Parasitology (1977), 74, 291-298
291
Babesia bigemina in Kenya: experimental transmission by Boophilus decoloratus and the production of tick-derived stabilates S. P. MORZARIA,* A. S. YOUNG.f and E. B. HUDSONJ Immunological Research on Tick-borne Cattle Diseases and Tick Control Project, East African Veterinary Research Organization, Muguga, P.O. Box 32, Kikuyu, Kenya {Received 12 October 1976) SUMMARY
A Babesia-free batch of laboratory reared Boophilus decoloratus ticks was infected with a Kenyan strain of Babesia bigemina by feeding them on a steer infected by inoculation of a blood stabilate. The engorged female ticks showed developing stages of B. bigemina in their haemolymph and subsequently their progeny transmitted the parasite to a susceptible splenectomized steer. Attempts were made to produce stabilates from pre-fed larvae and nymphs derived from infected batches of B. decoloratus. Only the stabilates derived from the nymphs pre-fed either on cattle or rabbits produced B. bigemina infection when inoculated intravenously into susceptible cattle. INTBODTJCTION
Babesia bigemina is a widely distributed haemoprotozoan parasite which causes red water fever of cattle with resultant serious economic losses. In many areas of the world the tick vectors of B. bigemina have been identified (Riek, 1966). In most areas of Africa, however, the tick vectors have yet to be established. Several tick species, including the one-host tick Boophilus decoloratus, have been reported to transmit B. bigemina in South Africa. Throughout East Africa, B. decoloratus and Rhipicephalus appendiculatus have been implicated as vectors of B. bigemina but there is no experimental evidence for this. Purnell, Branagan & Brown (1970) attempted to transmit B. bigemina with R. appendiculatus, but without success. In this paper we report the transmission of a Kenyan strain of B. bigemina by B. decoloratus and we describe the production of stabilates derived from ticks infected with B. bigemina. * Present address: Central Veterinary Laboratory, Department of Virology, New Haw, Weybridge, Surrey, U.K. t FAO Staff member. % ODM Staff member, seconded from Central Veterinary Laboratory, Weybridge, U.K.
292
S. P. MORZARIA, A. S. Y O U N G AND E. B. H U D S O N MATERIALS AND M E T H O D S
Cattle used were of the Bos taurus type and were over 6 months of age. For 7 days prior to experiments, blood smears taken from the cattle were stained with Giemsa and examined for the presence of Babesia parasites. In addition, sera from the cattle were tested for the presence of Babesia antibodies using the indirect fluorescent-antibody (IFA) test with B. bigemina piroplasm antigen (Ross & Lohr, 1968). Only those cattle negative for both Babesia parasites and antibodies (a titre of 1/40 or less) were used in experiments. In certain cases cattle were splenectomized before experiments. The rabbits were chinchilla crosses from the East African Veterinary Research Organization (EAVRO) small animal unit. The B. decoloratus colony was established from engorged female ticks collected from two cattle held at EAVRO. They had been fed for 2 generations on susceptible cattle and did not transmit B. bigemina to them. These ticks were designated a clean (uninfected with B. bigemina) strain of B. decoloratus. The B. bigemina strain was obtained from a calf which died of babesiosis at EAVRO. Before the calf died, blood from the jugular vein was collected in heparin and was cryopreserved as a stabilate by Dr K. F. Lohr using glycerol as a cryoprotectant (Lumsden, 1972). It was then stored in liquid nitrogen. The strain was designated B. bigemina (Muguga). During experiments rectal temperatures of the cattle were taken daily and a rectal temperature of above 39-4 °C associated with Babesia infection was considered indicative of a febrile response. Also, blood smears were made daily from the ear veins, stained in Giemsa and examined for the presence of intraerythrocytic piroplasms. If blood slides of experimental cattle remained Babesianegative for 100 days, examinations were discontinued. For haematology, experimental cattle were bled 3 times a week from the jugular vein, starting a week prior to the experiments. Blood was collected with the potassium salt of ethylenediaminotetra-acetic acid (EDTA) as an anticoagulant. The haematology was determined using a Coulter counter Model ZBI and a Coulter haemoglobinometer (Coulter Electronics Inc., Florida, USA). Blood was also obtained from the jugular veins of the experimental cattle at weekly intervals, allowed to clot and the serum was separated for the IFA test. The B. bigemina parasitaemia was expressed as the number of infected cells per 103 red blood cells (RBC). B. decoloratus were fed on cattle by sprinkling larvae on the backs of the animals. During tick feeding the cattle were housed in a pen which was surrounded by a water moat. B. decoloratus were fed on rabbits by applying larvae to their ears according to the methods of Bailey (1960). The development of B. decoloratus ticks feeding on experimental cattle was checked by removing samples from the host at regular intervals and examining the ticks under a dissecting microscope. Replete females were collected 4 times daily and kept at 24 °C and 70% relative humidity (rel. hum.) during oviposition and hatching of eggs. Unfed larvae were maintained at 20 °C and at 80 % rel. hum. The infection rate of B. bigemina vermicules (Riek, 1964) in replete females was determined
Tick transmission of Babesia bigemina
293
