EXPERIMENTAL
Babesia
PARASITOLOGY
74,
117-126 (1992)
bigemina: Quantitation of infection in Nymphal Boophilus microplus Using a DNA Probe
and Adult
JENNIFER L. HODGSON,*" DAVID STILLER,? DOUGLAS P. JASMER,* GERALD M. BUENING,$ GALE G. WAGNER,§ AND TRAVIS C. MCGUIRE*~* *Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, U.S.A.; fAnima1 Disease Research Unit, USDA-ARS, Moscow, Idaho, U.S.A.; SDepartment of Veterinary Microbiology, University of Missouri-Columbia, Columbia, Missouri, U.S.A.: and Kenter for Tropical Animal Health, College of Veterinary Medicine, Texas A & M University, College Station, Texas, U.S.A. HODGSON, J. L., STILLER, D., JASMER, MCGUIRE, T. C. 1992. Babesia bigemina:
D. P., BUENING,
G. M.,
WAGNER,
G. G., AND
Quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe. Experimental Parasitology 74, 117-126. Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia DNA probe. DNA from progeny of female Boophilus microplus infected bigemina-specific with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3 + ) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microp/us demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately lo6 infective forms. This number of infective forms is suitable for isolation and further characterization. 0 1992 Academic Press, Inc. INDEX DESCRIFTORS AND ABBREVIATIONS: Babesia bigemina; Boophilus microplus; Infective forms; DNA probe; Nymphs; Hemoparasites; Protozoa; Deoxyribonucleic acid (DNA); Tris(hydroxyethylaminoethane) (Tris); NaCVEDTAITris-HCI (NET); Ethylenediaminetetraacetic acid (EDTA); Sodium dodecyl sulfate (SDS); Sodium chloride/sodium citrate @SC); Room temperature (RT).
INTRODUCTION Babesia bigemina is an intraerythrocytic protozoan parasite which causes babesiosis in cattle. Significant economic losses are associated with this disease in countries where the tick vector, Boophilus microplus, is found (McCosker 1981). Live vaccines are currently used to reduce these losses, but have numerous inherent disadvantages (Callow 1977). The development ’ Present address: Department of Animal Health, University of Sydney, Private Mailbag 3, Camden, N.S.W. 2570, Australia. * To whom correspondence should be addressed.
of an antigenically defined vaccine containing only necessary immunogens would alleviate many of these problems (Hines et al. 1989). Antigens that could be incorporated into such a vaccine include proteins found on the surface of two parasite stages: (1) merozoites, which are found in erythrocytes of infected cattle, and (2) infective forms, which are found in salivary glands of tick vectors and are transmitted to the bovine host during tick feeding. To date, vaccine development has focused on the intraerythrocytic merozoites (Taylor 1989; Montenegro-James et al. 1989; Goodger 1989), and very little is known about the 117 0014-4894/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All tights of reproduction in any form reserved.
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ability of surface proteins of infective forms thropod vectors is the use of DNA probes (Greig and Ashall 1987; Macina et al. 1987; to induce a protective immune response. To investigate infective form proteins as Kukla et al. 1987; Goff et al. 1988; Ambrovaccine candidates, large numbers of infec- sio et al. 1988; Delves et al. 1989). The tive forms, and hence infected ticks, are re- goals of this study were to (1) detect B. bigemina in Bo. microplus with a specific quired. The only stage of Bo. microplus DNA probe, (2) identify conditions which that can become infected with B. bigemina is adult female ticks (Callow and Hoyte resulted in the highest prevalence of infection in progeny of infected female ticks, and 1961). Infected female ticks transovarially transmit B. bigemina to their progeny, but (3) quantitate B. bigemina infective forms infective forms do not develop in salivary within salivary glands under optimized conglands until the ticks have molted to the ditions. nymphal and adult stages (Callow and MATERIALSAND METHODS Hoyte 1961; Riek 1964). In earlier studies, prevalence of the bovine babesiae within Strains ofSo. microplus and B. bigemina. The three progeny of infected female ticks was found strains of Bo. microplus used in these studies (Mexico, to be very low (Riek 1964, 1966; Mahoney Santa Fe, and Martinez) were obtained from cattle in and Mirre 1971). In addition, a large num- Mexico. A noninfected colony of each strain was ber of variables were found to influence maintained under normal laboratory conditions prevalence of babesial infection of Bo. mi- (Thompson 1976) at the USDA Animal Disease Research Unit in Moscow, Idaho. croplus (Riek 1964; Dalgliesh and Stewart The strain of B. bigeminu used in these studies was 1976; Mahoney and Mirre 1977; Dalgliesh isolated from a parasitemic cow in Mexico (McElwain and Stewart 1979, 1982; Friedhoff and et al. 1987). This strain was passaged twice through Smith 1981; Friedhoff 1988). Whether these splenectomized calves, once through Bo. microplus variables also influence the numbers of in- (Mexico), and once more through a splenectomized calf. When parasitemia reached 2% in this calf, blood fective forms within salivary glands is not stabilates were made. known as infection of ticks in these studies In all experiments, 4- to 5-month-old Holstein steers was determined by the presence of kinetes were used. The animals were obtained from a Babesiawithin tick or egg squashes or by inocula- free area and maintained under conditions that pretion of tick homogenates into susceptible cluded natural infestation with ticks. Calves were monitored weekly for the absence of hemoparasitic calves. There was no direct demonstration infection by examination of Giemsa-stained smears of infective forms in salivary glands. and serology (Goff et al. 1985; Hodgson et al. 1989). In contrast, conditions causing high prev- All calves were splenectomized before use and during alence of infection within salivary glands of the experiments were maintained in individual pens tick vectors have been investigated for surrounded by moats containing acaricide. Infection ofSo. microplus with B. bigemina. To obother piroplasms including B. microti and tain infected ticks for analysis, adult female ticks were Theileria parva (Purnell et al. 1971; infected with B. bigemina as described previously Btischer and Tangus 1986; Lewengrub et (Thompson 1976; Hodgson et al. 1989)and their progal. 