Veterinary Microbiology, 31 ( 1 9 9 2 ) 8 1 - 8 7 Elsevier Science Publishers B.V., A m s t e r d a m
81
The effect of oxytetracycline treatment on immunity induced by Anaplasma centrale E. Pipano, Y. Krigel, A. Markovics and Varda Shkap Division of Parasitology, The Kimron Veterinary Institute, Bet Dagan 50250, Israel (Accepted 30 August 1991 )
ABSTRACT Pipano, E., Krigel, Y., Markovics, A and Shkap, V., 1991. The effect of oxytetracycline treatment on immunity induced by Anaplasma centrale. Vet. Microbiol., 31: 81-87. Calves vaccinated with Anaplasma centrale were treated with 20 mg/kg of long-acting oxytetracycline ( O T C / L A ) before or simultaneously with vaccination or up to seven months later. Of 40 animals given one or two of O T C / L A from 3 to 13 days before vaccination, 23 become patent after vaccination, with an average prepatent period almost twice as long as that in non-treated vaccinated controls. Upon challenge with 2 X 108 A. centrale per dose all 17 previously non-patent calves showed average maximum parasitemias of 2 to 3.8%. Out of 30 calves treated with two to four doses of O T C / L A from one to four weeks after vaccination, 29 remained negative for A. centrale and reacted to challenge infection with average maximum parasitemias of 6.9-7.8%. Five out of 10 calves receiving O T C / L A simultaneously with the vaccination, and all of a separate group of 10 calves treated with a single dose seven days after vaccination, become patent an average of 51.6 and 63.5 d, respectively, after vaccination. Upon challenge, the five previously non-patent calves showed an average of 5.2% maximum parasitemia. In all groups, only rare parasites were seen in previously patent calves after challenge. Thirty calves treated with 2 - 4 doses of O T C / L A about six months after vaccination showed no or only a few parasites upon challenge. The above results show that treatment with single or multiple doses o f O T C / L A a few weeks before or after administration of live A. centrale vaccine can interfere with elaboration of immunity.
INTRODUCTION
In some regions enzootic for anaplasmosis, cattle are protected against Anaplasma marginale by vaccination with living parasites of the relatively benign species, A. centrale (Anon, 1984). Immunity induced by this procedure is considered to be dependent on the continuous presence of live parasites in the vaccinated animal. Young cattle destined for vaccination often suffer from intestinal or respiratory infections, and broad spectrum tetracycline derivatives are among the drugs of choice for treating such infections. However, A. centrale is also sensitive to tetracyclines (Pipano et al., 1985a). Thus, animals vaccinated with 0 3 7 8 - 1 1 3 5 / 9 2 / $ 0 5 . 0 0 © 1992 Elsevier Science Publishers B.V. All rights reserved.
82
E. PIPANO ET AL.
A. centrale that receive tetracyclines for other infections may be unintentionally disinfected of the A. centrale infection, and may lose their immunity against anaplasmosis. At present, there is no published information on the time or dosage relationships that may affect immunity to anaplasmosis in A. centrale-vaccinated animals that have been treated with tetracyclines. The present work was planned to investigate the influence of single and multiple treatments with oxytetracycline (OTC) on the immunity induced by vaccination with A. centrale. MATERIALS AND METHODS
General procedures Friesian female calves maintained on an anaplasmosis-free farm were used in the experiments. The calves in groups 1 to 13 (Tables 1 and 2) were 9-12 months old at the beginning of the experiment, and 12-15 months on the day of challenge. The calves in groups 14 to 17 (Table 3) were 3-4 months old upon vaccination and 10-11 months old when challenged. Calves were vaccinated with a single dose of a cryopreserved A. centrale vaccine containing 108 organisms (Pipano et al., 1986). A double dose (2 × 108 ) was used as the challenge infection. A long-acting formulation of oxytetracycline ( O T C / L A ) (Dabicycline 15 Vet., Oxytetracycline HCL 150 m g / m l , ABIC LTD, Israel) was administered intramuscularly at two sites in the neck area of the calves at the time periods described below. Results of initial and challenge infections were monitored by examination of Giemsa-stained thin blood films for presence ofA. centrale. Experimental design The experiments were performed in three stages. In the first stage, animals received O T C / L A before vaccination with A. centrale. In the second stage, animals received the drug simultaneously with the vaccination or at intervals up to 28 days later. In the third stage, animals were given the drug six months or longer after vaccination. In the first two stages, animals were challenged with live A. centrale 100 days after the vaccination. In stage 3, the animals were challenged 98 to 112 days after the last treatment with the drug. At each stage, appropriate controls of non-treated and non-vaccinated calves were included in the experiment. Calves that did not show parasitemia during the prechallenge period, and that showed a m a x i m u m red blood cell infection rate of 2% or more after challenge, were classified as "susceptible" animals.
