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Preliminary development of a live drug-controlled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus
Veterinary Parasitology, 42 (1992) 179-188 Elsevier Science Publishers B.V., Amsterdam
179
Preliminary development of a live drugcontrolled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus J.S. Gray and P. Gannon ERM, Faculty of Agriculture, University College, Dublin, Ireland (Accepted 15 November 1991 )
ABSTRACT Gray, J.S. and Gannon, P., 1992. Preliminary development of a live drug-controlled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus. Vet. Parasitol., 42:179-188. This study investigated the practicality and potential of the gerbil, Meriones unguiculatus, as a source of live Babesia divergens vaccine and also as a model for the use of the vaccine in cattle. A series of experiments with gerbils concerningvaccine infectivity, immunogenicityand safety were carried out. It was concluded that the use of RPMI medium/40% foetal calf serum as a diluent improved vaccine infectivity, but that the parasitaemia of the blood obtained from donor gerbils had little or no effect. The immunostimulantslevamisole and killed Corynebacteriurn parvum improved vaccine immunogenicity and it was also shown that the subcutaneous route of infection resulted in the greatest host response. Control of vaccine virulence with drugs was only possible when drugs with prophylactic properties, such as imidocarb and long-acting oxytetracycline, were used. More studies are required on all these topics, particularly with regard to their applicability to cattle, and also concerning the possible attenuation of the parasite by manipulation in the gerbil host.
INTRODUCTION
Bovine babesiosis, caused by Babesia divergens, is widespread in Europe and has a particularly high prevalence in Ireland (Gray and Harte, 1985). Vaccination remains the best control option, but there have been considerable difficulties in the development of useable vaccines. These are the poor immunogenicityof non-living preparations, the difficulty of attenuating live vaccines and the reliance on a bovine source for such vaccines, with the attendant problems of transmission of other pathogens and of auto-immunity (Wright, 1991 ). The gerbil, Meriones unguiculatus, is highly susceptible to B. divergens and Correspondence to: J.S. Gray, Department of Environmental Resource Management, Faculty of Agriculture, University College, Belfield, Dublin 4, Ireland.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
180
J.S. GRAY A N D P. G A N N O N
this fact has been exploited in studies that sought to vaccinate cattle with gerbil-derived parasites (Friedhoff et al., 1989; Gray et al., 1989). Friedhoff et al. (1989) used only calves, the least susceptible age group, in extensive field studies without prophylactic drugs. However, this approach has limited applicability in Ireland, where it is necessary to vaccinate older cattle because of considerable movement of cattle. Gray et al. (1989) showed that it was possible to vaccinate cattle over 1 year of age when imidocarb was used as a prophylactic, resulting in long-lasting resistance to heterologous challenge. The only apparent weakness of the system was variable infectivity, and thus immunogenicity, of vaccine inocula for some cattle. It is evident that this aspect could be improved and the system otherwise optimised. The subjects addressed in these experiments were infectivity of the inocula, immunoresponsiveness of the host and chemoimmunisation. MATERIALS AND METHODS
Experimental animals The gerbils were bred in the University animal facility from a stock of five pairs obtained from Bantin and Kingman Ltd. (Hull, UK). The first progeny of these animals were examined and were found to be free of parasites and other pathogens. The animals were used in groups of five when 12-16 weeks old, the females weighing at least 50 g and the males 60 g.
Parasites The B. divergens strain used was originally isolated in Ireland (Purnell et al., 1976) and designated CE2. Following use in experimental cattle it was passaged in gerbils and when used here it had undergone more than 80 gerbil passages.
General procedures Unless otherwise stated, parasite infections were initiated by intraperitoneal (i.p.) injections of infected red blood cells (IRBC) in 0.1 ml of phosphate buffered saline (PBS). For routine maintenance of the parasite, 10 7 IRBC were injected i.p., but for experimental infections the dose level ranged from 104 to 108 IRBC, depending on the particular experiment and the route of infection used. Blood parasitaemias were monitored daily from thin blood smears prepared from tail blood and stained in Giemsa. Blood for serum antibody analysis was obtained by cardiac puncture (0.2 ml) under halothane anaesthesia. Antibody levels were measured with the indirect immunofluorescent antibody test (IFAT) using a triple sandwich
B. DIVERGENS VACCINEDEVELOPMENT
181
technique described in detail by Langley and Gray ( 1987 ). The method consists essentially of incubation of antigen on multiwell slides with test sera, followed by incubation with anti-gerbil IgG (raised in rabbits) at 1 : 1000 dilution and finally incubation with a goat anti-rabbit fluorescein-isothiocyanate IgG (Miles Laboratories, Bridgend, UK) at 1:80 dilution. After washing in PBS the slides were examined under blue light with a Leitz Dialux epifluorescent microscope system and a titre obtained for each sample. In some experiments readings were obtained from a white light rheostat which was used to match the intensity of the fluorescence (Gray and Kaye, 1991 ). Other procedures are described where the particular experiments in which they were used are considered.
Experimental design A series of experiments were carried out to investigate three different topics: ( 1 ) infectivity of the vaccine, determined by varying blood parasitaemias in donor gerbils and by different inoculum diluents; (2) immunogenicity of the vaccine, determined by non-specific immunostimulation and by different routes of infection; (3) control of parasite virulence with drugs.
Statistical analysis Unless otherwise indicated, results from experimental groups were compared with controls using Student's t-test. When more than one group were being compared, analysis of variance and Duncan's multiple range test were carried out. Percentage data were transformed to arcsines before analysis. RESULTS
Effects of different donor parasitaemias on infectivity Blood was obtained from three gerbils with parasitaemias of 6.6, 26.8 and 72.1%. Inocula of 105 IRBC per 0.1 ml were prepared from each source after dilution with PBS and injected intraperitoneally into each of five gerbils which had been pretreated subcutaneously with 0.5 mg kg-l dexamethasone (Dexafort, Intervet Laboratories Ltd., Cambridge, UK) in order to reduce variations caused by resistance mechanisms. When the mean times taken to 1.0% parasitaemias (4.2 days, 4.0 days and 4.0 days, respectively) and the mean parasitaemias on Day 4 of infection (3.62%, 2.84% and 2.20% respectively) were compared, no significant differences between the three groups were evident.