1987; Ochanda et al. 1988; Blouin and eny were evaluated for prevalence of infection. IndiStolz 1989). Using direct microscopic ex- vidual tick strains were first applied under separate cloth patches on the same calf so that strains could be amination of salivary gland preparations, collected separately. At an appropriate time, parasiinfection of individual salivary gland acini temia was induced in the calves either by inoculation was determined. However, this technique of a blood stabilate or by concomitant feeding of inis labor intensive and quantitation of infec- fected Bo. microplus. Parasitemias were monitored tive forms within whole salivary glands is daily (Mahoney and Saal 1961) and engorged female ticks were collected at peak parasitemia and placed in very difficult. individual glass vials during oviposition. Infection of An alternate technique for detection and engorged female ticks was determined on Day 10 of quantitation of hemoparasites within ar- oviposition by examination of Giemsa-stained he-
B. bigemina: QUANTITATION OF INFECTION IN B. microplus molymph smears for the presence of kinetes (Riek 1964). Intensity of infection (Margolis et al. 1982) of adult female ticks was scored according to numbers of kinetes present in hemolymph smears: 0 = no kinetes found in 50 fields (1000x); 1+ = lO kinetes in 50 fields but 109 cpm/ Fg, 5 x IO5 cpm/ml of hybridization solution) were added to filters and hybridized overnight at 65°C. Filters were washed twice in 100 ml of 2x SSC (IX SSC : 0.15 M NaCI, 0.015 M Na citrate) at RT for 5 min, twice in 200 ml of 2X SSC at 6s”C for 30 min, and twice in 100 ml of 0.1 X SSC at RT for 30 min. Filters were exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY) with Cronex high speed XF intensifying screens (DuPont, Wilmington, DE) and were developed after 4, 24, and 48 hr. The DNA probe used in these experiments has been described (Buening et al. 1990).The probe used in our assays was a 4.8-kb AvaI and BstEII fragment of the B. bigemina insert isolated by electroelution. Probe DNA was radiolabeled by random primer labeling (Feinberg and Vogelstein 1983) using a commercially available kit (BRL, Gaithersburg, MD) and [a-32P]dCTP at 3000 Ci/mmol sp act (New England Nuclear). Statistics. A logistical regression analysis was used to evaluate the prevalence of infection in nymphal and adult progeny of infected female ticks (Agreshi 1990).
RESULTS
Speci$city and sensitivity of the DNA probe. The DNA probe was first evaluated by hybridizing to known amounts of B. bigemina merozoite DNA and control
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HODGSON ET AL.
DNAs. The probe was specific for B. bigemina DNA as evidenced by lack of hybridization to Bo. microplus DNA, bovine DNA, and B. bovis DNA. The maximal sensitivity of the probe was 10 pg of B. bigemina DNA, 1 pg of unlabeled probe DNA, and DNA extracted from lo3 B. bigemina merozoites (Fig. 1). The signal observed for 10 pg of B. bigemina DNA was approximately equivalent to the signal for lo3 merozoites, which suggests that 1 merozoite nucleus contains approximately 0.01 pg of DNA. This is a similar amount to Plasmodium spp. (Zolg et al. 1987), another member of the phylum Apicomplexa (Levine 1988). To define probe sensitivity for the study of infected ticks, a radiolabeled probe was first hybridized to DNA extracted from noninfected ticks and ticks to which known quantities (102-106) of B. bigeminu merozoites were added. No hybridization occurred with DNA from noninfected ticks, and tick DNA did not decrease the signal intensity of B. bigeminu merozoite DNA (data not shown). Nymphal and adult progeny of infected female ticks were designated as infected if the hybridization signal was equal to or greater than the signal caused by lo3 abcde
f
FIG. 1. Sensitivity and specificity of B. bigemina DNA probe. Row 1, B. bigemina probe DNA (1 ng-10 fg); Row 2, B. bigemina DNA (10 ng-100 fg); Row 3, DNA from lo6 B. bigemina merozoites; Row 4, E. bovis DNA (100 ng-1 pg); Row 5, bovine DNA (100 ng-1 pg); and Row 6, Bo. microplus DNA (100 ng-1 pg). Lanes (a-f) are IO-fold dilutions of DNA samples.
merozoites or 10 pg of B. bigeminu DNA (Figs. 2a and 2b). Prevalence of infection. Prevalence of B. bigeminu in nymphal and adult progeny of infected female Bo. microplus was determined using the DNA probe in a dot blot analysis (Tables I and II). Several variables were evaluated to define conditions that produce the highest prevalence of infection in ticks available for these studies. The first variables examined were the strain of the tick and the infection intensity of parent ticks (Tables I and II, Experiment 1). There was no significant difference in prevalence among the three strains of Bo. microplus at the three levels of infection intensity of parent ticks, except for the Santa Fe strain where the adult progeny of 2 + parent ticks had a significantly lower prevalence than the other two strains (P < 0.05). The comparison of prevalence within the Mexico and Martinez strains, however, showed that significantly higher prevalences were observed for nymphal and adult progeny of females scored as 2+ and 3 + than for progeny of females scored as If (Tables I and II, Experiment 1). For the Santa Fe strain, prevalence was higher only in progeny of females graded as 3 + . In all groups of ticks examined in this first experiment, however, prevalence of infection was low (Tables I and II, Experiment 1; Fig. 2a). The next variable examined was the method of induction of parasitemia in the bovine host (Tables 1 and II, Experiment 2), where either a blood stabilate or infected ticks were used to induce parasitemia in calves for parent tick feeding. In this experiment, only the Martinez strain was used as previous results showed this strain consistently had the highest prevalence of infection in both parent female ticks (data not shown) and their progeny (Tables I and II, Experiment 1). Although higher prevalences were observed in nymphal and adult progeny of parent ticks that had engorged on a calf with a tick-
B. bigemina:
QUANTITATION
abcdefghij
OF INFECTION
b
abcdef
IN B.
