Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in Male Dermacentor andersoni (Acari: Ixodidae) Transferred Successively from Infected to Susceptible Calves KATHERINE M. KOCAN, W. L. GOFF,* DAVID STILLER,* P. L. CLAYPOOL,3 WANDA EDWARDS, S. A. EWING, JAKIE A. HAIR,4 AND SELWYN J. BARRON College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078
J. Med. Entomol. 29(4): 657-668 (1992) ABSTRACT The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10 initial body equivalents, confirming heavy infections. A. marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods. KEY WORDS Arachnida, Dermacentor andersoni, anaplasmosis, rickettsia
Anaplasma marginale Theiler is a unique rickettsia that develops within certain ixodid ticks, Although the organism can be transmitted to cattie mechanically by blood-contaminated mouthparts of biting flies and fomites, it is transmitted biologically by ticks. Approximately 20 species of ticks have been incriminated as vectors worldwide (Dikmans 1950, Ristic 1968, Bram 1975, Ewing 1981). Transstadial transmission (larva to nymph and nymph to adult) has been demonstrated repeatedly (Rees 1934; Anthony & Roby 1962; Kocan et al. 1980a, 1981; Kocan 1986). Recently, transmission has been shown to occur by adults infected as larvae without re-exposure as nymphs (Stich et al. 1989) and by male ticks transferred from infected to susceptible hosts —rr—:—rrr.
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(Potgieter 1979, Stiller & Johnson 1983, Zaugg et al. 1986, Coan et al. 1987, Stiller et al. 1989a). Morphological features of A. marginale have been studied extensively in the midgut of Dermacentor spp., and the organism has been shown to undergo a complex developmental cycle involving several morphologically distinct stages (Kocan et al. 1980a, b; Oberst et al. 1981; Kocan et al. 1983, 1984, 1986, 1989). Development of the final, infective stage of A. marginale in ticks occurs in salivary glands (Kocan et al. 1988, Stiller et al. 1989b). The developmental cycle of A. marginale in male ticks transferred from infected to susceptible calves has been reported recently (Kocan et al. 1992a, b). Males exposed as adults were found to be the most highly infected compared with ticks exposed in other stages; colonies developed in the midgut epithelial cells, ,
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State University, Pullman, Wash. 99164. gut muscle cells, and salivary gland acini. 2 Animal Disease Research Unit, USDA-ARS, Moscow, The current Study was undertaken to docuIdaho 83843. ment the persistence of A. marginale in male 3 Department of Statistics, Oklahoma State University, Still- Dermacentor andersoni Stiles during successive water, Okla. 74078. ,. .,, , ° , , c « Department of Entomology, Oklahoma State University, teedingS On susceptible calves. Anaplasma deStillwater, Okla. 74078. velopment and intensity of infection in ticks 0022-2585/92/0657-0668$02.00/0 © 1992 Entomological Society of America
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Repeated Tick Feeding
Infection of Ticks
Acquisition Feeding
^ Held * Ticks
7 Days
5 Days
Transmission ~ Feeding
10 Days
5 Days
5 Days
5 Days
5 Days
5 Days
Fig. 1. Experimental design used to study development and transmission of A. marginale by D. andersoni males transferred successively to susceptible calves.
were assessed by light microscopy, a DNA probe, and infectivity for calves during a 35-d feeding period. Microscopic examination documented parasite development, whereas the DNA probe, which detects genomic equivalents, was used to quantify DNA and to estimate parasite numbers within tick tissues. The efficacy of microscopy and the DNA probe for detection of infected ticks were compared.
bodies were detected in blood smears, the calves were monitored daily. Calves that did not develop anaplasmosis within 120 d were challengeexposed by inoculation with blood from a known carrier calf. Exposure of Ticks. Dermacentor andersoni males (1,500) were placed in orthopedic stockinettes attached to the donor calves when parasitemias reached 3-5%. The ticks were allowed to feed for 7 d, after which they were removed from the calves and placed in a humidity chamMaterials and Methods ber for 5 d (Fig. 1). The ticks (500) were selected Agent. The Virginia isolate of A. marginale randomly from those infected on the two calves, (VAM) was used to infect donor calves by whole- then placed in stockinettes on susceptible calf blood transfusion. This isolate has been used PA-59 and allowed to feed for 10 d. Ten ticks successfully in other studies of A. marginale in- were removed by hand on each of the 10 d for volving ticks and cattle in our laboratory (Kocan sampling, with the remainder transferred succeset al. 1980a, b, c, 1981; 1982; 1983; 1984; 1988; sively to five calves (Rl to R5). On each calf, ticks 1989; 1992a, b; Oberst et al. 1981; Kocan 1986; were allowed to feed for 5 d, with 10 ticks reStich et al. 1989). moved each day for sampling. Tick Propagation. Dermacentor andersoni Collection of Tissues and Light Microscopy. nymphs and adults were reared at the Oklahoma State University, Medical Entomology Labora- Midgut and salivary gland tissues were dissected tory (Patrick & Hair 1975). Larvae and nymphs from each of 10 ticks on each day of the 35-d were fed on rabbits and sheep and allowed to experiment. From each tick, four individual sammolt to the subsequent stage. Males selected ples were collected and processed: 1 salivary from among the resulting adults were held in a and V2 gut for DNA hybridization and 1 salivary humidity chamber (90-98% RH) at 25°C and a gland and V2 gut for microscopy. Uninfected 14-h photoperiod until they were used for this ticks (five per day) were collected from calf PA-64 and processed in the same manner to study. Experimental Calves. Nine splenectomized serve as controls for microscopy and DNA hycalves (2-4 mo old), determined to be free of bridization studies. The dissected tissues were anaplasmosis by the complement-fixation (CF) placed immediately in cold 2% glutaraldehyde test, were used for these studies. Two calves in a 0.1-M sodium cacodylate buffer and proc(PA-33 and PA-51) were inoculated IV with essed according to the procedures of Kocan et al. VAM-infected bovine blood and served as do- (1980b). Thick sections (1 /im) were stained with nors for infection of ticks. One susceptible calf Mallory's stain (Richardson et al. 1960) for 2 min (PA-59) was used to feed exposed ticks to test for at 60°C and were examined by light microscopy transmission of anaplasmosis, and five calves (Rl for colonies of A. marginale. through R5) were used for successive tick feedNucleic Acid Hybridization Analysis of Tick ings. Another susceptible calf (PA-64) was used Salivary Gland and Midgut Tissues. An A. marto feed uninfected control ticks. All calves were gina/e-specific DNA probe, described previously monitored three times a week by examination of in terms of specificity, sensitivity, and utility Wright-stained blood smears and determination with tick tissues, was used for this study (Goffet of packed cell volume (PCV). Once marginal al. 1988). Salivary glands and midgut were proc-
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essed individually for nucleic acid hybridization as described by Kocan et al. (1992a, b). The level of sensitivity of the probe was established from the genomic DNA standard dilutions. The amount of DNA detected was equal to the amount calculated. Based on a genome size of =1,500 Kb, the probe has consistently detected =6,000 A. marginale initial bodies. For analysis, the number of positive samples from each group was recorded and, for each positive sample, the probe signal was compared with standard genomic DNA for estimating the number of organisms in each tissue. Each probe sample consisted of Vio of the entire tick tissue (midgut or salivary glands). Therefore, after comparing each positive signal with the standard, the number of genomic equivalents was adjusted for the entire tissue (x 10) and the estimated number of organisms recorded. Samples producing a probe signal greater than the highest standard were re-evaluated after 10-fold dilutions of the samples were probed. The estimated number of organisms from each tissue sample was converted to log lo . For statistical analysis, the mean number of organisms and standard deviations for each group of tissue samples per day were established. The means were obtained using n = the number of samples producing a positive signal rather than uniformly using 10. This was done because a negative sample may indicate only Decrease of packed cell volume 61.0 60.0 66.0 51.0 56.0 57.0
Ticks were infected as adults on calves PA 33 and PA 51 that developed peak parasitemias of 74.0 and 71.5%, respectively, during tick feeding.