EFFECT OF OXYTETRACYCLINETREATMENT ON IMMUNITY
83
RESULTS
Table 1 summarizes the results of stage 1 experiments in which OTC/LA was administered 13-3 days before vaccination. Among the calves receiving two consecutive treatments with OTC/LA before vaccination, on days 13 and 6, or days 10 and 3, (groups 1 and 2), 5 and 3 animals, respectively, became patent. After a single administration of OTC/LA on day 6 or 3 before vaccination (groups 3 and 4) resulted in 9 and 6 animals, respectively, developing parasitemias after vaccination. The average prepatent periods in calves showing parasitemia after receiving OTC/LA was considerably longer (47.2 to 60.8 days) than in the vaccinated, non-treated calves of group 5 (32.8 days). Following the challenge inoculation, all previously negative calves, ("SUSceptibles"), showed average maximum parasitemias of 2.0-3.8%, while only a few parasites were seen in non-treated, vaccinated calves (group 5), or in the previously patent calves of groups 1-4. The average parasitemia in the non-vaccinated controls (group 6) was 8.6%. In stage 2 experiments (Table 2), animals received OTC/LA simultaneously with the vaccination (group 7 ) or at weekly intervals therefore for 1-4 weeks (groups 8-11 ). Five out of 10 animals receiving OTC/LA at the time of vaccination, and all 10 animals treated 7 days later developed parasitemias (groups 7 and 8 ). By contrast, none of the animals (with a single exception) receiving two or more weekly doses of OTC/LA after the vaccination became patent (groups 9, 10, 11 ). The average prepatent periods in groups 7, 8 and 9 TABLEI Parasitemia and immunity in calves treated with 20 mg/kg of long-acting oxytetracycline (OTC/LA) before vaccination with A. centrale Groups ( n = 10 )
1 2 3 4 5 6
OTC/LA given on day No.
13, 6 10, 3 6 3 Vaccinated, non-treated Non-vaccinated controls
Response to vaccination
Response to challenge
No, of calves with parasitemia
Average prepatent period (d)
No. of susceptible animals ~
5 3 9 6 10
60.8 57.3 47.2 53.0 32.8
5 7 1 4 0
3.8 3.7 2.0 3.5 few
-
-
10
8.6
Average maximum PPE 2
~Animals that did not show parasitemia in the prechallenge period, and that showed a maximum red blood cell infection rate of 2% or more after challenge. 2percent parasitised erythrocytes
84
E. PIPANO ET AL.
TABLE 2 Parasitemia and immunity in calves treated with 20 mg/kg OTC/LA simultaneously with and after vaccination with A. centrale Groups ( n = 10 )
7 8 9 l0 11 12
13
OTC/LA given on day No.
0 7 7,14 7,14,21 7,14,21,28 Vaccinated, non-treated Non-vaccinated controls
Response to vaccination
Response to challenge
No. of calves with parasitemia
Average prepatent period (d)
No. of susceptible animals j
Average maximum PPE 2
5 10 1 0 0 10
51.6 63.5 81.0 29.2
5 0 9 10 10 0
5.2 few 7.8 7.1 6.9 few
-
-
10
10.8
~'2See Table 1 for details. TABLE 3 Susceptibility to challenge ofA. centrale vaccinated calves treated with 20 mg/kg OTC/LA six months or more after vaccination. Group (n=10)
OTC/LA given on day No.
