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Babesia bovis: Evidence for selection of subpopulations during attenuation
EXPERIMENTALPARASITOLOOY
Babesia
70,404-410(1990)
bovis: Evidence for Selection during Attenuation
of Subpopulations
C . A . CARSON,**~ P. TIMMS,* A. F. COWMAN,? AND N. P. STEWART+ *Animal Research Institute, Department of Primary Industries, Queensland 4105, Australia; tThe Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia; and #Tick Fever Research Centre, Wacol, Queensland 4076, Australia CARSON,~. A.,TIMMs,P.,COWMAN,A. F.,AND STEWART, N. P.1990. Babesiabovis: Evidence for selection of subpopulations during attenuation. Experimental Parasitology 70. 404-410. DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the fust was a basic profde typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent E. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profde of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative “markers” or actual elements responsible for these biological characteristics. 8 ma Academic press,hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Babesia bovis; Protozoa, parasitic; Babesiosis, bovine; Virulence; Attenuation process; Subpopulations; Vaccination; Cattle; Ticks; Boophilus microplus; Deoxyribonucleic acid (DNA), Complementary DNA (cDNA); Probes; Radiolabeling, biosynthetic; Electrophoresis, agarose; Southern blotting; Hybridization.
analysis of the vaccine (Ka) strain after passage through the tick vector suggested that Bovine babesiosis is a malaria-like disa subpopulation was being selected which ease, caused by intraerythrocytic protomore closely resembles Kv than Ka (Cowzoan organisms of the genus Babesiu. Two man er al. 1984b). species, B. bovis and B. bigemina, are reAnalysis of parasite genomic DNA by sponsible for substantial economic losses in hybridization with cDNA clones has shown parts of the world where the tick vector, distinguishing characteristics of various Boophilus microplus, occurs. Vaccination isolates and indicated that each is comhas been successfully accomplished for posed of a heterogeneous mixture of submany years in Australia (Callow 1979) populations (Cowman et al. 1984b). Isothrough the use of a live avirulent derivalates cloned by in vitro cultivation have furtive (designated Ka) of the K-virulent (Kv) ther substantiated the existence of an array isolate, attenuated by multiple syringe pasof individual parasite populations (Rodsage in splenectomized calves (Callow and riguez et al. 1983). However, an actual Mellors 1966), and passages 23 through 28 “profile,” characteristic of either virulent are routinely used for vaccine production. or avirulent organisms, has not been Attenuation of B. bovis can be reversed by shown, nor have the changes occurring durrapid syringe passage through intact cattle ing syringe passage been elucidated. Anal(Callow er al. 1979) and genomic DNA ysis of the Ka isolate after tick passage showed antigenic variation but it was not ’ Present address: Department of Veterinary Microbiology, College of Veterinary Medicine, University of clear whether this resulted from differential gene expression within a population and/or Missouri, Columbia, Missouri 65211. INTRODUCTION
404 OO144!W9O $3.00 cOpy&htQ 19!70 byAcadcmicF’rcss,Inc. All rights ofrcproduction h any foml rcmrvcd.
B.
bOViS:
VARIATION IN SUBPOPULATIONS
selection of subpopulations that express variable B. bovis protein antigens (Kahl et al. 1983). Previous work (Kahl et al. 1982) has shown that there are antigenic differences between virulent (Kv) and avirulent (Ka) forms of the K strain of B. bovis. We have used cDNA clones to probe a sequential array of B. bovis parasites in the progression from virulence to avirulence. Two cDNA clones, designated pK4 (B. bovis “rearranging” or BabR locus) and pK5, were chosen for this study. The former was encoded by a highly polymorphic and complex locus and variation in fragment intensity and patterns of hybridization with genomic DNA were related to the existence of heterogeneous mixtures of subpopulations (Cowman et al. 1984a, b). Analysis of the cloned genomic fragments, corresponding to BabR, indicated that rearrangements had occurred, creating genes that share homology regions but encode RNA species of different size. Hybridization with the pK5 probe suggested an abundance of the respective gene in Ka or avirulent isolates and was chosen as an indicator for advancing attenuation (Cowman 1983; Cowman et al. 1984b). A third cDNA probe, pK21, known to hybridize to an equal intensity with Ka and Kv isolates and presumably detecting a single copy gene, was used for quantitative determination of parasite genomic DNA delivered into gel slots. We present data which show that attenuation of K strain B. bovis becomes evident with serial passage in splenectomized cattle and that the selection of minor initial subpopulations is related to the attenuation process. Reversion to the virulent form in which Ka is transmitted by the tick vector, Boophilus microplus, or needle-passaged through intact cattle, is also examined. MATERIALSANDMETHODS Serial samples of B. bovis-infected bovine blood, collected during passage of a field isolate (K strain) through splenectomized calves in the process of atten-
405
uation for vaccine development (Callow and Mellors 1!266),were chosenfor comparison and possible evidence of genomic change related to the progression from Kv to Ka. After attenuation in this manner the K strain is successfully used commercially as a live avirulent vaccine. Previous work (Cowman er al. 1984b) suggested that this process involves selection of a subpopulation of avirulent parasites from the virulent form. To understand the conversion to avirulence, we probed genomic DNA isolated from early (3P), middle (6P, 9P, 14P), and late (2OP, 22P) passages. Samples previously described as characteristic of Kv, prepared by passaging Ka through ticks (Cowman et al. 1984a) or intact cattle (Callow et al. 1979), were also used for comparison. High passage Ka (2OP) was inoculated into a splenectomized calf, upon which B. microplus ticks were fed, and larval progeny were then allowed to feed on and transmit the disease to another splenectomized calf, J3. bovis-infected blood was then collected from the latter animal. In the second procedure used for reversion to Kv the attenuated (Ka) strain was syringe-passaged sequentially through three intact cattle.
cDNA clones Isolation of cDNA clones pK4, pK5, and pK21 and their use as probes have been previously described by Cowman et al. (1984b).