tissues was determined by measuring the area of gut cross-section using a calibrated ocular grid. Each colony was assigned to one of two categories (gut epithelial or gut muscle cell) based on location of the colonies as seen with light microscopy. For each tick, the colony density in gut cells (number of colonies/0.1 mm 2 of gut tissue examined) was computed. For salivary glands, the percentage of infected acini in a cross-section was determined. Statistical Analysis. All data were analyzed using an analysis of variance (ANOVA) procedure for a completely randomized design, a trend analysis based on orthogonal polynomials to determine the presence of linear or quadratic trends, and a linear regression analysis for determining the regression and correlation coefficients over specified feeding periods. Results Infection of Ticks and Transmission of Anaplasmosis. Male ticks used for this study were from a larger group of ticks exposed to A. marginale on calves PA 33 and PA 51 that developed peak parasitemias during tick feeding of 74.0 and 71.5%, respectively. After being held in the humidity chamber, the ticks fed for 10 d on calf PA 59. This calf developed anaplasmosis with an incubation period of 26 d and a peak parasitemia of 34.7% (Table 1). The five calves (R1-R5) on which ticks fed successively developed anaplasmosis with incubation periods that ranged from 19 to 23 d (Table 1). Parasitemias ranged from 19 to 40%, and the percentage reduction in PCV ranged from 51 to 66%. Calf PA 64, used for feeding of uninfected ticks, did not develop anaplasmosis and was confirmed to be susceptible to A. marginale by challenge-exposure. Microscopy Studies. Throughout the 35-d experiment, colonies of A. marginale were present in midgut epithelial cells, with the exception of day 28, when none was seen (Fig. 2A and 5A). No pattern of colony density was apparent while ticks were feeding on individual calves, even though there were significant differences among
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DAYS OF TICK FEEDING Fig. 2. Colony densities of A. marginale in midgut and percentage infected salivary gland acini determined with light microscopy in male D. andersoni that were transferred, successively, to susceptible calves.
days of feeding. Colonies were also present in percentage of ticks examined was infected in eisalivary glands throughout the experiment (Fig. ther gut or salivary glands, or both (Fig. 5). 2B and 5B). A quadratic trend in the average DNA Hybridization Studies. A high percentpercentage infected salivary gland acini was age of tick midgut and salivary glands contained present while ticks fed on each calf, and the A. marginale DNA throughout the entire 35-d pattern differed for each of the six calves (P = experiment (Fig. 6), although the amount varied 0.0071). During the first part of the experiment, considerably among ticks (Fig. 7 and 8). When colonies were most frequently observed in mid- samples (both midgut and salivary gland) progut epithelial cells (Fig. 3). However, from days duced probe signals comparable with the maxi8 to 35, A. marginale colonies occurred in gut mum standard signal, they were serially diluted muscle cells, and on days 12-35, these cells were and reprobed, but none exceeded an estimated the predominant site of Anaplasma develop- 107 organisms per tissue sample. ment, resulting in greatly enlarged cells (Fig. 3 During the first 10 d of tick feeding on calf and 4). On most days of the experiment, a large PA-59, there was a linear increase in the esti-
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mated number of organisms in midgut tissues (P = 0.0001; r = 0.84); the highest number occurred on day 7 (Fig. 9A). From this point throughout the remainder of the 35-d experiment, during which ticks were transferred to the five other calves, the estimated number of organisms decreased. The decrease was significant (P = 0.0085) but not linear. The correlation coefficient from day 11 (first day on calf Rl) through day 35 was r = —0.64. On day 35, a great decrease in detectable DNA occurred, and only 1 of the 10 midgut samples produced a positive signal (Fig. 9A). The estimated number of organisms increased in salivary glands throughout the first 10 d. However, no DNA was detected on day 1, and only one sample was probe-positive on day 2; this sample produced a strong signal, with an estimated 6 x 106 organisms. The increase in estimated infection from day 3 through 10 was linear with a correlation coefficient of regression of r = 0.68. In contrast to that of midgut tissues, estimated infections in salivary glands remained essentially constant throughout the remaining 25-d period of successive feeding (r = 0.21) (Fig. 9B). However, there was a pronounced, although statistically insignificant, decrease in salivary gland infection intensities of ticks feeding during the last 5 d on calf R5 (Fig. 9B). Table 2 shows the estimated total number of organisms present in salivary glands of all feeding ticks per day throughout the successive 25-d period. This population represents the total available organisms potentially infective for the calf on each day of feeding. Despite daily removal of 10 ticks, each calf was exposed to rather large numbers of organisms.
Comparison of Microscopy and DNA Hybridization. Microscopy and the DNA probe results were compared for their efficacy in detecting ticks infected with A. marginale (Fig. 10). The two tests were in agreement on 68.6% of the total samples. However, microscopy appeared to be more sensitive in detecting salivary gland infections, whereas the DNA probe detected considerably more infected midgut samples. When both methods were compared to determine the percentage positive samples for the entire experimental periods, there was no correlation for midgut tissues (r = 0.35), but there was a positive correlation for salivary gland tissues (r = 0.75). Discussion The current study clearly demonstrated that male D. andersoni infected as adults can readily reattach and transmit A. marginale to successive calves, causing clinical disease. Microscopy and DNA hybridization confirmed that the majority of these males can acquire and retain high infections for extended periods. It is probable that 100% of the ticks in this study were infected with A. marginale because DNA probe-negative tick tissues may have been infected with less than the probe-detectable number of 6,000 organisms. The persistent infection of midgut tissues represents a different pattern of pathogen development than that described for protozoan parasites, such as Theileria and Babesia, in which midgut infections clear as the parasites move into salivary glands. Persistence of A. marginale in midgut cells appears to be a source of organisms for infection of salivary glands over a protracted period. The predominant gut cell infected with A.
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Fig. 4. Photomicrographs of colonies of A. marginale in gut tissues of D. andersoni that were transferred to successive, susceptible calves. (A) Colonies (C) were seen in gut epithelial cells (GE) near the host nucleus (Nu) and in gut muscle cells (IMC). (B) Infected muscle cells (IMC) were seen adjacent to muscle fibers (M) on the hemocoel side of the basement membrane and were the predominant cell infected with A. marginale from days 8 to 35 of tick feeding (x 2,500, reduced to 90%).
