parasite resistance and genetic variation
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Animal Conservation (2001) 4, 103–109 © 2001 The Zoological Society of London Printed in the United Kingdom Parasite resistance and genetic variation in the endangered Gila topminnow Philip W. Hedrick, Timothy J. Kim and Karen M. Parker1 Department of Biology, Arizona State University, Tempe, AZ 85287, USA 1 Present address: Inst. de Zoologie et d’Ecologie Anim., Université de Lausanne, Bâtiment de Biologie, Lausanne 1015, Switzerland (Received 22 May 2000; accepted 8 November 2000) Abstract In recent years, it has become apparent that introduced or novel pathogens or parasites may have a sig- niﬁcant negative impact on endangered species. Here we examine experimentally the effect of an exotic ﬂuke from guppies on the endangered Gila topminnow. Populations from different sources showed vari- able responses (although statistically non-signiﬁcant) to the ﬂuke and, in particular, the most homozy- gous population had high ﬂuke infections and high subsequent mortality. Homozygotes for a MHC (major histocompatibility complex) gene had lower (although statistically non-signiﬁcant) survival when infected with ﬂukes than did heterozygotes. An inbred line from one of the populations had statistically signiﬁcant lower survival and higher ﬂuke infection than did a simultaneous outbred control. Overall, Gila topminnows appear quite susceptible to infection by the non-native ﬂuke compared to other related species. In addition, it was shown that Gila topminnows can be infected by casual contact with infected guppies. This is another example of the potential detrimental effects of a parasite on an endangered species, a threat that may constitute a particular problem for species with low genetic variation, either in general, for important MHC genes, or for populations with a past history of inbreeding. INTRODUCTION Resistance to HIV and hepatitis in humans appears to be higher for MHC heterozygotes than for homozygotes Threats to endangered species include human mediated (Thurz et al., 1997; Carrington et al., 1999). However, effects of habitat alteration and destruction, hunting and the level of genetic variation in the MHC, and other pollution, and the biotic effects of introduced competi- genes that inﬂuence host resistance, may be lower in tors and predators. In recent years, it has become widely endangered species because of past or present small pop- recognized that endangered species may be also threat- ulation size. This may make endangered species even ened by exposure to parasites (O’Brien & Evermann, more susceptible to the effects of parasites than com- 1988; Lyles & Dobson, 1993), many of them exotic and mon species that have large population sizes and con- novel to the endangered species. Here we will deﬁne sequently higher levels of genetic variation. In addition, parasites broadly to include viruses, bacteria, protozoa, inbred animals within a population (inbreeding may be helminths and arthropods, which can cause a reduction of higher frequency in the small populations of endan- in ﬁtness to the host (Anderson & May, 1979). Often gered species) may have higher susceptibility than out- parasites may not be able to maintain themselves in an bred individuals (Coltman et al., 1999). endangered species because of low host density. The Gila topminnow (Poeciliopsis o. occidentalis) However, if the parasites can infect multiple hosts, they was once the most common ﬁsh species in the Gila River may have a reservoir in more common related taxa, such watershed, which drains much of southeastern and east as introduced or domesticated species, including live- central Arizona, United States, but now it is present in stock or pets (Woodroffe, 1999). only a few isolated headwaters and springs (Minckley, Resistance of a host to parasites often has an impor- 1999). The major reasons for this decline are thought to tant genetic component. In particular, genes in the MHC be loss of habitat and the introduction of the non-native (major histocompatibility complex) (Edwards & western mosquito ﬁsh (Gambusia afﬁnis). The Gila Hedrick, 1998) play an important role in disease resis- topminnow was listed in the United States as endangered tance to parasites in vertebrates (Hedrick & Kim, 2000). in 1967 and has since been the subject of extensive For example, the MHC in humans has been shown to be translocation and research efforts