Journal of Vector Ecology 171

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1 Vol. 30, no. 2 Journal of Vector Ecology 171 Tick infestations of the eastern cottontail rabbit (Sylvilagus floridanus) and small rodentia in northwest Alabama and implications for disease transmission Joseph C. Cooney 1, Willy Burgdorfer 2, Martin K. Painter 3, and Cynthia L. Russell 3 1 Northwest Shoals Community College, P. O. Box 101 George Wallace Blvd., Muscle Shoals, AL 35662, U.S.A. 2 U.S. Department of Health, Education, and Welfare, Public Health Service National Institutes of Health, National Institute of Allergy and Infectious Diseases, Epidemiology Branch, Rocky Mountain Laboratories, Hamilton, MT 59840, U.S.A Guntersville Street, Sheffield, AL 35660, U.S.A. Received 27 October 2005; Accepted 13 April 2005 ABSTRACT: Studies were conducted over a four-county area of northwest Alabama to determine the association of eastern cottontail rabbits with Dermacentor variabilis, the eastern United States vector of Rocky Mountain spotted fever. A secondary objective was to compare infestations of this tick on rabbits with infestations on commonly encountered rodent species as a means of determining the relative importance of each in the disease transmission cycle. These epidemiologic surveys were conducted in response to reported fatal cases of Rocky Mountain Spotted Fever in two counties of the study area. From 202 eastern cottontail rabbits, 3,956 ticks were collected. Of this total, 79.87% were Haemphysalis leporispalustris, 9.15% Amblyomma americanum, 8.22% Ixodes dentatus, and 2.76% D. variabilis. Only immature stages of D. variabilis were collected from cottontail rabbits. Ticks were collected on rabbits in all months except November, and only one specimen was taken in January. Based on the average number of ticks per host collected in each month, April was the peak month for D. variabilis and I. dentatus. High values for H. leporispalustris also occurred at this time, but even higher values occurred in October and December. The heaviest infestation of A. americanum occurred during the month of August and coincides with the activity period for the larvae of this species. Two hundred sixty-nine of the smaller Rodentia, comprising 13 species, yielded 264 ticks, all D. variabilis, and all but two were immature stages. Five rodent species, Microtus ochragaster, Orozomys palustris, Peromyscus gossypinus, Peromyscus leucopus, and Sigmodon hispidus accounted for 95.83% of the ticks collected, and appeared to be preferred hosts for D. variabilis; all five had higher infestation levels per host than did the eastern cottontail rabbit. Data on host relationships in association with seasonal activity are presented. Journal of Vector Ecology 30 (2): Keyword Index: Tick/host relationships, rodents, lagomorphs. INTRODUCTION Investigations into the natural history of the spotted fever agent, Rickettsia rickettsii, at the Tennessee Valley Authority s Land Between The Lakes recreation and conservation area (170,000 acre facility) between 1969 and 1973, provided serologic evidence that this agent was widely distributed among a large variety of medium-sized mammals (Burgdorfer et al. 1974). The eastern cottontail rabbit (Sylvilagus floridanus), as shown by complement fixation tests, had the highest number (32 of 66) of seropositives. In the same studies, Dermacentor variabilis was the only species of tick from which R. rickettsii was isolated, although numerous specimens of Amblyomma americanum and D. albipictus were examined, along with considerably smaller numbers of Haemphysalis leporispalustris and Ixodes spp. Other studies, (Hechemy et al. 1990), in Ohio yielded similar results regarding rickettsial infection in ticks. These findings tend to support the hypothesis of earlier investigators (Parker 1923, Jellison 1945, Parker et al. 1951), who suggested a strong relationship between lagomorphs and the spotted fever agent, R. rickettsii. D. variabilis is also an established vector of Francisella tularensis, the causative agent of tularemia, which is of special interest in national security because of its potent pathogenicity to man and its potential use as a bioterrorism agent (Burgdorfer et al. 1974, Tarnvik and Berglund 2003). Although the incidence of tularemia has declined from peak numbers in 1939, it remains throughout the United States and occurs in endemic foci in Arkansas, Missouri, and Oklahoma, with occasional outbreaks on Martha s Vineyard Island, Massachusetts (Feldman et al. 2001, Hornick 2001). In addition to its association with R. rickettsia and F. tularensis, D. variabilis has been studied for competence as a potential vector of several other pathogenic agents. Investigations to assess its potential for vectoring Borrelia burgdorferi based on laboratory transmission trials or examination of field-collected specimens, demonstrated that D. variabilis was not a competent vector (Piesman and Sinsky 1988, Mather and Mather 1990, Levine et al. 1991, Lindsay et al. 1991, Mukolwe et al. 1992, Sanders and Oliver 1995, Piesman and Happ 1997). Dermacentor variabilis has also been evaluated for its potential to vector Ehrlichia organisms. Studies indicate that it is not a competent vector of either human granulocytic ehrlichiosis or Ehrlichia chaffeensis