KOCAN ET
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marginale during most of the successive feedings was the muscle cell, and in histologic sections these greatly enlarged cells were often seen adjacent to salivary gland acini where transfer of the organism could easily be effected. The current study confirms a previous one in which the DNA probe and microscopy demonstrated that, in male ticks exposed as adults, an initial replicative site of A. marginale occurs in midgut tissue, followed by infection and multiplication in salivary glands (Kocan et al. 1992a, b). In the previous study, the probe was used semiquantitatively, assigning relative strengths of signal rather than estimating numbers of organisms. The linear increase of infection seen in both gut and salivary glands suggested that the parasite replicates and that ticks may remain infected with A. marginale for long periods. The current study is the first in which we were able to quantify A. marginale in tick tissues. Most studies concerning the development of A. marginale in ticks have been limited to microscopic techniques that allow for relative estimation of parasite numbers and for determination of the developmental cycle. In the present study, microscopy has been used to define further the
role of midgut muscle cells as a transition site between midgut epithelial cells and salivary glands. However, microscopy was not useful for quantifying infection intensities because Anaplasma colonies vary greatly in size and in number of organisms they contain. Furthermore, as we found in another study (Kocan et al. 1992b), A. marginale may be present in tick tissues in a form not yet recognized by microscopy. Colonies were not observed in salivary glands of adult ticks infected as nymphs, even though salivary glands from the same ticks proved to be infective for calves and contained increasing amounts of A. marginale DNA as ticks fed. At present, accurate estimates of the number of organisms in ticks can be derived using only a DNA probe with standard DNA dilutions made from known numbers of organisms as was done in this study. It is estimated that many male ticks in this study contained between 10 and 100 million organisms. It was assumed that each day's group of 10 ticks from each calf was representative of the total feeding tick population. Thus, the calculated number of organisms from salivary glands was adjusted to estimate the total number of organisms in salivary glands from the entire feed-
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EXPOSED SALIVARY GLANDS
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Fig. 8. DNA probe hybridizations of individual salivary gland samples from male D. andersoni (A) infected with A. marginale as adults and transferred to the third of the five susceptible calves, and (B) unexposed control salivary glands collected from calf PA-64 on the same days.
July 1992
KOCAN ET AL.: PERSISTENCE OF Anaplasma
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DAYS OF TICK FEEDING Fig. 9. Mean estimated infections of A. marginale in (A) midgut and (B) salivary glands from D. andersoni males transferred successively to susceptible calves. Estimates of organisms were determined by comparison of DNA hybridization signals with standard signals from known numbers of erythrocytic initial bodies. The standard deviations and number of positive ticks are marked on each column.
ing tick population for that day. If salivary glandderived organisms are assumed to be the infective forms for cattle, then each calf was exposed to large numbers. Although we estimated the total number of organisms in salivary glands rather than the number transmitted, the potential exposure dose was large and could explain the short incubation periods observed. During the 25-d extended and successive transfer feeding period, there was a decrease in the infection level in midgut tissues, most pronounced on the last day of feeding; infection levels in salivary glands were lowest in samples taken from calf R5 on the last 5 d of feeding. However, the regression analysis did not provide evidence for a linear relationship over this interval of time. The trend toward reduction in infec-
tion in both tissues, particularly near the end of the experimental period, suggests that infections might have been depleted eventually. The length of time required to reach depletion, and whether or not at some point the decrease becomes linear, is not known. Under present experimental conditions, and using the regression line for midgut tissues, a projected time of clearance would be 194 d. This estimated persistence of infection correlates with findings in which live male ticks were recovered from cattle 176 d after application (Wilkinson 1979). Fed, exposed males were recovered live from experimental packets (simulating off-host field conditions) 216 d later and reattached and transmitted A. marginale up to 120 d later (Coan et al. 1989). Under actual field conditions, interhost transfer
666
Table 2. Summary of estimated number of organisms present in tick salivary glands for exposure of each calf during the 25-d period when ticks were transferred successively to susceptible calves Calf
Day
No. ticks"
Estimated no. positive6
E T (log)c
Rl
11 12 13 14 15
400 390 380 370 360
160 312 266 259 288
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350 340 330 320 310
315 272 297 320 310
8.51 8.93 8.70 9.34 9.45
300 290 280 270 260 250 240 230 220 210
270 290 280 216 234
9.64 9.20 8.94 8.99 8.98
250 216 207 220 210
9.39 9.37 8.86 9.25 8.83
31 32 33 34 35
200 190 180 170 160
140 133 144 136 144
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" Total number of ticks feeding on the calf for each day, accounting for removal of 10 ticks per day for light microscopy and DNA probe studies. b Number of samples estimated detectable (>6,000) organisms by the DNA probe in salivary glands based on the percent positive of the 10-tick sample. c Log10 estimates of the number of organisms in salivary glands of total infected male ticks feeding each day, based on the equation stated in the text.
might involve successive feeding on either susceptible or carrier cattle. The effect on the level and duration of infection from re-exposure due to feeding on carriers is not known, but the present data suggest that even without re-exposure, male Dermacentor may remain infected and serve as a source of infection to ruminant hosts for the tick's entire life expectancy. Light microscopy and DNA hybridization proved to be useful techniques for identification of ticks infected with A. marginale. When estimates derived by the two methods were compared to determine the percentage of tick midgut tissues that were positive, there was no correlation. However, the two methods did compare favorably when results from salivary glands were evaluated. This result may derive from the fact that microscopic representation of an entire salivary gland is much easier than for midgut, and consequently it may be easier to identify A. marginale colonies in lightly infected salivary gland tissue than in midguts. In contrast, the DNA probe identified considerably more infected midgut tissues. There may be a form of A. mar-
MIDGUTS
SALIVARY GLANDS
DNA PROBE
DNA PROBE
19 115 I + 37 174
27 15
O
50 253
TOTAL SAMPLES ONA PROBE
46 130 87 427 Fig. 10. Comparison of efficacy of light microscopy
and the DNA probe for detection of A. marginale in gut and salivary gland tissues of male D. andersoni transferred successively to susceptible calves. ginale in midgut cells that is not readily detected microscopically. Development of in situ hybridization would allow visualization of the specific location of Anaplasma DNA in these tissues. It appears from these and other studies that male ticks may play a major role in transmission of anaplasmosis. It has been demonstrated that male ticks freely transfer among hosts in the field and can remain off the host in the environment for long periods while maintaining high infection levels and rates (Coan et al. 1989; Stiller et al. 1989a). Thus, males have the potential to transmit anaplasmosis to multiple cattle in a short time. The current study provides additional evidence that male Dermacentor species are efficient vectors able to support replication of A. marginale over a long time. Ongoing studies involve the acquisition of A. marginale from lowlevel carrier cattle in efforts to determine their amplification potential and to compare the persistence of such acquired infection with that of ticks infected under laboratory conditions. Acknowledgment The authors thank Dollie Clawson and Roger W. Stich (Oklahoma State University) and Carl Johnson (USDA-ARS, Animal Disease Research Unit) for their excellent technical assistance, and Travis McGuire for constructive criticism of the manuscript. Published as journal series article 91-010 of the Oklahoma Agricultural Experiment Station, Stillwater. Supported by Oklahoma Agricultural Experiment Station projects 1669 and 1761; USDA Science and Education Administration, Animal Health Program, grant 88-341163633; and USDA-ARS, Animal Disease Research Unit project 5348-34000-008-00D.