by the examination of Giemsa-stained haemolymph smears produced by severing legs of the ticks 10 days after repletion. Suspensions of B. bigemina parasites for cryopreservation were produced from infected B. decoloratus larvae and nymphs which had been pre-fed on cattle or rabbits for 3-4 days. These ticks were removed from the hosts, ground up in a mortar and pestle with washed sand as an abrasive and Eagle's Minimum Essential Medium (MEM) with 3-4 % bovine plasma albumin (BPA) as a diluent (Cunningham, Brown, Purnell & Burridge, 1973). The suspensions were centrifuged at 50g for 5 min and the supernatant fluid removed. An equal volume of MEM/BPA with 20 % dimethyl sulphoxide (Me2SO) was added gradually to the supernatant fluid to give a final proportion by volume of 10% Me2SO as a cryoprotectant. The final suspension was dispensed in 2-5 ml volume glass ampoules insulated by a thin sheet of corrugated paper. The ampoules were frozen in an ultra-low refrigerator (Revco Inc., South Carolina, USA) to — 80 °C at a cooling rate of approximately 1 °C/min (M. P. Cunningham, unpublished results). Before use ampoules were removed and thawed rapidly. Experiment 1 The purpose of this experiment was to determine whether clean B. decoloratus ticks would become infected with B. bigemina when they engorged on a parasitaemic animal. A susceptible steer E325 was infested with approximately 2 x 103 B. decoloratus larvae obtained from the clean colony. Nineteen days later when most of the ticks had moulted into adults and had re-attached, the steer was inoculated subcutaneously with 7 ml of B. bigemina (Muguga) blood stabilate and monitored for Babesia infection. The replete females were collected and their haemolymph smears examined for B. bigemina infection. To test the infectivity of the larvae, 5 x 103 3-month-old larvae derived from the above females were applied to a susceptible splenectomized animal K46. The animal was examined for the development of a Babesia infection and the engorged females from this animal were examined for developing parasites. Experiment 2 Attempts were made to produce B. bigemina stabilates derived from infected B. decoloratus ticks pre-fed on cattle. Approximately 103 larvae and 400 nymphs were removed 3—4 days after attachment to K46 and ground up as described above to produce final volumes of 20 ml of larval suspension and 18 ml of nymphal suspension after adding MEM/BPA and Me2SO. Two susceptible steers K171 and K47 were inoculated intravenously (i.v.) with 10 ml of the larval suspension and 9 ml of the nymphal suspension respectively. The steers were examined for the development of B. bigemina infections. The remainder of the suspension was cryopreserved as described above. Forty-one days after freezing, two ampoules of the frozen nymphal suspension (St. 122) were thawed rapidly and 4 ml inoculated i.v. into animal K774 and 1 ml i.v. into animal J138. 2O
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Experiment 3 The purpose of this experiment was to produce B. bigemina stabilates from infected B. decoloratus nymphs pre-fed on rabbits. B. decoloratus larvae (2 x 104) derived from infected female ticks which had engorged during the period of patent parasitaemia in K46, were applied to 5 rabbits. After the larvae had developed into nymphs and had been feeding on the rabbits for 3—4 days, they were scraped off the rabbits' ears and processed as described previously. The final volume of the suspension containing 10 % Me2SO was 134 ml. The suspension was cryopreserved. Twelve days after freezing the contents of two ampoules of the cryopreserved suspension (St. 126) were inoculated i.v. into 2 susceptible steers K332 and K338. A susceptible steer J867, which had been splenectomized previously, was inoculated i.v. with 5 ml of St. 126 3 months after freezing. Nine months after freezing, 5 ml of St. 126 was inoculated into a susceptible splenectomized calf L580. RESULTS
Experiment 1 E325 developed a patent B. bigemina infection 5 days after inoculation of the stabilate (24 days after the application of uninfected ticks). By this time, the ticks on the steer had developed into B. decoloratus adults and many of the engorging females were dropping. The parasitaemia remained patent at levels of 1 per 103 RBC's or less for 9 days during which time all the ticks had completed engorgement. The engorged females which dropped from the steer during different days of the parasitaemia showed Babesia infection rates in their haemolymph Tanging from 20% to 100%. E325 showed no clinical symptoms associated with Babesia infection, but showed an IFA response (1/2560 titre by day 35). Steer K46, to which the larvae derived from the above females were applied, developed a patent B. bigemina infection 19 days after the application of the ticks. By day 22 the parasitaemia had reached 150 per 103 RBC's and the steer showed a marked degree of anaemia (packed cell volume of 10%). The animal also developed a febrile response; the maximum rectal temperature being 41-0 °C on day 21 post-tick application. The steer was treated with diminazene aceturate (Berenil, Hoechst Pharmaceuticals, Frankfurt(M), W. Germany) on day 23 to prevent death. The steer developed antibodies to B. bigemina reaching a maximum titre of 1/2560 on day 35. The engorged females obtained from K46 pre-treatment showed a Babesia infection rate of 80%. Experiment 2 The unfrozen nymphal suspension inoculated into K47 produced a B. bigemina infection while the unfrozen larval suspension failed to infect K171. K47 developed an antibody titre and anaemia was apparent (Table 1). However, no febrile response occurred. The infectivity of the cryopreserved larval suspension was not tested since
St. 126
St. 126
St. 126
St. 126
St. 122
of
Nymphs prefed on K46 Nymphs prefed on K46 Nymphs prefed on rabbits Nymphs prefed on rabbits Nymphs prefed on rabbits Nymphs prefed on rabbits
K46
Pre-fed on
K46
ticks Pre-fed on
Source
or
stabilate (St.) no. Larval suspension Nymphal suspension St. 122
Suspension
285
90
12
12
41
41
1 1 5 5
K332 J867* L580*
4
1
9
K338
K774
J138
K47
K171
Cattle no. inoculated
7
0
11
8
7
6
4
18
12f
11
11
10
10
12
Figures in parentheses are days after inoculation. * Spleneotomized animal, t Day of death.