2 172 Journal of Vector Ecology December 2005 (Anderson et al. 1993, Des Vignes et al. 1999). However, in the first documented trials in the laboratory, D. variabilis effectively transmitted Ehrlichia canis to dogs (Johnson et al. 1998). This transmission represented the first by a species other than Rhipicephalus sanguineus. In addition to the human vector importance of D. variabilis, the other species included in our studies having significant human vector concern is Amblyomma americanum. Although it has been found naturally infected with Borrelia burgdorferi in a Missouri study (Oliver et al. 1998), other investigations of its vector competency based on both field and laboratory assessments suggest that it is of only minor concern to human transmission (Piesman and Sinsky 1988, Mather and Mather 1990, Sanders and Oliver 1995, Piesman and Happ 1997). It has, however, been found naturally infected with Ehrlichia chaffeensis in pools of ticks collected from five states, (Anderson et al. 1993), and more recently, Borrelia lonestari, the causative agent of southern tick associated rash illness, was detected in this tick in specimens taken in southern Alabama (Burkot et al. 2001). Although the remaining two species included in our collections, Haemaphysalis leporispalustris and Ixodes dentatus, are not known to feed on man, they were the most prevalent species to occur on cottontail rabbits. The importance of H. leporispalustris in the natural cycle of Rocky Mountain Spotted Fever has been stated previously, and I. dentatus has been documented in laboratory studies as being capable of transmitting B. burgdorferi to laboratory rabbits (Telford and Spielman 1989). I. dentatus has also been found naturally infected with a new genospecies, B. andersoni (Marconi et al. 1995). To further define the seasonal tick-host relationships of these species, studies were conducted to evaluate more precisely the role of the eastern cottontail rabbit as a host for ticks. Concurrent with these studies were complementary investigations into the seasonal host-parasite relationships of Ixodid ticks infesting 13 species of small rodents occurring in the study area. The smaller Rodentia are reportedly the preferred hosts for D. variabilis, and these studies would further clarify preference among the 13 collected rodent species. These data would also demonstrate the relative importance of rodents and lagomorphs as hosts for Ixodid ticks, especially as hosts for D. variabilis. MATERIALS AND METHODS Eastern cottontail rabbits were collected by gun from a contiguous four county area in northwest Alabama from 1970 through 1973, with most of the specimens taken in 1972 and the first half of Collections were made in all months of the year although the greatest proportions (76%) were made from April through August, the period of greatest activity and abundance for both cottontail rabbits and ticks. The seasonal abundance of cottontail rabbits was determined through personal observations of their relative numbers during collection periods throughout the year, and the period of greatest activity for the ticks included in this study was determined from personal observations and published research (Bishopp and Trembley 1945, Cooley 1946, Sonenshine and Stout 1970, Cooney and Hayes 1972, Cooney and Burgdorfer 1974, Zimmerman et al. 1988). Small rodents were collected by live trapping in the same four county area of northwest Alabama from 1970 through 1973 with the greatest proportion of specimens taken during the spring and summer of 1972 and 1973; no trapping occurred during January and February each year. All collected specimens (both rabbits and rodents) were immediately placed in tightly sealed plastic bags to prevent the escape of ticks. A chloroform saturated cotton ball was placed in each bag to kill live rodents and to facilitate the removal of live ticks which were combed from the animal fur or removed with forceps. All collected animal species were handled as humanely as possible in the process. Removed tick species were all identified to species. RESULTS Cottontail rabbit/tick relationships Infestation levels Of 202 eastern cottontail rabbits collected, 158 (78%) were infested with a total of 3,956 ticks comprising the four species Haemphysalis leporispalustris, Amblyomma americanum, Ixodes dentatus, and