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Kocan, K. M., T. N. Yellin, S. A. Ewing, J. A. Hair & S. J. Barron. 1984. Morphology of colonies of Anaplasma marginale in nymphal Dermacentor Anthony, D. W. & T. O. Roby. 1962. Anaplasmosis andersoni. Am. J. Vet. Res. 45: 1434-1440. studies with Dermacentor variabilis (Say) and Dermacentor andersoni Stiles (D. venustus Marx) as Kocan, K. M., K. B. Wickwire, S. A. Ewing, J. A. Hair & S. J. Barron. 1988. Preliminary studies of the deexperimental vectors, pp. 78-81. In Proceedings, velopment of Anaplasma marginale in salivary 4th National Anaplasmosis Research Conference, glands of adult, feeding Dermacentor andersoni. Reno, Nev. Am. J. Vet. Res. 49: 1010-1013. Bram, R. A. 1975. Tick-borne livestock diseases and their vectors. 1. The global problem. World Anim. Kocan, K. M., R. W. Stich, P. L. Claypool, S. A. Ewing, J. A. Hair & S. J. Barron. 1989. Intermediate site Rev. 6: 1-5. of development of Dermacentor andersoni ticks Coan, M. E., J. L. Zaugg & D. Stiller. 1987. Probthat were infected as nymphs. Am. J. Vet. Res. 51: able anaplasmosis transmission by natural intrasta128-132. dial interhost transfer of adult 3-host ticks, p. 34. In 8th Annual Western Conference Food Animal Dis- Kocan, K. M., D. Stiller, W. L. Goff, P. L. Claypool, W. Edwards, S. A. Ewing, T. C. McGuire, J. A. Hair & ease Research, University of Idaho, Boise. S. J. Barron. 1992a. Development of Anaplasma Coan, M. E., D. Stiller & G. B. Schoeler. 1989. Permarginale in male Dermacentor andersoni transsistence of Anaplasma marginale infectivity and ferred from infected to susceptible cattle. Am. J. tick survival in intrastadially infected Dermacentor Vet. Res. (in press). andersoni held in the field, pp. 161-176. In Proceedings of the 8th National Veterinary Hemopara- Kocan, K. M., W. L. Goff, D. Stiller, W. Edwards, S. A. Ewing, P. L. Claypool, T. C. McGuire, J. A. Hair & site Disease Conference. S. J. Barron. 1992b. Development of Anaplasma Dikmans, G. 1950. The transmission of anaplasmomarginale in salivary glands of male Dermacentor sis. Am. J. Vet. Res. 11: 5-16. andersoni. Am. J. Vet. Res. (in press). Ewing, S. A. 1981. Transmission of Anaplasma marginale by arthropods, pp. 395-423. In Proceed- Oberst, R. D., K. M. Kocan, J. A. Hair & S. A. Ewing. 1981. Staining characteristics of colonies of Anaings 7th National Anaplasmosis Conference, Missisplasma marginale Theiler in Dermacentor andersippi State University. soni Stiles. Am. J. Vet. Res. 42: 2006-2009. Goff, W. L., A. Barbet, D. Stiller, G. Palmer, D. Knowles, K. M. Kocan, J. Gorham & T. C. McGuire. Patrick, C. D. & J. A. Hair. 1975. Laboratory rearing procedures and equipment for multi-host ticks 1988. Detection of Anaplasma margino/e-infected (Acarina: Ixodidae). J. Med. Entomol. 12: 389-390. tick vectors by using a cloned DNA probe. Proc. Potgieter, F. T. 1979. Epizootiology and control of Natl. Acad. Sci. U.S.A. 85: 919-923. anaplasmosis in South Africa. J. South Afr. Vet. AsKocan, K. M. 1986. Development of Anaplasma soc. 50: 367-372. marginale in ixodid ticks: coordinated development of a rickettsial organism and its tick host, pp. 472- Rees, C. W. 1934. Transmission of anaplasmosis by various species of ticks. USDA Tech. Bull. 418. 505. In J. R. Sauer & J. A. Hair [eds.], Morphology, physiology and behavioral ecology of ticks. Hor- Richardson, K. C , L. Jarrett & E. H. Finke. 1960. Embedding in epoxy resins for ultrathin sectioning wood, Chichester, England. in electron microscopy. Stain Technol. 35: 313-323. Kocan, K. M., K. D. Teel & J. A. Hair. 1980a. Demonstration of Anaplasma marginale Theiler in ticks Ristic, M. 1968. Anaplasmosis, pp. 478-542. In D. Weinman & M. Ristic [eds.], Infectious blood disby tick transmission, animal inoculation and fluoeases of man and animals. Academic, New York. rescent antibody studies. Am. J. Vet. Res. 41: 183Stich, R. W., K. M. Kocan, G. H. Palmer, S. A. Ewing, 186. J. A. Hair & S. J. Barron. 1989. Transstadial and Kocan, K. M., J. A. Hair & S. A. Ewing. 1980b. attempted transovarial transmission of Anaplasma Ultrastructure of Anaplasma marginale Theiler in marginale by Dermacentor variabilis. Am. J. Vet. Dermacentor andersoni Stiles and Dermacentor Res. 50: 1377-1380. variabilis (Say). Am. J. Vet. Res. 41: 1966-1976. Kocan, K. M., K. C. Hsu, J. A. Hair & S. A. Ewing. Stiller, D. & L. W. Johnson. 1983. Experimental transmission of Anaplasma marginale Theiler by 1980c. Demonstration of Anaplasma marginale males of Dermacentor albipictus (Packard) and Theiler in Dermacentor variabilis (Say) by ferritin Dermacentor occidentalis Marx (Acari: Ixodidae), conjugated antibody technique. Am. J. Vet. Res. 41: pp. 59-65. In Proceedings of the 87th Meeting of 1077-1981. the U.S. Animal Health Association Las Vegas, Nev. Kocan, K. M., J. A. Hair, S. A. Ewing & L. G. Stratton. 1981. Transmission of Anaplasma marginale Stiller, D., M. E. Coan, W. L. Goff, L. W. Johnson & T. C. McGuire. 1989a. The importance and puTheiler by Dermacentor andersoni Stiles and Dertative role of Dermacentor spp. males in anaplasmacentor variabilis (Say). Am. J. Vet. Res. 42: 15mosis epidemiology: transmission of Anaplasma 18. marginale to cattle by ad libitum interhost transfer Kocan, K. M., S. J. Barren, D. Holbert, S. A. Ewing & of D. andersoni males under semi-natural condiJ. A. Hair. 1982. Influence of increased tempertions (abstract), p. 206. In Proceedings, 8th National ature on Anaplasma marginale Theiler in the gut of Veterinary Hemoparasite Disease Conference, St. Dermacentor andersoni Stiles. Am. J. Vet. Res. 43: Louis, Mo. 32-35. Kocan, K. M., S. A. Ewing, D. Holbert & J. A. Hair. Stiller, D., K. M. Kocan, W. Edwards, S. A. Ewing, J. A. Hair & S. J. Barron. 1989b. Detection of colonies 1983. Morphologic characteristics of colonies of of Anaplasma marginale Theiler in salivary glands Anaplasma marginale Theiler in midgut epithelial of three Dermacentor spp. infected as nymphs or cells of Dermacentor andersoni Stiles. Am. J. Vet. adults. Am. J. Vet. Res. 50: 1381-1386. Res. 43: 586-593.