Pre-freeze
No. days of oryopreservation Pre-freeze
Volume
71
34
29
31
< 1 260
33
29
32
34
30
7
30
60
—
7(11) 9(18)
1/2560 (28)
1/10240 (35)
23 (14) 25 (13)
1/2560 (35)
1/2560 (28)
18(12) 24 (13)
1/640 (28)
16(14)
Peak Pre- Days to para- Packed Packed cell inpatent peak sitaemia cell vol. vol. lowest per % pre- level % post- Peak IFA titre oculated period para(days) sitaemia KPRBC infection infection (ml) post-infection < 1/40 31 28 (15) 10
Table 1. Infectivity for cattle of suspensions derived from Boophilus decoloratus larvae and nymphs infected with Babesia bigemina
V
CD
IsS
eg" 8 B"
o*
82.
CD
w §•
&
I
296
S. P. MORZARIA, A. S. YOUNG AND E. B. HUDSON
the initial viability test was negative. Thawed nymphal suspension (St. 122) which was inoculated into steers K774 and J138 41 days after cryopreservation produced B. bigemina infection in both cattle. Anaemia developed in both animals but a febrile response was only observed in K774. The cattle showed a rise in B. bigemina antibody titres and recovered without treatment. (Table 1). Experiment 3
K338 and K332 which were inoculated with cryopreserved nymphal suspensions (St. 126) derived from ticks pre-fed on rabbits developed B. bigemina infection. No febrile response was observed but both developed a mild anaemia and showed significant rises in IFA titres (Table 1). After 3 months of cryopreservation St. 126 was inoculated into a splenectomized animal J867 and a fatal B. bigemina infection was produced associated with a febrile response and acute anaemia (Table 1). Similarly, L580 which was inoculated with St. 126 9 months after cryopreservation underwent a fatal Babesia infection. DISCUSSION
We have demonstrated that the experimental transmission of a Kenyan strain of B. bigemina by B. decoloratus was efficient. High percentages of ticks became infected after feeding on cattle with low Babesia parasitaemias. It is therefore probable that B. decoloratus is a natural vector of B. bigemina in Kenya. It would be of importance to identify the tick vectors of B. bigemina in East Africa so that red water fever could be controlled more efficiently. The method of transmission of the Kenyan strain of B. bigemina by B. decoloratus is similar to the transmission of B. bigemina by B. microplus in Australia (Riek, 1964). The female ticks became infected during feeding on an animal with a patent B. bigemina parasitaemia. Examination of the haemolymph smears from the engorged female Tevealed both a high Babesia infection rate and a high grade of infection in many ticks. Subsequently larvae derived from the female ticks produced B. bigemina infection in a susceptible steer during the course of their feeding and development into adult ticks. Thus trans-ovarian transmission was demonstrated. We obtained evidence that the nymphal stages of the ticks were responsible for the transmission of B. bigemina to cattle. Suspensions from nymphal ticks produced infections in cattle but larval suspensions did not. This agrees with the findings of Callow & Hoyte (1961) who reported that they were unable to transmit B. bigemina by the larvae of B. microplus. However, further work is required to demonstrate whether the larval or nymphal stages of B. decoloratus can acquire B. bigemina and whether adults can transmit the infection. This is the first time that B. bigemina stabilates derived from ticks have been produced. The nymphal ticks used had to be pre-fed since B. bigemina undergoes a maturation cycle in salivary glands of the nymphal tick to produce a form infective to cattle (Riek, 1964). Previously, Mahoney & Mirre (1974), Potgieter & Van Vuuren (1974) and Morzaria, Brocklesby, Harradine & Luther (1977) nave been able to produce tick-derived B. argentina, B. bovis and B. major
Tick transmission of Babesia bigemina
297
