen used for running the IFA tests was A. marginale infected erythrocytes in thin blood smears since A. centrale and A. marginale share immunodominant epitopes, and consequently serological cross-reaction, The low A. centrale antibody titres might be explained by this partial crossed immunity. On this point, a better approach was recently reported from Australia where a monoclonal antibody, that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test which will be useful for confirming species identity in patent infections (Molloy et al., 2001). Live vaccines against rickettsial or protozoan organisms have the advantage of eliciting a life long or permanent immunity. However their storage is expensive and may occasionally cause post-vaccination reactions. Field and laboratory failures of A. centrale vaccines are not uncommon (Brizuela et al., 1998; Guglielmone and Vanzini, 1999). In addition, the vaccine strain has been reported to cause severe anaplasmosis in splenectomized adult cattle (Kuttler, 1966; Pipano et al., 1985), and in particular in high performance dairy cows (Pipano, 1976). In this experiment the live A. centrale vaccine was tested under a greater risk group of cattle: pure bred pregnant Jersey cows. Nonetheless, after evaluating and monitoring the vaccinated group during 4 months no case of abortion or clinical anaplasmosis was diagnosed. These results show the safety of this live vaccine in cows, although the use of the vaccine is recommended in bovines below 8 months old, and not in adult cattle.
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In conclusion, a live A. centrale vaccine was experimentally evaluated in pure bred pregnant Jersey cows and after monitoring the immune response, the hematological parameters, and the clinical conditions of these cows, it is concluded that a humoral immune response is elicited by the vaccine with no changes in the blood values tested or clinical anaplasmosis, and without causing abortion in pregnant cows. Acknowledgements To Anacent Co., Valencia, Venezuela, for the financial support of this research; to Mirian Rojas for her technical assistance, and to the personnel of “La Ponderosa” dairy farm at Cua, Miranda, Venezuela. References Abdala, A.A., Pipano, E., Aguirre, D.H., Gaido, A.B., Zurbriggen, M.A., Mangold, A.J., Guglielmone, A.A., 1990. Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection. Rev. Elev. Méd. Vet. Pays Trop. 43, 155–158. Anziani, O.S., Tarabla, H.D., Ford, C.A., Galleto, C., 1987. Vaccination with Anaplasma centrale: response after experimental challenge with Anaplasma marginale. Trop. Anim. Health Prod. 19 (2), 83–87. Bock, R.E., de Vos, A.J., 2001. Immunity following use of Australian tick fever vaccine: a review of the evidence. Aust. Vet. J. 79, 832–839. Brizuela, C.M., Ortellado, C.A., Sanabria, A., Torres, A., Ortigosa, D., 1998. The safety and efficacy of Australian tick-borne disease vaccine strains in cattle in Paraguay. Vet. Parasitol. 76, 27–41. Guglielmone, A.A., Vanzini, V.R., 1999. Análisis de fracasos en la prevención de la anaplasmosis y la babesiosis en bovinos inoculados con vacunas vivas. Rev. Med. Vet. (Buenos Aires) 80, 66–68. Kuttler, K.L., 1966. Clinical and hematologic comparison of Anaplasma marginale and Anaplasma centrale infections in cattle. Am. J. Vet. Res. 27, 941–946. Molloy, J.B., Bock, R.E., Templeton, J.M., Bruyeres, A.G., Bowles, P.M., Blight, G.W., Jorgensen, W.K., 2001. Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale. Int. J. Parasitol. 31 (2), 179–186. Montenegro-James, S., James, M.A., Ristic, M., 1985. Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle. Am. J. Vet. Res. 46, 2401–2403. Nakamura, Y., Kawazu, S., Minami, T., 1991. Analysis of protein composition and surface protein epitopes of Anaplasma centrale and Anaplasma marginale. Vet. Med. Sci. 53 (1), 73–79. Palmer, G.H., Rurangirwa, F.R., Kocan, K.M., Brown, W.C., 1999. Molecular bases for vaccine development against the ehrlichial pathogen Anaplasma marginale. Parasitol. Today 15, 281–286. Palmer, G.H., Rurangirwa, F.R., McElwain, T.F., 2001. Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission. J. Clin. Microbiol. 39, 631–635. Pipano, E., 1976. Control of bovine theileriosis and anaplasmosis in Israel. Bull. Off. Int. Epizootiol. 86, 55–59. Pipano, E., Mayer, E., Frank, M., 1985. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Br. Vet. J. 141, 174–178. Potgieter, F., Van Rensburg, L., 1983. Infectivity, virulence and immunogenicity of Anaplasma centrale live blood vaccine. Onderstepoort J. Vet. Res. 50, 29–31. Ristic, M., 1977. Bovine anaplasmosis. In: Kreier, J. (Ed.), Parasitic Protozoa, vol. 4. Academic Press, New York, pp. 235–249. Shkap, V., Pipano, E., McGuire, T.C., Palmer, G.H., 1991. Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale. Vet. Immun. Immunopath. 29, 31–40. Shkap, V., Molad, T., Brayton, K.A., Brown, W.C., Palmer, G.H., 2002. Expression of major surface protein 2 variants with conserved T-cell epitopes in Anaplasma centrale vaccinates. Infect. Immun. 70, 642–648. Theiler, A., 1911. Further investigations into anaplasmosis of South African cattle. First Report of the Director of Veterinary Research, Union of South Africa, pp. 7–46.
Humoral immune response and hematologic evaluation of pregnant Jersey cows after vaccination with Anaplasma centrale R.D. Meléndez a,∗ , M. Toro Ben´ıtez b , G. Niccita c , J. Moreno b , S. Puzzar a , J. Morales c a
c
Decanato de Ciencias Veterinarias, Area de Parasitolog´ıa, Universidad Centroccidental “Lisandro Alvarado” (UCLA), Apartado Postal 665, Barquisimeto, Lara, Venezuela b ANACENT Co., Naguanagua, Carabobo, Venezuela Centro de Investigaciones Médicas y Biotecnológicas (CIMBUC), Universidad de Carabobo (UC), Naguanagua, Carabobo, Venezuela Received 9 August 2002; received in revised form 7 April 2003; accepted 8 April 2003
Abstract The main objective of this work was to evaluate the safety of an Anaplasma centrale vaccine in pregnant pure bred Jersey cows selected from a herd located at Miranda State, Venezuela. Ten cows of 3–5 months of gestation were chosen and previous vaccination all cows were tested for Anaplasma antibodies by the indirect immunofluorescence assay (IFA), so only seronegative cows were included in the group, and for blood parameters, rectal temperature, and pregnancy. Selected cows were vaccinated intramuscularly with 1 ml of an A. centrale live vaccine which had 108 A. centrale per ml. Over the next 2 months cows were checked weekly for hematological parameters and Anaplasma antibodies, and then for the next 2 months these evaluations were performed monthly. Among the values monitored were: A. centrale parasitemia, hematocrit, hemoglobin, and white blood cells (WBCs) (neutrophil, lymphocyte and eosinophil counts). Levels of Anaplasma antibodies were measured by IFA. Anaplasma were observed for the first time in blood films of two vaccinated cows at 14 days post-vaccination (PV), 6 out of 10 cows were A. centrale positive at 30 days PV, and all cows were A. centrale positive at 42 days PV. A. centrale often showed low parasitemia, 1–3%. Anaplasma antibodies were detected at day 14 PV in all vaccinated cows with a mean group titre of 360 (range: 80–1280). All vaccinated cows showed few changes in their hematologic parameters or in rectal temperature, and all gave birth to healthy calves. In conclusion, adult pregnant cows were
∗
Corresponding author. Tel./fax: +58-251-259-2440. E-mail address: [email protected] (R.D. Mel´endez). 0378-1135/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1135(03)00128-7
336
R.D. Mel´endez et al. / Veterinary Microbiology 94 (2003) 335–339
safely vaccinated with this live A. centrale vaccine, which may help to develop a cross-protective immunity against field strains of A. marginale. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Anaplasma centrale; Vaccination; Cattle; Rickettsia
