Aftermath

The Sverdlovsk Anthrax Outbreak of 1979 Author(s): Matthew Meselson, Jeanne Guillemin, Martin Hugh-Jones, Alexander Langmuir, Ilona Popova, Alexis Shelokov and Olga Yampolskaya Source: Science, New Series, Vol. 266, No. 5188 (Nov. 18, 1994), pp. 1202-1208 Published by: American Association for the Advancement of Science Stable URL: http://www.jstor.org/stable/2885382 . Accessed: 16/09/2013 14:56

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A 20~> 25 mK

3.0-

v1/2 1.

2.0

9.0 9.5

0.0 0.0 0.2 Magnetic field (T)

Fig. 4. The resistance R = V34/121 for the magnet- ic focusing sample shown in the inset. (A) Focus- ing peaks of electrons near B = 0, and (B) focus- ing peaks of composite fermions near B* = 0 (that is, near v = 1/2). The scales of B and B* differ by a factor of about \/. A qualitative difference be- tween the positive and negative B* (that is, be- tween v > 1/2 and v < 1/2) is evident, as is the one-to-one correspondence between several composite fermion and electron focusing peaks. [Reprinted from (30) with permission of Goldman etal.]

the lattice; some of the most relevant com- mensurate orbits are shown in the figure. Similar dimensional resonances of compos- ite fermions show up near B* = 0. Goldman et al. (30) observed magnetic focusing of composite fermions near v = 1/2. The ex- perimental setup is shown in Fig. 4.; the current flows from 1 to 2, and the voltage is measured between 3 and 4. Near B = 0, a number of quasi-periodic peaks are observed (Fig. 4B), which occur at those values of B where the electrons coming straight out of the left constriction are focused into the right constriction, possibly after several specular reflections from the gate. Similar quasi-periodic structure was observed near B* = 0 (Fig. 4A). The close correspon- dence between the electron and the com- posite fermion peaks is evident in both Figs. 3 and 4. These experiments confirm the existence of composite fermions in the compressible region near v = 1/2 by dem- onstrating that the dynamics of the charge carriers are described by the effective field B* rather than the external field B. Thus, the composite fermion framework has not only provided a simple "one-step" explana- tion of the FQHE, it has also helped reveal the nontrivial nature of the metallic state at even-denominator fractions.

Conclusion

The following picture has finally emerged. First, electrons form LLs because of quan- tization of their kinetic energy. This re- sults in the JQHE. Within the lowest LL, in a range of filling factor, electrons min- imize their interaction energy by capturing vortices and transforming into composite

fermions. Even though the composite fer- mions are quantum mechanical particles with a true many-body character, they may be treated, for most purposes, as or- dinary noninteracting fermions moving in an effective magnetic field. They form quasi-LLs, execute cyclotron motion, and fill a Fermi sea. The formation of compos- ite fermions lies at the root of the FQHE and several other fascinating experimental phenomena.

REFERENCES AND NOTES

1. E. H. Hall, Am. J. Math 2, 287 (1879). 2. K. von Klitzing, G. Dorda, M. Pepper, Phys. Rev. Lett.

45, 494 (1980). 3. D. C. Tsui et al., ibid. 48, 1559 (1982). 4. See L. D. Landau and E. M. Lifshitz, Quantum Me-

chanics (Addison-Wesley, Reading, MA, 1965), pp. 424-426.

5. R. B. Laughlin, Phys. Rev. B 23, 5632 (1981). 6. , Phys. Rev. Lett. 50, 1395 (1983). 7. For example, G. Fano, F. Ortolani, E. Colombo, Phys.

Rev. B 34, 2670 (1986); F. D. M. Haldane and E. H. Rezayi, Phys. Rev. Lett. 54, 237 (1985).

8. F. D. M. Haldane, Phys. Rev. Lett. 51, 605 (1983). 9. B. I. Halperin, ibid. 52, 1583 (1984).

10. See J. K. Jain, Adv. Phys. 41, 105 (1992). 11. S. M. Girvin and A. H. MacDonald, Phys. Rev. Lett.

58, 1252 (1987). 12. S. C. Zhang et al., ibid. 62, 82 (1989). 13. N. Read, ibid., p. 86.

14. J. K. Jain, ibid. 63, 199 (1989). 15. To see how multiplication by the Jastrow factor at-

taches 2m vortices to each electron, fix all z, in the Jastrow factor except z1I As z1 traverses in a closed loop around any other electron, it acquires a phase of 4m7r, that is, it sees 2m vortices on all other electrons.

16. N. Trivedi and J. K. Jain, Mod. Phys. Lett. B 5, 503 (1 991).

17. G. Dev and J. K. Jain, Phys. Rev. Lett. 69, 2843 (1992).

18. X. G. Wu, G. Dev, J. K. Jain, ibid. 71, 153 (1993); X. G. Wu and J. K. Jain, preprint.

19. E. H. Rezayi and A. H. MacDonald, Phys. Rev. B 44, 8395 (1991); M. Kasner and W. Apel, ibid. 48,11435 (1993); E. H. Rezayi and N. Read, Phys. Rev. Lett. 72, 900 (1994).

20. V. J. Goldman etal., Phys. Rev. Lett. 65, 907 (1990). 21. J. K. Jain, S. A. Kivelson, N. Trivedi, ibid. 64, 1297

(1 990). 22. B. I. Halperin, P. A. Lee, N. Read, Phys. Rev. B 47,

7312 (1993). 23. R. R. Du et al., Phys. Rev. Lett. 70, 2944 (1993). 24. Also see, I. V. Kukushkin, R. J. Haug, K. von Klitzing,

K. Ploog, ibid. 72, 736 (1994). 25. D. R. Leadley et al., ibid., p. 1906. 26. R. R. Du, H. L. Stormer, D. C. Tsui, L. N. Pfeiffer, K.