Preparation
of cDNA Probes
Clones pK4 (BabR 9.7), pKS, and pK21, in the form of recombinant pBR 322, were digested with PsfI. The respective clones were labeled by random priming 200 ng of each clone, using a commercial kit for oligonucleotide-labeling DNA restriction fragments with 5 pCi of [‘*P]dCTP (2000-3000 Ci/mmole, Biotechnology Research Enterprises, S.A. Pty Ltd., Adelaide, South Australia).
Strains of B. bovis K-isolate. Stabilates of this isolate had been stored in liquid nitrogen at the Tick Fever Research Centre, Wacol, Queensland. Kvltick. Obtained by injecting 20P (Ka) blood into a splenectomized calf infested 20 days previously with parasite-free larval ticks. Maturation of the ticks coincided with patent infection. Larval progeny from replete female ticks were released on other splenectomized calves, from which blood was collected for this study. Kvlintacr. 20P (Ka) after serial syringe passage through three intact cattle to revert to Kv. Approximately 1 x 10’ babesia-infected erythrocytes were inoculated intravenously; usually drawn from the donor on postinfection Days 9 or 10 when the parasitemia was between 0.5 and 5.0%.
406
CARSONET AL.
Ku. Derived by 20 or more syringe passages of Kv through a series of splenectomized calves.
DNA Preparation and Hybridization Analysis The following B. bovis isolates, mixed 1:l with 2.0 M DMSO and stored in liquid nitrogen for many years, were selected to represent Kv: Passage three (3P), since no previous material remained available from earlier work; Ka (20P) after tick passage; and 20P after three syringe passes through intact cattle. Samples intermediate between Kv and Ka (6P, 9P, and 14P)were chosen from stored blood specimens, not leukocyte free, that had been held at - 196°Cup to 11years prior to use. Samples of freshly collected 20P and 22P (representing Ka) and 20P after tick passage (Kv) were collected in heparin, washed twice in phosphatebuffered saline (PBS) (20 m&f sodium phosphate, 125 mM NaCl, pH 7.3), and centrifuged at 1OOOgfor 10 min. Supematant was removed, buffy coat was discarded, and erythrocytes, suspended in PBS, were passed through a column of cellulose powder (Whatman CF 11, Whatman International Ltd., Maidstone, England) to remove remaining leukocyte DNA. Parasitized erythrocytes were stored at - 20°C prior to use. Each blood sample, from storage or recently collected, was processed in the same manner from this point. Packed cells were thawed and mixed with an equal volume of lysis buffer containing 10 mM Tris Cl, pH 7.5, 10 r&f EDTA, 10 mM NaCI, and 1% SDS (buffer A). After incubation at 37°C for 15 min, proteinase K was added to 50 pg ml-’ and the mixture was incubated at 50°C for 3 hr. The lysate was extracted twice with phenol, once with chloroform/isoamylalcohol, and twice with ether. Genomic DNA was diluted in TE buffer [lo mM Tris Cl (pH 7.4), 1 mM EDTA (pH 8.0)1 at pH 7.4 and stored at 4°C. DNA from an array of B. bovis-infected erythrocytes was extracted and quantitated in an effort to standardize samples for comparison. A source of error proved to be contributed by contaminating leukocyte DNA, particularly in samples collected and stored years earlier for other purposes. Samples were digested with PsrI and delivered into wells in agarose gels (0.6%) so that sequential tracks were arranged in a presumed progression from virulent to avirulent. Track a contained a DNA sample from Kv/intact; track b represented Kv/tick; track c received Kv/tick DNA from another tick passage of Ka (2OP); and tracks d-i contained DNA from serial passages of the original K isolate through splenectomized calves as 3P, 6P, 9P, 14P, 2OP,and 22P, respectively. After electrophoretic separation the patterns were transferred to nylon filters (Hybond-N, Amersham Australia Pty Ltd., Sydney) according to the method of Southern (1975). DNA probes were used sequentially to: (I) detect
the presence of heterogenous mixtures of subpopulations (probe pK4); (2) preferentially bind to DNA from avirulent babesia (probe pK5); and (3) quantify B. bovis DNA loading in the gels (probe pK21). Hybridizations were performed in Hybrid-Ease chambers (Hoeffer Scientific Instruments, San Francisco, CA) at 65°C in 5x SSCE(lx SSCE = 0.15M NaCl, 15 m&f Na Citrate, 10 mM EDTA), 0.1% SDS, 10x Denhardt’s solution, and 240 pg ml-’ salmon sperm DNA. Filters were washed at 65°C in 0.1 X SSCE containing 0.1% SDS. Autoradiography was for g-36 hr at - 70°C using fast tungstate screens. Filters were washed with 0.1 M NaOH, followed by neutralization with 1.5 M NaCVO.5 M Tris, pH 7.5, and finally soaked in 20x SSC to remove previous probe prior to reuse.
RESULTS
DNA extracted from nearly every isolate had six or sevenfragments, associated with the BabR locus, detected by probe pK4. In Fig. lA, track a Kv/intact DNA, from p20 after passage three times in intact calves, shows fragments of 10.5, 6.4, 4.1, 4.0, 2.7, and 1.7 kb. Kv/tick DNA, from the reversion procedure performed by tick-transmitting p20 (lanes b and c), shows fragments at 10.4, 6.4, 4.1, 4.0, 1.7, and 1.2 kb in track b and an additional fragment at 2.7 kb in track c. The earliest available sample in the attenuation series (3P) shows fragments in track d at 10.5, 6.4, 4.1, 4.0, 1.8, 1.7, and 1.2 kb. Patterns of fragments evident in tracks a-c constitute a basic pattern with consistent fragments in the range of 10.5,6.4,4.1,4.0, and 1.7 kb. Tracks a and c have fragments at 2.7 kb while tracks b and d do not. Passages3,6,9,14,20, and 22 (tracks d through i, respectively) show a tendency toward great variation in intensity of hybridization compared to the basic pattern evident in the first three tracks. Passage 3 (track d) shows a doublet at 1.7/1.8 kb and a strong fragment at 1.2 kb. Track e, which contained 6P DNA, has only three fragments located at 7.3, 3.2, and 1.5 kb. The 9P fragments (track f) are located near 10.0,7.3,6.4,3.2, and 1.7 kb. Fragments in track g, related to 14P, are located at 10.0, 7.3, 6.4, 4.0, and 1.7 kb. At the point when the passagedB. bovis is designated as the
B.