182
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Effect of vaccine diluent on infectivity Inocula containing 105 IRBC were stored in five different diluents for 24 h at 4 ° C before intraperitoneal inoculation into groups of five gerbils. The diluents were PBS, Eagle's minimum essential medium (EMEM), RPMI- 1640 culture medium with 25 mM Hepes and L-glutamine (RPMI), RPMI plus 10% foetal calf serum (RPMI/10FCS ) and RPMI plus 40% foetal calf serum (RPMI/40FCS). A sixth group of gerbils were infected with 105 fresh IRBC in PBS. The results showed that the best diluent was RPMI/40FCS and furthermore, that there was no significant difference in the infectivity of parasites in this diluent compared with fresh parasites (Table 1 ).
Route of infection studies Previous studies established that M. unguiculatus resists primary infections inoculated subcutaneously more readily than those inoculated intraperitoneally or by the cardiac route (Langley, 1985 ). A series of experiments was carried out to investigate this phenomenon further and it was found that greater splenomegaly occurred following subcutaneous infection compared with the intraperitoneal route and was accompanied by the enlargement of the lymph nodes draining the site of the subcutaneous inoculations (Table 2 ). The importance of the subcutaneous route in relation to splenic activity and resistance was also indicated by the fact that dexamethasone treatment only increased susceptibility if infections were given subcutaneously and furthermore, that whereas splenectomy has no effect in adult gerbils when the intraperitoneal infection route is used (Lewis et al., 1981; Langley, 1985), TABLE1 Effect of diluent on B. divergens inoculum infectivity in gerbils (24 h after 4 °C storage, 1 X 105 IRBC, i.p.)
Mean days to 1.0% parasitaemia _+SE
PBS
EMEM
RPMI
RPMI/ 10FCS
RPM1/ 40FCS
PBS (Day 0)
7.8 +0.288
9.5 + 1.584
7.2 +0.374
6.1 +0.510
4.8 +0.583
4.2 _+0.10
Values underscored by the same line are not significantly different at P < 0 . 0 5 (Duncan's multiple range test). PBS, phosphate buffered saline; EMEM, Eagle's m i n i m u m essential medium; RPMI, standard medium, 25 m m Hepes, L-glutamine; FCS, foetal calf serum.
183
B. DIVERGENSVACCINEDEVELOPMENT TABLE 2 The effect ofB. divergens route of infection on splenomegaly and lymph node enlargement Infection route
Infection level Mean spleen weight (mg+SE)
Subcutaneous
5X10 7
Intraperitoneal
5× 105
169.2+16.52 113.9+ 9.05
Student's t
2.93 P < 0.02
Mean axillary lymph Student's t node weight (mg+SE) 5.15+1.14 1.59+0.13
2.79 P < 0.05
TABLE 3 Effect ofsplenectomy and dexamethasone on the susceptibility of gerbils to intraperitoneal (i.p.) and subcutaneous (s.c.) B. divergens infections Mean days to 1.0% parasitaemia_+ SE Splenectomised ( 106 IRBC s.c. ) Intact ( 106 IRBC s.c.) Dexamethasone ( 106 IRBC s.c. ) Untreated ( 10 6 IRBC s.c. ) Dexamethasone ( 10 4 IRBC i.p. ) Untreated ( 104 IRBC i.p. )
Student's t
4.83 + 0.48 3.16
P < 0.02
4.747
P < 0.005
0.681
P > 0.1
6.50 + 0.22 3.80 + 0.200 6.40 + 0.510 3.75 + 0.414 4.17 + 0.597
splenectomised animals were more susceptible than intact ones when infections were given subcutaneously (Table 3 ). The antibody response to infections induced by subcutaneous and intraperitoneal infections were compared in an experiment in which blood for IFA serology was obtained from gerbils 6 days after subcutaneous and intraperitoneal infection with 107 and 105 IRBC, respectively, when parasitaemias had reached 40%. Although the antibody levels were much lower than would be obtained in recovered gerbils, those that received subcutaneous infections showed significantly higher fluorescence extinction values than those that received intraperitoneal infections (Student's t= 3.53, P< 0.005). A second experiment involving gerbils, in which infections were terminated with 6.0 mg kg -~ of the babesicide imidocarb dipropionate (Imizol, Coopers PitmanMoore, Berkhamstead, UK), failed to reproduce this effect. This was probably because any difference was obscured by the high antibody levels resulting from the effect of the babesicide on the parasites. Although maximum paras-
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J.S. GRAY AND P. GANNON
itaemias of the challenge infections in this second experiment were lower in the subcutaneously infected gerbils, this difference was not statistically significant.