microplus ghi
121 j
FIG. 2. (a) DNA probe analysis of adult progeny with low prevalence of infection. DNA was extracted from adult (Day 15) progeny of female i?o. microplus (Martinez) which had fed on a blood stabilate-induced parasitemia. Row 1 (lanes a-f), IO-fold dilutions of DNA extracted from lo6 B. bigemina merozoites; Row 2 (lanes a-j), DNA extracted from 10 progeny of noninfected ticks; Row 3 (lanes a-j), DNA extracted from 10 progeny of 1+ infected ticks; Row 4 (lanes a-j), DNA extracted from 10 progeny of 2 + infected ticks; Row 5 (lanes a-j), DNA extracted from 10 progeny of 3 + infected ticks; Row 6, no samples; Row 7 (lanes a-t), IO-fold dilution of B. bigemina DNA (100 ng-1 pg); Row 8 (lanes a-f), IO-fold dilution of unlabeled DNA probe (10 rig-O. 1 pg). (b) DNA probe analysis of adult progeny with high prevalence of infection. DNA was extracted from adult (Day 15) progeny of a 3 + infected female Bo. microplus (Martinez) which had fed on a parasitemia induced by infected ticks. Only eggs laid after 120 hr postengorgement were selected. Row 1 (lanes a-f), IO-fold dilutions of DNA extracted from lo6 B. bigemina merozoites; Rows 2-6 (lanes a-j), DNA extracted from 50 progeny of infected ticks; Row 7 (lanes a-j), DNA extracted from 10 progeny of noninfected ticks; Row 8 (lanes a-f), lo-fold dilution of unlabeled DNA probe (10 ng-O.1 pg), (lanes g-j) IO-fold dilutions of B. bigemina DNA (100 ng-100 pg).
transmitted parasitemia, this was not statistically significant at the P = 0.05 level. The last variable examined was the timing of egg selection (Tables I and II, Experiment 3). Prevalence in nymphal and adult progeny derived from all eggs laid by infected female ticks was compared with progeny derived from eggs laid after 120 hr postengorgement. Nymphal and adult progeny from eggs collected after 120 hr had significantly higher prevalences of infection and this higher prevalence is reflected in Fig. 2b. Quantitation of B. bigemina within salivary glands and remaining tick tissues. To determine numbers of organisms within tick tissues collected at various times after attachment, the signal intensity of log dilutions of DNA extracted from infected tick tissues was compared with DNA from
known numbers of B. bigemina merozoites (Fig. 3, Table III). Numbers of parasites in preparations of 100 pooled salivary glands were highest in Day 9 and 10 nymphal ticks and represented approximately lo6 organisms. In addition, nymphal ticks had higher numbers of B. bigemina organisms in their salivary glands than in the remaining tissues, whereas in adult ticks more organisms were found in remaining tissues than in salivary glands on the days examined. DISCUSSION
It is not known whether B. bigemina infective forms can induce a protective immune response against tick-transmitted babesiosis. However, studies on other hemoparasites have demonstrated that analogous stages can protect the mammalian host against arthropod-transmitted disease
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HODGSON ET AL.
TABLE I Prevalence of Babesiu bigemina Infection in Nymphal Progeny of Infected Female Boophilus microplus Intensity” of infection in parents Strain
Parasitemiab
Eg&
1+
2+
3+
Prevalenced in nymphal progeny Experiment 1 Mexico Martinez Santa Fe
Blood Blood Blood
Total Total Total
0 0 0
2 4 0
4 4 8
Experiment 2 Martinez Martinez
Blood Tick
Total Total
0 nd
4 nd
4 8
Experiment 3 Martinez Martinez
Tick Tick
Total >120
nd nd
nd nd
8 19
LIIntensity (1 + , 2 + , and 3 +) of infection in parent defined in text. ’ Parasitemia induced in calf by blood stabilate or infected ticks. c Eggs collected from infected female ticks; either total eggs laid or only eggs laid ~120 hr postengorgement. d Prevalence of infection in 50 nymphal ticks per condition examined expressed as a percentage. e nd, not determined.
(Nussenzweig and Nussenzweig 1989; Williamson et al. 1989). A significant deterrent to studies of B. bigemina infective forms is the low prevalence of infection in progeny of infected female Bo. microplus and low numbers of infective forms within
salivary glands of infected ticks. To resolve these issues, we used a DNA probe to define conditions that maximized numbers of B. bigemina infective forms within the salivary glands of Bo. microplus The DNA probe used in these studies
TABLE II Prevalence of Bnbesia bigemina Infection in Adult Progeny of Infected Female Boophilus microplus Intensity” of infection in parents Strain
Parasitemiab
Eggs’
I+
2+
3+
Prevalenced in nymphal progeny Experiment 1 Mexico Martinez Santa Fe
Blood Blood Blood
Total Total Total
0 2 0
8 10 0
8 10 14
Experiment 2 Martinez Martinez
Blood Tick
Total Total
2 nd’
10 nd
10 18
Experiment 3 Martinez Martinez
Tick Tick
Total >120
nd nd
nd nd
18 32
a Intensity (I+ , 2 + , and 3 +) of infection in parent defined in text. b Parasitemia induced in calf by blood stabilate or infected ticks. c Eggs collected from infected female ticks; either total eggs laid or only eggs laid > 120 hr postengorgement. d Prevalence of infection in 50 nymphal ticks per condition examined expressed as a percentage. e nd, not determined.
B. bigemina: QUANTITATION
OF INFECTION
NYMPH ’------ 6 I
RT
SG 9 N
I
N
lo”13 I
N
Q N
I
I
’
10 N
I
N
I
N
1
ADULT -1 15 -----IN
RT
SG 18 IN
17 IN
15 I
16 N
I
17 N
FIG. 3. Probe hybridization to DNA from salivary gland (SG) and remaining tick tissue (RT) preparations of infected (I) and noninfected (N) nymphal and adult Bo. microplus (Martinez). Row 1 is DNA extracted from nymphal ticks collected on Days 8,9, and 10 and adult ticks collected onDays 15, 16, and 17. Control (C) is DNA extracted from lo5 B. bigemina merozoites. Rows 2-5 are IO-fold dilutions of DNAs.