stabilates respectively. The use of tick-derived stabilates for experimental infection of cattle has certain advantages. For example, most of the experimental studies on Babesia infection in cattle have been blood-induced, which is an unnatural method ot infection. Infections produced by tick feeding are difficult to monitor since the feeding ticks may emit parasites over a long period (Morzaria et al. 1977), and the success of tick feeding on individual animals may vary considerably. Also, if the parasites are maintained continuously in ticks or by alternate tick/ animal passage they may change their characteristics. The tick-derived stabilates produced in the present experiments remained infective for at least 9 months and should remain infective for several years without change. Mahoney & Mirre (1974) suggested that tick-derived Babesia stabilates may provide material for the vaccination of cattle against babesiosis. The present method of vaccination as practised in Australia (Callow, 1976), and to a limited extent in Kenya, depends on the inoculation of cattle with blood infected with the relevant Babesia species. This method of vaccination involves dangers, including the contamination of blood with other organisms infective to cattle and the induction of haemolytic disease. It should be possible to immunize cattle with a controlled inoculum of tick-derived Babesia stabilates produced from infected ticks pre-fed on rabbits. This would reduce the dangers inherent in vaccination with bovine blood. It is also possible that Babesia immunization could be combined with immunization against theileriosis by chemoprophylaxis which is being developed at EAVRO (Radley, Brown, Cunningham, Kimber, Musisi, Payne, Purnell, Stagg & Young, 1975). This project is supported by the United Nations Development Programme with the Food and Agriculture Organization of the United Nations as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Ministry of Overseas Development of the United Kingdom (Research Projects R 2396, R 2492 & R 2845 A & B), the United States Department of Agriculture, the Rockefeller Foundation, the International Atomic Energy Agency and the Pfizer Corporation Inc. We are grateful to our colleagues on the Project for advice and assistance. We would especially like to thank Dr K. F. Lohr of the Veterinary Laboratories, Kabete, for providing us with B. bigemina blood stabilate. We would also like to thank Dr G. Maxie of the IDRC Project, EAVRO, for supervision of the haematology. This paper is published by kind permission of the Director of EAVRO, Muguga, Kenya. REFERENCES
K. P. (1960). Notes on the rearing of Rhipicephaltcs appendiculatua and their infection with Theileria parva for experimental transmission. Bulletin of Epizootic Diseases of Africa 8, 33-43. CALLOW, L. L. (1976). Tick-borne livestock diseases and their vectors. 3. Australian methods of vaccination against anaplasmosis and babesiosis. World Animal Review 18, 9-15. CALLOW, L. L. & HOYTE, H. M. D. (1961). Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick Boophilua microplus. Australian Veterinary Journal 37, 381-90. CUNNINGHAM, M. P., BROWN, C. G. D., BURRIDGE, M. J. & PURNELL, R. E. (1973). Cryopreservation of the infective particles of Theileria parva. International Journal for Paraaitology 3, 583-7. LUMSDEN, W. H. R. (1972). Principles of viable preservation of parasitic protozoa. International Journal for Parasitology 2, 327-32. BAJXEY,
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MiHONEY, D. F. & MIBBE, G. B. (1974). Babesia argentina: the infection of splenectomized calves with extracts of larval ticks (BoophUus microplus). Research in Veterinary Science 16, 112-14. MOBZABIA, S. P., BBOCELESBY, D. W., HABBADINE, DENISE & LUTHEB, P. D. (1977). Babesia major in Britain: infectivity for cattle of cryopreserved parasites derived from Haemaphysalis pundata nymphs. Research in Veterinary Science 22, 190-3. POTGIETEB, P. T. & VAN "VITOBEN, A. S. (1974). Transmission of Babesia bovis using frozen infective material obtained from BoophUus microplus larvae. Onderstepoort Journal of Veterinary Research 41, 79-80. PUBNELL, R. E., BBANAGAN, D. & BBOWN, C. G. D. (1970). Attempted transmission of some piroplasms by Rhipicephalid ticks. Tropical Animal Health and Production 2, 146-50. RADLEY, D. E., BBOWN, C. G. D., CUNNINGHAM, M. P., KIMBEB, C. D., MUSISI, F. L., PAYNE, R. C, PUBNELL, R. E., STAGG, S. M. & YOUNG, A. S. (1975). East Coast fever. 3.
Chemoprophylactic immunization of cattle using oxytetracycline and a combination of theilerial strains. Veterinary Parasitology 1, 51-60. REEK, R. F. (1964). The life cycle of Babesia bigemina (Smith & Kilborne 1893) in the tick vector BoophUus microplus (Canestrini). Australian Journal of Agricultural Research 15, 802-21. R I E K , R. F. (1966). The development of Babesia spp. and Theileria spp. in ticks with special reference to those occurring in cattle. In Biology of Parasites (ed. E. J. L. Soulsby), pp. 15—32. London: Academic Press. Ross, J. P. J.