Non-specific imrnunostimulation The object of this experiment was to increase host immune responsiveness without affecting the viability of the infective inoculum. This was done by treating two groups of animals with two immunostimulants; a killed preparation of Corynebacterium parvum (The Wellcome Foundation Ltd., Beckenham, UK) and the anthelmintic, levamisole (Levacide, Norbrook Laboratories Ltd., Newry, Northern Ireland) ), at infection and again 7 days later. A third group of gerbils received PBS injections only. The infections of 2 × 10 7 IRBC were administered intraperitoneally and then terminated on Day 3 by a subcutaneous injection of 6.0 mg kg-1 imidocarb. The level of immunostimulation was determined by measuring seroconversion using the IFAT on serum samples obtained on Day 10 after infection. On Day 17 all gerbils were challenged with 10 8 IRBC by intraperitoneal injection. The results show significantly higher antibody levels in both C. parvum and levamisole treated gerbils than in the untreated animals. All gerbils survived the challenge infection. In the C. parvum treated animals the infections did not become patent, whereas in the other two groups low level patency was reached (Table 4). TABLE4
I mmunostimulation of drug-terminated B. divergens i n f e c t i o n s (2 × l07 I R B C i.p. terminated on Day 3, 6.0 m g kg -~ imidocarb) lmmunostimulants Median IFA titre ( D a y s 0,7 after (7 days after infection ) termination )
Mean time (days) to clear after challenge ( 1 X 10 s I R B C 14 days after termination )
Mann-Whitney test
Duncan's multiple range test
P
P
Corynebacterium parvum ( 70.0 mg kg - i )
evamso,e
( 3.0 m g k g - i ) PBS
N o patency
1280 -1
J
4Ool
< 0.05* < 0.05*
<0.05* < 0.001*** 3.1 _+ 0.367 <0.01"
>0.05 4.2_+0.644
B. DIVERGENS VACCINEDEVELOPMENT
185
Virulence control with anti-babesia drugs These experiments sought to evaluate the gerbil/B, divergens model as a means of identifying drugs that could be used to control vaccine virulence. In the first experiment, a specific babesicide, diminazene aceturate (Berenil, Hoechst AG, Frankfurt am Main, Germany), with limited prophylactic propTABLE 5 Chemovaccination of gerbils against B. divergens using diminazene aceturate (Berenil, Hoechst ) with 1 × 108 IRBC, s.c. Drug dose (mg kg-~ )
0 5 10 20 40
Infection on day after treatment
Infection on day of treatment Mean max. % P~
Infection mortality
Immunity to challenge
Mean max. % P
Infection mortality
Immunity to challenge
50.0 40.0 0.0 0.0 -
5/5 4/5 0/5 0/5 -
NA 0/5 0/5 1/5 -
50.0 40.5 30.6 0.21
5/5 4/5 3/5 0/5
NA 2 1/5 2/5 2/5
JP, parasitaemia.
2NA,not applicable. TABLE 6 Chemovaccination of gerbils against B. divergens using imidocarb dipropionate (Imizol, Coopers Pitman-Moore) and long-acting oxytetracycline (Terramycin LA, Pfizer) with 1 × 106 IRBC, i,p. Drug treatment group
Mean max. % P'
Imidocarb (1.5 mg kg - l on Day 0) Long-acting oxytetracycline (75.0 mgkg - l on Day 0) Untreated infected controls Unvaccinated challenge controls
4.06
0/5
5/5
3.90
0/5
5/5
50.0
10/10
NA 2
NA
NA
0/ 6
~P, parasitaemia.
2NA,not applicable.
Infection mortality
Immunity to challenge
186
J.S. GRAYANDP. GANNON
erties was used at doses ranging from 5.0 to 40.0 mg kg- 1 on the day of infection and 1 day before, with 108 IRBC given subcutaneously. Surviving animals were challenged with 10 s IRBC given intraperitoneally. It can be seen from Table 5 that this drug was unable to suppress the infection adequately at the lower doses and prevented the development of immunity at the higher doses. A second experiment was carried out with imidocarb, a recognised babesicide, and long-acting oxytetracycline (Terramycin LA, Pfizer, Sandwich, U K ) , which has no therapeutic activity, but has some prophylactic activity against B. divergens in cattle (Taylor et al., 1986). Infections of 1 0 6 IRBC were given by intraperitoneal injection to three groups of gerbils and the drugs were injected subcutaneously on the same day, at the rate of 1.5 mg kg- ~ for imidocarb and 75.0 mg kg- 1 for oxytetracycline. The third group of gerbils was left untreated as controls for the immunising infection. After 14 days all surviving gerbils plus a fourth group of previously uninfected and untreated controls were given a heavy intraperitoneal challenge of 108 IRBC to determine whether they were immune. The results show that both drugs suppressed the immunising infection, but allowed immunity to develop, so that all treated animals were resistant to challenge. All the controls for both the immunising and the challenge infections developed fulminating infections (Table 6). DISCUSSION
In this study the gerbil was investigated both as a source ofB. divergens live vaccine and as a model for the use of the vaccine in cattle. Infectivity was investigated in relation to donor parasitaemia and vaccine diluents. The infectivity of the vaccine did not vary significantly when obtained from gerbils with different parasitaemias, although the data suggested a trend of higher infectivity with higher parasitaemias. The best yield of blood is usually obtained at about 30% parasitaemia. A good diluent, R P M I / F C S 4 0 , was identified and it is unlikely that this could be improved upon, as it is essentially an in vitro culture medium. Cheaper diluents may, however, be developed with the substitution of the expensive foetal calf serum with a non-ruminant serum. Sera from young or adult ruminants cause lysis and agglutination of gerbil red blood cells. Although B. divergens infections in gerbils tend to be much more acute than in cattle, several studies suggest that the B. divergens/gerbil model may be quite relevant to many aspects of bovine babesiosis (Gray, 1983; Gray et al., 1985; Langley and Gray, 1987; Langley and Gray, 1989 ), more so in fact than rodent babesias such as Babesia microti, which are now acknowledged to be more distantly related to bovine babesias than previously thought (Friedhoff and Smith, 1981 ). Gerbils were used here to investigate ways of increasing the response of animals to the live vaccine. It was found that the host may
B. DIVERGENS VACCINEDEVELOPMENT
18 7