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Wilkinson, P. R. 1979. Ecological aspects of pest management of ixodid ticks, pp. 25-33. In J. G. Rodriquez [ed.], Recent advances in acarology, vol. II. Academic, New York. Zaugg, J. L., D. Stiller, M. E. Coan & S. D. Lincoln. 1986. Transmission of Anaplasma marginale
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Theiler by males of Dermacentor andersoni Stiles fed on an Idaho field-infected chronic carrier cow. Am. J. Vet. Res. 47: 2269-2271. Received for publication 20 November 1991; accepted 18 February 1992.
J. Med. Entomol. 29(4): 657-668 (1992) ABSTRACT The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10 initial body equivalents, confirming heavy infections. A. marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods. KEY WORDS Arachnida, Dermacentor andersoni, anaplasmosis, rickettsia
Anaplasma marginale Theiler is a unique rickettsia that develops within certain ixodid ticks, Although the organism can be transmitted to cattie mechanically by blood-contaminated mouthparts of biting flies and fomites, it is transmitted biologically by ticks. Approximately 20 species of ticks have been incriminated as vectors worldwide (Dikmans 1950, Ristic 1968, Bram 1975, Ewing 1981). Transstadial transmission (larva to nymph and nymph to adult) has been demonstrated repeatedly (Rees 1934; Anthony & Roby 1962; Kocan et al. 1980a, 1981; Kocan 1986). Recently, transmission has been shown to occur by adults infected as larvae without re-exposure as nymphs (Stich et al. 1989) and by male ticks transferred from infected to susceptible hosts —rr—:—rrr.
„ u IT •,. TTCI-.A ADO m? u- ^.— 'Animal Disease Research Unit, UbDA-ARS, Washington
(Potgieter 1979, Stiller & Johnson 1983, Zaugg et al. 1986, Coan et al. 1987, Stiller et al. 1989a). Morphological features of A. marginale have been studied extensively in the midgut of Dermacentor spp., and the organism has been shown to undergo a complex developmental cycle involving several morphologically distinct stages (Kocan et al. 1980a, b; Oberst et al. 1981; Kocan et al. 1983, 1984, 1986, 1989). Development of the final, infective stage of A. marginale in ticks occurs in salivary glands (Kocan et al. 1988, Stiller et al. 1989b). The developmental cycle of A. marginale in male ticks transferred from infected to susceptible calves has been reported recently (Kocan et al. 1992a, b). Males exposed as adults were found to be the most highly infected compared with ticks exposed in other stages; colonies developed in the midgut epithelial cells, ,
Ir 11
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State University, Pullman, Wash. 99164. gut muscle cells, and salivary gland acini. 2 Animal Disease Research Unit, USDA-ARS, Moscow, The current Study was undertaken to docuIdaho 83843. ment the persistence of A. marginale in male 3 Department of Statistics, Oklahoma State University, Still- Dermacentor andersoni Stiles during successive water, Okla. 74078. ,. .,, , ° , , c « Department of Entomology, Oklahoma State University, teedingS On susceptible calves. Anaplasma deStillwater, Okla. 74078. velopment and intensity of infection in ticks 0022-2585/92/0657-0668$02.00/0 © 1992 Entomological Society of America
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Repeated Tick Feeding
Infection of Ticks
Acquisition Feeding
^ Held * Ticks
7 Days
5 Days
Transmission ~ Feeding
10 Days
5 Days
5 Days
5 Days
5 Days
5 Days
Fig. 1. Experimental design used to study development and transmission of A. marginale by D. andersoni males transferred successively to susceptible calves.
were assessed by light microscopy, a DNA probe, and infectivity for calves during a 35-d feeding period. Microscopic examination documented parasite development, whereas the DNA probe, which detects genomic equivalents, was used to quantify DNA and to estimate parasite numbers within tick tissues. The efficacy of microscopy and the DNA probe for detection of infected ticks were compared.
bodies were detected in blood smears, the calves were monitored daily. Calves that did not develop anaplasmosis within 120 d were challengeexposed by inoculation with blood from a known carrier calf. Exposure of Ticks. Dermacentor andersoni males (1,500) were placed in orthopedic stockinettes attached to the donor calves when parasitemias reached 3-5%. The ticks were allowed to feed for 7 d, after which they were removed from the calves and placed in a humidity chamMaterials and Methods ber for 5 d (Fig. 1). The ticks (500) were selected Agent. The Virginia isolate of A. marginale randomly from those infected on the two calves, (VAM) was used to infect donor calves by whole- then placed in stockinettes on susceptible calf blood transfusion. This isolate has been used PA-59 and allowed to feed for 10 d. Ten ticks successfully in other studies of A. marginale in- were removed by hand on each of the 10 d for volving ticks and cattle in our laboratory (Kocan sampling, with the remainder transferred succeset al. 1980a, b, c, 1981; 1982; 1983; 1984; 1988; sively to five calves (Rl to R5). On each calf, ticks 1989; 1992a, b; Oberst et al. 1981; Kocan 1986; were allowed to feed for 5 d, with 10 ticks reStich et al. 1989). moved each day for sampling. Tick Propagation. Dermacentor andersoni Collection of Tissues and Light Microscopy. nymphs and adults were reared at the Oklahoma State University, Medical Entomology Labora- Midgut and salivary gland tissues were dissected tory (Patrick & Hair 1975). Larvae and nymphs from each of 10 ticks on each day of the 35-d were fed on rabbits and sheep and allowed to experiment. From each tick, four individual sammolt to the subsequent stage. Males selected ples were collected and processed: 1 salivary from among the resulting adults were held in a and V2 gut for DNA hybridization and 1 salivary humidity chamber (90-98% RH) at 25°C and a gland and V2 gut for microscopy. Uninfected 14-h photoperiod until they were used for this ticks (five per day) were collected from calf PA-64 and processed in the same manner to study. Experimental Calves. Nine splenectomized serve as controls for microscopy and DNA hycalves (2-4 mo old), determined to be free of bridization studies. The dissected tissues were anaplasmosis by the complement-fixation (CF) placed immediately in cold 2% glutaraldehyde test, were used for these studies. Two calves in a 0.1-M sodium cacodylate buffer and proc(PA-33 and PA-51) were inoculated IV with essed according to the procedures of Kocan et al. VAM-infected bovine blood and served as do- (1980b). Thick sections (1 /im) were stained with nors for infection of ticks. One susceptible calf Mallory's stain (Richardson et al. 1960) for 2 min (PA-59) was used to feed exposed ticks to test for at 60°C and were examined by light microscopy transmission of anaplasmosis, and five calves (Rl for colonies of A. marginale. through R5) were used for successive tick feedNucleic Acid Hybridization Analysis of Tick ings. Another susceptible calf (PA-64) was used Salivary Gland and Midgut Tissues. An A. marto feed uninfected control ticks. All calves were gina/e-specific DNA probe, described previously monitored three times a week by examination of in terms of specificity, sensitivity, and utility Wright-stained blood smears and determination with tick tissues, was used for this study (Goffet of packed cell volume (PCV). Once marginal al. 1988). Salivary glands and midgut were proc-
July 1992
KOCAN ET AL.:
PERSISTENCE
essed individually for nucleic acid hybridization as described by Kocan et al. (1992a, b). The level of sensitivity of the probe was established from the genomic DNA standard dilutions. The amount of DNA detected was equal to the amount calculated. Based on a genome size of =1,500 Kb, the probe has consistently detected =6,000 A. marginale initial bodies. For analysis, the number of positive samples from each group was recorded and, for each positive sample, the probe signal was compared with standard genomic DNA for estimating the number of organisms in each tissue. Each probe sample consisted of Vio of the entire tick tissue (midgut or salivary glands). Therefore, after comparing each positive signal with the standard, the number of genomic equivalents was adjusted for the entire tissue (x 10) and the estimated number of organisms recorded. Samples producing a probe signal greater than the highest standard were re-evaluated after 10-fold dilutions of the samples were probed. The estimated number of organisms from each tissue sample was converted to log lo . For statistical analysis, the mean number of organisms and standard deviations for each group of tissue samples per day were established. The means were obtained using n = the number of samples producing a positive signal rather than uniformly using 10. This was done because a negative sample may indicate only Decrease of packed cell volume 61.0 60.0 66.0 51.0 56.0 57.0
Ticks were infected as adults on calves PA 33 and PA 51 that developed peak parasitemias of 74.0 and 71.5%, respectively, during tick feeding.