to promote recovery. important in resistance to HIV, hepatitis and malaria. Research includes genetic studies to determine the All correspondence to: Philip W. Hedrick. Tel: (480) 965-0799; amount and pattern of molecular genetic variation and Fax: (480) 965-2519; E-mail: email@example.com. ﬁtness differences over the extant populations 104 P. W. HEDRICK ET AL. (Vrijenhoek, Douglas & Meffe, 1985; Quattro & heavily infected guppy was anaesthetized with MS 222 Vrijenhoek, 1989; Sheffer, 1997; Parker, Sheffer & (23 mg/l). Flukes from this guppy were used to infect Hedrick, 1999; Sheffer, Hedrick & Velasco, 1999). In topminnows with two ﬂukes each, an average standard addition, Hedrick & Parker (1998) investigated variation used in other investigations (Scott, 1982; Leberg & for a class II MHC gene in Gila topminnow populations Vrijenhoek, 1994) (a pilot study on Gila topminnow from the four watersheds in which they remain. Relevant indicated inoculation with ﬁve ﬂukes quickly over- to our study here, they found no variation at this MHC whelmed the ﬁsh). The topminnow to receive ﬂukes was gene in the Bylas Spring population while they found also anaesthetized with MS 222 and, using two ﬁne for- MHC variation in samples from Cienega Creek, Monkey ceps, an infected guppy ﬁn was placed on the caudal ﬁn Spring and Sharp Spring. of the topminnow under a dissecting microscope We investigated resistance of the Gila topminnow to (25–30×). After two ﬂukes were transferred, the top- the exotic ﬂuke, Gyrodactylus turnbulli, a short-lived, minnow was placed in a plastic container with 500 ml native parasite on wild guppies, Poecilia reticulata, in of water. Each was fed Tetramin daily and was on a Trinidad (Harris, 1986). This ﬂuke has been a subject of 10/14 dark/light cycle. Topminnows were sedated at study both in guppies (Scott, 1982; Lyles, 1990) and in 5-day intervals and the ﬂuke numbers assayed until day Mexican Poeciliopsis species (Leberg & Vrijenhoek, 25. By this time, virtually all of the ﬁsh either had elim- 1994) closely related to P. o. occidentalis. It is a mono- inated the ﬂukes or had died from heavy ﬂuke infesta- genean helminth living on body surfaces, mainly gills, tion. During experiments, a topminnow from each test and multiplying viviparously and generally asexually. category, such as different populations, received ﬂukes Guppies are native to northern South America and from the same guppy to reduce any differences that may nearby Caribbean islands, but have been spread world- occur by using ﬂukes from different source guppies. wide in suitable habitat and persist in warm springs in Generation length of the ﬂuke is short, on average 4.2 several western states of the United States, including days on guppies (Scott, 1982) and Leberg & Vrijenhoek Arizona. As a result, guppy parasites, such as G. turn- (1994) stated that generation length at 25°C was 2.4 bulli, have increased in geographic distribution and may days. Although we did not speciﬁcally measure genera- potentially become an exotic parasite on populations of tion length with life-history information, under our hus- Gila topminnows. A related ﬂuke, G. salaris, has bandry and temperature (average 25°C) conditions, we become a major source of mortality in Atlantic salmon estimated that it was about 3 to 4 days. in Norway (Johnsen & Jensen, 1986). Our speciﬁc goals were to determine if resistance in RESULTS Gila topminnows to the exotic ﬂuke (1) varies among individuals from the four remaining watersheds, (2) dif- During the infection of the ﬁrst 20 ﬁsh, ﬁve from each fers between heterozygotes and homozygotes at the of the four populations, ﬁve control ﬁsh from each pop- MHC gene, and (3) differs between inbred and outbred ulation were also handled in the same manner except for topminnows. infection with ﬂukes. All but one control ﬁsh survived. In other words, there appeared to be no effect of han- dling and anesthesia and it seemed unnecessary to con- MATERIALS AND METHODS tinue controls with uninfected ﬁsh for other parts of the Gila topminnows were sampled from stocks originally study. However, in all cases there were simultaneous collected from four Arizona sites with natural popula- infections of ﬁsh from different populations or groups tions, Bylas Spring, Cienega Creek, Monkey Spring and so that effects were measured relative to those in other Sharp Spring, under US Fish and Wildlife Services per- individuals. For different MHC