respond more vigorously to the vaccine when it is injected subcutaneously than when injected intraperitoneally and it is likely that draining lymph nodes as well as the spleen are involved in this effect. Taylor et al. (1983a) also reported a more vigorous response with a subcutaneous rather than intravenous injection ofB. divergens antigen in cattle. Immunostimulation seems to be a promising area for further research in cattle, as in gerbils C. parvum immunostimulation was especially successful and levamisole partially so. It is not possible to use C. parvum in cattle, but more work on levamisole and on other appropriate immunostimulants seems justified if avirulent vaccine strains of limited immunogenicity are used. With any live vaccine it is desirable to use an attenuated strain or to suppress the immunising infection in some way. Babesia divergens has not proved amenable to attenuation by rapid passage in cattle (Taylor et al., 1983b) or in gerbils (Murphy et al., 1986). However, the gerbil still has potential for the manipulation of parasite virulence by, for example, rapid passage in immunosuppressed animals using the subcutaneous infection route and it should also be possible to use gerbils to clone avirulent strains. For the present, however, it will probably be necessary to use drugs to control the virulence of B. divergens vaccines in older animals. Although immunisation may be achieved by treating symptoms caused by artificial infections with specific babesicides, this is not practical at the farm level. From this study it is evident that babesicides are not likely to be effective in immunisation programmes when given with or shortly before infections, unless they have marked prophylactic activity. At present the choice of suitable chemoprophylactics is limited in that imidocarb is not available throughout Europe, whereas long-acting oxytetracyclines are only effective at high concentrations and have not been sufficiently studied in cattle. Further study on chemoprophylactics, particularly with regard to alternative formulations and compounds, would be desirable. As a bovine source of live vaccine is not desirable, gerbils probably provide the best alternative. Rats can also be infected with B. divergens, but they must be splenectomised and the parasites require a period of adaptation (Phillips, 1984 ). In contrast, observations in this laboratory suggest that unsplenectomised gerbils are almost always susceptible to field strains, so that it should be possible to use gerbils at a local level. It is estimated that a single gerbil can provide between 150 and 200 vaccine inocula, which is quite adequate for small-scale vaccination programmes and the studies described here suggest that a robust reliable system using this source of vaccine could be developed. ACKNOWLEDGEMENTS
We are grateful to Coopers Pitman-Moore (Ireland) Ltd. and to Hoechst Ireland Ltd. for providing the babesicide drugs for this study and also to The Wellcome Foundation Ltd. (Beckenham, UK) for supplying C. parvum.
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J.S. G R A Y A N D P. G A N N O N
REFERENCES Friedhoff, K.T. and Smith, R.D., 1981. Transmission of Babesia by ticks. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 267-321. Friedhoff, K.T., Ganse-Dumrath, D., Weber, C. and Muller, I., 1989. Epidemiology and control of Babesia divergens infections in northern Germany. Proceedings of the 8th National Veterinary Hemoparasite Disease Conference, pp. 441-449. Gray, J.S., 1983. Chemotherapy ofBabesia divergens in the Mongolian gerbil, Meriones unguiculatus. Res. Vet. Sci., 35:318-324. Gray, J.S. and Harte, L.N., 1985. An estimation of the prevalence and economic importance of clinical babesiosis in the Republic of Ireland. Ir. Vet. J., 39: 75-78. Gray, J.S. and Kaye, B., 1991. Studies on the use of gerbil-derived Babesia divergens antigen for the diagnosis of bovine babesiosis. Vet. Parasitol., 39:215-224. Gray, J.S., Langley, R.J. and Murphy, T.M., 1985. Morphological comparisons of the bovine piroplasm, Babesia divergens, in cattle and jird (Meriones unguiculatus) erythrocytes. J. Parasitol., 71: 799-802. Gray, J.S., Langley, R.J., Brophy, P. and Gannon, P., 1989. Vaccination against bovine babesiosis with drug-controlled live parasites. Vet. Rec., 125: 369-372. Langley, R.J., 1985. The host-parasite relationship of the cattle piroplasm, Babesia divergens in the Mongolian gerbil, Meriones unguiculatus. Ph.D. Thesis, National University of Ireland, Dublin. Langley, R.J. and Gray, J.S., 1987. Age related susceptibility of the gerbil, Meriones unguiculatus, to the bovine parasite, Babesia divergens. Exp. Parasitol., 64: 466-473. Langley, R.J. and Gray, J.S., 1989. Non-specific resistance to Babesia divergens in the Mongolian gerbil (Meriones unguiculatus). Int. J. Parasitol., 19: 265-269. Lewis, D., Young, E.R., Baggot, D.G. and Osborn, G.D., 1981. Babesia divergens infection of the Mongolian gerbil: titration of infective dose and preliminary observations on the disease produced. J. Comp. Pathol., 91: 565-572. Murphy, T.M., Gray, J.S. and Langley, R.J., 1986. Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves. Res.Vet. Sci., 40: 285-287. Phillips, R.S., 1984. Babesia divergens in splenectomised rats. Res. Vet. Sci., 36:251-255. Purnell, R.E., Brocklesby, D.W., Hendry, D.J. and Young, E.R., 1976. Separation and recombination of Babesia divergens and Ehrlichia phagocytophila from a field case of redwater from Eire. Vet. Rec., 99: 415-417. Taylor, S.M., Kenny, J. and Mallon, T., 1983a. The effect of route of administration of a Babesia divergens inactivated vaccine on protection against homologous challenge. J. Comp. Pathol., 93: 423-428. Taylor, S.M., Kenny, J. and Mallon, T., 1983b. The effects of multiple rapid passage on strains of Babesia divergens: a comparison of the clinical effects on juvenile and adult cattle ofpassaged and irradiated parasites. J. Comp. Pathol., 93:391-396. Taylor, S.M., Elliott, C.T. and Kenny, J., 1986. Inhibition of Babesia divergens in cattle by oxytetracycline. Vet. Rec., 118: 98-102. Wright, I.A., 1991. Towards a synthetic Babesia vaccine. Int. J. Parasitol., 21:155-159.