Dermacentor variabilis. The American dog tick, D. variabilis, ranked last in numbers of ticks collected although 17.82% of all cottontail rabbits were infested. Only 109 ticks, all immature stages, were collected from 36 infested rabbits yielding a mean intensity index, (average number per infested host), of The lone star tick, A. americanum, was the second most abundant species in terms of total number of ticks collected from cottontail rabbits; however, only 9.41% were infested, (prevalence index). The percentage of cottontail rabbits infested with A. americanum would probably have been much higher if rabbit collections had been concentrated in the months of August and September, the peak active season for the larvae of A. americanum. However, only 13% of all the rabbits collected were taken during these two months. The rabbit tick, H. 1eporispalustris, was the dominant species in terms of density per host and frequency of occurrence on cottontail rabbits (77.23%). Ixodes dentatus was third in terms of numbers of specimens taken, however, it was the second most frequently occurring species infesting 30.30% of all cottontail rabbits. Of the total number of ticks collected from cottontail rabbits, 79.87% were H. leporispalustris, 9.15% A. americanum, 8.22% I. dentatus, and 2.76% D. variabilis. Seasonal prevalence of ticks Based on values of average numbers of total ticks (all species) per host, cottontail rabbits were most heavily infested from March through October with a mid-summer slump during June and July. Peak numbers occurred in April and October and again in December. The high value obtained in December represented almost exclusively the presence of H. leporispalustris, especially larvae. No specimens of any species were collected during November and values for

3 Vol. 30, no. 2 Journal of Vector Ecology 173 January and February were very low, represented by only four nymphs of H. leporispalustris in January from four cottontail rabbits, and 35 H. leporispalustris and 10 I. dentatus from six cottontail rabbits in February. In proportion to the number of cottontail rabbits collected per month, June produced the lowest average infestation of ticks, with the exception of January and November which are considered to be months of inactivity for both D. variabilis and A. americanum. Of the total number, including all species of ticks collected, peak numbers occurred during April in which 33.3% of specimens were collected, followed by May with 18.2%. Three species, H. leporispalustris, I. dentatus, and D. variabilis, accounted for the high infestation levels recorded during these months. A. americanum was only minimally represented in the collections until August and September when the larval stages appeared, although 23 nymphs were obtained during April and May. With the exception of November in which no ticks were collected, and January in which only four specimens were obtained, cottontail rabbits appear to be heavily infested with one or more of four species of ticks throughout the year in the southeastern United States. Dermacentor variabilis was collected on rabbits from March through October (Figure 1a). About 70% of all specimens collected were taken in April and were represented by seven larvae and 68 nymphs. Occurrence of specimens remained low for the next two months and was followed by a slight resurgence in July during which time about 15% of the total numbers collected were taken. Of the total 109 specimens collected, 32 were larvae and 77 were nymphs; the adult stage did not occur. Figure 1a illustrates that rabbits were most heavily infested with D. variabilis larvae in March while nymphal infestation was greatest in April. Infestation of both life stages on cottontail rabbits dropped sharply after the peak months and then gradually declined to zero by the first of November. The cottontail rabbit most heavily infested with D. variabilis was collected in April and contained 17 nymphs. Two other cottontail rabbits contained 12 D. variabilis each. One of these collected in April contained 11 nymphs and one larva, the other collected in July contained one nymph and 11 larvae. Infestations were generally low and usually were no more than one per infested host. Amblyomma americanum infestations on cottontail rabbits were highly variable with peak averages of the number of ticks per host occurring in August and a lesser peak in September (Figure 1b). Of the 362 total specimens collected, 73% were taken in August and 19% in September. These highest values coincide with the time of year in which larval specimens of A. americanum enter the population. Of the 306 specimens collected during the two peak months, 92% were larvae. Figure 1b illustrates the seasonal infestation of A. americanum on cottontail rabbits. It should be noted that, as with