W. West, Solid State Commun. 90, 71 (1994). 27. H. W. Jiang, H. L. Stormer, D. C. Tsui, L. N. Pfeiffer,

K. W. West, Phys. Rev. B 40, 12013 (1989); R. L. Willett et al., Phys. Rev. Lett. 65, 112 (1990).

28. R. L. Willett, R. R. Ruel, K. W. West, L. N. Pfeiffer, Phys. Rev. Lett. 71, 3846 (1993).

29. W. Kang et al., ibid., p. 3850. 30. V. J. Goldman et al., ibid. 72, 2065 (1994). 31. V. J. Goldman, unpublished figure. 32. Financial support from the National Science Founda-

tion under grant DMR93-18739 is acknowledged.

The Sverdlovsk Anthrax Outbreak of 1979

Matthew Meselson,* Jeanne Guillemin, Martin Hugh-Jones, Alexander Langmuir,t Ilona Popova, Alexis Shelokov,

Olga Yampolskaya

In April and May 1979, an unusual anthrax epidemic occurred in Sverdlovsk, Union of Soviet Socialist Republics. Soviet officials attributed it to consumption of contaminated meat. U.S. agencies attributed it to inhalation of spores accidentally released at a military microbiology facility in the city. Epidemiological data show that most victims worked or lived in a narrow zone extending from the military facility to the southern city limit. Farther south, livestock died of anthrax along the zone's extended axis. The zone paralleled the northerly wind that prevailed shortly before the outbreak. It is concluded that the escape of an aerosol of anthrax pathogen at the military facility caused the outbreak.

Anthrax is an acute disease that primarily affects domesticated and wild herbivores and is caused by the spore-forming bacteri- um Bacillus anthracis. Human anthrax re- sults from cutaneous infection or, more rarely, from ingestion or inhalation of the pathogen from contaminated animal prod- ucts (1). Anthrax has also caused concern as a possible agent of biological warfare (2).

Early in 1980, reports appeared in the Western press of an anthrax epidemic in Sverdlovsk, a city of 1.2 million people 1400 km east of Moscow (3, 4). Later that year, articles in Soviet medical, veterinary,

and legal journals reported an anthrax out- break among livestock south of the city in the spring of 1979 and stated that people developed gastrointestinal anthrax after eating contaminated meat and cutaneous anthrax after contact with diseased animals (5-7). The epidemic has occasioned intense international debate and speculation as to whether it was natural or accidental and, if accidental, whether it resulted from activi- ties prohibited by the Biological Weapons Convention of 1972 (8).

In 1986, one of the present authors (M.M.) renewed previously unsuccessful re-

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30 -

25

en 20;

15-

10-4 z

5-d

1 2 3 4 5 6 Time (weeks)

Fig. 1. Time course of the epidemic: onsets of fatal cases by week. The first week begins on 4 April 1979, the date of the first onset we recorded. Ughter shading represents cases for which the onset date is unknown and is estimated by sub- tracting 3 days from the date of death.

quests to Soviet officials to bring indepen- dent scientists to Sverdlovsk to investigate. This resulted in an invitation to come to Moscow for discussions with four physicians who had gone to Sverdlovsk to deal with the outbreak (including another of the present authors, O.Y., who was a clinician in the intensive care unit set aside to treat the victims). In 1988, two of these Soviet phy- sicians visited the United States, where they gave formal presentations and participated in discussions with private and government specialists. According to their account, con- taminated animals and meat from an epizootic south of the city starting in late March 1979 caused 96 cases of human an- thrax with onsets from 4 April to 18 May. Of these cases, 79 were said to be gastrointesti- nal and 17 cutaneous, with 64 deaths among the former and none among the latter (9).

The impression left on those of the present authors who took part in the U.S. meetings (J.G., A.L., M.M., and A.S.) was that a plausible case had been made but that additional epidemiological and patho- anatomical evidence was needed. Further requests by M.M. for an invitation led to an on-site study in Sverdlovsk, initiated there in June 1992, and a return visit in August 1993.

Starting in 1990, several articles about the epidemic appeared in the Russian press

M. Meselson is in the Department of Molecular and Cel- lular Biology, Harvard University, Cambridge, MA 02138, USA. J. Guillemin is in the Department of Sociology, Bos- ton College, Chestnut Hill, MA 02167, USA. M. Hugh- Jones is in the School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA. A. Lang- muir is in the School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA. I. Pop- ova is in the Social and Political Sciences DMsion, Ural State University, Ekaterinburg 620083, Russia. A. She- lokov is in the Govemment Services Division, Salk Insti- tute, San Antonio, TX 78228, USA. 0. Yampolskaya is in the Botkin Hospital, Moscow 125101, Russia.

*To whom correspondence should be addressed. tDeceased 22 November 1993.

Table 1. Case data. Case numbers for fatalities are as they appear on the administrative list. Case numbers for survivors are arbitrary. Days of onset and death are counted from 1 April 1979. Abbrevia- tions: 0, onset; D, death; R, residence; W, workplace; *, unidentified man; ?, not known; ma, mid-April; s, survivor; c, cutaneous survivor; +, in high-risk zone; -, outside high-risk zone; a, had two residences, one in Compound 32; p, pensioner; r, daytime military reservist at Compound 32; u, unemployed. Patients 25, 29, 48, and 87 were home on vacation during the first week of April.

Case Age/sex O/D R/W Case Age/sex O/D R/W no. no.