bovis: VARIATION IN SUBPOPULATIONS
A
407
C abcdefghi
abcdefghi
I
I
abcdefghi
10.5 6.4 4.1 -
2.5 -
PROBE
me
PK4
FIG. 1. Southern hybridization of identical babesia DNA patterns in tracks a-i using probe pK4 in (A), probe pK5 in (B), and pK21 in (C). Tracks a-i contain: a (Kv/intact), 20P after passage three times through intact cattle; b (Kv/tick), 20P after tick transmission; c (Kv/tick), another tick-transmitted 20P sample; d, 3P; e, 6P; f, 9P; g, 14P; h, 20P; i, 22P. Probe used in (A) was removed from filter and other probes were applied sequentially for (B) and (C). Gel loading varied according to probe pK21 (C) but tracks c, d, f, h, and i exhibit signal of reasonably equal intensity; variation of hybridization pattern in these tracks in (A) is, therefore, not due to quantitative differences in DNA applied to wells.
attenuated strain (Ka) the 20P fragments in trackhappearat 10.0,6.4,4.0,4.1, 1.7, and 1.2 kb. The sample from 22P (track i) shows major fragments at 4.1, 1.9, 1.7, and 1.2 kb; lesser fragments appear at 10.0 and 6.4 kb. The composite of patterns in tracks e through i is a highly variable array generally failing to conform to the typical Kv pattern of five or six consistent fragments at the range of 10.0, 6.4, 4.1, and 1.7 kb. A second cDNA clone, pK5, has been previously suggested (Cowman et al. 1984a) as a marker of attenuation since it hybridized to a subpopulation in avirulent strains that increases as the isolate is passaged through splenectomized calves. We used pK5 to probe the DNA from different serially passaged parasites. Probe pK5 shows virtually no signal in tracks a and b (Fig. 1B) and increasing intensity at the 2.5 kb level in tracks c through i (with the exception of track e) in the progression from Kv to Ka. It is clearly a marker of aviru-
lence as the amount of hybridization increases with attenuation. The pK21 probe (Fig. lC), which hybridizes to a single copy gene represented by a 6.0-kb band in all EcoRI-digested genomic DNA isolates (Cowman et al. 1984a), was previously used to confirm that equal amounts of DNA were loaded into each track. In the case of &I-digested DNA, used in the present study, multiple bands were detected in three locations, presumably as a result of restriction length polymorphisms within the uncloned parasite population. No bands are evident in track a but one band appeared at 4.3 kb in track b; a band at 4.3 and 2.5 kb in track c; an intense band at 4.3 and a very weak band at 4.0 kb in track d; a single indistinct band at 0.75 kb in track e; a more intense band at 0.75 kb in track f; one weak band at 4.3 and a second at 0.75 in track g; and single intense 4.3-kb bands in tracks h and i. These hybridization patterns indicate approxi-
408
CARSON ET AL.
mately equal amounts of DNA in tracks c, d, f, h, and i; lesser amounts were loaded into tracks b, e, and g; and an extremely small quantity in track a. Although loading is not constant, the observation that a typical Kv hybridization pattern for probe pK4 becomes variable with progression toward Ka (Fig. lA, tracks d through i) remains valid since these major differences are evident in tracks that contain similar amounts of DNA (e.g., Fig. 1C series including c, d, f, h, and i; also b, e, and g). In Fig. lA, track a presents a particularly weak pattern due to the extremely small amount of DNA present (no band visible in Fig. lC, track a) but the typical Kv pattern is evident nonetheless. The observed increase in intensity of pK5 fragments in Fig, 1B is validated by observing differences in tracks c, d, f, and h; also the virtual lack of signal in track d, which appears to have the largest amount of DNA. DISCUSSION
Previous analysis of genomic DNA and poly(A)+ RNA from various isolates suggested that some babesia isolates are composed of a heterogenousmixture of subpopulations (Cowman et al. 1984b).Analysis of a polymorphic gene family (BabR) indicated that the Ka isolate of B. bovis consisted of at least two subpopulations (Cowman et al. 1984a). Different-sized BabR fragments were not present in equimolar amounts and the intensity of hybridization of a certain species corresponded to less than one copy per genome. Probe pK4 detected major fragments of 5.7 and 7.7 kb in genomic DNA from Ka digested with EcoRI and only trace levels in Kv (Cowman et al. 1984a).It was proposed, therefore, that parasites containing these restriction length polymorphisms occur only rarely in the mixed parasite population that constitutes Kv and that the increase in certain fragments in Ka results from selection of minor subpopulations during the process of attenuation. Reversion of Ka to a pattern
similar to Kv, after passage through the tick, was also considered to result by selection of certain subpopulations from the mixture in Ka. The present study was designed to study possible subpopulation selection during the traditional process of attenuation by serial passage through splenectomized calves. Hybridization studies using cDNA probes, which hybridized to hypervariable loci (pK4) or preferentially to DNA from Ka parasites (pKS), indicate that during serial passagethe typical pattern of babesia parasites in Kv shows early evidence of shifting to a more variable pattern related to attenuation. Distinct genomic DNA fragments of the Kv form (i.e., 10.5, 6.4, 4.1, 4.0, 2.7, and 1.7), representing the BabR locus, exhibit variability with respect to presence, fragment size, and molar ratio as attenuation progressesfrom pass 3 through 22. Previous hybridization of probe pK4 with PvuII-digested genomic DNA from B. bovis isolates revealed differences between virulent and attenuated isolates, including Kv and Ka (Cowman et al. 1984b).The Ka pattern of fragments was more complex than Kv, with bands varying in intensity and the appearance of additional bands; two small fragments were present in quantities less than one copy per genome, indicating the presence of subpopulations. Additional information from other hybridization studies showed that composition and complexity of fragments from different Ka preparations varies and putative clones, produced by an in vivo method, implied the presence of different Ka subpopulations (Gill et al. 1987). These studies indicated that variation in fragment size probably represents subpopulation shifts, although concurrent genomic changes such as deletion, recombination, or rearrangement are possible. In the present study, babesia subpopulations, present in low numbers in Kv isolates, appear to become predominant with increased passage through splenectomized calves and variation in specific frag-
B. bovis: VARIATION IN SUBPOPULATIONS