D. variabilis, adults did not occur on any of the cottontail rabbit hosts. Larvae were the dominant life stage of A. americanum comprising about 86% of the total infestation. Although only 9.41% of all cottontail rabbits collected were infested with A. americanum, one specimen alone taken in August contained 247 larvae and one nymph. This same rabbit was the most heavily infested rabbit seen during the course of these studies and contained a combined total of 322 ticks. Frequency of cottontail rabbit infestation with A. americanum would be expected to be much higher, however, distribution of A. americanum in Alabama is irregular and usually coincides with high populations of deer, the preferred host for the adult stage. With the exception of a small portion of Colbert and Lauderdale Counties, most rabbit collections were made in areas known to be sparsely inhabited by deer. This situation has changed rapidly since these studies were conducted, and now virtually all portions of the previous study area have harvestable populations of white-tailed deer. Consequently A. americanum and Ixodes scapularis are now occurring in tick collections from these areas, and A. americanum has now become the object of numerous tick complaints throughout the study area (unpublished data). Haemaphysalis leporispalustris, by far the dominant species based on density/host and frequency of occurrence, appeared on cottontail rabbits in all months of the year except November. Based on infestations per rabbit, rates were highest in October followed by December and April. One hundred seventy-seven was the highest number of H. leporispalustris recorded on a single cottontail rabbit. All life stages of H. leporispalustris occurred on cottontail rabbits in the 11 months in which they were infested, except that nymphs were not present in December and larvae and adults were absent in January. The adult stage of H. leporispalustris was considerably more prevalent than either the larva or nymph, occurring 1.6 to 1.4 times more than either respectively; larvae and nymphs were about equally represented. Figure 1c illustrates the seasonal distribution of the various life stages of H. leporispalustris on cottontail rabbits. Based on average number of ticks per host, the adult and nymphal stages had graphic coincidental population peaks during April which dropped abruptly during June. Adults resurged and produced a secondary peak in August, and nymphs had a secondary peak in September. Their numbers then declined to zero by November and essentially remained there through December and January. Conversely, larval infestations were low from February through September, but then abruptly peaked in October at a level much higher than either of the other two life stages. Larval infestation levels then dropped to zero in November as did both the nymphal and adult levels. However, a rapid rise immediately followed so that December larval levels were again very high. Sampling in November may have been inadequate to accurately assess infestation levels. Ixodes dentatus was collected on cottontail rabbits throughout most of the year although no specimens were taken in January, September, and November. All life stages were taken on cottontail rabbits with the greatest number of specimens recorded during April, followed by May and March. Eighty-six percent of the total number of I. dentatus collected were adults while larvae and nymphs occurred in about equal proportions. Based on average number of ticks per host, infestations were highest during March followed closely by April. Infestations were generally light for any one cottontail rabbit with the highest number, 35, taken from a

4 174 Journal of Vector Ecology December 2005 a b c d Figure 1. Seasonal infestation of four species of Ixodid ticks on 202 eastern cottontail rabbits collected from June 1970 to September 1973 in northwest Alabama.

5 Vol. 30, no. 2 Journal of Vector Ecology 175 Table 1. Seasonal infestation of Dermacentor variabilis on 269 rodent hosts collected from June 1970 through September 1973 in northwest Alabama. Number Of Rodents Collected/Month Rodentia Species Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Total Blarina brevicauda Microtus ochragaster Mus musculus Neotoma floridana Oryzomys palustris Peromyscus gossypinus Peromyscus leucopus Peromyscus nuttalli Pitymys pinetorum Rattus norvegicus Sciurus carolinensis Sigmodon hispidus Tamias striatus Total Average Number of Dermacentor variabilis Collected/Host/Month Dermacentor variabilis Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Overall Average Larvae Nymphs Adults Composite Average

6 176 Journal of Vector Ecology December 2005 Figure 2. Average number of Dermacentor variabilis collected from 15 mammalian hosts, June 1970 to September 1973, in northwest Alabama.