specifically detected B. bigemina within infected Bo. microplus as demonstrated by the lack of hybridization to DNA from Bo. microplus, calf thymus, and B. bovis merozoites. Sensitivity of the probe was determined to be 10 pg of B. bigemina DNA or lo3 merozoites. Thus, in this study, individual ticks containing fewer than lo3 parasites were designated as noninfected. Although this level of sensitivity was sufficient for studies to increase parasite numbers, it may account for the lower prevalence of infection observed for nymphal ticks when compared to adult ticks taken from the same group (Tables I and II). Also, the precision for detecting low parasite numbers in tick tissues may be enhanced by improving DNA extraction procedures and quantitat-
IN
B. microplus
123
ing the amount of DNA assayed from each sample. Four variables were investigated to determine conditions for the highest prevalence of infection within progeny of known infected female ticks. In this first experiment, three strains of Bo. microplus that had fed on a calf with a blood stabilateinduced parasitemia were compared. There was no statistical difference in prevalence of infection in progeny of the three strains in this study, although strain variation has been reported previously for Bo. microplus (Riek 1964; Hoffman 1971; Friedhoff and Smith 1981; Friedhoff 1988; Biischer 1989). However, prevalence of infection was increased in progreny of infected female ticks with a higher intensity of hemolymph infection for all strains of Bo. microplus investigated. This is in contrast to an earlier report (Friedhoff and Smith 1981) where it was concluded that the prevalence of infection was similar in progeny of infected female ticks regardless of the intensity of hemolymph infection. The use of different methods to quantify prevalence or variation among strains of Bo. microplus may account for this difference in results. The greatest increase in the prevalence of infection in progeny was achieved by selection of eggs during oviposition. Infection of ova increases during oviposition for both B. bigemina (Friedhoff and Smith 1981) and B. bovis (Mahoney and Mirre 1977). Thus, exclusion of eggs laid during the first 120 hr postengorgement results in a higher prevalence of infection of the remaining eggs. This result was reflected in the increased prevalence of infection in nymphal and adult ticks (Tables I and II). When this selection criterion was used in conjunction with a tick-transmitted parasitemia and selection of progeny from females with a hemolymph intensity score of 3 + , up to 33% of all progeny were infected with at least lo3 parasites. In the final experiment, the timing of maximal numbers of B. bigemina infective
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HODGSON ET AL.
TABLE III Number of Babesia bigeminn Organisms in Pooled Salivary Gland and Remaining Tick Tissue Preparations of 10 Nymphal and Adult Boophilus microplus Adult
Nymph
Day 8
Day 9
Infected Salivary glands Remaining tissue
lo5 10’
lo6 lo5
Noninfected Salivary gland Remaining tissue
0 0
0 0
forms in salivary glands was determined. Day 9 and 10 nymphal ticks had lo- to lOOfold more parasites than other ticks examined and this observation quantitatively supports a previous study (Potgieter and Els 1977) where more B. bigemina infective forms were detected in nymphal rather than adult salivary glands by light and electron microscopy. However, in both studies, only young adult ticks were examined (Days 15, 16, and 17). As male Bo. micro&s can live up to 70 days (Dalgliesh et al. 1978), higher numbers of infective forms may develop later in these ticks. The pooled salivary gland preparations from 9- and IO-day-old nymphal ticks contained approximately lo6 infective forms. Since the salivary gland preparations contained 10 salivary gland equivalents, and the prevalence of infection in this group of ticks was 33%, individual infected ticks could have approximately 3 x lo5 infective forms within their salivary glands. We anticipate that these numbers of infective forms would be adequate to isolate infective forms from tick tissues. In this final experiment we also examined parasite numbers within remaining tick tissues to determine if infective forms (in salivary glands) or kinetes (in remaining tick tissues) are the predominant stage of B. bigemina within nymphal and adult ticks. Day 9 and 10 nymphal ticks had approximately IO-fold more parasites in their salivary glands than in remaining tick tissues,
Day 10
Day 15
Day 16
Day 17
0 0
0 0
0 0
0 0
which suggests that whole nymphal ticks may be an alternative and easier source of infective forms for isolation. In contrast, adult ticks had higher numbers of parasites in remaining tick tissues than in salivary glands. Thus, other B. bigemina stages could contaminate infective forms if whole adult tick preparations were used for isolation. The increase in numbers of parasites observed in the remaining tissues of adult ticks is probably due to multiplication of kinetes and may result in prolonged infection of adult ticks. This could be of epidemiological importance in male ticks which live longer (Dalgliesh et al. 1978) and may transfer between bovine hosts (Friedhoff and Smith 1981). In conclusion, this study provides a quantitative analysis of B. bigemina infection of Bo. microplus and identifies conditions for maximal production of infective forms. These results will be used for isolation and antigenic analysis of infective forms. ACKNOWLEDGMENTS We thank Alberta Brassfield, Greg Sun, Mary Inman, Ralph Horn, and Emma Karel for excellent technical assistance. Also, we thank Stephen Hines and Alberta Brassfield for critical assessment of the manuscript. This work was supported by U.S. Agency for International Development Grant DAN-4178-A-007056-o. REFERENCES AGRESHI, A. 1990. Logistical regression analysis. In
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bi@?minU:
QUANTITATION
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miCrO#lS
125
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MAHONEY, D. F., AND MIRRE, G. B. 1971. Bovine babesiasis: Estimation of infection rates in the tick vector Boophilus microplus (Canestrini). Annals of Tropical Medicine and Parasitology 65, 309-317. MAHONEY, D. F., AND MIRRE, G. B. 1977.The selection of larvae of Boophilus microplus infected with Babesia bovis (syn B. argentina). Research in Veterinary Science 23, 126-127. MAHONEY, D. F., AND SAAL, J. R. 1961. Bovine babesiosis: Thick blood films for the detection of parasitemia. Australian Veterinary Journal 37, 4441. MANIATIS, T., FRISCH, E. F., AND SAMBROOK, J. 1982. “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MARGOLIS,I.., ESCH, G. W., HOLMES, J. C., KURIS, A. M ., *ND SCHAD, G. A. 1982. The use of ecological terms in parasitology (report of an ad hoc committee of the American Society of Parasitologists). Journal ofParasitology 68, 131-133. MCCOSKER,P. J. 1981.The global importance of babesiosis. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. l-24. Academic Press, New York. MCELWAIN, T. F., PERRYMAN, L. E., DAVIS, W. C., AND MCGUIRE, T. C. 1987. Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites. Journal of Zmmunology 138, 22912304. MONTENEGRO-JAMES, S., KAKOMA, I., AND RISTIC, M. 1989. Culture derived Babesia exoantigens as immunogens. In “Veterinary Protozoan and Hemoparasite Vaccines” (I. G. Wright, Ed.), pp. 61-97. CRC Press, Boca Raton, FL. NUSSENZWEIG,R. S., AND NLJSSENZWEIG,V. 1989. Antisporozoite vaccine for malaria: Experimental basis and current status. Reviews of Infectious Diseases ll(Supp1. 3), 579-585. OCHANDA, H., YOUNG, A. S., MUTUGI, J. J., MUMO,
J., AND OMWOYO,P. L. 1988.The effect of temperature on the rate of transmission of Theileria parva parva infection to cattle by its tick vector, Rhipicephalus appendiculatus. Parasitology 91, 23%245.