* ?/m ?/? ?/? 51 31/m 10/15 -/? 32 40/m 7/? -/? 40 37/m 12/15 +/+ 67 26/m ?/? +/+ 36 68/f 7/16 a/p 68 32/f ?/? +/+ 35 52/m 13/16 +/+

8 60/f ?/8 +/+ 34 43/m 14/16 -/+ 18 38/m 6/8 -M/ 38 69/f 14/16 +/p 16 40/m 7/9 +/+ 39 49/m 14/16 +/+ 66 55/f ?/9 -/? 41 41 /f ?/1 7 +/p

1 44/m 6/9 +/+ 42 43/m 15/18 -/+ 2 46/m 6/9 +/+ 43 39/m 15/19 +/u 5 66/m 7/9 -/+ 44 47/m 15/21 -M

49 51/m 8/9 +/+ 45 45/m ?/22 +/+ 21 49/m ?7/10 -/? 46 39/m 20/23 -/+

4 54/f 5/10 +/+ 47 41/m 21/24 -/- 6 40/m 7/10 -M/ 52 42/m 21/24 -/-

20 39/m 7/10 -/- 53 47/m 22/24 -/- 17 67/f 8/10 +/+ 48 57/f 15/25 +/- 9 72/f 9/10 +/p 54 50/f 17/25 -/? 7 52/f ?/11 +/+ 55 31/m 23/25 +/+

19 64/f 7/1 1 -/ 57 31//m 27/28 -/r 22 27/m ?/1 1 +/+ 58 32/m 29/30 -/+ 23 43/m ?/11 -/r 59 55/m 27/31

3 48/f 4/11 -/+ 60 33/m 25/33 -/r 10 27/m 9/11 +/- 61 42/m 34/40 -/+ 65 72/m 9/11 +/p 62 29/m 39/40 +/+ 15 48/f 6/12 +/+ 63 25/m 37/42 -/+ 25 46/m 10/12 -/- 64 28/m 42/46 -/ 12 38/m 11/12 -/+ 90 28/m ?/s 1 1 27/m 12/12 -/r 82 68/f 13/s +/+ 26 67/m 9/13 +/p 80 49/m 14/c 13 24/f 10/13 +/+ 84 55/f ma/c 24 65/f 10/13 +/p 85 40/f ma/s +/+ 28 47/m 11/13 +/+ 89 50/f 34/s 14 49/m 12/13 -/+ 86 28/m 37/s 27 64/m 10/14 +/+ 81 29/m 38/s +/+ 31 42/m 11/14 -/r 83 45/m 41/s +/+ 30 52/m 12/14 +/+ 87 41/m 42/s +/- 29 45/m 13/14 +/+ 88 37/m 45/s +/+ 50 72/f 7/15 +/p

(10). These included interviews with Sver- dlovsk physicians who questioned the food- bome explanation of the epidemic and with officials at the military microbiology facili- ty. These officials said that in 1979 they had been developing an improved vaccine against anthrax but knew of no escape of anthrax pathogen. Late in 1991, Russian President Boris Yeltsin, who in 1979 was the chief Communist Party official of the Sverdlovsk region, directed his Counsellor for Ecology and Health to determine the origin of the epidemic (1 1). In May 1992, Yeltsin was quoted as saying that "the KGB admitted that our military developments were the cause" (12). No further informa- tion was provided. Subsequently, the chair- man of the committee created by Yeltsin to oversee biological and chemical disarma- ment expressed doubt that the infection originated at the military facility and stated that his committee would conduct its own

investigation (13). The results of that in- vestigation have not yet appeared.

Pathoanatomical evidence that the fatal cases were inhalatory, recently published by Russian pathologists who performed autop- sies during the epidemic (14-16), is sum- marized in an earlier report from the present study (17). Here we report epidemiological findings that confirm that the pathogen was airborne, and we identify the location and date of its escape into the atmosphere.

Sources of Information

Local medical officials told us that hospital and public health records of the epidemic had been confiscated by the KGB. We nev- ertheless were able to assemble detailed in- formation on many patients from a variety of sources. (i) An administrative list giving names, birth years, and residence addresses of 68 people who died, compiled from KGB

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records and used by the Russian govern- ment to compensate families of the de- ceased (18). Comparison with other sources of information, including those listed be- low, indicates that the administrative list may include most or all of those who died of anthrax. (ii) Household interviews with rel- atives and friends of 43 people on the ad- ministrative list and with 9 survivors or their relatives (or both). The interviews (directed by J.G.) were designed to identify the workplaces and other whereabouts of patients before their illness. (iii) Grave

markers, giving names and dates of birth and death, that we inspected in the ceme- tery sector set aside for the anthrax victims. These include 61 markers with names that are also on the administrative list and 5 with illegible or missing name plates. (iv) Pathologists' notes regarding 42 autopsies that resulted in a diagnosis of anthrax (14- 17). All but 1 of the 42, an unidentified man, are on the administrative list. The notes include name, age, and dates of onset, admission, death, and autopsy. (v) Various hospital lists, with names, residence ad-

dresses, and, in some cases, workplaces or diagnoses (or both) of approximately 110 patients who were apparently screened for anthrax, 48 of whom are indicated to have died. Of the latter, 46 are on the adminis- trative list. (vi) Full clinical case histories of 5 survivors hospitalized in May 1979.

Current street and regional maps were purchased in Sverdlovsk, which is known again by its prerevolutionary name of Eka- terinburg. The city is the seat of an admin- istrative region, or oblast, named Sverd- lovskaya. The city itself is divided among a number of districts, or rayon, the most southerly being Chkalovskiy rayon. A satel- lite photograph of the city taken 31 August 1988 was purchased from SPOT Image Cor- poration (Reston, Virginia). Archived me- teorological data from the city's Koltsovo airport were obtained from the National Center for Atmospheric Research (Boulder, Colorado).

Case Data

Table 1 presents information on 66 pa- tients who died and 11 who survived. The fatalities include the unidentified man and all people named on the administra- tive list, except for three patients for whom recent reexamination of preserved autopsy specimens does not support a di- agnosis of anthrax (19). For survivors, di- agnoses of anthrax are supported by clin- ical case histories or hospital lists or both and by household interviews.