7 Vol. 30, no. 2 Journal of Vector Ecology 177 Figure 3. Seasonal infestation of Dermacentor variabilis on a total of 269 rodent hosts collected in northwest Alabama, June 1970 to September *More than 99% of the total ticks collected were immatures. cottontail rabbit collected in April (29 adults and 6 nymphs). The average number of ticks recorded for infested rabbits was 5.33, however the most frequent infestation level was 1-2 per host. Figure 1d illustrates the seasonal infestation cycle for I. dentatus based on average number of ticks collected per host per month. Adults were frequently observed in copulo on the host and most often occurred in even sex ratios of males to females. Adult infestations peaked in March and then gradually receded to low levels through mid-to-late summer, followed by virtually no infestations from fall through midwinter. In contrast, larval infestations peaked in mid-fall as did nymphal density per host. The presence of larvae, nymphs, and adults on hosts in December, and adults again in February, suggests that this species remains on the host year round. The lack of specimens in January may be attributed to the paucity (4) of cottontail rabbits collected during this month. Considering the seasonal infestation data of all four species of ticks including all life stages during each month of collection, it appears that peak infestations of D. variabilis, I. dentatus, and H. leporispalustris all occurred during the spring months although H. leporispalustris produced additional peaks of even greater magnitude in October and December. A. americanum occurred at relatively low levels until August when infestations per host became high as the larval stage entered the activity cycle. When considering the total number of ticks collected, (3,956), about 35% were taken on rabbits during April. Rodent/tick relationships Infestation levels Thirteen species of the smaller Rodentia were collected concurrently with the cottontail rabbit studies and totaled 269 specimens. Table 1 lists the species and numbers collected of each. Sigmodon hispidus and Peromyscus leucopus were the dominant species represented in our collections, followed by Mus musculus, Microtus ochragaster and Tamias striatus, the latter three at much reduced levels. From all of the rodents collected, 264 D. variabilis ticks were obtained (the only species represented), and of these, more than 99% were immature stages; only one adult specimen was recorded. With the exception of Oryzomys palustris, which was represented in our collections by only one specimen, M. ochragaster was the most frequently infested species (44%), followed by Peromyscus nuttalli, P. leucopus, Peromyscus gossypinus, M. musculus, S. hispidus, and T. striatus. Although the percentage of hosts infested value for P. nuttalli was recorded as 33.33%, this figure tends to inflate the importance of this species as a host for D. variabilis, especially when considering that only three P. nuttalli were collected; only one D. variabilis was taken from these three hosts yielding an average infestation per host value of Figure 2 illustrates the relative value of 13 rodent and two lagomorph species as hosts for D. variabilis in northwest Alabama. It is apparent from this chart that at least five rodent species are more preferred hosts for D. variabilis than is Sylvilagus floridanus based on average infestation levels per host.

8 178 Journal of Vector Ecology December 2005 Rodents were collected in all months except January and February (Table 1). However collections during November and December included only three rodent specimens, demonstrating that essentially all rodent hosts were taken during the period of greatest activity for most hard ticks. Seasonal prevalence of ticks Ticks were generally collected from March through September although none were collected in June. Infestations on infested hosts (intensity) were relatively low and averaged 4.89 with one to three ticks occurring most often. Two specimens of S. hispidus however harbored 55 and 32 ticks respectively, and one P. leucopus contained 27 ticks. The composite average of ticks collected per rodent hosts (269 rodents) was Although the largest numbers of ticks were collected in March and September, per host infestation rates in March were lower than September, May, and July. Based on average infestation rates per rodent, September yielded the highest value followed by May. The seasonal activity period for immature D. variabilis based on average infestation values per host, although fluctuating monthly, displayed an increasing trend from spring to autumn, the period of highest activity (Figure 3). DISCUSSION The present observations on the significance of the eastern cottontail rabbits as a tick host are in general agreement with previously published records (Cooley and Kohls 1944, Bishop and Trembley 1945, Cooley 1946, Clifford et al. 1961, Clymer et al. 1970, Cooney and Hayes 1972, Cooney and Burgdorfer 1974, Zimmerman et al. 1988). The data also support previous findings indicating the preference of the immature stages of D. variabilis for rodents, and considerably less for lagomorphs (Cooney and Hayes 1972, Cooney and Burgdorfer 1974, Sonenshine and Stout 1970, Zimmerman et al.1987). Out of a total of 202 rabbits, only 