ment signal strength reflects gene contributions from shifting proportions of subpopulations. Less than molar quantities of two fragments in 22P also indicate the presence of minor subpopulations of parasites. It is unlikely that these observed differences relate to genomic changes since major differencesin hybridization patterns occur over a short time period and would require programmed gene rearrangement in populations. Fragments at 7.3,3.2, and 1.2 kb, not pronounced in Kv but intermittent during attenuation, may result from such population shifts or may represent the tendency toward hypervariability of subpopulations emerging during passage. Differences observed with probe pK4 are not due simply to quantitative differences of DNA, although loading was apparently not uniform. Probe pK21, used for quantitation, identified more than one fragment in P&I digests compared to the single band/single copy gene identified in EcoRI digests of genomic DNA, but it is obvious that loading was similar in certain tracks and that these proved useful in substantiating findings. Different strains of attenuated babesia isolates all have exhibited the particular propensity to hybridize with probe pK5, compared to the relative absence of this quality in virulent isolates (Cowman et al. 1984b). There is no evidence that the pK5 gene is specifically related to avirulence, but the present study’s correlation between the gradual increase in hybridization intensity with serial passage,known to gradually produce attenuation, is remarkable. This pattern substantiates the serial emergence of a subpopulation of attenuated organisms with pK5 fragments and the probe seemsat least a likely marker for avirulence. It has now been shown that Ka and Kv subpopulations differ both qualitatively and quantitatively with respect to their pK4 and pK5 sequences. Evidence has been advanced in the present study for genomic similarity between the product of Ka passage through
409
ticks and syringe passage through intact cattle. The attenuated subpopulations, which emerge in serial passagetoward development of Ka, therefore, revert to a basically virulent form (Kv) as previously indicated by Cowman et al. (1984b). In vitro-cultivated clones of B. bovis differ in both degree of virulence (C. A. Carson et al., unpublished data) and rate of growth in culture (Rodriguez et al. 1983). Since attenuation by passage of babesia through splenectomized calves involves serial needle passageof blood, it seemslikely that the mechanism provides a progressive enrichment for rapidly growing subpopulations. Further characterization of the dynamics of B. bovis subpopulations will require the examination of organisms cloned in vitro from heterogenous isolates, including the K strain. Employment of procedures used in this study, to derive genomic patterns for homogenous clones, should fully characterize the subpopulations of Kv and Ka. ACKNOWLEDGMENTS The authors deeply appreciate the outstanding technical assistance of Ms. Van Le and the contribution of fresh blood samples by Dr. I. Shiels, frozen blood samples with the approval of Dr. A. J. deVos, and expert advice from Dr. R. J. Dalgliesh. Special thanks to Dr. L. L. Callow, Director, Animal Research Institute, Yeerongpilly, Queensland, for making the senior author’s participation in this investigation possible. Support was provided by the Department of Primary Industries, Queensland, Australia. A. F. Cowman is an Australian Wellcome Senior Research Fellow.
REFERENCES CALLOW, L. L. 1979. Some aspects of the epidemiology and control of bovine babesiosis in Australia. Journal of the South Afn’can Veterinary Association 50, 353-356. CALLOW, L. L., AND MELLORS, L. T. 1966. A new vaccine for Eabesia argentina infection produced in splenectomized calves. Australian Veterinuty Journal 42,464465. CALLOW, L. L., MELLORS, L. T., AND MCGREOOR, W. 1979. Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle. Znternational Journal of Parasitology 9, 333-338.
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COWMAN, A. F. 1983. “Genes of the Protozoan Parasite Babesiu bovis.” PhD thesis, University of Melbourne, Australia. COWMAN, A. F., BERNARD, O., STEWART, N., AND KEMP, D. J. 1984a.Genes of the protozoan parasite Eabesiu bovis that rearrange to produce RNA species with different sequences. Cell 37, 653-660. COWMAN,A. F., TIMMS, P., AND KEMP, D. J. 1984b. DNA polymorphisms and subpopulations in Babesin bovis. Molecular and Biochemical Parasitology 1, 91-103. GILL, A. C., COWMAN, A. F., STEWART, N. P., KEMP, D. J., AND TIMMS, P. 1987. Babesiu bovis: Molecular and biological characteristics of cloned parasite lines. Experimental Parasitology 63, 180188. KAHL, L. P., ANDERS, R. F., RODWELL, B. J., TIMMS, P., AND MITCHELL, G. F. 1982. Variable and common antigens ofBabesiu bovis parasites dif-
fering in strain and virulence. Journal of Immunology 129, 1700-1705. KAHL, L. P., MITCHELL, G. F., DALGLIESH, R. J., STEWART, N. P., RODWELL, B. J., MELLORS, L. T., TIMMS, P., AND CALLOW, L. L. 1983. Babesia bovis: Proteins of virulent and avimlent parasites passaged through ticks and splenectomized or intact calves. Experimental Parasitology 56, 222-235. RODRIGUEZ,S. D., BUENING, G. M., GREEN, T. J., AND CARSON,C. A. 1983. Cloning of Babesia bovis by in vitro cultivation. Infection and Immunity 42, 15-18. SOUTHERN, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503-517. Received 26 May 1989; accepted with revision 20 November 1989
Babesia
70,404-410(1990)
bovis: Evidence for Selection during Attenuation
of Subpopulations
C . A . CARSON,**~ P. TIMMS,* A. F. COWMAN,? AND N. P. STEWART+ *Animal Research Institute, Department of Primary Industries, Queensland 4105, Australia; tThe Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia; and #Tick Fever Research Centre, Wacol, Queensland 4076, Australia CARSON,~. A.,TIMMs,P.,COWMAN,A. F.,AND STEWART, N. P.1990. Babesiabovis: Evidence for selection of subpopulations during attenuation. Experimental Parasitology 70. 404-410. DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the fust was a basic profde typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent E. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profde of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative “markers” or actual elements responsible for these biological characteristics. 8 ma Academic press,hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Babesia bovis; Protozoa, parasitic; Babesiosis, bovine; Virulence; Attenuation process; Subpopulations; Vaccination; Cattle; Ticks; Boophilus microplus; Deoxyribonucleic acid (DNA), Complementary DNA (cDNA); Probes; Radiolabeling, biosynthetic; Electrophoresis, agarose; Southern blotting; Hybridization.