Responsetochallenge No. of animals with parasitemia
14 15 16 17
181,188 181,188,195 181,188,195,202 Non-vaccinated, non-treated controls
Average maximum PPE j
2
~w
3 3 10
~w ~w 8.1
tPercent parasitised erythrocytes.
were 51.6, 63.5 and 81.0 days, respectively, compared to 29.2 days in vaccinated, non-treated animals (group 12). Following the challenge infection, the previously negative calves in groups 7 and 9, as well as all calves in groups 10 and 11, became patent with average parasitemias of 5.2-7.8%. The calves in group 8, as well as those in group 12 that did not receive OTC/LA, showed only rare parasites in blood smears during the whole period of the post challenge examinations. The non-vaccinated control calves showed a maximum average parasitemia of 10.8%. In stage 3 experiments (Table 3), vaccinated calves received 2-4 weekly doses of OTC/LA, starting six months after the vaccination (groups 14, 15
EFFECT OF OXYTETRACYCLINE TREATMENT ON IMMUNITY
85
and 16). They were then challenged 98 to 112 days after the last treatment, at which time none of the animals was patent. Only two of three calves in each group become patent after challenge, showing rare parasites on blood smears. In the non-vaccinated, non-treated controls (group 17 ), all 10 calves become patent with an average parasitemia of 8.1%. DISCUSSION
It is evident from the above results that treating calves with O T C / L A shortly before vaccination with A. centrale, as well as treating them during the prepatent period after vaccination, may interfere with the elaboration of immunity in a significant percentage of the animals. On the other hand, repeated weekly treatments during the chronic stage of the infection did not lower the resistance to challenge with the homologous parasite three months later. Blood concentrations of OTC in cattle following the inoculation of 20mg/ kg of long acting formulations, decreased to about 0.5/tg/ml after 60 to 86 hours (Davey et al., 1985; Toutain and Raynaud, 1983; Xia et al., 1983). This is generally considered to be the lowest concentration having a therapeutic effect. In the present trials, the latter low concentration presumably present at the time of vaccination, prevented the establishment of the parasite in 4 out of 10 calves (group 4), and also caused a considerable extension of the prepatent period in the remaining calves (Table 1 ). Furthermore, even the presumably trace quantity of OTC remaining 6 d after the inoculation, prevented parasitemia in one animal. The antianaplasma activity was enhanced when two treatments at 7-d intervals were given before vaccination. This phenomenon might be due to some cumulative influence of OTC, since it seems unlikely that parasiticidal concentrations of the drug could remain by the 10th or 13th days following the first injection. Observations in this laboratory on bovine Babesia infections have indicated that repeated OTC treatments may trigger resistance to the multiplication of the babesial parasites (Pipano et al., 1985b). A single treatment administered simultaneously with the A. centrale vaccination (group 7 ) was no more effective in preventing the infection than treatment given before vaccination, despite the higher presumed blood levels of the drug in the former case. This observation reinforces the assumption that the influence of the drug is not only due to a direct parasiticidal effect. None of the calves receiving a single OTC dose 7 d post vaccination became parasite-free (group 8 ), although considerably lower serum concentrations of OTC in groups 1-4 prevented infection in a portion of the calves. A possible explanation for this anomaly might be that on day 7 post vaccination the parasites had already become established within the red blood cells of the inoculated animals, whereas in calves receiving treatment before infection, the parasites might be affected by the drug during the transfer from donor to re-