tissues was determined by measuring the area of gut cross-section using a calibrated ocular grid. Each colony was assigned to one of two categories (gut epithelial or gut muscle cell) based on location of the colonies as seen with light microscopy. For each tick, the colony density in gut cells (number of colonies/0.1 mm 2 of gut tissue examined) was computed. For salivary glands, the percentage of infected acini in a cross-section was determined. Statistical Analysis. All data were analyzed using an analysis of variance (ANOVA) procedure for a completely randomized design, a trend analysis based on orthogonal polynomials to determine the presence of linear or quadratic trends, and a linear regression analysis for determining the regression and correlation coefficients over specified feeding periods. Results Infection of Ticks and Transmission of Anaplasmosis. Male ticks used for this study were from a larger group of ticks exposed to A. marginale on calves PA 33 and PA 51 that developed peak parasitemias during tick feeding of 74.0 and 71.5%, respectively. After being held in the humidity chamber, the ticks fed for 10 d on calf PA 59. This calf developed anaplasmosis with an incubation period of 26 d and a peak parasitemia of 34.7% (Table 1). The five calves (R1-R5) on which ticks fed successively developed anaplasmosis with incubation periods that ranged from 19 to 23 d (Table 1). Parasitemias ranged from 19 to 40%, and the percentage reduction in PCV ranged from 51 to 66%. Calf PA 64, used for feeding of uninfected ticks, did not develop anaplasmosis and was confirmed to be susceptible to A. marginale by challenge-exposure. Microscopy Studies. Throughout the 35-d experiment, colonies of A. marginale were present in midgut epithelial cells, with the exception of day 28, when none was seen (Fig. 2A and 5A). No pattern of colony density was apparent while ticks were feeding on individual calves, even though there were significant differences among
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days of feeding. Colonies were also present in percentage of ticks examined was infected in eisalivary glands throughout the experiment (Fig. ther gut or salivary glands, or both (Fig. 5). 2B and 5B). A quadratic trend in the average DNA Hybridization Studies. A high percentpercentage infected salivary gland acini was age of tick midgut and salivary glands contained present while ticks fed on each calf, and the A. marginale DNA throughout the entire 35-d pattern differed for each of the six calves (P = experiment (Fig. 6), although the amount varied 0.0071). During the first part of the experiment, considerably among ticks (Fig. 7 and 8). When colonies were most frequently observed in mid- samples (both midgut and salivary gland) progut epithelial cells (Fig. 3). However, from days duced probe signals comparable with the maxi8 to 35, A. marginale colonies occurred in gut mum standard signal, they were serially diluted muscle cells, and on days 12-35, these cells were and reprobed, but none exceeded an estimated the predominant site of Anaplasma develop- 107 organisms per tissue sample. ment, resulting in greatly enlarged cells (Fig. 3 During the first 10 d of tick feeding on calf and 4). On most days of the experiment, a large PA-59, there was a linear increase in the esti-
July 1992
661
KOCAN ET AL.: PERSISTENCE OF Anaplasma IN D. andersoni
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mated number of organisms in midgut tissues (P = 0.0001; r = 0.84); the highest number occurred on day 7 (Fig. 9A). From this point throughout the remainder of the 35-d experiment, during which ticks were transferred to the five other calves, the estimated number of organisms decreased. The decrease was significant (P = 0.0085) but not linear. The correlation coefficient from day 11 (first day on calf Rl) through day 35 was r = —0.64. On day 35, a great decrease in detectable DNA occurred, and only 1 of the 10 midgut samples produced a positive signal (Fig. 9A). The estimated number of organisms increased in salivary glands throughout the first 10 d. However, no DNA was detected on day 1, and only one sample was probe-positive on day 2; this sample produced a strong signal, with an estimated 6 x 106 organisms. The increase in estimated infection from day 3 through 10 was linear with a correlation coefficient of regression of r = 0.68. In contrast to that of midgut tissues, estimated infections in salivary glands remained essentially constant throughout the remaining 25-d period of successive feeding (r = 0.21) (Fig. 9B). However, there was a pronounced, although statistically insignificant, decrease in salivary gland infection intensities of ticks feeding during the last 5 d on calf R5 (Fig. 9B). Table 2 shows the estimated total number of organisms present in salivary glands of all feeding ticks per day throughout the successive 25-d period. This population represents the total available organisms potentially infective for the calf on each day of feeding. Despite daily removal of 10 ticks, each calf was exposed to rather large numbers of organisms.
Comparison of Microscopy and DNA Hybridization. Microscopy and the DNA probe results were compared for their efficacy in detecting ticks infected with A. marginale (Fig. 10). The two tests were in agreement on 68.6% of the total samples. However, microscopy appeared to be more sensitive in detecting salivary gland infections, whereas the DNA probe detected considerably more infected midgut samples. When both methods were compared to determine the percentage positive samples for the entire experimental periods, there was no correlation for midgut tissues (r = 0.35), but there was a positive correlation for salivary gland tissues (r = 0.75). Discussion The current study clearly demonstrated that male D. andersoni infected as adults can readily reattach and transmit A. marginale to successive calves, causing clinical disease. Microscopy and DNA hybridization confirmed that the majority of these males can acquire and retain high infections for extended periods. It is probable that 100% of the ticks in this study were infected with A. marginale because DNA probe-negative tick tissues may have been infected with less than the probe-detectable number of 6,000 organisms. The persistent infection of midgut tissues represents a different pattern of pathogen development than that described for protozoan parasites, such as Theileria and Babesia, in which midgut infections clear as the parasites move into salivary glands. Persistence of A. marginale in midgut cells appears to be a source of organisms for infection of salivary glands over a protracted period. The predominant gut cell infected with A.