analysis of the vaccine (Ka) strain after passage through the tick vector suggested that Bovine babesiosis is a malaria-like disa subpopulation was being selected which ease, caused by intraerythrocytic protomore closely resembles Kv than Ka (Cowzoan organisms of the genus Babesiu. Two man er al. 1984b). species, B. bovis and B. bigemina, are reAnalysis of parasite genomic DNA by sponsible for substantial economic losses in hybridization with cDNA clones has shown parts of the world where the tick vector, distinguishing characteristics of various Boophilus microplus, occurs. Vaccination isolates and indicated that each is comhas been successfully accomplished for posed of a heterogeneous mixture of submany years in Australia (Callow 1979) populations (Cowman et al. 1984b). Isothrough the use of a live avirulent derivalates cloned by in vitro cultivation have furtive (designated Ka) of the K-virulent (Kv) ther substantiated the existence of an array isolate, attenuated by multiple syringe pasof individual parasite populations (Rodsage in splenectomized calves (Callow and riguez et al. 1983). However, an actual Mellors 1966), and passages 23 through 28 “profile,” characteristic of either virulent are routinely used for vaccine production. or avirulent organisms, has not been Attenuation of B. bovis can be reversed by shown, nor have the changes occurring durrapid syringe passage through intact cattle ing syringe passage been elucidated. Anal(Callow er al. 1979) and genomic DNA ysis of the Ka isolate after tick passage showed antigenic variation but it was not ’ Present address: Department of Veterinary Microbiology, College of Veterinary Medicine, University of clear whether this resulted from differential gene expression within a population and/or Missouri, Columbia, Missouri 65211. INTRODUCTION
404 OO144!W9O $3.00 cOpy&htQ 19!70 byAcadcmicF’rcss,Inc. All rights ofrcproduction h any foml rcmrvcd.
B.
bOViS:
VARIATION IN SUBPOPULATIONS
selection of subpopulations that express variable B. bovis protein antigens (Kahl et al. 1983). Previous work (Kahl et al. 1982) has shown that there are antigenic differences between virulent (Kv) and avirulent (Ka) forms of the K strain of B. bovis. We have used cDNA clones to probe a sequential array of B. bovis parasites in the progression from virulence to avirulence. Two cDNA clones, designated pK4 (B. bovis “rearranging” or BabR locus) and pK5, were chosen for this study. The former was encoded by a highly polymorphic and complex locus and variation in fragment intensity and patterns of hybridization with genomic DNA were related to the existence of heterogeneous mixtures of subpopulations (Cowman et al. 1984a, b). Analysis of the cloned genomic fragments, corresponding to BabR, indicated that rearrangements had occurred, creating genes that share homology regions but encode RNA species of different size. Hybridization with the pK5 probe suggested an abundance of the respective gene in Ka or avirulent isolates and was chosen as an indicator for advancing attenuation (Cowman 1983; Cowman et al. 1984b). A third cDNA probe, pK21, known to hybridize to an equal intensity with Ka and Kv isolates and presumably detecting a single copy gene, was used for quantitative determination of parasite genomic DNA delivered into gel slots. We present data which show that attenuation of K strain B. bovis becomes evident with serial passage in splenectomized cattle and that the selection of minor initial subpopulations is related to the attenuation process. Reversion to the virulent form in which Ka is transmitted by the tick vector, Boophilus microplus, or needle-passaged through intact cattle, is also examined. MATERIALSANDMETHODS Serial samples of B. bovis-infected bovine blood, collected during passage of a field isolate (K strain) through splenectomized calves in the process of atten-
405
uation for vaccine development (Callow and Mellors 1!266),were chosenfor comparison and possible evidence of genomic change related to the progression from Kv to Ka. After attenuation in this manner the K strain is successfully used commercially as a live avirulent vaccine. Previous work (Cowman er al. 1984b) suggested that this process involves selection of a subpopulation of avirulent parasites from the virulent form. To understand the conversion to avirulence, we probed genomic DNA isolated from early (3P), middle (6P, 9P, 14P), and late (2OP, 22P) passages. Samples previously described as characteristic of Kv, prepared by passaging Ka through ticks (Cowman et al. 1984a) or intact cattle (Callow et al. 1979), were also used for comparison. High passage Ka (2OP) was inoculated into a splenectomized calf, upon which B. microplus ticks were fed, and larval progeny were then allowed to feed on and transmit the disease to another splenectomized calf, J3. bovis-infected blood was then collected from the latter animal. In the second procedure used for reversion to Kv the attenuated (Ka) strain was syringe-passaged sequentially through three intact cattle.
cDNA clones Isolation of cDNA clones pK4, pK5, and pK21 and their use as probes have been previously described by Cowman et al. (1984b).