genotypes, individual mit. These stocks are now maintained at Arizona State ﬁsh that were heterozygous or homozygous were not University (ASU) in large raceways at adult population known at the time of infection. Therefore, although numbers averaging around 1000. We also used an inbred infection for all groups by ﬂukes from the same guppy line derived from our Cienega Creek stock and main- was not controlled, infection was unrelated (at random) tained by brother–sister mating for seven generations. to MHC genotype. The ﬂuke stock was originally acquired from a feral We observed three general patterns of infection by population of guppies in a water treatment pond at Davis, ﬂukes (Table 1) and Madhavi & Anderson (1985) have California. To reduce genetic variation in the ﬂukes and given host susceptibility designations for these three cat- thereby standardize virulence of the parasite over the dif- egories. First, resistant hosts are those ‘on which the par- ferent hosts, ﬂukes were passed through ten bottleneck asite either fails to establish or fails to reproduce after generations. Speciﬁcally, to initiate the stock, two ﬂukes temporary establishment’ (see Bylas 13 in Table 1). For were used to manually infect a guppy. Once this guppy Bylas 13, because ﬂukes were checked after experi- was heavily infected, two ﬂukes from it were used to mental transfer for attachment, it is assumed that ﬂukes infect another. This was done serially for ten guppy died over the ﬁrst 5 days without reproducing. In addi- infections and the resulting ﬂukes were used in the tion, if there were one or two ﬂukes on day 5 and no experiments. ﬂukes on day 10, then the host is also categorized as In the challenge protocol, as approved by the ASU resistant. Second, moderately susceptible hosts (see Institutional Animal Care and Use Committee, ﬁrst a Cienega 5 in Table 1) are ones ‘on which the parasite Parasite resistance in topminnows 105 Table 1. Description of different typical courses of infection of the ﬂuke on topminnows observed at 5-day intervals and the categoriza- tion of the host susceptibility type where – indicates no ﬁsh Number of ﬂukes on day Individual 0 5 10 15 20 25 Host susceptibility Bylas 13 2 0 0 > 0 0 0 Resistant Cienega 5 2 13 6 > 0 0 0 Moderately susceptible Sharp 13 2 4 14 >50 Dead – Highly susceptible population builds up by reproduction but the host slowly recovers and the parasite is eliminated’. Because the life expectancy of the ﬂuke is short (as discussed above), observation of more than two ﬂukes at day 5 or any ﬂukes at day 10 demonstrates reproduction. Finally, highly susceptible hosts are ones ‘on which the parasite Fig. 1. The mean number of ﬂukes for different census days population grows rapidly and the infection results in host for ﬁsh from the four populations. death’. For some of these ﬁsh, as for Sharp 13 in Table 1, the number of ﬂukes increased greatly, generally to more than 50 (at this point it became difﬁcult to accu- rapid with only ﬁve infected on day 15 and only one ﬁsh rately count all ﬂukes), and the ﬁsh died. In a few oth- infected at day 20 (out of 35 infected on day 5). Second, ers, the numbers of ﬂukes increased but were not this the number of ﬂukes was highest for either Bylas Spring high during our counts. Because we had extremely low or Sharp Spring for all sampling days except day 20 or mortality in our controls, if these ﬁsh died, we classiﬁed 25 when few ﬁsh remained infected. In particular, a them as highly susceptible. very high medium number of 25 ﬂukes was observed on the 15 infected ﬁsh in the Bylas sample at day 15. Although there are these trends, all t-tests between Comparison between populations different populations for the different census times are We compared 41 ﬁsh from each of the four populations non-signiﬁcant. for resistance to ﬂukes (actually one ﬁsh was eliminated The impact of infection can also be described by com- from further consideration from both Monkey and Sharp paring the proportion of ﬁsh with different numbers of Springs because they died though no ﬂukes were ﬂukes (no ﬂukes, 1–10 ﬂukes, >10 ﬂukes) or where the observed on day 5). Let us ﬁrst examine the number of ﬁsh are dead. Again, infection appeared most severe, ﬂukes observed at different days by considering the based on the number of ﬂukes in the different categories mean and medium number of ﬂukes over the course of and the proportion of mortality, in either the Bylas or the