291
Babesia bigemina in Kenya: experimental transmission by Boophilus decoloratus and the production of tick-derived stabilates S. P. MORZARIA,* A. S. YOUNG.f and E. B. HUDSONJ Immunological Research on Tick-borne Cattle Diseases and Tick Control Project, East African Veterinary Research Organization, Muguga, P.O. Box 32, Kikuyu, Kenya {Received 12 October 1976) SUMMARY
A Babesia-free batch of laboratory reared Boophilus decoloratus ticks was infected with a Kenyan strain of Babesia bigemina by feeding them on a steer infected by inoculation of a blood stabilate. The engorged female ticks showed developing stages of B. bigemina in their haemolymph and subsequently their progeny transmitted the parasite to a susceptible splenectomized steer. Attempts were made to produce stabilates from pre-fed larvae and nymphs derived from infected batches of B. decoloratus. Only the stabilates derived from the nymphs pre-fed either on cattle or rabbits produced B. bigemina infection when inoculated intravenously into susceptible cattle. INTBODTJCTION
Babesia bigemina is a widely distributed haemoprotozoan parasite which causes red water fever of cattle with resultant serious economic losses. In many areas of the world the tick vectors of B. bigemina have been identified (Riek, 1966). In most areas of Africa, however, the tick vectors have yet to be established. Several tick species, including the one-host tick Boophilus decoloratus, have been reported to transmit B. bigemina in South Africa. Throughout East Africa, B. decoloratus and Rhipicephalus appendiculatus have been implicated as vectors of B. bigemina but there is no experimental evidence for this. Purnell, Branagan & Brown (1970) attempted to transmit B. bigemina with R. appendiculatus, but without success. In this paper we report the transmission of a Kenyan strain of B. bigemina by B. decoloratus and we describe the production of stabilates derived from ticks infected with B. bigemina. * Present address: Central Veterinary Laboratory, Department of Virology, New Haw, Weybridge, Surrey, U.K. t FAO Staff member. % ODM Staff member, seconded from Central Veterinary Laboratory, Weybridge, U.K.
292
S. P. MORZARIA, A. S. Y O U N G AND E. B. H U D S O N MATERIALS AND M E T H O D S
Cattle used were of the Bos taurus type and were over 6 months of age. For 7 days prior to experiments, blood smears taken from the cattle were stained with Giemsa and examined for the presence of Babesia parasites. In addition, sera from the cattle were tested for the presence of Babesia antibodies using the indirect fluorescent-antibody (IFA) test with B. bigemina piroplasm antigen (Ross & Lohr, 1968). Only those cattle negative for both Babesia parasites and antibodies (a titre of 1/40 or less) were used in experiments. In certain cases cattle were splenectomized before experiments. The rabbits were chinchilla crosses from the East African Veterinary Research Organization (EAVRO) small animal unit. The B. decoloratus colony was established from engorged female ticks collected from two cattle held at EAVRO. They had been fed for 2 generations on susceptible cattle and did not transmit B. bigemina to them. These ticks were designated a clean (uninfected with B. bigemina) strain of B. decoloratus. The B. bigemina strain was obtained from a calf which died of babesiosis at EAVRO. Before the calf died, blood from the jugular vein was collected in heparin and was cryopreserved as a stabilate by Dr K. F. Lohr using glycerol as a cryoprotectant (Lumsden, 1972). It was then stored in liquid nitrogen. The strain was designated B. bigemina (Muguga). During experiments rectal temperatures of the cattle were taken daily and a rectal temperature of above 39-4 °C associated with Babesia infection was considered indicative of a febrile response. Also, blood smears were made daily from the ear veins, stained in Giemsa and examined for the presence of intraerythrocytic piroplasms. If blood slides of experimental cattle remained Babesianegative for 100 days, examinations were discontinued. For haematology, experimental cattle were bled 3 times a week from the jugular vein, starting a week prior to the experiments. Blood was collected with the potassium salt of ethylenediaminotetra-acetic acid (EDTA) as an anticoagulant. The haematology was determined using a Coulter counter Model ZBI and a Coulter haemoglobinometer (Coulter Electronics Inc., Florida, USA). Blood was also obtained from the jugular veins of the experimental cattle at weekly intervals, allowed to clot and the serum was separated for the IFA test. The B. bigemina parasitaemia was expressed as the number of infected cells per 103 red blood cells (RBC). B. decoloratus were fed on cattle by sprinkling larvae on the backs of the animals. During tick feeding the cattle were housed in a pen which was surrounded by a water moat. B. decoloratus were fed on rabbits by applying larvae to their ears according to the methods of Bailey (1960). The development of B. decoloratus ticks feeding on experimental cattle was checked by removing samples from the host at regular intervals and examining the ticks under a dissecting microscope. Replete females were collected 4 times daily and kept at 24 °C and 70% relative humidity (rel. hum.) during oviposition and hatching of eggs. Unfed larvae were maintained at 20 °C and at 80 % rel. hum. The infection rate of B. bigemina vermicules (Riek, 1964) in replete females was determined
Tick transmission of Babesia bigemina
293