POTGIETER,F. T., AND ELS, H. J. 1977. Light and electron microscopic observations on the development of Babesia bigemina in larvae, nymphae, and non-replete females of Boophilus decoloratus. Onderstepoort
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213-232. PURNELL, R. E., BOARER, C. D. H., AND PIERCE, M. A. 1971. Theileria parva: Comparative infection rates of adults and nymphal Rhipicephaius uppendiculatus.
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RIEK, R. F. 1964. The life cycle of Bnbesia bigemina (Smith and Kilbome, 1893) in the tick vector Boophilus microplus (Canestrini). Australian Journal of Agricultural Research 15, 802-821. RIEK, R. F. 1966. The life cycle of Babesia argentina (Lignieres, 1903) (Sporozoa: Piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Australian Journal of Agricultural Research 17, 247254. TAYLOR, S. M. 1989. Babesia vaccines attenuated by blood passage and irradiation. In “Veterinary Protozoan and Hemoparasite Vaccines” (I. G. Wright, Ed.), pp. 43-59. CRC Press, Boca Raton, FL. THOMPSON,K. C. 1976. A technique to establish a laboratory colony of Boophilus microplus infected with Babesia bigemina. Veterinary Parasitology 2, 223-228. VEGA, C. A., BUENING, G. M., GREEN, T. J., AND CARSON,C. A. 1985. In vitro cultivation of Babesia bigemina. American search 46, 41w20.
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WILLIAMSON,S., TAIT, A., BROWN,D., WALKER, A., BECK, P., SHIELS, B., FLETCHER, J., AND HALL, R. 1989. Theileria annulatn sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody. Proceedings of rhe National Academy of Sciences USA 86, 4639-4643.
ZOLG, J. W., ANDRADE, L. E., AND SCOTT, E. D. 1987.Detection of Plasmodium falciparum DNA using repetitive DNA clones as species specific probes. Molecular and Biochemical Parasitology 22, 145-151. Received 22 May 1991; accepted with revision 2 August 1991
Babesia
PARASITOLOGY
74,
117-126 (1992)
bigemina: Quantitation of infection in Nymphal Boophilus microplus Using a DNA Probe
and Adult
JENNIFER L. HODGSON,*" DAVID STILLER,? DOUGLAS P. JASMER,* GERALD M. BUENING,$ GALE G. WAGNER,§ AND TRAVIS C. MCGUIRE*~* *Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, U.S.A.; fAnima1 Disease Research Unit, USDA-ARS, Moscow, Idaho, U.S.A.; SDepartment of Veterinary Microbiology, University of Missouri-Columbia, Columbia, Missouri, U.S.A.: and Kenter for Tropical Animal Health, College of Veterinary Medicine, Texas A & M University, College Station, Texas, U.S.A. HODGSON, J. L., STILLER, D., JASMER, MCGUIRE, T. C. 1992. Babesia bigemina:
D. P., BUENING,
G. M.,
WAGNER,
G. G., AND
Quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe. Experimental Parasitology 74, 117-126. Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia DNA probe. DNA from progeny of female Boophilus microplus infected bigemina-specific with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3 + ) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microp/us demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately lo6 infective forms. This number of infective forms is suitable for isolation and further characterization. 0 1992 Academic Press, Inc. INDEX DESCRIFTORS AND ABBREVIATIONS: Babesia bigemina; Boophilus microplus; Infective forms; DNA probe; Nymphs; Hemoparasites; Protozoa; Deoxyribonucleic acid (DNA); Tris(hydroxyethylaminoethane) (Tris); NaCVEDTAITris-HCI (NET); Ethylenediaminetetraacetic acid (EDTA); Sodium dodecyl sulfate (SDS); Sodium chloride/sodium citrate @SC); Room temperature (RT).
INTRODUCTION Babesia bigemina is an intraerythrocytic protozoan parasite which causes babesiosis in cattle. Significant economic losses are associated with this disease in countries where the tick vector, Boophilus microplus, is found (McCosker 1981). Live vaccines are currently used to reduce these losses, but have numerous inherent disadvantages (Callow 1977). The development ’ Present address: Department of Animal Health, University of Sydney, Private Mailbag 3, Camden, N.S.W. 2570, Australia. * To whom correspondence should be addressed.
of an antigenically defined vaccine containing only necessary immunogens would alleviate many of these problems (Hines et al. 1989). Antigens that could be incorporated into such a vaccine include proteins found on the surface of two parasite stages: (1) merozoites, which are found in erythrocytes of infected cattle, and (2) infective forms, which are found in salivary glands of tick vectors and are transmitted to the bovine host during tick feeding. To date, vaccine development has focused on the intraerythrocytic merozoites (Taylor 1989; Montenegro-James et al. 1989; Goodger 1989), and very little is known about the 117 0014-4894/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All tights of reproduction in any form reserved.