179
Preliminary development of a live drugcontrolled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus J.S. Gray and P. Gannon ERM, Faculty of Agriculture, University College, Dublin, Ireland (Accepted 15 November 1991 )
ABSTRACT Gray, J.S. and Gannon, P., 1992. Preliminary development of a live drug-controlled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus. Vet. Parasitol., 42:179-188. This study investigated the practicality and potential of the gerbil, Meriones unguiculatus, as a source of live Babesia divergens vaccine and also as a model for the use of the vaccine in cattle. A series of experiments with gerbils concerningvaccine infectivity, immunogenicityand safety were carried out. It was concluded that the use of RPMI medium/40% foetal calf serum as a diluent improved vaccine infectivity, but that the parasitaemia of the blood obtained from donor gerbils had little or no effect. The immunostimulantslevamisole and killed Corynebacteriurn parvum improved vaccine immunogenicity and it was also shown that the subcutaneous route of infection resulted in the greatest host response. Control of vaccine virulence with drugs was only possible when drugs with prophylactic properties, such as imidocarb and long-acting oxytetracycline, were used. More studies are required on all these topics, particularly with regard to their applicability to cattle, and also concerning the possible attenuation of the parasite by manipulation in the gerbil host.
INTRODUCTION
Bovine babesiosis, caused by Babesia divergens, is widespread in Europe and has a particularly high prevalence in Ireland (Gray and Harte, 1985). Vaccination remains the best control option, but there have been considerable difficulties in the development of useable vaccines. These are the poor immunogenicityof non-living preparations, the difficulty of attenuating live vaccines and the reliance on a bovine source for such vaccines, with the attendant problems of transmission of other pathogens and of auto-immunity (Wright, 1991 ). The gerbil, Meriones unguiculatus, is highly susceptible to B. divergens and Correspondence to: J.S. Gray, Department of Environmental Resource Management, Faculty of Agriculture, University College, Belfield, Dublin 4, Ireland.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
180
J.S. GRAY A N D P. G A N N O N
this fact has been exploited in studies that sought to vaccinate cattle with gerbil-derived parasites (Friedhoff et al., 1989; Gray et al., 1989). Friedhoff et al. (1989) used only calves, the least susceptible age group, in extensive field studies without prophylactic drugs. However, this approach has limited applicability in Ireland, where it is necessary to vaccinate older cattle because of considerable movement of cattle. Gray et al. (1989) showed that it was possible to vaccinate cattle over 1 year of age when imidocarb was used as a prophylactic, resulting in long-lasting resistance to heterologous challenge. The only apparent weakness of the system was variable infectivity, and thus immunogenicity, of vaccine inocula for some cattle. It is evident that this aspect could be improved and the system otherwise optimised. The subjects addressed in these experiments were infectivity of the inocula, immunoresponsiveness of the host and chemoimmunisation. MATERIALS AND METHODS
Experimental animals The gerbils were bred in the University animal facility from a stock of five pairs obtained from Bantin and Kingman Ltd. (Hull, UK). The first progeny of these animals were examined and were found to be free of parasites and other pathogens. The animals were used in groups of five when 12-16 weeks old, the females weighing at least 50 g and the males 60 g.
Parasites The B. divergens strain used was originally isolated in Ireland (Purnell et al., 1976) and designated CE2. Following use in experimental cattle it was passaged in gerbils and when used here it had undergone more than 80 gerbil passages.
General procedures Unless otherwise stated, parasite infections were initiated by intraperitoneal (i.p.) injections of infected red blood cells (IRBC) in 0.1 ml of phosphate buffered saline (PBS). For routine maintenance of the parasite, 10 7 IRBC were injected i.p., but for experimental infections the dose level ranged from 104 to 108 IRBC, depending on the particular experiment and the route of infection used. Blood parasitaemias were monitored daily from thin blood smears prepared from tail blood and stained in Giemsa. Blood for serum antibody analysis was obtained by cardiac puncture (0.2 ml) under halothane anaesthesia. Antibody levels were measured with the indirect immunofluorescent antibody test (IFAT) using a triple sandwich
B. DIVERGENS VACCINEDEVELOPMENT
181
technique described in detail by Langley and Gray ( 1987 ). The method consists essentially of incubation of antigen on multiwell slides with test sera, followed by incubation with anti-gerbil IgG (raised in rabbits) at 1 : 1000 dilution and finally incubation with a goat anti-rabbit fluorescein-isothiocyanate IgG (Miles Laboratories, Bridgend, UK) at 1:80 dilution. After washing in PBS the slides were examined under blue light with a Leitz Dialux epifluorescent microscope system and a titre obtained for each sample. In some experiments readings were obtained from a white light rheostat which was used to match the intensity of the fluorescence (Gray and Kaye, 1991 ). Other procedures are described where the particular experiments in which they were used are considered.
Experimental design A series of experiments were carried out to investigate three different topics: ( 1 ) infectivity of the vaccine, determined by varying blood parasitaemias in donor gerbils and by different inoculum diluents; (2) immunogenicity of the vaccine, determined by non-specific immunostimulation and by different routes of infection; (3) control of parasite virulence with drugs.
Statistical analysis Unless otherwise indicated, results from experimental groups were compared with controls using Student's t-test. When more than one group were being compared, analysis of variance and Duncan's multiple range test were carried out. Percentage data were transformed to arcsines before analysis. RESULTS
Effects of different donor parasitaemias on infectivity Blood was obtained from three gerbils with parasitaemias of 6.6, 26.8 and 72.1%. Inocula of 105 IRBC per 0.1 ml were prepared from each source after dilution with PBS and injected intraperitoneally into each of five gerbils which had been pretreated subcutaneously with 0.5 mg kg-l dexamethasone (Dexafort, Intervet Laboratories Ltd., Cambridge, UK) in order to reduce variations caused by resistance mechanisms. When the mean times taken to 1.0% parasitaemias (4.2 days, 4.0 days and 4.0 days, respectively) and the mean parasitaemias on Day 4 of infection (3.62%, 2.84% and 2.20% respectively) were compared, no significant differences between the three groups were evident.