1. Introduction Bovine anaplasmosis is an arthropod-borne hemoparasitic disease caused by the intraerythrocytic rickettsia Anaplasma marginale. This hemoparasite causes severe anaemia, fever, high levels of rickettsemia and even death, particularly in animals aged over 2 years (Ristic, 1977). Cattle that survive to acute anaplasmosis develop a chronic carrier state characterized by persistent low level infection, which generally is asymptomatic (“premunition stage”). Anaplasma centrale is another hemoparasite of cattle which was also discovered in South Africa by Theiler (1911). Its current taxonomic classification is A. marginale ss. centrale, which is considered less pathogenic than A. marginale, since it causes only mild anaplasmosis. In addition, A. centrale shows a central location in the erythrocytes on Giemsa-stained blood smears (Theiler, 1911; Ristic, 1977). After Theiler isolated A. centrale in the early 1900s he took advantage of the biological properties of this subspecies to develop a live A. centrale vaccine, which is the most widely used live vaccine strain for control of bovine anaplasmosis (Theiler, 1911), since cattle infected or vaccinated with A. centrale develop protective immunity against A. marginale infections. The A. centrale strain continues to be used for vaccine production in several regions of the world including Africa, Australia, Israel and some Latin America countries, i.e., Argentina, Brazil, Uruguay, and Venezuela (Pipano et al., 1985; Abdala et al., 1990). Both A. centrale and A. marginale share immunodominant epitopes that may play a role in the protection induced by A. centrale (Nakamura et al., 1991; Shkap et al., 1991). Recent studies have demonstrated that antigenic variation of major surface protein 2 (MSP2) occurs during persistent A. centrale infections in a manner similar to that described for A. marginale (Palmer et al., 1999). In addition, CD4+ T-cell epitopes were conserved between the two species, which may contribute to the cross-protection afforded by the A. centrale live vaccine (Shkap et al., 2002). Recent studies suggest that cattle can become infected with one genotype of A. marginale since only one was found per animal in an area where many genotypes were diagnosed (Palmer et al., 2001). Field and laboratory failures of A. centrale vaccines have been reported and are not uncommon (Brizuela et al., 1998; Guglielmone and Vanzini, 1999). In addition, the vaccine strain has been reported to cause severe anaplasmosis in splenectomized adult cattle (Kuttler, 1966; Pipano et al., 1985); by contrast, the vaccine has been most successfully used in young cattle (Pipano et al., 1985). In a recent study carried out in Australia (Bock and de Vos, 2001) use of the live A. centrale vaccine appeared to be justified because, although the bovine response was variable, protection against challenge-exposure was sufficient in most cases to prevent disease. In spite of these facts, A. centrale vaccines have been used for more than 90 years, and it appears that cattlemen and veterinarians promote the use of this live vaccine for prevention
R.D. Mel´endez et al. / Veterinary Microbiology 94 (2003) 335–339
337
of anaplasmosis outbreaks in the field. This is a reality in countries like Argentina, Australia, Brazil, South Africa, Uruguay and Venezuela, where several thousands doses are sold yearly, and use of the vaccine will most likely continue until more effective Anaplasma vaccines become available. In Venezuela, a live A. centrale vaccine was introduced from Australia in 1997 and it has been annually produced through a joint agreement between University of Carabobo and Anacent Co. So far, more than 200,000 doses have been sold and applied both in dairy and beef cattle. In order to officially register this live vaccine in Venezuela several research projects were undertaken. In this case, the main objective was to evaluate the safety of the live A. centrale vaccine in pregnant pure bred Jersey cows.