86
E. PIPANOETAL.
cipient red cells. The course of infection was, however, affected to some degree, since a considerable extension of the prepatent period occurred. A similar effect was observed also in A. marginale-infected cattle treated shortly after infection (Lincoln et al., 1982 ). Infection with A. centrale did not occur in 29 out of 30 animals receiving 2-4 post-vaccination treatments (groups 9 to 11 ). A similar weekly treatment regime for cattle infected with A. marginale about two months previously caused disinfection (Roby et al., 1978), while in another report (Lincoln et al., 1982 ) 1-3 treatments give 7-21 days after infection did not prevent A. marginale from establishing itself. No comparative studies on the sensitivity ofA. centrale and A. marginale to OTC have been published, but it appears from the above results that A. centrale may be more sensitive to this drug. The results presented in Table 3 show clearly that calves vaccinated with A. centrale and treated vigorously six months later, retained their immunity to reinfection with the homologous A. centrale parasites. Studies with A. marginale have shown that cattle in which chronic infections have been eliminated have nevertheless remained i m m u n e to reinfection with the same organism (Richney et al., 1977; Magonigle and Newby, 1984). In practice however, vaccination with A. centrale is intended to protect against the more virulent heterologous species, A. marginale, and the question remains whether calves receiving vigorous OTC treatments during the chronic stage of the A. centrale infection (possibly leading to disinfection) are still protected against field A. marginale infection. This question is not answered by the present study. Until this issue is clarified by further experiments, the practical implication of the work shown here is that revaccination with A. centrale can be carried out safely in older animals even after heavy treatment with O T C / L A . This will restore the A. centrale carrier state if the animals became A. marginale-negative by the drug treatment. ACKNOWLEDGEMENT
This study was partially supported by the Binational Agricultural Research and Development Fund (BARD) projects US-846-84C and US-1561-88. The authors thank Mrs. Leah Fish for her excellent technical assistance.
REFERENCES Anon, 1984. Immunization against bovine anaplasmosis. In: Ticks and tick-born disease control. A practical field manual, Food and Agriculture Organization of the United Nations, Rome, vol. II, pp. 444-456. Davey, L.A., Ferber, M.T. and Kaye, B., 1985. Comparison of the serum pharmacokinetics of a long acting and conventional oxytetracycline injection. Vet. Rec., I 17: 426-429. Lincoln, S., Eckblad, P. and Mangonigle, R., 1982. Bovine anaplasmosis: clinical, hematologic,
EFFECT OF OXYTETRACYCLINE TREATMENT ON IMMUNITY
87
and serologic manifestations in cows given a long-acting oxytetracycline formulation in the prepatent period. Am. J. Vet. Res., 43:1360-1363. Mangonigle, R. and Newly, T., 1984. Response of cattle upon re-exposure to Anaplasrna marginale after elimination of chronic carrier infections. Am. J.Vet. Res., 45: 695-697. Pipano, E., Mayer, E. and Frank, M., 1985a. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Br. Vet. J., 141: 174-178. Pipano, E., Krigel, Y. and Markovics, A., 1985b. Oxytetracycline-induced resistance to Babesia bovis infection in splenectomized calves. Trop. Anim. Hlth. Prod., 17:153-154. Pipano, E., Krigel, Y., Frank, M. and Markovics, A., 1986. Frozen Anaplasma centrale vaccine against anaplasmosis in cattle. Br. Vet. J., 142: 553-556. Richney, E., Brock, W., Kliewer, I. and Jones, E., 1977. Resistance to anaplasmosis after elimination of latent Anaplasma marginale infections. Am. J. Vet. Res., 38, 2:169-170. Roby, T., Simpson, J. and Amerault, T., 1978. Elimination of the carrier state of bovine anaplasmosis with a long-acting oxytetracycline. Am. J. Vet. Res., 39:1360-1363. Toutain, P. and Raynaud, J., 1983. Pharmacokinetics of oxytetracycline in young cattle: Comparison of conventional vs. long-acting formulations. Am. J. Vet. Res., 44: 1203-1209. Xia, W., Nielsen, P. and Gyrd-Hansen, N., 1983. Oxytetracyclines in cattle. A comparison between a conventional and a long-acting preparation. Acta Vet. Scand., 24: 120-128.