182
J.S. GRAY ANDP. GANNON
Effect of vaccine diluent on infectivity Inocula containing 105 IRBC were stored in five different diluents for 24 h at 4 ° C before intraperitoneal inoculation into groups of five gerbils. The diluents were PBS, Eagle's minimum essential medium (EMEM), RPMI- 1640 culture medium with 25 mM Hepes and L-glutamine (RPMI), RPMI plus 10% foetal calf serum (RPMI/10FCS ) and RPMI plus 40% foetal calf serum (RPMI/40FCS). A sixth group of gerbils were infected with 105 fresh IRBC in PBS. The results showed that the best diluent was RPMI/40FCS and furthermore, that there was no significant difference in the infectivity of parasites in this diluent compared with fresh parasites (Table 1 ).
Route of infection studies Previous studies established that M. unguiculatus resists primary infections inoculated subcutaneously more readily than those inoculated intraperitoneally or by the cardiac route (Langley, 1985 ). A series of experiments was carried out to investigate this phenomenon further and it was found that greater splenomegaly occurred following subcutaneous infection compared with the intraperitoneal route and was accompanied by the enlargement of the lymph nodes draining the site of the subcutaneous inoculations (Table 2 ). The importance of the subcutaneous route in relation to splenic activity and resistance was also indicated by the fact that dexamethasone treatment only increased susceptibility if infections were given subcutaneously and furthermore, that whereas splenectomy has no effect in adult gerbils when the intraperitoneal infection route is used (Lewis et al., 1981; Langley, 1985), TABLE1 Effect of diluent on B. divergens inoculum infectivity in gerbils (24 h after 4 °C storage, 1 X 105 IRBC, i.p.)
Mean days to 1.0% parasitaemia _+SE
PBS
EMEM
RPMI
RPMI/ 10FCS
RPM1/ 40FCS
PBS (Day 0)
7.8 +0.288
9.5 + 1.584
7.2 +0.374
6.1 +0.510
4.8 +0.583
4.2 _+0.10
Values underscored by the same line are not significantly different at P < 0 . 0 5 (Duncan's multiple range test). PBS, phosphate buffered saline; EMEM, Eagle's m i n i m u m essential medium; RPMI, standard medium, 25 m m Hepes, L-glutamine; FCS, foetal calf serum.
183
B. DIVERGENSVACCINEDEVELOPMENT TABLE 2 The effect ofB. divergens route of infection on splenomegaly and lymph node enlargement Infection route
Infection level Mean spleen weight (mg+SE)
Subcutaneous
5X10 7
Intraperitoneal
5× 105
169.2+16.52 113.9+ 9.05
Student's t
2.93 P < 0.02
Mean axillary lymph Student's t node weight (mg+SE) 5.15+1.14 1.59+0.13
2.79 P < 0.05
TABLE 3 Effect ofsplenectomy and dexamethasone on the susceptibility of gerbils to intraperitoneal (i.p.) and subcutaneous (s.c.) B. divergens infections Mean days to 1.0% parasitaemia_+ SE Splenectomised ( 106 IRBC s.c. ) Intact ( 106 IRBC s.c.) Dexamethasone ( 106 IRBC s.c. ) Untreated ( 10 6 IRBC s.c. ) Dexamethasone ( 10 4 IRBC i.p. ) Untreated ( 104 IRBC i.p. )
Student's t
4.83 + 0.48 3.16
P < 0.02
4.747
P < 0.005
0.681
P > 0.1
6.50 + 0.22 3.80 + 0.200 6.40 + 0.510 3.75 + 0.414 4.17 + 0.597
splenectomised animals were more susceptible than intact ones when infections were given subcutaneously (Table 3 ). The antibody response to infections induced by subcutaneous and intraperitoneal infections were compared in an experiment in which blood for IFA serology was obtained from gerbils 6 days after subcutaneous and intraperitoneal infection with 107 and 105 IRBC, respectively, when parasitaemias had reached 40%. Although the antibody levels were much lower than would be obtained in recovered gerbils, those that received subcutaneous infections showed significantly higher fluorescence extinction values than those that received intraperitoneal infections (Student's t= 3.53, P< 0.005). A second experiment involving gerbils, in which infections were terminated with 6.0 mg kg -~ of the babesicide imidocarb dipropionate (Imizol, Coopers PitmanMoore, Berkhamstead, UK), failed to reproduce this effect. This was probably because any difference was obscured by the high antibody levels resulting from the effect of the babesicide on the parasites. Although maximum paras-
184
J.S. GRAY AND P. GANNON
itaemias of the challenge infections in this second experiment were lower in the subcutaneously infected gerbils, this difference was not statistically significant.