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ability of surface proteins of infective forms thropod vectors is the use of DNA probes (Greig and Ashall 1987; Macina et al. 1987; to induce a protective immune response. To investigate infective form proteins as Kukla et al. 1987; Goff et al. 1988; Ambrovaccine candidates, large numbers of infec- sio et al. 1988; Delves et al. 1989). The tive forms, and hence infected ticks, are re- goals of this study were to (1) detect B. bigemina in Bo. microplus with a specific quired. The only stage of Bo. microplus DNA probe, (2) identify conditions which that can become infected with B. bigemina is adult female ticks (Callow and Hoyte resulted in the highest prevalence of infection in progeny of infected female ticks, and 1961). Infected female ticks transovarially transmit B. bigemina to their progeny, but (3) quantitate B. bigemina infective forms infective forms do not develop in salivary within salivary glands under optimized conglands until the ticks have molted to the ditions. nymphal and adult stages (Callow and MATERIALSAND METHODS Hoyte 1961; Riek 1964). In earlier studies, prevalence of the bovine babesiae within Strains ofSo. microplus and B. bigemina. The three progeny of infected female ticks was found strains of Bo. microplus used in these studies (Mexico, to be very low (Riek 1964, 1966; Mahoney Santa Fe, and Martinez) were obtained from cattle in and Mirre 1971). In addition, a large num- Mexico. A noninfected colony of each strain was ber of variables were found to influence maintained under normal laboratory conditions prevalence of babesial infection of Bo. mi- (Thompson 1976) at the USDA Animal Disease Research Unit in Moscow, Idaho. croplus (Riek 1964; Dalgliesh and Stewart The strain of B. bigeminu used in these studies was 1976; Mahoney and Mirre 1977; Dalgliesh isolated from a parasitemic cow in Mexico (McElwain and Stewart 1979, 1982; Friedhoff and et al. 1987). This strain was passaged twice through Smith 1981; Friedhoff 1988). Whether these splenectomized calves, once through Bo. microplus variables also influence the numbers of in- (Mexico), and once more through a splenectomized calf. When parasitemia reached 2% in this calf, blood fective forms within salivary glands is not stabilates were made. known as infection of ticks in these studies In all experiments, 4- to 5-month-old Holstein steers was determined by the presence of kinetes were used. The animals were obtained from a Babesiawithin tick or egg squashes or by inocula- free area and maintained under conditions that pretion of tick homogenates into susceptible cluded natural infestation with ticks. Calves were monitored weekly for the absence of hemoparasitic calves. There was no direct demonstration infection by examination of Giemsa-stained smears of infective forms in salivary glands. and serology (Goff et al. 1985; Hodgson et al. 1989). In contrast, conditions causing high prev- All calves were splenectomized before use and during alence of infection within salivary glands of the experiments were maintained in individual pens tick vectors have been investigated for surrounded by moats containing acaricide. Infection ofSo. microplus with B. bigemina. To obother piroplasms including B. microti and tain infected ticks for analysis, adult female ticks were Theileria parva (Purnell et al. 1971; infected with B. bigemina as described previously Btischer and Tangus 1986; Lewengrub et (Thompson 1976; Hodgson et al. 1989)and their progal. 1987; Ochanda et al. 1988; Blouin and eny were evaluated for prevalence of infection. IndiStolz 1989). Using direct microscopic ex- vidual tick strains were first applied under separate cloth patches on the same calf so that strains could be amination of salivary gland preparations, collected separately. At an appropriate time, parasiinfection of individual salivary gland acini temia was induced in the calves either by inoculation was determined. However, this technique of a blood stabilate or by concomitant feeding of inis labor intensive and quantitation of infec- fected Bo. microplus. Parasitemias were monitored tive forms within whole salivary glands is daily (Mahoney and Saal 1961) and engorged female ticks were collected at peak parasitemia and placed in very difficult. individual glass vials during oviposition. Infection of An alternate technique for detection and engorged female ticks was determined on Day 10 of quantitation of hemoparasites within ar- oviposition by examination of Giemsa-stained he-
B. bigemina: QUANTITATION OF INFECTION IN B. microplus molymph smears for the presence of kinetes (Riek 1964). Intensity of infection (Margolis et al. 1982) of adult female ticks was scored according to numbers of kinetes present in hemolymph smears: 0 = no kinetes found in 50 fields (1000x); 1+ = lO kinetes in 50 fields but 109 cpm/ Fg, 5 x IO5 cpm/ml of hybridization solution) were added to filters and hybridized overnight at 65°C. Filters were washed twice in 100 ml of 2x SSC (IX SSC : 0.15 M NaCI, 0.015 M Na citrate) at RT for 5 min, twice in 200 ml of 2X SSC at 6s”C for 30 min, and twice in 100 ml of 0.1 X SSC at RT for 30 min. Filters were exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY) with Cronex high speed XF intensifying screens (DuPont, Wilmington, DE) and were developed after 4, 24, and 48 hr. The DNA probe used in these experiments has been described (Buening et al. 1990).The probe used in our assays was a 4.8-kb AvaI and BstEII fragment of the B. bigemina insert isolated by electroelution. Probe DNA was radiolabeled by random primer labeling (Feinberg and Vogelstein 1983) using a commercially available kit (BRL, Gaithersburg, MD) and [a-32P]dCTP at 3000 Ci/mmol sp act (New England Nuclear). Statistics. A logistical regression analysis was used to evaluate the prevalence of infection in nymphal and adult progeny of infected female ticks (Agreshi 1990).
RESULTS
Speci$city and sensitivity of the DNA probe. The DNA probe was first evaluated by hybridizing to known amounts of B. bigemina merozoite DNA and control
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DNAs. The probe was specific for B. bigemina DNA as evidenced by lack of hybridization to Bo. microplus DNA, bovine DNA, and B. bovis DNA. The maximal sensitivity of the probe was 10 pg of B. bigemina DNA, 1 pg of unlabeled probe DNA, and DNA extracted from lo3 B. bigemina merozoites (Fig. 1). The signal observed for 10 pg of B. bigemina DNA was approximately equivalent to the signal for lo3 merozoites, which suggests that 1 merozoite nucleus contains approximately 0.01 pg of DNA. This is a similar amount to Plasmodium spp. (Zolg et al. 1987), another member of the phylum Apicomplexa (Levine 1988). To define probe sensitivity for the study of infected ticks, a radiolabeled probe was first hybridized to DNA extracted from noninfected ticks and ticks to which known quantities (102-106) of B. bigeminu merozoites were added. No hybridization occurred with DNA from noninfected ticks, and tick DNA did not decrease the signal intensity of B. bigeminu merozoite DNA (data not shown). Nymphal and adult progeny of infected female ticks were designated as infected if the hybridization signal was equal to or greater than the signal caused by lo3 abcde
f
FIG. 1. Sensitivity and specificity of B. bigemina DNA probe. Row 1, B. bigemina probe DNA (1 ng-10 fg); Row 2, B. bigemina DNA (10 ng-100 fg); Row 3, DNA from lo6 B. bigemina merozoites; Row 4, E. bovis DNA (100 ng-1 pg); Row 5, bovine DNA (100 ng-1 pg); and Row 6, Bo. microplus DNA (100 ng-1 pg). Lanes (a-f) are IO-fold dilutions of DNA samples.