Overall, 55 of the 77 tabulated patients are men, whose mean age was 42. The mean

I

2 4

d

Fig. 2. Probable locations of patients when ex- posed. The part of the city shown in the photo- graph is enclosed bya rectangle in the inset. Case numbers, in red, correspond to those in Table 1

4 w D Y al and indicate probable daytime locations of pa- tients during the period 2 to 6Apr1l 1979. Of the 66 patients mapped as explained in the text, 62

* _ mapped in the area shown. This distribution may [5jn 3 be somewhat biased against residence locations,

62 3121420 13 j __because daytime workers not on vacation who 3442 both resided and worked in the high-risk zone are

mapped at their workplaces. Proceeding from north to south, Compound 19, Compound 32, and the ceramics factory are outlined in yellow. The five patients residing in Compound 32 are mapped at their apartments. Within the com- pound, the placement of an additional, part-time resident and of the five reservists is arbitrary, as is that of the five residents and a nonresident em- ployee in Compound 19. Patients known to have worked in the ceramics pipe shop are mapped in the eastem part of thefactoryarea, where the pipe shop is located. Calculated contours of constant dosage are shown in black. Approximately 7000 people lived in the area bounded by the outermost contour of constant dosage, Compound 32, and the ceramics factory. The terrain slopes gently downward by about 40 m from Compound 19 to the ceramics factory.

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age for women was 55. No man was younger than 24, and only two women, aged 24 and 32, were under 40. Recorded onsets span a period of nearly 6 weeks, 4 April to 15 May, with a mean time between onset and death of 3 days (Table 1 and Fig. 1).

Approximately 60% of the 33 men for whom we have relevant information were described as moderate or heavy smokers and nearly half as moderate or heavy drinkers. None of the women was said to have smoked or to have consumed alcohol more than occasionally. Few patients were report- ed to have had serious preexisting medical conditions. Among the 35 men whose oc- cupation in 1979 we could determine, the most common occupation was welder, ac- counting for 7.

In descending order of frequency, symp- toms reported in household interviews in- cluded fever, dyspnea, cough, headache, vomiting, chills, weakness, abdominal pain, and chest pain. Two of the survivors inter- viewed reported having had cutaneous an- thrax, one on the back of the neck, the other on the shoulder. Hospitalized pa- tients were treated with penicillin, ceph- alosporin, chloramphenicol, anti-anthrax globulin, corticosteroids, osmoregulatory solutions, and artificial respiration. The average hospital stay was 1 to 2 days for fatal cases and approximately 3 weeks for survivors. To the best of our knowledge, no human anthrax has occurred in the Sverdlovsk region since 1979.

Public Health Response

Public health measures were initially direct- ed by an emergency commission formed in Chkalovskiy rayon, where most patients lived and worked. On or about 10 April, overall direction was assumed by a commis- sion that was constituted at oblast level and included the USSR Deputy Minister of Health. Military personnel participated lit- tle if at all in the implementation of med- ical and public health measures.

Before the bacteriological confirmation of anthrax, on 11 April (14), patients were taken to hospitals served by the ambulance or polyclinic of first contact. Starting on 12 April, most patients presenting with high fever or other indications of possible an- thrax or who died at home or elsewhere of suspected anthrax were taken to city hospi- tal No. 40, where separate areas were des- ignated for screening suspect cases and for treating nonsystemic cutaneous cases, for intensive care, and for autopsy. Bodies of those who died were placed in coffins with chlorinated lime and buried in a single sec- tor of a city cemetery. Medical and sanita- tion teams recruited from local hospitals and factories visited homes of suspected and confirmed cases throughout the city, where they conducted medical interviews, dis- pensed prophylactic tetracycline to pa- tients' households, disinfected kitchens and sick rooms, and took meat and environmen- tal samples for bacteriological testing. Hu-

man anthrax is not considered contagious, nor was there any evidence of person-to- person transmission. In the part of Chka- lovskiy rayon where most patients resided, building exteriors and trees were washed by local fire brigades, stray dogs were shot by police, and several previously unpaved streets were asphalted. Newspaper articles and posters wamed of the risk of anthrax from consumption of uninspected meat and contact with sick animals. Uninspected meat in vehicles entering the city from the south was confiscated and bumed at high- way checkpoints.

Starting in mid-April, a voluntary im- munization program using a live nonencap- sulated spore vaccine (designated STI) was carried out for healthy persons 18 to 55 years old served by clinics in Chkalovskiy rayon. Posters urged citizens to obtain "pro- phylactic immunization against anthrax" at designated times and places. Of the 59,000 people considered eligible, about 80% were vaccinated at least once.

Geographical Distribution of Human Cases

Most of the 77 tabulated patients lived and worked in the southern area of the city shown in Fig. 2. Of the 66 patients for whom we have both residence and work- place locations, 9 lived and regularly worked outside of this area. Interviews with relatives and friends revealed that five of these nine had attended military reserve classes during the first week of April 1979 at Compound 32, an army base in the affected area. Respondents stated and, in one case, showed diary notes establishing that the first day of attendance was Monday, 2 April, that classes began at 0830, and that participants retumed home each evening. Assuming that the reservists were exposed while at or near Compound 32, this must have occurred during the daytime in the week of 2 April.

In order to locate the high-risk area more precisely, we prepared a map showing probable daytime locations of the 66 pa- tients during the week of 2 April. Those with residence or work addresses in military compounds or attending reserve classes were placed in the appropriate military compound; night workers, pensioners, un- employed people, and vacationers were placed at their homes; and all other workers were placed at their workplaces. This mapped 57 patients in a narrow zone ap- proximately 4 km long, extending from the military microbiology facility to the south- ern city limit. The remaining nine worked outside this zone, but three of them resided within it. Placing the latter at their resi- dences gives the distribution shown in Fig. 2, with 60 of the 66 mapped cases in the

Fig. 3. Villages with animal 60030' 61?000' anthrax. Six villages where *-. livestock died of anthrax in April 1979 are A, Rudniy; B, Bolshoye Sedelnikovo; C, ~ Maloye Sedelnikovo; D, Per- vomaiskiy; E, Kashino; and F, . . Abramovo. Settled areas are- shown in gray, roads in white, lakes in blue, and calculated contours of constant dosage in black.A

560401 B

D *......