Non-specific imrnunostimulation The object of this experiment was to increase host immune responsiveness without affecting the viability of the infective inoculum. This was done by treating two groups of animals with two immunostimulants; a killed preparation of Corynebacterium parvum (The Wellcome Foundation Ltd., Beckenham, UK) and the anthelmintic, levamisole (Levacide, Norbrook Laboratories Ltd., Newry, Northern Ireland) ), at infection and again 7 days later. A third group of gerbils received PBS injections only. The infections of 2 × 10 7 IRBC were administered intraperitoneally and then terminated on Day 3 by a subcutaneous injection of 6.0 mg kg-1 imidocarb. The level of immunostimulation was determined by measuring seroconversion using the IFAT on serum samples obtained on Day 10 after infection. On Day 17 all gerbils were challenged with 10 8 IRBC by intraperitoneal injection. The results show significantly higher antibody levels in both C. parvum and levamisole treated gerbils than in the untreated animals. All gerbils survived the challenge infection. In the C. parvum treated animals the infections did not become patent, whereas in the other two groups low level patency was reached (Table 4). TABLE4
I mmunostimulation of drug-terminated B. divergens i n f e c t i o n s (2 × l07 I R B C i.p. terminated on Day 3, 6.0 m g kg -~ imidocarb) lmmunostimulants Median IFA titre ( D a y s 0,7 after (7 days after infection ) termination )
Mean time (days) to clear after challenge ( 1 X 10 s I R B C 14 days after termination )
Mann-Whitney test
Duncan's multiple range test
P
P
Corynebacterium parvum ( 70.0 mg kg - i )
evamso,e
( 3.0 m g k g - i ) PBS
N o patency
1280 -1
J
4Ool
< 0.05* < 0.05*
<0.05* < 0.001*** 3.1 _+ 0.367 <0.01"
>0.05 4.2_+0.644
B. DIVERGENS VACCINEDEVELOPMENT
185
Virulence control with anti-babesia drugs These experiments sought to evaluate the gerbil/B, divergens model as a means of identifying drugs that could be used to control vaccine virulence. In the first experiment, a specific babesicide, diminazene aceturate (Berenil, Hoechst AG, Frankfurt am Main, Germany), with limited prophylactic propTABLE 5 Chemovaccination of gerbils against B. divergens using diminazene aceturate (Berenil, Hoechst ) with 1 × 108 IRBC, s.c. Drug dose (mg kg-~ )
0 5 10 20 40
Infection on day after treatment
Infection on day of treatment Mean max. % P~
Infection mortality
Immunity to challenge
Mean max. % P
Infection mortality
Immunity to challenge
50.0 40.0 0.0 0.0 -
5/5 4/5 0/5 0/5 -
NA 0/5 0/5 1/5 -
50.0 40.5 30.6 0.21
5/5 4/5 3/5 0/5
NA 2 1/5 2/5 2/5
JP, parasitaemia.
2NA,not applicable. TABLE 6 Chemovaccination of gerbils against B. divergens using imidocarb dipropionate (Imizol, Coopers Pitman-Moore) and long-acting oxytetracycline (Terramycin LA, Pfizer) with 1 × 106 IRBC, i,p. Drug treatment group
Mean max. % P'
Imidocarb (1.5 mg kg - l on Day 0) Long-acting oxytetracycline (75.0 mgkg - l on Day 0) Untreated infected controls Unvaccinated challenge controls
4.06
0/5
5/5
3.90
0/5
5/5
50.0
10/10
NA 2
NA
NA
0/ 6
~P, parasitaemia.
2NA,not applicable.
Infection mortality
Immunity to challenge
186
J.S. GRAYANDP. GANNON
erties was used at doses ranging from 5.0 to 40.0 mg kg- 1 on the day of infection and 1 day before, with 108 IRBC given subcutaneously. Surviving animals were challenged with 10 s IRBC given intraperitoneally. It can be seen from Table 5 that this drug was unable to suppress the infection adequately at the lower doses and prevented the development of immunity at the higher doses. A second experiment was carried out with imidocarb, a recognised babesicide, and long-acting oxytetracycline (Terramycin LA, Pfizer, Sandwich, U K ) , which has no therapeutic activity, but has some prophylactic activity against B. divergens in cattle (Taylor et al., 1986). Infections of 1 0 6 IRBC were given by intraperitoneal injection to three groups of gerbils and the drugs were injected subcutaneously on the same day, at the rate of 1.5 mg kg- ~ for imidocarb and 75.0 mg kg- 1 for oxytetracycline. The third group of gerbils was left untreated as controls for the immunising infection. After 14 days all surviving gerbils plus a fourth group of previously uninfected and untreated controls were given a heavy intraperitoneal challenge of 108 IRBC to determine whether they were immune. The results show that both drugs suppressed the immunising infection, but allowed immunity to develop, so that all treated animals were resistant to challenge. All the controls for both the immunising and the challenge infections developed fulminating infections (Table 6). DISCUSSION
In this study the gerbil was investigated both as a source ofB. divergens live vaccine and as a model for the use of the vaccine in cattle. Infectivity was investigated in relation to donor parasitaemia and vaccine diluents. The infectivity of the vaccine did not vary significantly when obtained from gerbils with different parasitaemias, although the data suggested a trend of higher infectivity with higher parasitaemias. The best yield of blood is usually obtained at about 30% parasitaemia. A good diluent, R P M I / F C S 4 0 , was identified and it is unlikely that this could be improved upon, as it is essentially an in vitro culture medium. Cheaper diluents may, however, be developed with the substitution of the expensive foetal calf serum with a non-ruminant serum. Sera from young or adult ruminants cause lysis and agglutination of gerbil red blood cells. Although B. divergens infections in gerbils tend to be much more acute than in cattle, several studies suggest that the B. divergens/gerbil model may be quite relevant to many aspects of bovine babesiosis (Gray, 1983; Gray et al., 1985; Langley and Gray, 1987; Langley and Gray, 1989 ), more so in fact than rodent babesias such as Babesia microti, which are now acknowledged to be more distantly related to bovine babesias than previously thought (Friedhoff and Smith, 1981 ). Gerbils were used here to investigate ways of increasing the response of animals to the live vaccine. It was found that the host may
B. DIVERGENS VACCINEDEVELOPMENT
18 7