experiment (Fig. 1, Table 2). Because the growth of Sharp Spring samples. Infection in Cienega Creek and the ﬂuke appears exponential and, as a result, the cate- Monkey Spring seemed less severe, with lower numbers gory > 50 ﬂukes (called 50 here) greatly increases the of ﬂukes in most categories and a lower proportion of mean, examining the medium number is also useful. mortality. The cumulative mortality over time for the Note that the sample size declined over days, either four populations is given in Fig. 2. χ2 tests showed no because ﬁsh eliminated the ﬂuke or because ﬁsh died. signiﬁcant heterogeneity for any of these comparisons. First, the number of ﬂukes is lowest for the Monkey A complementary pattern was seen for ﬁsh that had Spring sample at the 5-, 10- and 15-day censuses. Also recovered from initial infection with ﬂukes, i.e., the the course of infection for the Monkey Spring ﬁsh was cumulative proportion of ﬁsh that had no ﬂukes was Table 2. The mean (± standard error) and medium number of ﬂukes, on ﬁsh that had ﬂukes, for the four different populations at 5-day intervals (N is the sample number of ﬁsh). – indicates that there were no ﬁsh with ﬂukes Number of ﬂukes on day Population Measure 5 10 15 20 25 Bylas Spring Mean 12.2 ± 2.4 13.0 ± 1.7 28.3 ± 5.1 6.0 – Medium 6 14 25 6 – N 36 21 15 2 – Cienega Creek Mean 5.8 ± 0.7 14.0 ± 2.8 14.3 ± 4.1 10.0 ± 4.9 2.0 Medium 5 9 9 2 2 N 39 27 15 5 1 Monkey Spring Mean 5.6 ± 0.4 7.2 ± 2.2 13.0 ± 9.4 10 – Medium 3 2 2 10 – N 35 19 5 1 – Sharp Spring Mean 8.7 ± 1.1 20.6 ± 3.0 16.7 ± 5.5 – – Medium 7 23 6 – – N 38 27 13 – – 106 P. W. HEDRICK ET AL. the SSCP (single-stranded conﬁrmation polymorphism) approach as given by Hedrick & Parker (1998) for this class II MHC locus. For various technical reasons, we were able to determine genotypes of only 77 of the 121 ﬁsh from Cienega, Monkey and Sharp Springs (all Bylas were assumed homozygous; see Hedrick & Parker, 1998). Overall, there were 87 ﬁsh homozygous and 31 heterozygous (Table 3). For all three populations in which both heterozygotes and homozygotes were present, survival of heterozygotes was higher. The over- all survival of heterozygotes was 15.5% higher than that of homozygotes. However, χ2 = 1.64 (1 df) and is non- signiﬁcant. Observed MHC heterozygosity was 0.00, 0.41, 019 and 0.60 for Bylas, Cienega, Monkey and Sharp sam- ples, respectively, similar to that found by Hedrick & Fig. 2. The cumulative mortality for different census days for Parker (1998). Although Bylas had both the lowest MHC ﬁsh from the four populations. heterozygosity and a low survival, Sharp had the high- est MHC heterozygosity and low survival. In addition, higher (although non-signiﬁcantly so) for days 15 Monkey had the second lowest heterozygosity but sur- through 25 for Cienega Creek and Monkey Spring than vival nearly as high as Cienega Creek. In other words, for Bylas and Sharp Springs. there appears no positive association between MHC het- The overall effect of ﬂuke infection is summarized for erozygosity and survival from ﬂuke infection. the three categories, resistant, moderately susceptible and highly susceptible, in Fig. 3. Note the Bylas and Comparison of inbred versus outbred ﬁsh Sharp Springs samples have the lowest proportion of resistant ﬁsh (0.15 in both populations) and the highest The largest difference in the apparent effect of ﬂuke proportion of highly susceptible ﬁsh (0.46 in Bylas and infection was observed between the 13 inbred and 13 0.45 in Sharp). The highest proportion of resistant ﬁsh outbred ﬁsh we examined simultaneously from Cienega was in Monkey Spring (0.30). However, χ2 = 6.7 (6 df) Creek. Inbred ﬁsh had a much higher infection of ﬂukes and is non-signiﬁcant. Proportions of ﬁsh that survived than the simultaneously assessed outbred ﬁsh (and the the infection were 0.54, 0.71, 0.68 and 0.55 for the previously examined population sample discussed above Bylas, Cienega, Monkey and Sharp samples, respec- in Table 2). In addition, mortality of the inbred ﬁsh, 69%, tively. In other words, survival in Cienega Creek ﬁsh was much higher than in the outbred control, 31% (χ2 was 31%, 4% and 29% higher than in Bylas, Monkey = 3.84, 1 df, P = 0.05). In other words, survival of inbred and Sharp, respectively. ﬁsh was more than twice as high as that of the simulta- neously assessed outbred sample (the survival of the pre- viously examined population sample, 0.71, was even Comparison of MHC heterozygotes