by the examination of Giemsa-stained haemolymph smears produced by severing legs of the ticks 10 days after repletion. Suspensions of B. bigemina parasites for cryopreservation were produced from infected B. decoloratus larvae and nymphs which had been pre-fed on cattle or rabbits for 3-4 days. These ticks were removed from the hosts, ground up in a mortar and pestle with washed sand as an abrasive and Eagle's Minimum Essential Medium (MEM) with 3-4 % bovine plasma albumin (BPA) as a diluent (Cunningham, Brown, Purnell & Burridge, 1973). The suspensions were centrifuged at 50g for 5 min and the supernatant fluid removed. An equal volume of MEM/BPA with 20 % dimethyl sulphoxide (Me2SO) was added gradually to the supernatant fluid to give a final proportion by volume of 10% Me2SO as a cryoprotectant. The final suspension was dispensed in 2-5 ml volume glass ampoules insulated by a thin sheet of corrugated paper. The ampoules were frozen in an ultra-low refrigerator (Revco Inc., South Carolina, USA) to — 80 °C at a cooling rate of approximately 1 °C/min (M. P. Cunningham, unpublished results). Before use ampoules were removed and thawed rapidly. Experiment 1 The purpose of this experiment was to determine whether clean B. decoloratus ticks would become infected with B. bigemina when they engorged on a parasitaemic animal. A susceptible steer E325 was infested with approximately 2 x 103 B. decoloratus larvae obtained from the clean colony. Nineteen days later when most of the ticks had moulted into adults and had re-attached, the steer was inoculated subcutaneously with 7 ml of B. bigemina (Muguga) blood stabilate and monitored for Babesia infection. The replete females were collected and their haemolymph smears examined for B. bigemina infection. To test the infectivity of the larvae, 5 x 103 3-month-old larvae derived from the above females were applied to a susceptible splenectomized animal K46. The animal was examined for the development of a Babesia infection and the engorged females from this animal were examined for developing parasites. Experiment 2 Attempts were made to produce B. bigemina stabilates derived from infected B. decoloratus ticks pre-fed on cattle. Approximately 103 larvae and 400 nymphs were removed 3—4 days after attachment to K46 and ground up as described above to produce final volumes of 20 ml of larval suspension and 18 ml of nymphal suspension after adding MEM/BPA and Me2SO. Two susceptible steers K171 and K47 were inoculated intravenously (i.v.) with 10 ml of the larval suspension and 9 ml of the nymphal suspension respectively. The steers were examined for the development of B. bigemina infections. The remainder of the suspension was cryopreserved as described above. Forty-one days after freezing, two ampoules of the frozen nymphal suspension (St. 122) were thawed rapidly and 4 ml inoculated i.v. into animal K774 and 1 ml i.v. into animal J138. 2O
PAR
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S. P. MORZARIA, A. S. YOUNG AND E. B. HUDSON
Experiment 3 The purpose of this experiment was to produce B. bigemina stabilates from infected B. decoloratus nymphs pre-fed on rabbits. B. decoloratus larvae (2 x 104) derived from infected female ticks which had engorged during the period of patent parasitaemia in K46, were applied to 5 rabbits. After the larvae had developed into nymphs and had been feeding on the rabbits for 3—4 days, they were scraped off the rabbits' ears and processed as described previously. The final volume of the suspension containing 10 % Me2SO was 134 ml. The suspension was cryopreserved. Twelve days after freezing the contents of two ampoules of the cryopreserved suspension (St. 126) were inoculated i.v. into 2 susceptible steers K332 and K338. A susceptible steer J867, which had been splenectomized previously, was inoculated i.v. with 5 ml of St. 126 3 months after freezing. Nine months after freezing, 5 ml of St. 126 was inoculated into a susceptible splenectomized calf L580. RESULTS
Experiment 1 E325 developed a patent B. bigemina infection 5 days after inoculation of the stabilate (24 days after the application of uninfected ticks). By this time, the ticks on the steer had developed into B. decoloratus adults and many of the engorging females were dropping. The parasitaemia remained patent at levels of 1 per 103 RBC's or less for 9 days during which time all the ticks had completed engorgement. The engorged females which dropped from the steer during different days of the parasitaemia showed Babesia infection rates in their haemolymph Tanging from 20% to 100%. E325 showed no clinical symptoms associated with Babesia infection, but showed an IFA response (1/2560 titre by day 35). Steer K46, to which the larvae derived from the above females were applied, developed a patent B. bigemina infection 19 days after the application of the ticks. By day 22 the parasitaemia had reached 150 per 103 RBC's and the steer showed a marked degree of anaemia (packed cell volume of 10%). The animal also developed a febrile response; the maximum rectal temperature being 41-0 °C on day 21 post-tick application. The steer was treated with diminazene aceturate (Berenil, Hoechst Pharmaceuticals, Frankfurt(M), W. Germany) on day 23 to prevent death. The steer developed antibodies to B. bigemina reaching a maximum titre of 1/2560 on day 35. The engorged females obtained from K46 pre-treatment showed a Babesia infection rate of 80%. Experiment 2 The unfrozen nymphal suspension inoculated into K47 produced a B. bigemina infection while the unfrozen larval suspension failed to infect K171. K47 developed an antibody titre and anaemia was apparent (Table 1). However, no febrile response occurred. The infectivity of the cryopreserved larval suspension was not tested since
St. 126
St. 126
St. 126
St. 126
St. 122
of
Nymphs prefed on K46 Nymphs prefed on K46 Nymphs prefed on rabbits Nymphs prefed on rabbits Nymphs prefed on rabbits Nymphs prefed on rabbits
K46
Pre-fed on
K46
ticks Pre-fed on
Source
or
stabilate (St.) no. Larval suspension Nymphal suspension St. 122
Suspension
285
90
12
12
41
41
1 1 5 5
K332 J867* L580*
4
1
9
K338
K774
J138
K47
K171
Cattle no. inoculated
7
0
11
8
7
6
4
18
12f
11
11
10
10
12
Figures in parentheses are days after inoculation. * Spleneotomized animal, t Day of death.