respond more vigorously to the vaccine when it is injected subcutaneously than when injected intraperitoneally and it is likely that draining lymph nodes as well as the spleen are involved in this effect. Taylor et al. (1983a) also reported a more vigorous response with a subcutaneous rather than intravenous injection ofB. divergens antigen in cattle. Immunostimulation seems to be a promising area for further research in cattle, as in gerbils C. parvum immunostimulation was especially successful and levamisole partially so. It is not possible to use C. parvum in cattle, but more work on levamisole and on other appropriate immunostimulants seems justified if avirulent vaccine strains of limited immunogenicity are used. With any live vaccine it is desirable to use an attenuated strain or to suppress the immunising infection in some way. Babesia divergens has not proved amenable to attenuation by rapid passage in cattle (Taylor et al., 1983b) or in gerbils (Murphy et al., 1986). However, the gerbil still has potential for the manipulation of parasite virulence by, for example, rapid passage in immunosuppressed animals using the subcutaneous infection route and it should also be possible to use gerbils to clone avirulent strains. For the present, however, it will probably be necessary to use drugs to control the virulence of B. divergens vaccines in older animals. Although immunisation may be achieved by treating symptoms caused by artificial infections with specific babesicides, this is not practical at the farm level. From this study it is evident that babesicides are not likely to be effective in immunisation programmes when given with or shortly before infections, unless they have marked prophylactic activity. At present the choice of suitable chemoprophylactics is limited in that imidocarb is not available throughout Europe, whereas long-acting oxytetracyclines are only effective at high concentrations and have not been sufficiently studied in cattle. Further study on chemoprophylactics, particularly with regard to alternative formulations and compounds, would be desirable. As a bovine source of live vaccine is not desirable, gerbils probably provide the best alternative. Rats can also be infected with B. divergens, but they must be splenectomised and the parasites require a period of adaptation (Phillips, 1984 ). In contrast, observations in this laboratory suggest that unsplenectomised gerbils are almost always susceptible to field strains, so that it should be possible to use gerbils at a local level. It is estimated that a single gerbil can provide between 150 and 200 vaccine inocula, which is quite adequate for small-scale vaccination programmes and the studies described here suggest that a robust reliable system using this source of vaccine could be developed. ACKNOWLEDGEMENTS
We are grateful to Coopers Pitman-Moore (Ireland) Ltd. and to Hoechst Ireland Ltd. for providing the babesicide drugs for this study and also to The Wellcome Foundation Ltd. (Beckenham, UK) for supplying C. parvum.
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J.S. G R A Y A N D P. G A N N O N
REFERENCES Friedhoff, K.T. and Smith, R.D., 1981. Transmission of Babesia by ticks. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 267-321. Friedhoff, K.T., Ganse-Dumrath, D., Weber, C. and Muller, I., 1989. Epidemiology and control of Babesia divergens infections in northern Germany. Proceedings of the 8th National Veterinary Hemoparasite Disease Conference, pp. 441-449. Gray, J.S., 1983. Chemotherapy ofBabesia divergens in the Mongolian gerbil, Meriones unguiculatus. Res. Vet. Sci., 35:318-324. Gray, J.S. and Harte, L.N., 1985. An estimation of the prevalence and economic importance of clinical babesiosis in the Republic of Ireland. Ir. Vet. J., 39: 75-78. Gray, J.S. and Kaye, B., 1991. Studies on the use of gerbil-derived Babesia divergens antigen for the diagnosis of bovine babesiosis. Vet. Parasitol., 39:215-224. Gray, J.S., Langley, R.J. and Murphy, T.M., 1985. Morphological comparisons of the bovine piroplasm, Babesia divergens, in cattle and jird (Meriones unguiculatus) erythrocytes. J. Parasitol., 71: 799-802. Gray, J.S., Langley, R.J., Brophy, P. and Gannon, P., 1989. Vaccination against bovine babesiosis with drug-controlled live parasites. Vet. Rec., 125: 369-372. Langley, R.J., 1985. The host-parasite relationship of the cattle piroplasm, Babesia divergens in the Mongolian gerbil, Meriones unguiculatus. Ph.D. Thesis, National University of Ireland, Dublin. Langley, R.J. and Gray, J.S., 1987. Age related susceptibility of the gerbil, Meriones unguiculatus, to the bovine parasite, Babesia divergens. Exp. Parasitol., 64: 466-473. Langley, R.J. and Gray, J.S., 1989. Non-specific resistance to Babesia divergens in the Mongolian gerbil (Meriones unguiculatus). Int. J. Parasitol., 19: 265-269. Lewis, D., Young, E.R., Baggot, D.G. and Osborn, G.D., 1981. Babesia divergens infection of the Mongolian gerbil: titration of infective dose and preliminary observations on the disease produced. J. Comp. Pathol., 91: 565-572. Murphy, T.M., Gray, J.S. and Langley, R.J., 1986. Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves. Res.Vet. Sci., 40: 285-287. Phillips, R.S., 1984. Babesia divergens in splenectomised rats. Res. Vet. Sci., 36:251-255. Purnell, R.E., Brocklesby, D.W., Hendry, D.J. and Young, E.R., 1976. Separation and recombination of Babesia divergens and Ehrlichia phagocytophila from a field case of redwater from Eire. Vet. Rec., 99: 415-417. Taylor, S.M., Kenny, J. and Mallon, T., 1983a. The effect of route of administration of a Babesia divergens inactivated vaccine on protection against homologous challenge. J. Comp. Pathol., 93: 423-428. Taylor, S.M., Kenny, J. and Mallon, T., 1983b. The effects of multiple rapid passage on strains of Babesia divergens: a comparison of the clinical effects on juvenile and adult cattle ofpassaged and irradiated parasites. J. Comp. Pathol., 93:391-396. Taylor, S.M., Elliott, C.T. and Kenny, J., 1986. Inhibition of Babesia divergens in cattle by oxytetracycline. Vet. Rec., 118: 98-102. Wright, I.A., 1991. Towards a synthetic Babesia vaccine. Int. J. Parasitol., 21:155-159.