versus slightly more different from the inbred). homozygotes The ﬁsh examined from the four populations were cat- CONCLUSIONS AND DISCUSSION egorized as either heterozygous or homozygous using More and more evidence is accumulating that exotic par- asites may be a major detrimental factor in extinction of endangered species. To evaluate this effect under con- Table 3. The survival status at 25 days for homozygotes and heterozygotes at the class II MHC locus for ﬁsh from the four populations (proportions in parentheses). Genotypes Population Status Homozygotes Heterozygotes Bylas Spring Alive 22 (0.54) – Dead 19 (0.46) – Cienega Creek Alive 12 (0.71) 9 (0.75) Dead 5 (0.29) 3 (0.25) Monkey Spring Alive 15 (0.71) 4 (0.80) Dead 6 (0.29) 1 (0.20) Sharp Spring Alive 3 (0.38) 7 (0.58) Fig. 3. The proportion of ﬁsh from the four populations that Dead 5 (0.62) 5 (0.42) were classiﬁed as resistant, moderately susceptible and highly Total Alive 52 (0.60) 22 (0.71) Dead 35 (0.40) 9 (0.29) susceptible. Parasite resistance in topminnows 107 trolled experimental conditions, we examined the impact homozygotes observed in the previous samples). In other of an exotic guppy parasite, G. turnbulli, on the endan- words, nearly a doubling of the population experiment gered Gila topminnow. A topminnow population from and more than a tripling of the genotype experiment Bylas Spring, earlier found to have lower genetic vari- would have been necessary to have statistical signiﬁ- ation than other samples, appears to have relatively low cance for the size of effects that we observed. resistance to the ﬂuke. However, a Sharp Spring sam- Overall, these studies demonstrate Gila topminnows ple, which has the highest genetic variation, also had as generally susceptible to infection by the exotic guppy similarly low resistance. Survival of Bylas and Sharp ﬂuke, with 42.6% of ﬁsh tested moderately susceptible samples (54.5%) was approximately 30% lower than that and 38.3% highly susceptible. Although it is difﬁcult to in the other two samples from Cienega Creek and compare susceptibility and mortality across various stud- Monkey Spring (69.5%) although none of these differ- ies because of differences in protocols, particularly the ences was signiﬁcantly different. number and strain of ﬂukes used to initiate infection, it In addition, MHC heterozygotes appeared to have appears that Gila topminnows suffer relatively high mor- higher resistance (although non-signiﬁcant) to the ﬂukes tality from such infection. For example, although Leberg than do MHC homozygotes in all three polymorphic & Vrijenhoek (1994) monitored their topminnows for populations. Overall, MHC heterozygotes had an only 9 days, when their ﬁsh were inoculated with two approximately 15% higher survival than MHC homozy- parasites only ﬁve (13.2%) were moderately susceptible gotes. We cannot tell whether this effect is the result of and only two (5.2%) highly susceptible. the MHC gene that we examined or effects of loci sta- In several simple experiments, we showed Gila top- tistically associated with the MHC locus. However, minnows can acquire the guppy ﬂuke by casual contact because we randomly drew these individuals from our with infected guppies and suffer signiﬁcant subsequent large raceway populations after about ten generations in mortality. This is contrary to earlier ﬁndings by Leberg captivity, the likelihood of such statistical association is & Vrijenhoek (1994) in related topminnows in which probably only signiﬁcant for closely linked loci (Houle, they found a low rate of infection and no evidence of 1989; Savolainen & Hedrick, 1995). transmission by casual contact. For example, we placed The strongest effect observed was a lower resistance, two ﬂuke-infected guppies in a 10-gallon tank with 25 as measured by both ﬂuke infection level and mortality, uninfected guppies, and after 2 weeks six topminnows of inbred ﬁsh from Cienega Creek as compared to simul- were placed in the tank (a density of poeciliids in nature taneous outbred Cienega control. Even though the sam- this high is not uncommon). After 3 more weeks two of ple size for this experiment was lower than for the the topminnows were lightly infected with ﬂukes, and population study, the difference in mortality was statis- after another week two topminnows were dead. Both tically signiﬁcant. In this case, survival of the outbred dead topminnows were infected with ﬂukes, one quite ﬁsh was more than