Preparation
of cDNA Probes
Clones pK4 (BabR 9.7), pKS, and pK21, in the form of recombinant pBR 322, were digested with PsfI. The respective clones were labeled by random priming 200 ng of each clone, using a commercial kit for oligonucleotide-labeling DNA restriction fragments with 5 pCi of [‘*P]dCTP (2000-3000 Ci/mmole, Biotechnology Research Enterprises, S.A. Pty Ltd., Adelaide, South Australia).
Strains of B. bovis K-isolate. Stabilates of this isolate had been stored in liquid nitrogen at the Tick Fever Research Centre, Wacol, Queensland. Kvltick. Obtained by injecting 20P (Ka) blood into a splenectomized calf infested 20 days previously with parasite-free larval ticks. Maturation of the ticks coincided with patent infection. Larval progeny from replete female ticks were released on other splenectomized calves, from which blood was collected for this study. Kvlintacr. 20P (Ka) after serial syringe passage through three intact cattle to revert to Kv. Approximately 1 x 10’ babesia-infected erythrocytes were inoculated intravenously; usually drawn from the donor on postinfection Days 9 or 10 when the parasitemia was between 0.5 and 5.0%.
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Ku. Derived by 20 or more syringe passages of Kv through a series of splenectomized calves.
DNA Preparation and Hybridization Analysis The following B. bovis isolates, mixed 1:l with 2.0 M DMSO and stored in liquid nitrogen for many years, were selected to represent Kv: Passage three (3P), since no previous material remained available from earlier work; Ka (20P) after tick passage; and 20P after three syringe passes through intact cattle. Samples intermediate between Kv and Ka (6P, 9P, and 14P)were chosen from stored blood specimens, not leukocyte free, that had been held at - 196°Cup to 11years prior to use. Samples of freshly collected 20P and 22P (representing Ka) and 20P after tick passage (Kv) were collected in heparin, washed twice in phosphatebuffered saline (PBS) (20 m&f sodium phosphate, 125 mM NaCl, pH 7.3), and centrifuged at 1OOOgfor 10 min. Supematant was removed, buffy coat was discarded, and erythrocytes, suspended in PBS, were passed through a column of cellulose powder (Whatman CF 11, Whatman International Ltd., Maidstone, England) to remove remaining leukocyte DNA. Parasitized erythrocytes were stored at - 20°C prior to use. Each blood sample, from storage or recently collected, was processed in the same manner from this point. Packed cells were thawed and mixed with an equal volume of lysis buffer containing 10 mM Tris Cl, pH 7.5, 10 r&f EDTA, 10 mM NaCI, and 1% SDS (buffer A). After incubation at 37°C for 15 min, proteinase K was added to 50 pg ml-’ and the mixture was incubated at 50°C for 3 hr. The lysate was extracted twice with phenol, once with chloroform/isoamylalcohol, and twice with ether. Genomic DNA was diluted in TE buffer [lo mM Tris Cl (pH 7.4), 1 mM EDTA (pH 8.0)1 at pH 7.4 and stored at 4°C. DNA from an array of B. bovis-infected erythrocytes was extracted and quantitated in an effort to standardize samples for comparison. A source of error proved to be contributed by contaminating leukocyte DNA, particularly in samples collected and stored years earlier for other purposes. Samples were digested with PsrI and delivered into wells in agarose gels (0.6%) so that sequential tracks were arranged in a presumed progression from virulent to avirulent. Track a contained a DNA sample from Kv/intact; track b represented Kv/tick; track c received Kv/tick DNA from another tick passage of Ka (2OP); and tracks d-i contained DNA from serial passages of the original K isolate through splenectomized calves as 3P, 6P, 9P, 14P, 2OP,and 22P, respectively. After electrophoretic separation the patterns were transferred to nylon filters (Hybond-N, Amersham Australia Pty Ltd., Sydney) according to the method of Southern (1975). DNA probes were used sequentially to: (I) detect
the presence of heterogenous mixtures of subpopulations (probe pK4); (2) preferentially bind to DNA from avirulent babesia (probe pK5); and (3) quantify B. bovis DNA loading in the gels (probe pK21). Hybridizations were performed in Hybrid-Ease chambers (Hoeffer Scientific Instruments, San Francisco, CA) at 65°C in 5x SSCE(lx SSCE = 0.15M NaCl, 15 m&f Na Citrate, 10 mM EDTA), 0.1% SDS, 10x Denhardt’s solution, and 240 pg ml-’ salmon sperm DNA. Filters were washed at 65°C in 0.1 X SSCE containing 0.1% SDS. Autoradiography was for g-36 hr at - 70°C using fast tungstate screens. Filters were washed with 0.1 M NaOH, followed by neutralization with 1.5 M NaCVO.5 M Tris, pH 7.5, and finally soaked in 20x SSC to remove previous probe prior to reuse.
RESULTS
DNA extracted from nearly every isolate had six or sevenfragments, associated with the BabR locus, detected by probe pK4. In Fig. lA, track a Kv/intact DNA, from p20 after passage three times in intact calves, shows fragments of 10.5, 6.4, 4.1, 4.0, 2.7, and 1.7 kb. Kv/tick DNA, from the reversion procedure performed by tick-transmitting p20 (lanes b and c), shows fragments at 10.4, 6.4, 4.1, 4.0, 1.7, and 1.2 kb in track b and an additional fragment at 2.7 kb in track c. The earliest available sample in the attenuation series (3P) shows fragments in track d at 10.5, 6.4, 4.1, 4.0, 1.8, 1.7, and 1.2 kb. Patterns of fragments evident in tracks a-c constitute a basic pattern with consistent fragments in the range of 10.5,6.4,4.1,4.0, and 1.7 kb. Tracks a and c have fragments at 2.7 kb while tracks b and d do not. Passages3,6,9,14,20, and 22 (tracks d through i, respectively) show a tendency toward great variation in intensity of hybridization compared to the basic pattern evident in the first three tracks. Passage 3 (track d) shows a doublet at 1.7/1.8 kb and a strong fragment at 1.2 kb. Track e, which contained 6P DNA, has only three fragments located at 7.3, 3.2, and 1.5 kb. The 9P fragments (track f) are located near 10.0,7.3,6.4,3.2, and 1.7 kb. Fragments in track g, related to 14P, are located at 10.0, 7.3, 6.4, 4.0, and 1.7 kb. At the point when the passagedB. bovis is designated as the
B.