2. Materials and methods 2.1. Animals The assay was carried out at a farm in Miranda State, Venezuela, located 110 km south of Caracas, on a herd of pregnant pure bred Jersey cows. Ten cows, 3–5 months of gestation were chosen to form the vaccinated group. Previously, all cows had been tested for Anaplasma antibodies by the immunofluorescence assay (IFA) in order to include only seronegative cows in the experimental group. Simultaneously, these selected cows were also checked for blood parameters, rectal temperature, and pregnancy. 2.2. Hematological samples The samples were collected as follows: (1) 1 day before vaccination; (2) weekly postvaccination (PV) for the next 2 months; (3) monthly for the final 2 months. Among the samples collected were: capillary blood for thin blood smears to monitor A. centrale parasitemia, hematocrit, hemoglobin (Hb), and white blood cells (WBCs) (neutrophil, lymphocyte, and eosinophil counts). Anaplasma antibodies were measured on sera by a modified IFA (Montenegro-James et al., 1985). 2.3. Pregnancy and clinical conditions Pregnancy was diagnosed through rectal palpation by a veterinarian expert in bovine reproduction, whereas clinical conditions and rectal temperature were evaluated with the same frequency established for blood samples. 2.4. Vaccination All pregnant Jersey cows in the experimental group were vaccinated intramuscularly (IM) with 1 ml of a live A. centrale vaccine, which had 108 A. centrale per ml. The vaccine is kept frozen in nitrogen tanks in vials with 1 ml of A. centrale infected blood. Before vaccination vials were thawed, infected blood was resuspended in 99 ml of medium 199, and 1 ml of this mixture was injected IM to each cow.
338
R.D. Mel´endez et al. / Veterinary Microbiology 94 (2003) 335–339
3. Results A. centrale infected erythrocytes were observed for the first time at 14 days PV in thin blood films from two vaccinated pregnant cows. In addition, 6 out of 10 cows were A. centrale positive at 30 days PV, and in general all cows showed low parasitemia to A. centrale (1–3%); all experimental cows were A. centrale positive at 42 days PV. After 2 months PV all cows in the vaccinated group showed low parasitemia to A. centrale. The mean Hb and the mean hematocrit for cows of the vaccinated group during the whole assay were 9.11 g/dl and 26.94%, whereas the mean values for the WBC counts were on normal levels. Fever was not diagnosed in the group of cows PV. These data show that the A. centrale vaccine only caused a mild infection in vaccinated pregnant cows, and that their hematological parameters were not altered PV. Anaplasma antibodies were detected for the first time by IFA at day 14 PV in all pregnant cows of the vaccinated group which had a mean group titre of 360 (range: 80–1280) for the whole assay. It is important to stress that in this experiment all pregnant cows in the vaccinated group gave birth to healthy calves.
4. Discussion All vaccinated cows showed a low parasitemia with A. centrale, that lasted for a short time, and these cows stayed free of symptoms of anaplasmosis. All these observations are consistent with the less pathogenic condition of A. centrale when compared with A. marginale infections. The fact that the hematological values evaluated during this assay were little altered in pregnant cows of the vaccinated group is also in agreement with the low virulence of A. centrale infections, and also confirms previous results (Potgieter and Van Rensburg, 1983; Anziani et al., 1987). The mean group titre of Anaplasma antibodies in the vaccinated group was only 360 which seems a low mean titre. Nonetheless, it is appropriate to mention that the antigen used for running the IFA tests was A. marginale infected erythrocytes in thin blood smears since A. centrale and A. marginale share immunodominant epitopes, and consequently serological cross-reaction, The low A. centrale antibody titres might be explained by this partial crossed immunity. On this point, a better approach was recently reported from Australia where a monoclonal antibody, that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test which will be useful for confirming species identity in patent infections (Molloy et al., 2001). Live vaccines against rickettsial or protozoan organisms have the advantage of eliciting a life long or permanent immunity. However their storage is expensive and may occasionally cause post-vaccination reactions. Field and laboratory failures of A. centrale vaccines are not uncommon (Brizuela et al., 1998; Guglielmone and Vanzini, 1999). In addition, the vaccine strain has been reported to cause severe anaplasmosis in splenectomized adult cattle (Kuttler, 1966; Pipano et al., 1985), and in particular in high performance dairy cows (Pipano, 1976). In this experiment the live A. centrale vaccine was tested under a greater risk group of cattle: pure bred pregnant Jersey cows. Nonetheless, after evaluating and monitoring the vaccinated group during 4 months no case of abortion or clinical anaplasmosis was diagnosed. These results show the safety of this live vaccine in cows, although the use of the vaccine is recommended in bovines below 8 months old, and not in adult cattle.