56020 10

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high-risk zone, 2 cases east of it, and 4 cases north or east of the area of the figure. Of these six patients who both worked and lived outside the high-risk zone, three had occupations (truck driver, pipe layer, and telephone worker) that might have taken them there, one was temporarily working in Chkalovskiy rayon, one was on vacation, and inadequate information was available for another.

At the northern end of the high-risk zone is the military microbiology facility, Compound 19, followed to the south by Compound 32. Both compounds include numerous buildings, with four- and five- story apartment houses for about 5000 peo- ple at the former and 10,000 at the latter. The administrative list includes five people who lived in Compound 19 and five who lived in Compound 32. All of the latter resided in four adjacent apartment buildings in the eastern part of the compound. Inter- views in Compound 32 indicated that all of its residents who died of anthrax are on the administrative list. Interviews were not conducted in Compound 19.

Adjacent to Compound 32 and extend- ing south-southeast for about 1.5 km is a residential neighborhood with a 1979 pop- ulation density of approximately 10,000 per square kilometer, composed of small single- story private houses and a few apartment houses, shops, and schools. Just south of this is a ceramics factory that had about 1500 daytime employees. Of the 18 tabulated patients who were employees there, 10 worked in a large unpartitioned building where ceramic pipe was made and which had a daytime work force of about 450. The attack rate at the ceramics factory therefore appears to be 1 to 2%. Still farther south are several smaller factories, apartment build- ings, private houses, schools, and shops, beyond which begins open countryside with patches of woodland.

Animal Anthrax

Anthrax has been enzootic in Sverd- lovskaya oblast since before the 1917 revo- lution (20). Local officials recalled an out- break of anthrax among sheep and cattle

south of the city in early spring 1979. A detailed report of a commission of veteri- narians and local officials describes the epizootic in Abramovo, a village of approx- imately 100 houses 50 km south-southeast of Compound 19. The report, dated 25 April 1979, records the deaths or forced slaughter of seven sheep and a cow with anthrax that was confirmed by veterinary examination. The first such losses were of two sheep on 5 April, followed by two more on each of the next 2 days, another on 8 April, and a cow on 10 April, all belonging to different private owners. These losses were substantiated by interviews we con- ducted with owners of six of the sheep that died. Respondents said there had been no human anthrax in the village. During a livestock immunization program started on 10 April, 298 sheep were given anti-an- thrax serum or vaccine or both. The attack rate among sheep at Abramovo therefore appears to have been approximately 2%.

In addition, we obtained veterinary re- ports of bacteriological tests positive for anthrax in samples from three sheep from three farms in the village of Kashino, one sheep from Pervomaisky, and a cow from Rudniy, the earliest samples being received for testing on 6 April. Although other doc- uments cite the forced slaughter of a sheep in Rudniy on 28 March and the death of another in Abramovo on 3 April, the ear- liest livestock losses for which we have documentation of a diagnosis of anthrax are those in Abramovo on 5 April.

Altogether, Soviet publications (6, 7) and the documents we obtained cite out- breaks of anthrax among livestock in six villages: Rudniy, Bolshoye Sedelnikovo, Maloye Sedelnikovo, Pervomaiskiy, Kash- ino, and Abramovo. All six villages lie along the extended axis of the high-risk zone of human anthrax (Fig. 3). The cen- terline of human and livestock cases has a compass bearing of 3300 ? 100.

Meteorology

Surface (10 m) observations reported at 3-hour intervals from Koltsovo airport, 10 km east of the ceramics factory, were exam-

ined in order to identify times when the wind direction was parallel to the center- line of human and animal cases. During the time that the reservists who contracted an- thrax were at Compound 32, but before the first recorded human onsets, this occurred only on Monday, 2 April, when northerly winds from the sector 3200 to 3500 were reported throughout the period 0400 to 1900 local time (Fig. 4).

During the rest of April, winds from this sector seldom occurred, accounting for few- er than 2% of reports. During the period of northerly wind on 2 April, which followed the passage of a cold front, the wind speed was 4 to 6 m s-1, the temperature -10? to -30C, the relative humidity 50 to 66%, the sky cloudless, and the midday sun 390 above the horizon. These conditions of insolation and wind speed indicate that the atmo- sphere near the surface was of neutral sta- bility (21). As is consistent with this, tem- perature measurements at 500 to 1000 m indicated a slightly stable atmosphere at 0400 and 1000 hours, becoming neutral by 1600.

Discussion

We have presented evidence that (i) most people who contracted anthrax worked, lived, or attended daytime military reserve classes during the first week of April 1979 in a narrow zone, with its northern end in a military microbiology facility in the city and its other end near the city limit 4 km to the south; (ii) livestock died of anthrax in villages located along the extended axis of this same zone, out to a distance of 50 km; (iii) a northerly wind parallel to the high- risk zone prevailed during most of the day on Monday, 2 April, the first day that the military reservists who contracted anthrax were within the zone; and (iv) the first cases of human and animal anthrax ap- peared 2 to 3 days thereafter.

We conclude that the outbreak resulted from the windborne spread of an aerosol of anthrax pathogen, that the source was at the military microbiology facility, and that the escape of pathogen occurred during the day on Monday, 2 April. The epidemic is the largest documented outbreak of human inhalation anthrax.

The narrowness of the zone of human and animal anthrax and the infrequency of northerly winds parallel to the zone after 2 April suggest that most or all infections resulted from the escape of anthrax patho- gen on that day. Owing to the inefficiency of aerosol deposition and resuspension (22, 23), few if any inhalatory infections are likely to have resulted from secondary aero- sols on subsequent days. A single date of inhalatory infection is also consistent with the steady decline of onsets of fatal cases in

Fig. 4. Wind directions 2 April 3 April 4 April and speeds reported from Koltsovo airport for 3600 the period 2 to 4 April 1979. Numbers at the /I/\ /19 \ 19 downwind end of each 2700 1 13, line are local standard 0 16, times. Inner and outer 22 1 22 concentric circles desig- 114,13 13 nate wind speeds of 2.5 16 10 and 5.0 m s-1, respec- 19 tively. Zero wind speed was reported for 0400 on 3 April and for 0100 and 0400 on 4 April. No data were reported for 0700.