81
The effect of oxytetracycline treatment on immunity induced by Anaplasma centrale E. Pipano, Y. Krigel, A. Markovics and Varda Shkap Division of Parasitology, The Kimron Veterinary Institute, Bet Dagan 50250, Israel (Accepted 30 August 1991 )
ABSTRACT Pipano, E., Krigel, Y., Markovics, A and Shkap, V., 1991. The effect of oxytetracycline treatment on immunity induced by Anaplasma centrale. Vet. Microbiol., 31: 81-87. Calves vaccinated with Anaplasma centrale were treated with 20 mg/kg of long-acting oxytetracycline ( O T C / L A ) before or simultaneously with vaccination or up to seven months later. Of 40 animals given one or two of O T C / L A from 3 to 13 days before vaccination, 23 become patent after vaccination, with an average prepatent period almost twice as long as that in non-treated vaccinated controls. Upon challenge with 2 X 108 A. centrale per dose all 17 previously non-patent calves showed average maximum parasitemias of 2 to 3.8%. Out of 30 calves treated with two to four doses of O T C / L A from one to four weeks after vaccination, 29 remained negative for A. centrale and reacted to challenge infection with average maximum parasitemias of 6.9-7.8%. Five out of 10 calves receiving O T C / L A simultaneously with the vaccination, and all of a separate group of 10 calves treated with a single dose seven days after vaccination, become patent an average of 51.6 and 63.5 d, respectively, after vaccination. Upon challenge, the five previously non-patent calves showed an average of 5.2% maximum parasitemia. In all groups, only rare parasites were seen in previously patent calves after challenge. Thirty calves treated with 2 - 4 doses of O T C / L A about six months after vaccination showed no or only a few parasites upon challenge. The above results show that treatment with single or multiple doses o f O T C / L A a few weeks before or after administration of live A. centrale vaccine can interfere with elaboration of immunity.
INTRODUCTION
In some regions enzootic for anaplasmosis, cattle are protected against Anaplasma marginale by vaccination with living parasites of the relatively benign species, A. centrale (Anon, 1984). Immunity induced by this procedure is considered to be dependent on the continuous presence of live parasites in the vaccinated animal. Young cattle destined for vaccination often suffer from intestinal or respiratory infections, and broad spectrum tetracycline derivatives are among the drugs of choice for treating such infections. However, A. centrale is also sensitive to tetracyclines (Pipano et al., 1985a). Thus, animals vaccinated with 0 3 7 8 - 1 1 3 5 / 9 2 / $ 0 5 . 0 0 © 1992 Elsevier Science Publishers B.V. All rights reserved.
82
E. PIPANO ET AL.
A. centrale that receive tetracyclines for other infections may be unintentionally disinfected of the A. centrale infection, and may lose their immunity against anaplasmosis. At present, there is no published information on the time or dosage relationships that may affect immunity to anaplasmosis in A. centrale-vaccinated animals that have been treated with tetracyclines. The present work was planned to investigate the influence of single and multiple treatments with oxytetracycline (OTC) on the immunity induced by vaccination with A. centrale. MATERIALS AND METHODS
General procedures Friesian female calves maintained on an anaplasmosis-free farm were used in the experiments. The calves in groups 1 to 13 (Tables 1 and 2) were 9-12 months old at the beginning of the experiment, and 12-15 months on the day of challenge. The calves in groups 14 to 17 (Table 3) were 3-4 months old upon vaccination and 10-11 months old when challenged. Calves were vaccinated with a single dose of a cryopreserved A. centrale vaccine containing 108 organisms (Pipano et al., 1986). A double dose (2 × 108 ) was used as the challenge infection. A long-acting formulation of oxytetracycline ( O T C / L A ) (Dabicycline 15 Vet., Oxytetracycline HCL 150 m g / m l , ABIC LTD, Israel) was administered intramuscularly at two sites in the neck area of the calves at the time periods described below. Results of initial and challenge infections were monitored by examination of Giemsa-stained thin blood films for presence ofA. centrale. Experimental design The experiments were performed in three stages. In the first stage, animals received O T C / L A before vaccination with A. centrale. In the second stage, animals received the drug simultaneously with the vaccination or at intervals up to 28 days later. In the third stage, animals were given the drug six months or longer after vaccination. In the first two stages, animals were challenged with live A. centrale 100 days after the vaccination. In stage 3, the animals were challenged 98 to 112 days after the last treatment with the drug. At each stage, appropriate controls of non-treated and non-vaccinated calves were included in the experiment. Calves that did not show parasitemia during the prechallenge period, and that showed a m a x i m u m red blood cell infection rate of 2% or more after challenge, were classified as "susceptible" animals.