merozoites or 10 pg of B. bigeminu DNA (Figs. 2a and 2b). Prevalence of infection. Prevalence of B. bigeminu in nymphal and adult progeny of infected female Bo. microplus was determined using the DNA probe in a dot blot analysis (Tables I and II). Several variables were evaluated to define conditions that produce the highest prevalence of infection in ticks available for these studies. The first variables examined were the strain of the tick and the infection intensity of parent ticks (Tables I and II, Experiment 1). There was no significant difference in prevalence among the three strains of Bo. microplus at the three levels of infection intensity of parent ticks, except for the Santa Fe strain where the adult progeny of 2 + parent ticks had a significantly lower prevalence than the other two strains (P < 0.05). The comparison of prevalence within the Mexico and Martinez strains, however, showed that significantly higher prevalences were observed for nymphal and adult progeny of females scored as 2+ and 3 + than for progeny of females scored as If (Tables I and II, Experiment 1). For the Santa Fe strain, prevalence was higher only in progeny of females graded as 3 + . In all groups of ticks examined in this first experiment, however, prevalence of infection was low (Tables I and II, Experiment 1; Fig. 2a). The next variable examined was the method of induction of parasitemia in the bovine host (Tables 1 and II, Experiment 2), where either a blood stabilate or infected ticks were used to induce parasitemia in calves for parent tick feeding. In this experiment, only the Martinez strain was used as previous results showed this strain consistently had the highest prevalence of infection in both parent female ticks (data not shown) and their progeny (Tables I and II, Experiment 1). Although higher prevalences were observed in nymphal and adult progeny of parent ticks that had engorged on a calf with a tick-
B. bigemina:
QUANTITATION
abcdefghij
OF INFECTION
b
abcdef
IN B.
microplus ghi
121 j
FIG. 2. (a) DNA probe analysis of adult progeny with low prevalence of infection. DNA was extracted from adult (Day 15) progeny of female i?o. microplus (Martinez) which had fed on a blood stabilate-induced parasitemia. Row 1 (lanes a-f), IO-fold dilutions of DNA extracted from lo6 B. bigemina merozoites; Row 2 (lanes a-j), DNA extracted from 10 progeny of noninfected ticks; Row 3 (lanes a-j), DNA extracted from 10 progeny of 1+ infected ticks; Row 4 (lanes a-j), DNA extracted from 10 progeny of 2 + infected ticks; Row 5 (lanes a-j), DNA extracted from 10 progeny of 3 + infected ticks; Row 6, no samples; Row 7 (lanes a-t), IO-fold dilution of B. bigemina DNA (100 ng-1 pg); Row 8 (lanes a-f), IO-fold dilution of unlabeled DNA probe (10 rig-O. 1 pg). (b) DNA probe analysis of adult progeny with high prevalence of infection. DNA was extracted from adult (Day 15) progeny of a 3 + infected female Bo. microplus (Martinez) which had fed on a parasitemia induced by infected ticks. Only eggs laid after 120 hr postengorgement were selected. Row 1 (lanes a-f), IO-fold dilutions of DNA extracted from lo6 B. bigemina merozoites; Rows 2-6 (lanes a-j), DNA extracted from 50 progeny of infected ticks; Row 7 (lanes a-j), DNA extracted from 10 progeny of noninfected ticks; Row 8 (lanes a-f), lo-fold dilution of unlabeled DNA probe (10 ng-O.1 pg), (lanes g-j) IO-fold dilutions of B. bigemina DNA (100 ng-100 pg).
transmitted parasitemia, this was not statistically significant at the P = 0.05 level. The last variable examined was the timing of egg selection (Tables I and II, Experiment 3). Prevalence in nymphal and adult progeny derived from all eggs laid by infected female ticks was compared with progeny derived from eggs laid after 120 hr postengorgement. Nymphal and adult progeny from eggs collected after 120 hr had significantly higher prevalences of infection and this higher prevalence is reflected in Fig. 2b. Quantitation of B. bigemina within salivary glands and remaining tick tissues. To determine numbers of organisms within tick tissues collected at various times after attachment, the signal intensity of log dilutions of DNA extracted from infected tick tissues was compared with DNA from
known numbers of B. bigemina merozoites (Fig. 3, Table III). Numbers of parasites in preparations of 100 pooled salivary glands were highest in Day 9 and 10 nymphal ticks and represented approximately lo6 organisms. In addition, nymphal ticks had higher numbers of B. bigemina organisms in their salivary glands than in the remaining tissues, whereas in adult ticks more organisms were found in remaining tissues than in salivary glands on the days examined. DISCUSSION
It is not known whether B. bigemina infective forms can induce a protective immune response against tick-transmitted babesiosis. However, studies on other hemoparasites have demonstrated that analogous stages can protect the mammalian host against arthropod-transmitted disease
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TABLE I Prevalence of Babesiu bigemina Infection in Nymphal Progeny of Infected Female Boophilus microplus Intensity” of infection in parents Strain
Parasitemiab
Eg&
1+
2+
3+
Prevalenced in nymphal progeny Experiment 1 Mexico Martinez Santa Fe
Blood Blood Blood
Total Total Total
0 0 0
2 4 0
4 4 8
Experiment 2 Martinez Martinez
Blood Tick
Total Total
0 nd
4 nd
4 8
Experiment 3 Martinez Martinez
Tick Tick
Total >120
nd nd
nd nd
8 19
LIIntensity (1 + , 2 + , and 3 +) of infection in parent defined in text. ’ Parasitemia induced in calf by blood stabilate or infected ticks. c Eggs collected from infected female ticks; either total eggs laid or only eggs laid ~120 hr postengorgement. d Prevalence of infection in 50 nymphal ticks per condition examined expressed as a percentage. e nd, not determined.
(Nussenzweig and Nussenzweig 1989; Williamson et al. 1989). A significant deterrent to studies of B. bigemina infective forms is the low prevalence of infection in progeny of infected female Bo. microplus and low numbers of infective forms within
salivary glands of infected ticks. To resolve these issues, we used a DNA probe to define conditions that maximized numbers of B. bigemina infective forms within the salivary glands of Bo. microplus The DNA probe used in these studies
TABLE II Prevalence of Bnbesia bigemina Infection in Adult Progeny of Infected Female Boophilus microplus Intensity” of infection in parents Strain
Parasitemiab
Eggs’
I+
2+
3+
Prevalenced in nymphal progeny Experiment 1 Mexico Martinez Santa Fe
Blood Blood Blood
Total Total Total
0 2 0
8 10 0
8 10 14
Experiment 2 Martinez Martinez
Blood Tick
Total Total
2 nd’
10 nd
10 18
Experiment 3 Martinez Martinez
Tick Tick
Total >120
nd nd
nd nd
18 32
a Intensity (I+ , 2 + , and 3 +) of infection in parent defined in text. b Parasitemia induced in calf by blood stabilate or infected ticks. c Eggs collected from infected female ticks; either total eggs laid or only eggs laid > 120 hr postengorgement. d Prevalence of infection in 50 nymphal ticks per condition examined expressed as a percentage. e nd, not determined.