36 (17.8%) were infested with D. variabilis, yielding 109 ticks and producing an overall average infestation of 0.54 ticks per rabbit. In contrast however, the intensity index (average number per infested host) was 3.03 suggesting the effect of other factors than merely host preference on tick density. Regardless of the cause, these infestation rates for rabbits were still considerably higher than those reported by other authors cited previously in this paper, and reflect primarily infestation with the nymphal stage (70.6% of infesting ticks). The much higher infestation rates for eastern cottontail rabbits reported in this study may be attributed to increased availability of this species as a host for D. variabilis, and inherently higher tick densities as well, associated with higher populations of the preferred hosts cohabiting the study area. Conversely, studies at the TVA s Land Between the Lakes (LBL) area in southwest Kentucky and northwest Tennessee by Cooney and Burgdorfer (1974) and Zimmerman et al. (1988) corroborate this theory. Their mammal collections included proportionately fewer lagomorphs and rodents than did the northwest Alabama collections and were derived from an area which is not frequented by domestic or feral dogs, the preferred host for the adult stage of D. variabilis. In contrast, several species of rodents had much higher frequencies of infestation and produced proportionately larger numbers of ticks per host as well. Oryzomys palustris, although represented by only one specimen, was infested with five ticks. Forty-four percent of all M.ochragaster were infested and produced an average infestation per host of 1.07, twice the rate for cottontail rabbits. Thirty-two percent of all P. leucopus were infested and produced an average infestation per host of 1.38, almost three times the rate for cottontail rabbits. Peromyscus gossypinus was infested 20% of the time and produced an average infestation rate of 0.67, still higher than the rate for rabbits. Only 12.99% of all S. hispidus were infested, however the average infestation per host was 1.60, about three times the rate of infestation of cottontail rabbits. In summarizing, five of 13 rodent species collected had higher average rates of infestation with D. variabilis than did cottontail rabbits, suggesting a preference for these hosts. Further evidence of preference was indicated by density infestation data per host. The most heavily infested rodent contained 55 ticks, whereas the most heavily infested rabbit contained only 17. Of the D. variabilis ticks infesting rodents, 73.8% were larvae, whereas infestations on rabbits were 70.6% nymphs. Each of the three tick species more commonly associated with eastern cottontail rabbits, namely H. leporispalustris, A. americanum, and I. dentatus, has been found naturally infected with rickettsiae and has been incriminated as an actual or potential vector of the spotted fever agent R. rickettsii (Burgdorfer 1975). However, recent advances in serologic procedures, particularly the application of the indirect microimmuno-fluorescence test to the typing of tick-borne rickettsiae (Philip et al. 1978), has revealed that in many instances the rickettsial agents associated with these ticks are not R. rickettsii but rather closely related agents, such as R. montana, R. rhipicephali, or hitherto unclassified agents. Because of similarities in antigenic makeup, these closely related agents elicit cross reactions with spotted fever antigens, especially in the complement fixation test. Thus, it appears likely that the high percentage of seropositive cottontail rabbits recorded in TVA s Land Between The Lakes (Burgdorfer et al. l974) reflects, in part at least, exposure to spotted fever group rickettsiae that are distinct from R. rickettsii and are transmitted by ticks with a greater predilection for rabbits than the American dog tick, D. variabilis. Ultimally it has been shown (Burgdorfer et al. 1980) that cottontail rabbits, although susceptible to R. rickettsii, are not efficient sources for infecting D. variabilis. Infection of this tick, therefore, has to originate from different sources, possibly mice and voles. In the laboratory, at least, these rodents develop infections with rickettsemias of sufficient concentration to infect simultaneously feeding larval and nymphal ticks (Burgdorfer et al. 1966).

9 Vol. 30, no. 2 Journal of Vector Ecology 179 Acknowledgments Sincere thanks are extended to Bobby McDuff and Ben Beard, former employees of the Tennessee Valley Authority, for their tabulation and computation of data and for their assistance in processing specimens. We are also indebted to the dedicated efforts of John Upton and Thomas L. Hill for their untiring work in obtaining specimens under rather rigorous conditions. These studies, and the cost of publication, were supported by Northwest Shoals Community College and the Tennessee Valley Authority. REFERENCES Anderson, B.E., K.G. Sims, J.G. Olson, J.E. Childs, J.F. Piesman, C.M. Happ, G.O. Maupin, and B.J.B. Johnson Amblyomma americanum: a potential vector of human ehrlichiosis. Am. J. Trop. Med. Hyg. 49: Bishopp, F. C. and H. L. Trembley Distribution and hosts of certain North American ticks. J. 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