EFFECT OF OXYTETRACYCLINETREATMENT ON IMMUNITY
83
RESULTS
Table 1 summarizes the results of stage 1 experiments in which OTC/LA was administered 13-3 days before vaccination. Among the calves receiving two consecutive treatments with OTC/LA before vaccination, on days 13 and 6, or days 10 and 3, (groups 1 and 2), 5 and 3 animals, respectively, became patent. After a single administration of OTC/LA on day 6 or 3 before vaccination (groups 3 and 4) resulted in 9 and 6 animals, respectively, developing parasitemias after vaccination. The average prepatent periods in calves showing parasitemia after receiving OTC/LA was considerably longer (47.2 to 60.8 days) than in the vaccinated, non-treated calves of group 5 (32.8 days). Following the challenge inoculation, all previously negative calves, ("SUSceptibles"), showed average maximum parasitemias of 2.0-3.8%, while only a few parasites were seen in non-treated, vaccinated calves (group 5), or in the previously patent calves of groups 1-4. The average parasitemia in the non-vaccinated controls (group 6) was 8.6%. In stage 2 experiments (Table 2), animals received OTC/LA simultaneously with the vaccination (group 7 ) or at weekly intervals therefore for 1-4 weeks (groups 8-11 ). Five out of 10 animals receiving OTC/LA at the time of vaccination, and all 10 animals treated 7 days later developed parasitemias (groups 7 and 8 ). By contrast, none of the animals (with a single exception) receiving two or more weekly doses of OTC/LA after the vaccination became patent (groups 9, 10, 11 ). The average prepatent periods in groups 7, 8 and 9 TABLEI Parasitemia and immunity in calves treated with 20 mg/kg of long-acting oxytetracycline (OTC/LA) before vaccination with A. centrale Groups ( n = 10 )
1 2 3 4 5 6
OTC/LA given on day No.
13, 6 10, 3 6 3 Vaccinated, non-treated Non-vaccinated controls
Response to vaccination
Response to challenge
No, of calves with parasitemia
Average prepatent period (d)
No. of susceptible animals ~
5 3 9 6 10
60.8 57.3 47.2 53.0 32.8
5 7 1 4 0
3.8 3.7 2.0 3.5 few
-
-
10
8.6
Average maximum PPE 2
~Animals that did not show parasitemia in the prechallenge period, and that showed a maximum red blood cell infection rate of 2% or more after challenge. 2percent parasitised erythrocytes
84
E. PIPANO ET AL.
TABLE 2 Parasitemia and immunity in calves treated with 20 mg/kg OTC/LA simultaneously with and after vaccination with A. centrale Groups ( n = 10 )
7 8 9 l0 11 12
13
OTC/LA given on day No.
0 7 7,14 7,14,21 7,14,21,28 Vaccinated, non-treated Non-vaccinated controls
Response to vaccination
Response to challenge
No. of calves with parasitemia
Average prepatent period (d)
No. of susceptible animals j
Average maximum PPE 2
5 10 1 0 0 10
51.6 63.5 81.0 29.2
5 0 9 10 10 0
5.2 few 7.8 7.1 6.9 few
-
-
10
10.8
~'2See Table 1 for details. TABLE 3 Susceptibility to challenge ofA. centrale vaccinated calves treated with 20 mg/kg OTC/LA six months or more after vaccination. Group (n=10)
OTC/LA given on day No.