bovis: VARIATION IN SUBPOPULATIONS
A
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C abcdefghi
abcdefghi
I
I
abcdefghi
10.5 6.4 4.1 -
2.5 -
PROBE
me
PK4
FIG. 1. Southern hybridization of identical babesia DNA patterns in tracks a-i using probe pK4 in (A), probe pK5 in (B), and pK21 in (C). Tracks a-i contain: a (Kv/intact), 20P after passage three times through intact cattle; b (Kv/tick), 20P after tick transmission; c (Kv/tick), another tick-transmitted 20P sample; d, 3P; e, 6P; f, 9P; g, 14P; h, 20P; i, 22P. Probe used in (A) was removed from filter and other probes were applied sequentially for (B) and (C). Gel loading varied according to probe pK21 (C) but tracks c, d, f, h, and i exhibit signal of reasonably equal intensity; variation of hybridization pattern in these tracks in (A) is, therefore, not due to quantitative differences in DNA applied to wells.
attenuated strain (Ka) the 20P fragments in trackhappearat 10.0,6.4,4.0,4.1, 1.7, and 1.2 kb. The sample from 22P (track i) shows major fragments at 4.1, 1.9, 1.7, and 1.2 kb; lesser fragments appear at 10.0 and 6.4 kb. The composite of patterns in tracks e through i is a highly variable array generally failing to conform to the typical Kv pattern of five or six consistent fragments at the range of 10.0, 6.4, 4.1, and 1.7 kb. A second cDNA clone, pK5, has been previously suggested (Cowman et al. 1984a) as a marker of attenuation since it hybridized to a subpopulation in avirulent strains that increases as the isolate is passaged through splenectomized calves. We used pK5 to probe the DNA from different serially passaged parasites. Probe pK5 shows virtually no signal in tracks a and b (Fig. 1B) and increasing intensity at the 2.5 kb level in tracks c through i (with the exception of track e) in the progression from Kv to Ka. It is clearly a marker of aviru-
lence as the amount of hybridization increases with attenuation. The pK21 probe (Fig. lC), which hybridizes to a single copy gene represented by a 6.0-kb band in all EcoRI-digested genomic DNA isolates (Cowman et al. 1984a), was previously used to confirm that equal amounts of DNA were loaded into each track. In the case of &I-digested DNA, used in the present study, multiple bands were detected in three locations, presumably as a result of restriction length polymorphisms within the uncloned parasite population. No bands are evident in track a but one band appeared at 4.3 kb in track b; a band at 4.3 and 2.5 kb in track c; an intense band at 4.3 and a very weak band at 4.0 kb in track d; a single indistinct band at 0.75 kb in track e; a more intense band at 0.75 kb in track f; one weak band at 4.3 and a second at 0.75 in track g; and single intense 4.3-kb bands in tracks h and i. These hybridization patterns indicate approxi-
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mately equal amounts of DNA in tracks c, d, f, h, and i; lesser amounts were loaded into tracks b, e, and g; and an extremely small quantity in track a. Although loading is not constant, the observation that a typical Kv hybridization pattern for probe pK4 becomes variable with progression toward Ka (Fig. lA, tracks d through i) remains valid since these major differences are evident in tracks that contain similar amounts of DNA (e.g., Fig. 1C series including c, d, f, h, and i; also b, e, and g). In Fig. lA, track a presents a particularly weak pattern due to the extremely small amount of DNA present (no band visible in Fig. lC, track a) but the typical Kv pattern is evident nonetheless. The observed increase in intensity of pK5 fragments in Fig, 1B is validated by observing differences in tracks c, d, f, and h; also the virtual lack of signal in track d, which appears to have the largest amount of DNA. DISCUSSION
Previous analysis of genomic DNA and poly(A)+ RNA from various isolates suggested that some babesia isolates are composed of a heterogenousmixture of subpopulations (Cowman et al. 1984b).Analysis of a polymorphic gene family (BabR) indicated that the Ka isolate of B. bovis consisted of at least two subpopulations (Cowman et al. 1984a). Different-sized BabR fragments were not present in equimolar amounts and the intensity of hybridization of a certain species corresponded to less than one copy per genome. Probe pK4 detected major fragments of 5.7 and 7.7 kb in genomic DNA from Ka digested with EcoRI and only trace levels in Kv (Cowman et al. 1984a).It was proposed, therefore, that parasites containing these restriction length polymorphisms occur only rarely in the mixed parasite population that constitutes Kv and that the increase in certain fragments in Ka results from selection of minor subpopulations during the process of attenuation. Reversion of Ka to a pattern
similar to Kv, after passage through the tick, was also considered to result by selection of certain subpopulations from the mixture in Ka. The present study was designed to study possible subpopulation selection during the traditional process of attenuation by serial passage through splenectomized calves. Hybridization studies using cDNA probes, which hybridized to hypervariable loci (pK4) or preferentially to DNA from Ka parasites (pKS), indicate that during serial passagethe typical pattern of babesia parasites in Kv shows early evidence of shifting to a more variable pattern related to attenuation. Distinct genomic DNA fragments of the Kv form (i.e., 10.5, 6.4, 4.1, 4.0, 2.7, and 1.7), representing the BabR locus, exhibit variability with respect to presence, fragment size, and molar ratio as attenuation progressesfrom pass 3 through 22. Previous hybridization of probe pK4 with PvuII-digested genomic DNA from B. bovis isolates revealed differences between virulent and attenuated isolates, including Kv and Ka (Cowman et al. 1984b).The Ka pattern of fragments was more complex than Kv, with bands varying in intensity and the appearance of additional bands; two small fragments were present in quantities less than one copy per genome, indicating the presence of subpopulations. Additional information from other hybridization studies showed that composition and complexity of fragments from different Ka preparations varies and putative clones, produced by an in vivo method, implied the presence of different Ka subpopulations (Gill et al. 1987). These studies indicated that variation in fragment size probably represents subpopulation shifts, although concurrent genomic changes such as deletion, recombination, or rearrangement are possible. In the present study, babesia subpopulations, present in low numbers in Kv isolates, appear to become predominant with increased passage through splenectomized calves and variation in specific frag-