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ARTICLES

successive weeks of the epidemic. Accepting 2 April as the only date of

inhalatory exposure, the longest incubation period for fatal cases was 43 days and the modal incubation period was 9 to 10 days. This is longer than the incubation period of 2 to 6 days that has been estimated from very limited data for humans (24). Experi- ments with nonhuman primates have shown, however, that anthrax spores can remain viable in the lungs for many weeks and that the average incubation period de- pends inversely on dose, with individual incubation periods ranging between 2 and approximately 90 days (25, 26).

The absence of inhalation anthrax pa- tients younger than 24 remains unex- plained. Although nothing suggests a lack of children or young adults in Chkalovskiy rayon in 1979, they may have been under- represented in the aerosol plume. Alterna- tively, older people may have been more susceptible, which may also explain the lack of young people in epidemics of inhalation anthrax early in this century in Russian rural communities (27).

It may be asked if the geographical dis- tribution of cases is consistent with the dis- tribution expected for an aerosol of anthrax spores released at Compound 19 under the daytime atmospheric conditions of 2 April 1979. Contours of constant dosage were cal- culated from a Gaussian plume model of atmospheric dispersion, with standard devi- ations given by Briggs for neutral atmospher- ic stability in open country (21), a wind speed of 5 m s-1, a nominal release height of 10 m, and no limit to vertical mixing (Figs. 2 and 3). The aerosol is assumed to consist of particles of diameter <5 pLm, as can be produced, for example, by a laboratory aero- sol generator (28), and to have a negligible infectivity decay rate (<0.001 min-1) (2) and a deposition velocity <0.5 cm s-1, which is insufficient to cause appreciable reduction of dosage at downwind distances less than 50 km (29-31). Dosage contours are not shown closer than 300 m to the putative source, as the dosage at shorter distances depends sensitively on the effec- tive release height of the aerosol and the configuration of nearby buildings.

People indoors will be exposed to the same total dosage as those outside if filtra- tion, deposition, and infectivity decay of the aerosol are negligible. The negligibility of these factors is supported by the absence of significant dosage reduction in field stud- ies of protection afforded by tightly con- structed buildings against an outside spore aerosol (32).

The calculated contours of constant dos- age, like the zone of high human and ani- mal risk, are long and narrow. Contours are shown at 10, 5, and 1 x 10-8 Q spore minutes per cubic meter (Fig. 2) and at 0.5,

0.2, and 0.1 x 10-8 Q spore minutes per cubic meter (Fig. 3), where Q is the number of spores released as aerosol at the source. The number of spores inhaled is the dosage multiplied by the breathing rate. On the innermost contour of Fig. 2, for example, a person breathing 0.03 m3 min-1, as for a man engaged in light work (33), would inhale 3 x 10-9 Q spores.

The calculated dosage at Abramovo is more than an order of magnitude lower than that at the ceramics factory. This sug- gests that sheep, reported to be more sus- ceptible to inhalation anthrax than are monkeys (34), are also more susceptible than humans.

It has been suggested that if Compound 19 was the source, there would have been many more cases in its close vicinity than farther downwind (13). This expectation may be misleading, for as a cloud moves downwind it also widens. The total cross- wind-integrated dosage will therefore de- crease more slowly with distance than does the dosage along the centerline. In the present case, whereas the calculated center- line dosage decreases by a factor of 40 be- tween 0.3 and 3 km downwind, the cross- wind-integrated dosage decreases by a factor of only 4. Depending on the dose-response relation, the crosswind-integrated attack rate may decrease even more slowly than this. Considering, in addition, the lack of information regarding the exact locations of people in Compounds 19 and 32 at the time of exposure, the distribution of cases is not inconsistent with a source at Compound 19.

More detailed comparison of the geo- graphical distribution of cases with the cal- culated distribution of dosage would require knowledge of the precise locations of indi- viduals in relation to the plume, the num- ber of spores released as aerosol, and the relation between dosage and response for the particular spore preparation, aerosol, and population at risk.

By far the largest reported study of the dose-response relation for inhalation an- thrax in primates used 1236 cynomolgus monkeys exposed to an aerosol of the Vol- lum 1B strain of B. anthracis (26, 35). This provided data that, when fitted to a log- normal distribution of susceptibility to in- fection, gave a median lethal dose (LD50) of 4100 spores and a slope of 0.7 probits per log dose (26, 36). This LD50 may be com- pared with an LD50 of 2500 spores obtained in an experiment done under identical con- ditions with 200 rhesus monkeys (35) and with a U.S. Defense Department estimate that the LD50 for humans is between 8000 and 10,000 spores (8). For a log-normal distribution with LD50 = 8000 and slope = 0.7, the dose causing 2% fatalities, as re- corded at the ceramics pipe shop, approxi- mately 2.8 km downwind of the source, is

nine spores. According to the Gaussian plume model we have used, this dose would be inhaled by individuals breathing 0.03 m3 min-1 at the pipe shop if the aerosol re- leased at the source contained 4 x 1 O0 spores. In contrast, a release 150 times larg- er is estimated if the calculation is based on an LD50 of 4.5 x 104 spores, which has been obtained for rhesus monkeys by other investigators (37), and if it is assumed that spores act independently in pathogenesis and that all individuals are equally suscep- tible (38). This estimate would be lowered if allowance were made for nonuniform sus- ceptibility. If these divergent estimates bracket the actual value, the weight (39) of spores released as aerosol could have been as little as a few milligrams or as much as nearly a gram.