Responsetochallenge No. of animals with parasitemia
14 15 16 17
181,188 181,188,195 181,188,195,202 Non-vaccinated, non-treated controls
Average maximum PPE j
2
~w
3 3 10
~w ~w 8.1
tPercent parasitised erythrocytes.
were 51.6, 63.5 and 81.0 days, respectively, compared to 29.2 days in vaccinated, non-treated animals (group 12). Following the challenge infection, the previously negative calves in groups 7 and 9, as well as all calves in groups 10 and 11, became patent with average parasitemias of 5.2-7.8%. The calves in group 8, as well as those in group 12 that did not receive OTC/LA, showed only rare parasites in blood smears during the whole period of the post challenge examinations. The non-vaccinated control calves showed a maximum average parasitemia of 10.8%. In stage 3 experiments (Table 3), vaccinated calves received 2-4 weekly doses of OTC/LA, starting six months after the vaccination (groups 14, 15
EFFECT OF OXYTETRACYCLINE TREATMENT ON IMMUNITY
85
and 16). They were then challenged 98 to 112 days after the last treatment, at which time none of the animals was patent. Only two of three calves in each group become patent after challenge, showing rare parasites on blood smears. In the non-vaccinated, non-treated controls (group 17 ), all 10 calves become patent with an average parasitemia of 8.1%. DISCUSSION
It is evident from the above results that treating calves with O T C / L A shortly before vaccination with A. centrale, as well as treating them during the prepatent period after vaccination, may interfere with the elaboration of immunity in a significant percentage of the animals. On the other hand, repeated weekly treatments during the chronic stage of the infection did not lower the resistance to challenge with the homologous parasite three months later. Blood concentrations of OTC in cattle following the inoculation of 20mg/ kg of long acting formulations, decreased to about 0.5/tg/ml after 60 to 86 hours (Davey et al., 1985; Toutain and Raynaud, 1983; Xia et al., 1983). This is generally considered to be the lowest concentration having a therapeutic effect. In the present trials, the latter low concentration presumably present at the time of vaccination, prevented the establishment of the parasite in 4 out of 10 calves (group 4), and also caused a considerable extension of the prepatent period in the remaining calves (Table 1 ). Furthermore, even the presumably trace quantity of OTC remaining 6 d after the inoculation, prevented parasitemia in one animal. The antianaplasma activity was enhanced when two treatments at 7-d intervals were given before vaccination. This phenomenon might be due to some cumulative influence of OTC, since it seems unlikely that parasiticidal concentrations of the drug could remain by the 10th or 13th days following the first injection. Observations in this laboratory on bovine Babesia infections have indicated that repeated OTC treatments may trigger resistance to the multiplication of the babesial parasites (Pipano et al., 1985b). A single treatment administered simultaneously with the A. centrale vaccination (group 7 ) was no more effective in preventing the infection than treatment given before vaccination, despite the higher presumed blood levels of the drug in the former case. This observation reinforces the assumption that the influence of the drug is not only due to a direct parasiticidal effect. None of the calves receiving a single OTC dose 7 d post vaccination became parasite-free (group 8 ), although considerably lower serum concentrations of OTC in groups 1-4 prevented infection in a portion of the calves. A possible explanation for this anomaly might be that on day 7 post vaccination the parasites had already become established within the red blood cells of the inoculated animals, whereas in calves receiving treatment before infection, the parasites might be affected by the drug during the transfer from donor to re-