B. bigemina: QUANTITATION
OF INFECTION
NYMPH ’------ 6 I
RT
SG 9 N
I
N
lo”13 I
N
Q N
I
I
’
10 N
I
N
I
N
1
ADULT -1 15 -----IN
RT
SG 18 IN
17 IN
15 I
16 N
I
17 N
FIG. 3. Probe hybridization to DNA from salivary gland (SG) and remaining tick tissue (RT) preparations of infected (I) and noninfected (N) nymphal and adult Bo. microplus (Martinez). Row 1 is DNA extracted from nymphal ticks collected on Days 8,9, and 10 and adult ticks collected onDays 15, 16, and 17. Control (C) is DNA extracted from lo5 B. bigemina merozoites. Rows 2-5 are IO-fold dilutions of DNAs.
specifically detected B. bigemina within infected Bo. microplus as demonstrated by the lack of hybridization to DNA from Bo. microplus, calf thymus, and B. bovis merozoites. Sensitivity of the probe was determined to be 10 pg of B. bigemina DNA or lo3 merozoites. Thus, in this study, individual ticks containing fewer than lo3 parasites were designated as noninfected. Although this level of sensitivity was sufficient for studies to increase parasite numbers, it may account for the lower prevalence of infection observed for nymphal ticks when compared to adult ticks taken from the same group (Tables I and II). Also, the precision for detecting low parasite numbers in tick tissues may be enhanced by improving DNA extraction procedures and quantitat-
IN
B. microplus
123
ing the amount of DNA assayed from each sample. Four variables were investigated to determine conditions for the highest prevalence of infection within progeny of known infected female ticks. In this first experiment, three strains of Bo. microplus that had fed on a calf with a blood stabilateinduced parasitemia were compared. There was no statistical difference in prevalence of infection in progeny of the three strains in this study, although strain variation has been reported previously for Bo. microplus (Riek 1964; Hoffman 1971; Friedhoff and Smith 1981; Friedhoff 1988; Biischer 1989). However, prevalence of infection was increased in progreny of infected female ticks with a higher intensity of hemolymph infection for all strains of Bo. microplus investigated. This is in contrast to an earlier report (Friedhoff and Smith 1981) where it was concluded that the prevalence of infection was similar in progeny of infected female ticks regardless of the intensity of hemolymph infection. The use of different methods to quantify prevalence or variation among strains of Bo. microplus may account for this difference in results. The greatest increase in the prevalence of infection in progeny was achieved by selection of eggs during oviposition. Infection of ova increases during oviposition for both B. bigemina (Friedhoff and Smith 1981) and B. bovis (Mahoney and Mirre 1977). Thus, exclusion of eggs laid during the first 120 hr postengorgement results in a higher prevalence of infection of the remaining eggs. This result was reflected in the increased prevalence of infection in nymphal and adult ticks (Tables I and II). When this selection criterion was used in conjunction with a tick-transmitted parasitemia and selection of progeny from females with a hemolymph intensity score of 3 + , up to 33% of all progeny were infected with at least lo3 parasites. In the final experiment, the timing of maximal numbers of B. bigemina infective
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HODGSON ET AL.
TABLE III Number of Babesia bigeminn Organisms in Pooled Salivary Gland and Remaining Tick Tissue Preparations of 10 Nymphal and Adult Boophilus microplus Adult
Nymph
Day 8
Day 9
Infected Salivary glands Remaining tissue
lo5 10’
lo6 lo5
Noninfected Salivary gland Remaining tissue
0 0
0 0
forms in salivary glands was determined. Day 9 and 10 nymphal ticks had lo- to lOOfold more parasites than other ticks examined and this observation quantitatively supports a previous study (Potgieter and Els 1977) where more B. bigemina infective forms were detected in nymphal rather than adult salivary glands by light and electron microscopy. However, in both studies, only young adult ticks were examined (Days 15, 16, and 17). As male Bo. micro&s can live up to 70 days (Dalgliesh et al. 1978), higher numbers of infective forms may develop later in these ticks. The pooled salivary gland preparations from 9- and IO-day-old nymphal ticks contained approximately lo6 infective forms. Since the salivary gland preparations contained 10 salivary gland equivalents, and the prevalence of infection in this group of ticks was 33%, individual infected ticks could have approximately 3 x lo5 infective forms within their salivary glands. We anticipate that these numbers of infective forms would be adequate to isolate infective forms from tick tissues. In this final experiment we also examined parasite numbers within remaining tick tissues to determine if infective forms (in salivary glands) or kinetes (in remaining tick tissues) are the predominant stage of B. bigemina within nymphal and adult ticks. Day 9 and 10 nymphal ticks had approximately IO-fold more parasites in their salivary glands than in remaining tick tissues,
Day 10
Day 15
Day 16
Day 17
0 0
0 0
0 0
0 0
which suggests that whole nymphal ticks may be an alternative and easier source of infective forms for isolation. In contrast, adult ticks had higher numbers of parasites in remaining tick tissues than in salivary glands. Thus, other B. bigemina stages could contaminate infective forms if whole adult tick preparations were used for isolation. The increase in numbers of parasites observed in the remaining tissues of adult ticks is probably due to multiplication of kinetes and may result in prolonged infection of adult ticks. This could be of epidemiological importance in male ticks which live longer (Dalgliesh et al. 1978) and may transfer between bovine hosts (Friedhoff and Smith 1981). In conclusion, this study provides a quantitative analysis of B. bigemina infection of Bo. microplus and identifies conditions for maximal production of infective forms. These results will be used for isolation and antigenic analysis of infective forms. ACKNOWLEDGMENTS We thank Alberta Brassfield, Greg Sun, Mary Inman, Ralph Horn, and Emma Karel for excellent technical assistance. Also, we thank Stephen Hines and Alberta Brassfield for critical assessment of the manuscript. This work was supported by U.S. Agency for International Development Grant DAN-4178-A-007056-o. REFERENCES AGRESHI, A. 1990. Logistical regression analysis. In
B.
bi@?minU:
QUANTITATION
OF INFECTION
IN
B.
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