twice as high as that for the simul- heavily. Three days later another topminnow was dead, taneous control. also infected by ﬂukes. One of the three survivors was Although there were differential effects between pop- infected while the other two living topminnows were not. ulations and MHC genotypes, these effects did not reach Overall, four out of six topminnows were infected and statistical signiﬁcance. As a result, it is important to three died, suggesting ﬂukes may present a strong selec- determine sample sizes that would have been necessary tive pressure on natural populations of topminnows in to statistically detect the level of effect found with our contact with ﬂuke-infected guppies. experimental design. For the population study, for sta- In mammals, all major genes in the MHC appear to tistical signiﬁcance (P < 0.05) for the level of hetero- be clustered in one relatively small linkage group geneity that we observed, the total sample would have (Edwards & Hedrick, 1998). However, in ﬁshes, the to have been 308, or 77 ﬁsh per population, instead of class I MHC genes (primarily involved in recognition of the 164 total ﬁsh that we used. For MHC heterozygotes intracellular pathogens, such as viruses) and the class II versus homozygotes, a sample size of 375 would have MHC genes, as the one that we investigated (primarily been necessary rather than the 118 that we assayed involved in recognition of extracellular parasites, such (assuming the same proportions of heterozygotes and as bacteria), are unlinked (Sato et al., 2000). Even the class II genes in ﬁshes appear to be in at least two link- Table 4. The mean (± standard error) and medium number of ﬂukes age groups (Bingulac-Popovic et al., 1997; McConnell on ﬁsh that had ﬂukes for the inbred sample and the simultaneous et al., 1998) spread out over more than ten map units in outbred control from Cienega Creek at 5-day intervals (N is the sam- cichlids (Malaga-Trillo et al., 1998) and 30 map units ple number of ﬁsh). – indicates that there were no ﬁsh with ﬂukes in sticklebacks (Sato et al., 2000). Relevant to our ﬁnd- Number of ﬂukes on day ings of only a 15% difference in survival between MHC Population Measure 5 10 15 20 25 heterozygotes and homozygotes is that genes unlinked to the class II locus we examined, or linked but in link- Inbred Mean 5.5 ± 1.5 12.3 ± 3.1 29.8 ± 9.1 35.3 ± 14.7 50 Medium 6.5 12 30 50 50 age equilibrium with the class II locus, may inﬂuence N 8 7 5 3 1 parasite resistance. As a result, the signal from examin- Outbred Mean 4.9 ± 0.9 5.9 ± 2.3 3.3 ± 1.2 – – ing MHC genetic variation at a single gene inﬂuencing Medium 5 3 4 – – parasite resistance would be expected to be less in ﬁsh N 11 9 3 – – than in mammals because of the lower level of linkage 108 P. W. HEDRICK ET AL. disequilibrium expected. Even so we did ﬁnd a trend in Acknowledgements the effect of MHC heterozygotes having a higher para- We thank Helen Rodd for providing the stock of ﬂukes site resistance than MHC homozygotes. from Davis, CA. We appreciate support for this research This study provides further evidence that inbred ani- from the National Science Foundation, Arizona Heritage mals may have lowered parasite resistance. In our study, Fund, Bureau of Reclamation and Ullman Distinguished the inbred line had undergone seven generations of Professorship. We also appreciate comments on the man- brother–sister mating, had an inbreeding coefﬁcient of uscript from W. L. Minckley and two anonymous greater than 0.7, and was ﬁxed for allele Pooc 1 (Hedrick reviewers and assistance from Richard Fredrickson in & Parker, 1998). Although the line had no noticeable producing the ﬁgures. This research was carried out loss of ﬁtness for several other traits (Sheffer et al., using appropriate US Fish and Wildlife Service and 1999), it apparently had become susceptible to ﬂuke Arizona Department of Game and Fish permits. infection. Lyles (1990) also found inbred guppies had increased susceptibility to infection by the same ﬂuke species. The recent study of Coltman et al. (1999) found REFERENCES Soay sheep with the lowest levels of heterozygosity (inferred highest levels of inbreeding) had the greatest Anderson, R. M. & May, R. M. (1979). Population biology of susceptibility to a gastrointestinal nematode. However, infectious diseases. Nature 280: 361–367. Bingulac-Popovic, J., Figueroa, F., Sato, A., Talgot, W. 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