Pre-freeze
No. days of oryopreservation Pre-freeze
Volume
71
34
29
31
< 1 260
33
29
32
34
30
7
30
60
—
7(11) 9(18)
1/2560 (28)
1/10240 (35)
23 (14) 25 (13)
1/2560 (35)
1/2560 (28)
18(12) 24 (13)
1/640 (28)
16(14)
Peak Pre- Days to para- Packed Packed cell inpatent peak sitaemia cell vol. vol. lowest per % pre- level % post- Peak IFA titre oculated period para(days) sitaemia KPRBC infection infection (ml) post-infection < 1/40 31 28 (15) 10
Table 1. Infectivity for cattle of suspensions derived from Boophilus decoloratus larvae and nymphs infected with Babesia bigemina
V
CD
IsS
eg" 8 B"
o*
82.
CD
w §•
&
I
296
S. P. MORZARIA, A. S. YOUNG AND E. B. HUDSON
the initial viability test was negative. Thawed nymphal suspension (St. 122) which was inoculated into steers K774 and J138 41 days after cryopreservation produced B. bigemina infection in both cattle. Anaemia developed in both animals but a febrile response was only observed in K774. The cattle showed a rise in B. bigemina antibody titres and recovered without treatment. (Table 1). Experiment 3
K338 and K332 which were inoculated with cryopreserved nymphal suspensions (St. 126) derived from ticks pre-fed on rabbits developed B. bigemina infection. No febrile response was observed but both developed a mild anaemia and showed significant rises in IFA titres (Table 1). After 3 months of cryopreservation St. 126 was inoculated into a splenectomized animal J867 and a fatal B. bigemina infection was produced associated with a febrile response and acute anaemia (Table 1). Similarly, L580 which was inoculated with St. 126 9 months after cryopreservation underwent a fatal Babesia infection. DISCUSSION
We have demonstrated that the experimental transmission of a Kenyan strain of B. bigemina by B. decoloratus was efficient. High percentages of ticks became infected after feeding on cattle with low Babesia parasitaemias. It is therefore probable that B. decoloratus is a natural vector of B. bigemina in Kenya. It would be of importance to identify the tick vectors of B. bigemina in East Africa so that red water fever could be controlled more efficiently. The method of transmission of the Kenyan strain of B. bigemina by B. decoloratus is similar to the transmission of B. bigemina by B. microplus in Australia (Riek, 1964). The female ticks became infected during feeding on an animal with a patent B. bigemina parasitaemia. Examination of the haemolymph smears from the engorged female Tevealed both a high Babesia infection rate and a high grade of infection in many ticks. Subsequently larvae derived from the female ticks produced B. bigemina infection in a susceptible steer during the course of their feeding and development into adult ticks. Thus trans-ovarian transmission was demonstrated. We obtained evidence that the nymphal stages of the ticks were responsible for the transmission of B. bigemina to cattle. Suspensions from nymphal ticks produced infections in cattle but larval suspensions did not. This agrees with the findings of Callow & Hoyte (1961) who reported that they were unable to transmit B. bigemina by the larvae of B. microplus. However, further work is required to demonstrate whether the larval or nymphal stages of B. decoloratus can acquire B. bigemina and whether adults can transmit the infection. This is the first time that B. bigemina stabilates derived from ticks have been produced. The nymphal ticks used had to be pre-fed since B. bigemina undergoes a maturation cycle in salivary glands of the nymphal tick to produce a form infective to cattle (Riek, 1964). Previously, Mahoney & Mirre (1974), Potgieter & Van Vuuren (1974) and Morzaria, Brocklesby, Harradine & Luther (1977) nave been able to produce tick-derived B. argentina, B. bovis and B. major
Tick transmission of Babesia bigemina
297