In sum, the narrow zone of human and animal anthrax cases extending downwind from Compound 19 shows that the out- break resulted from an aerosol that originat- ed there. It remains to be learned what activities were being conducted at the com- pound and what caused the release of the pathogen.

REFERENCES AND NOTES

1. P. S. Brachman, in Bacterial Infections of Humans: Epidemiology and Control, A. S. Evans and P. S. Brachman, Eds. (Plenum, New York, ed. 2, 1991), pp. 75-86.

2. Health Aspects of Chemical and Biological Weapons (World Health Organization, Geneva, Switzerland, 1970).

3. Posev (Frankfurt) 1, 7 (1980). 4. B. Gwertzman, New York Times, 19 March 1980, p.

1. 5. I. S. Bezdenezhnykh and V. N. Nikiforov, Zh. Mikro-

biol. Epidemiol. Immunobiol. 1980 (no. 5), 1 11 (1980).

6. Veterinariya 1980 (no. 10), 3 (1980). 7. Chelovek i Zakon 117 (no. 9), 70 (1980). 8. Defense Intelligence Agency, "Soviet biological war-

fare threat," DST-1610F-057-86 (U.S. Department of Defense, Washington, DC, 1986); L. H. Gelb, New York Times Magazine, 29 November 1981, p. 31; J. P. P. Robinson, Arms Control 3, 41 (1982).

9. R. J. Smith and P. J. Hilts, Washington Post, 13 April 1988, p. 1; M. Meselson, Fed. Am. Sci. Public Inter- est Rep. 41, 1 (September 1988); I. S. Bezdenezh- nykh, P. N. Burgasov, V. N. Nikiforov, unpublished manuscript, "An Epidemiological Analysis of an Out- break of Anthrax in Sverdlovsk" (U.S. Department of State Language Service, translation 126894, Sep- tember 1988).

10. N. Zhenova, Literatumaya Gazeta (Moscow), 22 Au- gust 1990, p. 12; ibid., 2 October 1991, p. 6; S. Parfenov, Rodina (Moscow), May 1990, p. 21; Pash- kov, Isvestiya (Moscow), 1 1 November 1991, p. 8; V. Chelikov, Komsomolskaya Pravda (Moscow), 20 November 1991, p. 4.

11. N. Zhenova, Literatumaya Gazeta (Moscow), 13 No- vember 1991, p. 2.

12. D. Muratov, Yu. Sorokin, V. Fronin, Komsomolskaya Pravda (Moscow), 27 May 1992, p. 2.

13. L. Chernenko, Rossiyskiye Vesti (Moscow), 22 Sep- tember 1992, p. 2.

14. A. A. Abramova and L. M. Grinberg, Arkh. Patol. 55 (no. 1), 12 (1993).

15. , ibid., p. 18. 16. L. M. Grinberg and A. A. Abramova, ibid., p. 23. 17. F. A. Abramova, L. M. Grinberg, 0. V. Yampolskaya,

D. H. Walker, Proc. Natl. Acad. Sci. U.S.A. 90, 2291 (1993).

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18. Russian Federation, statute 2667-1 (4 April 1992). 19. L. Grinberg, personal communication. 20. V. M. Popugaylo, R. P. Sukhanova, M. I. Kukhto, in

Current Problems of Anthrax Prophylaxis in the USSR (Moscow, 1974), p. 50.

21. S. R. Hanna, G. A. Briggs, R. P. Hosker, Handbook on Atmospheric Diffusion (U.S. Department of Ener- gy Report No. DOE/TIC-1 1223, Washington, DC, 1982).

22. D. E. Davids and A. R. Lejeune, Secondary Aerosol Hazard in the Field (Defence Research Establish- ment Suffield Report No. 321, Ralston, Alberta, Can- ada, 1981).

23. A. Birenzvige, Inhalation Hazard from Reaerosolized Biological Agents: A Review (U.S. Army Chemical Research, Development and Engineering Center Re- port No. TR-413, Aberdeen, MD, 1992).

24. P. S. Brachman, S. A. Plotkin, F. H. Bumford, M. M. Atchison, Am. J. Hyg. 72, 6 (1960).

25. D. W. Henderson, S. Peacock, F. C. Belton, J. Hyg. 54, 28 (1956); C. A. Gleiser, C. C. Berdjis, H. A. Hartman, W. S. Gochenour, Br. J. Exp. Pathol. 44, 416 (1963).

26. H. N. Glassman, Bacteriol. Rev. 30, 657 (1966). 27. A. V. El'kina, Zh. Mikrobiol. Epidemiol. Immunobiol.

1971 (no. 9), 112 (1971).

28. J. V. Jemski and G. B. Phillips, in Methods of Animal Experimentation, W. I. Gay, Ed. (Academic Press, New York, 1965), pp. 273-341.

29. A. C. Chamberlain, Proc. R. Soc. London A 296, 45 (1967).

30. T. W. Horst, in Atmospheric Sulfur Deposition, D. S. Shriner, C. R. Richmond, S. E. Lindberg, Eds. (Ann Arbor Science, Ann Arbor, Ml, 1980), pp. 275-283.

31. A. Bovallius and P. Anas, in Proceedings of the 1st Intemational Conference on Aerobiology, Interna- tional Association for Aerobiology, Munich, West Germany, 13 to 15 August 1978, A. W. Frankland, E. Stix, H. Ziegler, Eds. (Erich Schmidt, Berlin, 1980), pp. 227-231.

32. G. A. Cristy and C. V. Chester, Emergency Protec- tion Against Aerosols (Report No. ORNL-5519, Oak Ridge National Laboratory, Oak Ridge, TN, July 1981).

33. D. S. Ditmer and R. M. Grebe, Eds., Handbook of Respiration (Saunders, Philadelphia, 1958).

34. G. A. Young, M. R. Zelle, R. E. Lincoln, J. Infect. Dis. 79, 233 (1946).

35. J. V. Jemski, personal communication. 36. C. I. Bliss, Ann. Appl. Biol. 22,134 (1935). 37. H. A. Druett, D. W. Henderson, L. Packman, S. J.