86
E. PIPANOETAL.
cipient red cells. The course of infection was, however, affected to some degree, since a considerable extension of the prepatent period occurred. A similar effect was observed also in A. marginale-infected cattle treated shortly after infection (Lincoln et al., 1982 ). Infection with A. centrale did not occur in 29 out of 30 animals receiving 2-4 post-vaccination treatments (groups 9 to 11 ). A similar weekly treatment regime for cattle infected with A. marginale about two months previously caused disinfection (Roby et al., 1978), while in another report (Lincoln et al., 1982 ) 1-3 treatments give 7-21 days after infection did not prevent A. marginale from establishing itself. No comparative studies on the sensitivity ofA. centrale and A. marginale to OTC have been published, but it appears from the above results that A. centrale may be more sensitive to this drug. The results presented in Table 3 show clearly that calves vaccinated with A. centrale and treated vigorously six months later, retained their immunity to reinfection with the homologous A. centrale parasites. Studies with A. marginale have shown that cattle in which chronic infections have been eliminated have nevertheless remained i m m u n e to reinfection with the same organism (Richney et al., 1977; Magonigle and Newby, 1984). In practice however, vaccination with A. centrale is intended to protect against the more virulent heterologous species, A. marginale, and the question remains whether calves receiving vigorous OTC treatments during the chronic stage of the A. centrale infection (possibly leading to disinfection) are still protected against field A. marginale infection. This question is not answered by the present study. Until this issue is clarified by further experiments, the practical implication of the work shown here is that revaccination with A. centrale can be carried out safely in older animals even after heavy treatment with O T C / L A . This will restore the A. centrale carrier state if the animals became A. marginale-negative by the drug treatment. ACKNOWLEDGEMENT
This study was partially supported by the Binational Agricultural Research and Development Fund (BARD) projects US-846-84C and US-1561-88. The authors thank Mrs. Leah Fish for her excellent technical assistance.
REFERENCES Anon, 1984. Immunization against bovine anaplasmosis. In: Ticks and tick-born disease control. A practical field manual, Food and Agriculture Organization of the United Nations, Rome, vol. II, pp. 444-456. Davey, L.A., Ferber, M.T. and Kaye, B., 1985. Comparison of the serum pharmacokinetics of a long acting and conventional oxytetracycline injection. Vet. Rec., I 17: 426-429. Lincoln, S., Eckblad, P. and Mangonigle, R., 1982. Bovine anaplasmosis: clinical, hematologic,
EFFECT OF OXYTETRACYCLINE TREATMENT ON IMMUNITY
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and serologic manifestations in cows given a long-acting oxytetracycline formulation in the prepatent period. Am. J. Vet. Res., 43:1360-1363. Mangonigle, R. and Newly, T., 1984. Response of cattle upon re-exposure to Anaplasrna marginale after elimination of chronic carrier infections. Am. J.Vet. Res., 45: 695-697. Pipano, E., Mayer, E. and Frank, M., 1985a. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Br. Vet. J., 141: 174-178. Pipano, E., Krigel, Y. and Markovics, A., 1985b. Oxytetracycline-induced resistance to Babesia bovis infection in splenectomized calves. Trop. Anim. Hlth. Prod., 17:153-154. Pipano, E., Krigel, Y., Frank, M. and Markovics, A., 1986. Frozen Anaplasma centrale vaccine against anaplasmosis in cattle. Br. Vet. J., 142: 553-556. Richney, E., Brock, W., Kliewer, I. and Jones, E., 1977. Resistance to anaplasmosis after elimination of latent Anaplasma marginale infections. Am. J. Vet. Res., 38, 2:169-170. Roby, T., Simpson, J. and Amerault, T., 1978. Elimination of the carrier state of bovine anaplasmosis with a long-acting oxytetracycline. Am. J. Vet. Res., 39:1360-1363. Toutain, P. and Raynaud, J., 1983. Pharmacokinetics of oxytetracycline in young cattle: Comparison of conventional vs. long-acting formulations. Am. J. Vet. Res., 44: 1203-1209. Xia, W., Nielsen, P. and Gyrd-Hansen, N., 1983. Oxytetracyclines in cattle. A comparison between a conventional and a long-acting preparation. Acta Vet. Scand., 24: 120-128.