stabilates respectively. The use of tick-derived stabilates for experimental infection of cattle has certain advantages. For example, most of the experimental studies on Babesia infection in cattle have been blood-induced, which is an unnatural method ot infection. Infections produced by tick feeding are difficult to monitor since the feeding ticks may emit parasites over a long period (Morzaria et al. 1977), and the success of tick feeding on individual animals may vary considerably. Also, if the parasites are maintained continuously in ticks or by alternate tick/ animal passage they may change their characteristics. The tick-derived stabilates produced in the present experiments remained infective for at least 9 months and should remain infective for several years without change. Mahoney & Mirre (1974) suggested that tick-derived Babesia stabilates may provide material for the vaccination of cattle against babesiosis. The present method of vaccination as practised in Australia (Callow, 1976), and to a limited extent in Kenya, depends on the inoculation of cattle with blood infected with the relevant Babesia species. This method of vaccination involves dangers, including the contamination of blood with other organisms infective to cattle and the induction of haemolytic disease. It should be possible to immunize cattle with a controlled inoculum of tick-derived Babesia stabilates produced from infected ticks pre-fed on rabbits. This would reduce the dangers inherent in vaccination with bovine blood. It is also possible that Babesia immunization could be combined with immunization against theileriosis by chemoprophylaxis which is being developed at EAVRO (Radley, Brown, Cunningham, Kimber, Musisi, Payne, Purnell, Stagg & Young, 1975). This project is supported by the United Nations Development Programme with the Food and Agriculture Organization of the United Nations as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Ministry of Overseas Development of the United Kingdom (Research Projects R 2396, R 2492 & R 2845 A & B), the United States Department of Agriculture, the Rockefeller Foundation, the International Atomic Energy Agency and the Pfizer Corporation Inc. We are grateful to our colleagues on the Project for advice and assistance. We would especially like to thank Dr K. F. Lohr of the Veterinary Laboratories, Kabete, for providing us with B. bigemina blood stabilate. We would also like to thank Dr G. Maxie of the IDRC Project, EAVRO, for supervision of the haematology. This paper is published by kind permission of the Director of EAVRO, Muguga, Kenya. REFERENCES
K. P. (1960). Notes on the rearing of Rhipicephaltcs appendiculatua and their infection with Theileria parva for experimental transmission. Bulletin of Epizootic Diseases of Africa 8, 33-43. CALLOW, L. L. (1976). Tick-borne livestock diseases and their vectors. 3. Australian methods of vaccination against anaplasmosis and babesiosis. World Animal Review 18, 9-15. CALLOW, L. L. & HOYTE, H. M. D. (1961). Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick Boophilua microplus. Australian Veterinary Journal 37, 381-90. CUNNINGHAM, M. P., BROWN, C. G. D., BURRIDGE, M. J. & PURNELL, R. E. (1973). Cryopreservation of the infective particles of Theileria parva. International Journal for Paraaitology 3, 583-7. LUMSDEN, W. H. R. (1972). Principles of viable preservation of parasitic protozoa. International Journal for Parasitology 2, 327-32. BAJXEY,
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MiHONEY, D. F. & MIBBE, G. B. (1974). Babesia argentina: the infection of splenectomized calves with extracts of larval ticks (BoophUus microplus). Research in Veterinary Science 16, 112-14. MOBZABIA, S. P., BBOCELESBY, D. W., HABBADINE, DENISE & LUTHEB, P. D. (1977). Babesia major in Britain: infectivity for cattle of cryopreserved parasites derived from Haemaphysalis pundata nymphs. Research in Veterinary Science 22, 190-3. POTGIETEB, P. T. & VAN "VITOBEN, A. S. (1974). Transmission of Babesia bovis using frozen infective material obtained from BoophUus microplus larvae. Onderstepoort Journal of Veterinary Research 41, 79-80. PUBNELL, R. E., BBANAGAN, D. & BBOWN, C. G. D. (1970). Attempted transmission of some piroplasms by Rhipicephalid ticks. Tropical Animal Health and Production 2, 146-50. RADLEY, D. E., BBOWN, C. G. D., CUNNINGHAM, M. P., KIMBEB, C. D., MUSISI, F. L., PAYNE, R. C, PUBNELL, R. E., STAGG, S. M. & YOUNG, A. S. (1975). East Coast fever. 3.
Chemoprophylactic immunization of cattle using oxytetracycline and a combination of theilerial strains. Veterinary Parasitology 1, 51-60. REEK, R. F. (1964). The life cycle of Babesia bigemina (Smith & Kilborne 1893) in the tick vector BoophUus microplus (Canestrini). Australian Journal of Agricultural Research 15, 802-21. R I E K , R. F. (1966). The development of Babesia spp. and Theileria spp. in ticks with special reference to those occurring in cattle. In Biology of Parasites (ed. E. J. L. Soulsby), pp. 15—32. London: Academic Press. Ross, J. P. J.