Peacock, J. Hyg. 51, 359 (1953).

38. H. A. Druett, Nature 170, 288 (1952). 39. R. Scherrer and V. E. Shull, Can. J. Microbiol. 33,

304 (1987). 40. We thank A. V. Yablokov, Counsellor to the Presi-

dent of Russia for Ecology and Health, for letters of introduction; Ural State University and its then rec- tor, P. E. Suetin, for inviting us to Ekaterinburg; S. F. Borisov, V. A. Shchepetkin, and A. P. Tiutiunnik for assistance and advice; members of the Ekater- inburg medical community and the Sverdlovskaya Oblast Sanitary Epidemiological Service for discus- sions, notes, and documents; interview respon- dents for their cooperation; P. N. Burgasov, USSR Deputy Minister of Health at the time of the out- break, for documents regarding livestock deaths; and People's Deputy L. P. Mishustina for the ad- ministrative list of those who died. I. V. Belaeva assisted with interviews. We also thank B. Ring, W. H. Bossert, P. J. M. Cannone, M. T. Collins, S. R. Hanna, J. V. Jemski, D. Joseph, H. F. Judson, M. M. Kaplan, J. Medema, C. R. Replogle, R. Stafford, J. H. Steele, and E. D. Sverdlov. Supported by grants to M.M. from the John D. and Catherine T. MacArthur Foundation and the Carnegie Corpora- tion of New York. This article is dedicated to Alex- ander Langmuir.

Analysis and Expression of a Cloned Pre-T Cell Receptor Gene

Claude Saint-Ruf,* Katharina Ungewiss,* Marcus Groettrup, Ludovica Bruno, Hans Joerg Fehling, Harald von Boehmer

The T cell antigen receptor (TCR) 3 chain regulates early T cell development in the absence of the TCR:x chain. The developmentally controlled gene described here encodes the pre-TCR:x (pT:x) chain, which covalently associates with TCRf and with the CD3 proteins forms a pre-TCR complex that transduces signals in immature thymocytes. Unlike the X5 pre-B cell receptor protein, the pT:x chain is a type I transmembrane protein whose cytoplasmic tail contains two potential phosphorylation sites and a Src homology 3 (SH3)-domain binding sequence. Pre-TCR:x transfection experiments indicated that surface expression of the pre-TCR is controlled by addi- tional developmentally regulated proteins. Identification of the pT:x gene represents an essential step in the structure-function analysis of the pre-TCR complex.

T cell development takes place in discrete steps during which the TCR genes are rear- ranged and expressed in temporal order. During development of TCRo43-expressing cells the TCRI gene is rearranged and ex- pressed before the TCRot gene (1, 2). With- out TCR rearrangement the development of T cells is arrested at an early stage (3-5). By introducing TCRI transgenes into mice that are defective for rearrangement of an- tigen receptor genes, it was shown that TCRI proteins, in the absence of TCRt

C. Saint-Ruf is at the Unit6 INSERM 373, Institut Necker, 75730 Paris, Cedex 15, France. K. Ungewiss, M. Groettrup, L. Bruno, H. J. Fehling, and H. von Boe- hmer are at the Basel Institute for Immunology, CH- 4005 Basel, Switzerland.

*The first two authors contributed equally to this work.

chains, are sufficient to promote early T cell development (6-8). Although such mice are still rearrangement-defective, their im- mature thymocytes (which express neither the CD4 nor CD8 proteins) begin to express CD4 and CD8 coreceptors, transcripts of the TCRo locus become detectable (7), and the number of thymocytes increases (6-8). In- troduction of TCRI transgenes into normal mice suppresses rearrangement of endoge- nous TCRI genes (9, 10). The TCRI trans- gene is expressed on the cell surface in the absence of TCRo proteins in both normal (11) as well as in rearrangement-defective mice (7, 8, 12) in an 80-kD disulfied-linked complex and as a glycosyl-phosphatidylino- sitol (GPI)-linked 40-kD monomer.

The presence of the TCRI chain in the

80-kD complex suggested that either the complex was a homodimer or that an un- known TCR chain was involved that may affect T cell maturation. A glycosylated chain of 33 kD (gp33) is paired with TCRI proteins in a TCRI-transfected immature T cell line (SCB.29) from severe combined immunodeficient (SCID) mice (12), but could not be identified in normal thymo- cytes (12, 13). The gp33-TCR3 complex of SCB.29 cells is associated with CD3 pro- teins (8, 12) and cross-linking of TCR chains initiates Ca2+ mobilization. This suggested that this TCRI complex could be responsible for the developmental progres- sion observed in TCRI transgenic, rear- rangement-deficient mice, whereas the TCRI GPI-linked monomer could repre- sent a transgenic artifact (14, 15). We have now cloned the gene encoding gp33 and examined its structure and expression. Be- cause of its properties, the gp33 protein was named the pre-TCROL (pTaO) chain.

Pre-T cell receptor ot (pTot) expression in immature T cells. The pTa chain can be identified by two-dimensional (diagonal) gel electrophoresis, in which the disulfide- linked pTo protein under reducing condi- tions migrates away from the diagonal just undemeath the TCRI protein (12) (Figs. 1 and 2). The analytical method was scaled up to obtain sufficient amounts of pTot protein for microsequencing. In a first at- tempt a 20-amino-acid-long NH2-terminal sequence was obtained; a peptide of the 18 NH2-terminal residues was synthesized and injected into rabbits to obtain a pToL-spe- cific antiserum. The antiserum was tested for binding to the pTot protein. To this end lysates from the TCRoL-negative SCB.29 cell line as well as the TCRo3-expressing B6.2.16BW hybridoma (1 2) were precipi- tated with the monoclonal antibody (mAb) F23.1 to V38 proteins (16). Precipitates

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