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Fig. 4. Photomicrographs of colonies of A. marginale in gut tissues of D. andersoni that were transferred to successive, susceptible calves. (A) Colonies (C) were seen in gut epithelial cells (GE) near the host nucleus (Nu) and in gut muscle cells (IMC). (B) Infected muscle cells (IMC) were seen adjacent to muscle fibers (M) on the hemocoel side of the basement membrane and were the predominant cell infected with A. marginale from days 8 to 35 of tick feeding (x 2,500, reduced to 90%).
KOCAN ET
July 1992
AL.: PERSISTENCE OF
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marginale during most of the successive feedings was the muscle cell, and in histologic sections these greatly enlarged cells were often seen adjacent to salivary gland acini where transfer of the organism could easily be effected. The current study confirms a previous one in which the DNA probe and microscopy demonstrated that, in male ticks exposed as adults, an initial replicative site of A. marginale occurs in midgut tissue, followed by infection and multiplication in salivary glands (Kocan et al. 1992a, b). In the previous study, the probe was used semiquantitatively, assigning relative strengths of signal rather than estimating numbers of organisms. The linear increase of infection seen in both gut and salivary glands suggested that the parasite replicates and that ticks may remain infected with A. marginale for long periods. The current study is the first in which we were able to quantify A. marginale in tick tissues. Most studies concerning the development of A. marginale in ticks have been limited to microscopic techniques that allow for relative estimation of parasite numbers and for determination of the developmental cycle. In the present study, microscopy has been used to define further the
role of midgut muscle cells as a transition site between midgut epithelial cells and salivary glands. However, microscopy was not useful for quantifying infection intensities because Anaplasma colonies vary greatly in size and in number of organisms they contain. Furthermore, as we found in another study (Kocan et al. 1992b), A. marginale may be present in tick tissues in a form not yet recognized by microscopy. Colonies were not observed in salivary glands of adult ticks infected as nymphs, even though salivary glands from the same ticks proved to be infective for calves and contained increasing amounts of A. marginale DNA as ticks fed. At present, accurate estimates of the number of organisms in ticks can be derived using only a DNA probe with standard DNA dilutions made from known numbers of organisms as was done in this study. It is estimated that many male ticks in this study contained between 10 and 100 million organisms. It was assumed that each day's group of 10 ticks from each calf was representative of the total feeding tick population. Thus, the calculated number of organisms from salivary glands was adjusted to estimate the total number of organisms in salivary glands from the entire feed-
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JOURNAL OF MEDICAL ENTOMOLOGY
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Fig. 8. DNA probe hybridizations of individual salivary gland samples from male D. andersoni (A) infected with A. marginale as adults and transferred to the third of the five susceptible calves, and (B) unexposed control salivary glands collected from calf PA-64 on the same days.
July 1992
KOCAN ET AL.: PERSISTENCE OF Anaplasma
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1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829303132333435 CALF1
CALF 2
CALF 3
CALF 4
CALF 5
DAYS OF TICK FEEDING Fig. 9. Mean estimated infections of A. marginale in (A) midgut and (B) salivary glands from D. andersoni males transferred successively to susceptible calves. Estimates of organisms were determined by comparison of DNA hybridization signals with standard signals from known numbers of erythrocytic initial bodies. The standard deviations and number of positive ticks are marked on each column.
ing tick population for that day. If salivary glandderived organisms are assumed to be the infective forms for cattle, then each calf was exposed to large numbers. Although we estimated the total number of organisms in salivary glands rather than the number transmitted, the potential exposure dose was large and could explain the short incubation periods observed. During the 25-d extended and successive transfer feeding period, there was a decrease in the infection level in midgut tissues, most pronounced on the last day of feeding; infection levels in salivary glands were lowest in samples taken from calf R5 on the last 5 d of feeding. However, the regression analysis did not provide evidence for a linear relationship over this interval of time. The trend toward reduction in infec-
tion in both tissues, particularly near the end of the experimental period, suggests that infections might have been depleted eventually. The length of time required to reach depletion, and whether or not at some point the decrease becomes linear, is not known. Under present experimental conditions, and using the regression line for midgut tissues, a projected time of clearance would be 194 d. This estimated persistence of infection correlates with findings in which live male ticks were recovered from cattle 176 d after application (Wilkinson 1979). Fed, exposed males were recovered live from experimental packets (simulating off-host field conditions) 216 d later and reattached and transmitted A. marginale up to 120 d later (Coan et al. 1989). Under actual field conditions, interhost transfer
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Table 2. Summary of estimated number of organisms present in tick salivary glands for exposure of each calf during the 25-d period when ticks were transferred successively to susceptible calves Calf
Day
No. ticks"
Estimated no. positive6
E T (log)c
Rl
11 12 13 14 15
400 390 380 370 360
160 312 266 259 288
6.98 9.37 9.66 8.68 7.57
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
350 340 330 320 310
315 272 297 320 310
8.51 8.93 8.70 9.34 9.45
300 290 280 270 260 250 240 230 220 210
270 290 280 216 234
9.64 9.20 8.94 8.99 8.98
250 216 207 220 210
9.39 9.37 8.86 9.25 8.83
31 32 33 34 35
200 190 180 170 160
140 133 144 136 144
7.61 8.18 8.13 8.16 8.14
R2
R3
R4
R5
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" Total number of ticks feeding on the calf for each day, accounting for removal of 10 ticks per day for light microscopy and DNA probe studies. b Number of samples estimated detectable (>6,000) organisms by the DNA probe in salivary glands based on the percent positive of the 10-tick sample. c Log10 estimates of the number of organisms in salivary glands of total infected male ticks feeding each day, based on the equation stated in the text.
might involve successive feeding on either susceptible or carrier cattle. The effect on the level and duration of infection from re-exposure due to feeding on carriers is not known, but the present data suggest that even without re-exposure, male Dermacentor may remain infected and serve as a source of infection to ruminant hosts for the tick's entire life expectancy. Light microscopy and DNA hybridization proved to be useful techniques for identification of ticks infected with A. marginale. When estimates derived by the two methods were compared to determine the percentage of tick midgut tissues that were positive, there was no correlation. However, the two methods did compare favorably when results from salivary glands were evaluated. This result may derive from the fact that microscopic representation of an entire salivary gland is much easier than for midgut, and consequently it may be easier to identify A. marginale colonies in lightly infected salivary gland tissue than in midguts. In contrast, the DNA probe identified considerably more infected midgut tissues. There may be a form of A. mar-
MIDGUTS
SALIVARY GLANDS
DNA PROBE
DNA PROBE
19 115 I + 37 174
27 15
O
50 253
TOTAL SAMPLES ONA PROBE
46 130 87 427 Fig. 10. Comparison of efficacy of light microscopy
and the DNA probe for detection of A. marginale in gut and salivary gland tissues of male D. andersoni transferred successively to susceptible calves. ginale in midgut cells that is not readily detected microscopically. Development of in situ hybridization would allow visualization of the specific location of Anaplasma DNA in these tissues. It appears from these and other studies that male ticks may play a major role in transmission of anaplasmosis. It has been demonstrated that male ticks freely transfer among hosts in the field and can remain off the host in the environment for long periods while maintaining high infection levels and rates (Coan et al. 1989; Stiller et al. 1989a). Thus, males have the potential to transmit anaplasmosis to multiple cattle in a short time. The current study provides additional evidence that male Dermacentor species are efficient vectors able to support replication of A. marginale over a long time. Ongoing studies involve the acquisition of A. marginale from lowlevel carrier cattle in efforts to determine their amplification potential and to compare the persistence of such acquired infection with that of ticks infected under laboratory conditions. Acknowledgment The authors thank Dollie Clawson and Roger W. Stich (Oklahoma State University) and Carl Johnson (USDA-ARS, Animal Disease Research Unit) for their excellent technical assistance, and Travis McGuire for constructive criticism of the manuscript. Published as journal series article 91-010 of the Oklahoma Agricultural Experiment Station, Stillwater. Supported by Oklahoma Agricultural Experiment Station projects 1669 and 1761; USDA Science and Education Administration, Animal Health Program, grant 88-341163633; and USDA-ARS, Animal Disease Research Unit project 5348-34000-008-00D.