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339
In conclusion, a live A. centrale vaccine was experimentally evaluated in pure bred pregnant Jersey cows and after monitoring the immune response, the hematological parameters, and the clinical conditions of these cows, it is concluded that a humoral immune response is elicited by the vaccine with no changes in the blood values tested or clinical anaplasmosis, and without causing abortion in pregnant cows. Acknowledgements To Anacent Co., Valencia, Venezuela, for the financial support of this research; to Mirian Rojas for her technical assistance, and to the personnel of “La Ponderosa” dairy farm at Cua, Miranda, Venezuela. References Abdala, A.A., Pipano, E., Aguirre, D.H., Gaido, A.B., Zurbriggen, M.A., Mangold, A.J., Guglielmone, A.A., 1990. Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection. Rev. Elev. Méd. Vet. Pays Trop. 43, 155–158. Anziani, O.S., Tarabla, H.D., Ford, C.A., Galleto, C., 1987. Vaccination with Anaplasma centrale: response after experimental challenge with Anaplasma marginale. Trop. Anim. Health Prod. 19 (2), 83–87. Bock, R.E., de Vos, A.J., 2001. Immunity following use of Australian tick fever vaccine: a review of the evidence. Aust. Vet. J. 79, 832–839. Brizuela, C.M., Ortellado, C.A., Sanabria, A., Torres, A., Ortigosa, D., 1998. The safety and efficacy of Australian tick-borne disease vaccine strains in cattle in Paraguay. Vet. Parasitol. 76, 27–41. Guglielmone, A.A., Vanzini, V.R., 1999. Análisis de fracasos en la prevención de la anaplasmosis y la babesiosis en bovinos inoculados con vacunas vivas. Rev. Med. Vet. (Buenos Aires) 80, 66–68. Kuttler, K.L., 1966. Clinical and hematologic comparison of Anaplasma marginale and Anaplasma centrale infections in cattle. Am. J. Vet. Res. 27, 941–946. Molloy, J.B., Bock, R.E., Templeton, J.M., Bruyeres, A.G., Bowles, P.M., Blight, G.W., Jorgensen, W.K., 2001. Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale. Int. J. Parasitol. 31 (2), 179–186. Montenegro-James, S., James, M.A., Ristic, M., 1985. Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle. Am. J. Vet. Res. 46, 2401–2403. Nakamura, Y., Kawazu, S., Minami, T., 1991. Analysis of protein composition and surface protein epitopes of Anaplasma centrale and Anaplasma marginale. Vet. Med. Sci. 53 (1), 73–79. Palmer, G.H., Rurangirwa, F.R., Kocan, K.M., Brown, W.C., 1999. Molecular bases for vaccine development against the ehrlichial pathogen Anaplasma marginale. Parasitol. Today 15, 281–286. Palmer, G.H., Rurangirwa, F.R., McElwain, T.F., 2001. Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission. J. Clin. Microbiol. 39, 631–635. Pipano, E., 1976. Control of bovine theileriosis and anaplasmosis in Israel. Bull. Off. Int. Epizootiol. 86, 55–59. Pipano, E., Mayer, E., Frank, M., 1985. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Br. Vet. J. 141, 174–178. Potgieter, F., Van Rensburg, L., 1983. Infectivity, virulence and immunogenicity of Anaplasma centrale live blood vaccine. Onderstepoort J. Vet. Res. 50, 29–31. Ristic, M., 1977. Bovine anaplasmosis. In: Kreier, J. (Ed.), Parasitic Protozoa, vol. 4. Academic Press, New York, pp. 235–249. Shkap, V., Pipano, E., McGuire, T.C., Palmer, G.H., 1991. Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale. Vet. Immun. Immunopath. 29, 31–40. Shkap, V., Molad, T., Brayton, K.A., Brown, W.C., Palmer, G.H., 2002. Expression of major surface protein 2 variants with conserved T-cell epitopes in Anaplasma centrale vaccinates. Infect. Immun. 70, 642–648. Theiler, A., 1911. Further investigations into anaplasmosis of South African cattle. First Report of the Director of Veterinary Research, Union of South Africa, pp. 7–46.