B. bovis: VARIATION IN SUBPOPULATIONS
ment signal strength reflects gene contributions from shifting proportions of subpopulations. Less than molar quantities of two fragments in 22P also indicate the presence of minor subpopulations of parasites. It is unlikely that these observed differences relate to genomic changes since major differencesin hybridization patterns occur over a short time period and would require programmed gene rearrangement in populations. Fragments at 7.3,3.2, and 1.2 kb, not pronounced in Kv but intermittent during attenuation, may result from such population shifts or may represent the tendency toward hypervariability of subpopulations emerging during passage. Differences observed with probe pK4 are not due simply to quantitative differences of DNA, although loading was apparently not uniform. Probe pK21, used for quantitation, identified more than one fragment in P&I digests compared to the single band/single copy gene identified in EcoRI digests of genomic DNA, but it is obvious that loading was similar in certain tracks and that these proved useful in substantiating findings. Different strains of attenuated babesia isolates all have exhibited the particular propensity to hybridize with probe pK5, compared to the relative absence of this quality in virulent isolates (Cowman et al. 1984b). There is no evidence that the pK5 gene is specifically related to avirulence, but the present study’s correlation between the gradual increase in hybridization intensity with serial passage,known to gradually produce attenuation, is remarkable. This pattern substantiates the serial emergence of a subpopulation of attenuated organisms with pK5 fragments and the probe seemsat least a likely marker for avirulence. It has now been shown that Ka and Kv subpopulations differ both qualitatively and quantitatively with respect to their pK4 and pK5 sequences. Evidence has been advanced in the present study for genomic similarity between the product of Ka passage through
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ticks and syringe passage through intact cattle. The attenuated subpopulations, which emerge in serial passagetoward development of Ka, therefore, revert to a basically virulent form (Kv) as previously indicated by Cowman et al. (1984b). In vitro-cultivated clones of B. bovis differ in both degree of virulence (C. A. Carson et al., unpublished data) and rate of growth in culture (Rodriguez et al. 1983). Since attenuation by passage of babesia through splenectomized calves involves serial needle passageof blood, it seemslikely that the mechanism provides a progressive enrichment for rapidly growing subpopulations. Further characterization of the dynamics of B. bovis subpopulations will require the examination of organisms cloned in vitro from heterogenous isolates, including the K strain. Employment of procedures used in this study, to derive genomic patterns for homogenous clones, should fully characterize the subpopulations of Kv and Ka. ACKNOWLEDGMENTS The authors deeply appreciate the outstanding technical assistance of Ms. Van Le and the contribution of fresh blood samples by Dr. I. Shiels, frozen blood samples with the approval of Dr. A. J. deVos, and expert advice from Dr. R. J. Dalgliesh. Special thanks to Dr. L. L. Callow, Director, Animal Research Institute, Yeerongpilly, Queensland, for making the senior author’s participation in this investigation possible. Support was provided by the Department of Primary Industries, Queensland, Australia. A. F. Cowman is an Australian Wellcome Senior Research Fellow.
REFERENCES CALLOW, L. L. 1979. Some aspects of the epidemiology and control of bovine babesiosis in Australia. Journal of the South Afn’can Veterinary Association 50, 353-356. CALLOW, L. L., AND MELLORS, L. T. 1966. A new vaccine for Eabesia argentina infection produced in splenectomized calves. Australian Veterinuty Journal 42,464465. CALLOW, L. L., MELLORS, L. T., AND MCGREOOR, W. 1979. Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle. Znternational Journal of Parasitology 9, 333-338.
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COWMAN, A. F. 1983. “Genes of the Protozoan Parasite Babesiu bovis.” PhD thesis, University of Melbourne, Australia. COWMAN, A. F., BERNARD, O., STEWART, N., AND KEMP, D. J. 1984a.Genes of the protozoan parasite Eabesiu bovis that rearrange to produce RNA species with different sequences. Cell 37, 653-660. COWMAN,A. F., TIMMS, P., AND KEMP, D. J. 1984b. DNA polymorphisms and subpopulations in Babesin bovis. Molecular and Biochemical Parasitology 1, 91-103. GILL, A. C., COWMAN, A. F., STEWART, N. P., KEMP, D. J., AND TIMMS, P. 1987. Babesiu bovis: Molecular and biological characteristics of cloned parasite lines. Experimental Parasitology 63, 180188. KAHL, L. P., ANDERS, R. F., RODWELL, B. J., TIMMS, P., AND MITCHELL, G. F. 1982. Variable and common antigens ofBabesiu bovis parasites dif-
fering in strain and virulence. Journal of Immunology 129, 1700-1705. KAHL, L. P., MITCHELL, G. F., DALGLIESH, R. J., STEWART, N. P., RODWELL, B. J., MELLORS, L. T., TIMMS, P., AND CALLOW, L. L. 1983. Babesia bovis: Proteins of virulent and avimlent parasites passaged through ticks and splenectomized or intact calves. Experimental Parasitology 56, 222-235. RODRIGUEZ,S. D., BUENING, G. M., GREEN, T. J., AND CARSON,C. A. 1983. Cloning of Babesia bovis by in vitro cultivation. Infection and Immunity 42, 15-18. SOUTHERN, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503-517. Received 26 May 1989; accepted with revision 20 November 1989