July 1992
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Kocan, K. M., T. N. Yellin, S. A. Ewing, J. A. Hair & S. J. Barron. 1984. Morphology of colonies of Anaplasma marginale in nymphal Dermacentor Anthony, D. W. & T. O. Roby. 1962. Anaplasmosis andersoni. Am. J. Vet. Res. 45: 1434-1440. studies with Dermacentor variabilis (Say) and Dermacentor andersoni Stiles (D. venustus Marx) as Kocan, K. M., K. B. Wickwire, S. A. Ewing, J. A. Hair & S. J. Barron. 1988. Preliminary studies of the deexperimental vectors, pp. 78-81. In Proceedings, velopment of Anaplasma marginale in salivary 4th National Anaplasmosis Research Conference, glands of adult, feeding Dermacentor andersoni. Reno, Nev. Am. J. Vet. Res. 49: 1010-1013. Bram, R. A. 1975. Tick-borne livestock diseases and their vectors. 1. The global problem. World Anim. Kocan, K. M., R. W. Stich, P. L. Claypool, S. A. Ewing, J. A. Hair & S. J. Barron. 1989. Intermediate site Rev. 6: 1-5. of development of Dermacentor andersoni ticks Coan, M. E., J. L. Zaugg & D. Stiller. 1987. Probthat were infected as nymphs. Am. J. Vet. Res. 51: able anaplasmosis transmission by natural intrasta128-132. dial interhost transfer of adult 3-host ticks, p. 34. In 8th Annual Western Conference Food Animal Dis- Kocan, K. M., D. Stiller, W. L. Goff, P. L. Claypool, W. Edwards, S. A. Ewing, T. C. McGuire, J. A. Hair & ease Research, University of Idaho, Boise. S. J. Barron. 1992a. Development of Anaplasma Coan, M. E., D. Stiller & G. B. Schoeler. 1989. Permarginale in male Dermacentor andersoni transsistence of Anaplasma marginale infectivity and ferred from infected to susceptible cattle. Am. J. tick survival in intrastadially infected Dermacentor Vet. Res. (in press). andersoni held in the field, pp. 161-176. In Proceedings of the 8th National Veterinary Hemopara- Kocan, K. M., W. L. Goff, D. Stiller, W. Edwards, S. A. Ewing, P. L. Claypool, T. C. McGuire, J. A. Hair & site Disease Conference. S. J. Barron. 1992b. Development of Anaplasma Dikmans, G. 1950. 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AsKocan, K. M. 1986. Development of Anaplasma soc. 50: 367-372. marginale in ixodid ticks: coordinated development of a rickettsial organism and its tick host, pp. 472- Rees, C. W. 1934. Transmission of anaplasmosis by various species of ticks. USDA Tech. Bull. 418. 505. In J. R. Sauer & J. A. Hair [eds.], Morphology, physiology and behavioral ecology of ticks. Hor- Richardson, K. C , L. Jarrett & E. H. Finke. 1960. Embedding in epoxy resins for ultrathin sectioning wood, Chichester, England. in electron microscopy. Stain Technol. 35: 313-323. Kocan, K. M., K. D. Teel & J. A. Hair. 1980a. Demonstration of Anaplasma marginale Theiler in ticks Ristic, M. 1968. Anaplasmosis, pp. 478-542. In D. Weinman & M. Ristic [eds.], Infectious blood disby tick transmission, animal inoculation and fluoeases of man and animals. Academic, New York. rescent antibody studies. Am. J. Vet. Res. 41: 183Stich, R. W., K. M. Kocan, G. H. Palmer, S. A. Ewing, 186. J. A. Hair & S. J. Barron. 1989. Transstadial and Kocan, K. M., J. A. Hair & S. A. Ewing. 1980b. attempted transovarial transmission of Anaplasma Ultrastructure of Anaplasma marginale Theiler in marginale by Dermacentor variabilis. Am. J. Vet. Dermacentor andersoni Stiles and Dermacentor Res. 50: 1377-1380. variabilis (Say). Am. J. Vet. Res. 41: 1966-1976. Kocan, K. M., K. C. Hsu, J. A. Hair & S. A. Ewing. Stiller, D. & L. W. Johnson. 1983. Experimental transmission of Anaplasma marginale Theiler by 1980c. Demonstration of Anaplasma marginale males of Dermacentor albipictus (Packard) and Theiler in Dermacentor variabilis (Say) by ferritin Dermacentor occidentalis Marx (Acari: Ixodidae), conjugated antibody technique. Am. J. Vet. Res. 41: pp. 59-65. In Proceedings of the 87th Meeting of 1077-1981. the U.S. Animal Health Association Las Vegas, Nev. Kocan, K. M., J. A. Hair, S. A. Ewing & L. G. Stratton. 1981. Transmission of Anaplasma marginale Stiller, D., M. E. Coan, W. L. Goff, L. W. Johnson & T. C. McGuire. 1989a. The importance and puTheiler by Dermacentor andersoni Stiles and Dertative role of Dermacentor spp. males in anaplasmacentor variabilis (Say). Am. J. Vet. Res. 42: 15mosis epidemiology: transmission of Anaplasma 18. marginale to cattle by ad libitum interhost transfer Kocan, K. M., S. J. Barren, D. Holbert, S. A. Ewing & of D. andersoni males under semi-natural condiJ. A. Hair. 1982. Influence of increased tempertions (abstract), p. 206. In Proceedings, 8th National ature on Anaplasma marginale Theiler in the gut of Veterinary Hemoparasite Disease Conference, St. Dermacentor andersoni Stiles. Am. J. Vet. Res. 43: Louis, Mo. 32-35. Kocan, K. M., S. A. Ewing, D. Holbert & J. A. Hair. Stiller, D., K. M. Kocan, W. Edwards, S. A. Ewing, J. A. Hair & S. J. Barron. 1989b. Detection of colonies 1983. Morphologic characteristics of colonies of of Anaplasma marginale Theiler in salivary glands Anaplasma marginale Theiler in midgut epithelial of three Dermacentor spp. infected as nymphs or cells of Dermacentor andersoni Stiles. Am. J. Vet. adults. Am. J. Vet. Res. 50: 1381-1386. Res. 43: 586-593.
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Theiler by males of Dermacentor andersoni Stiles fed on an Idaho field-infected chronic carrier cow. Am. J. Vet. Res. 47: 2269-2271. Received for publication 20 November 1991; accepted 18 February 1992.
