JPRS ID: 9468 TRANSLATION ANTHRAX ED. BY S.G. KOLESOV

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JPRS L/9468
5 January 1981
Translation
ANTHRAX
Ed. by
S.G. Kolesov
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JPRS L/9468
5 January 1981
ANTHRAX
Moscow SIBIRSKAYA YAZVA in Russian 1976 signed to press 23 Jan 76
PP 1-287
[Text of book edited by S.G. Kolesov, Izdatel'stvo "Kolos," 19,000
copies, 287 pages, UDC 616.981.51]
CONTENTS
AnnotaY_ion 1
Introduction
1
Historical Survey
(S. G. Kolesov, G. I. Romanov)
3
Biology of the Pathogen
(G. V. Dunayev, S. G. Kolesov)
10
Epizootiology
(Kh. Kh. Abdullin)
41
Diagnosis
(S. G. Kolesov, et al.)
65
- Pathogenesis
- (G. V. Dunayev, Kh. Kh. Abdullin)
89
Disease Symptoms and Course
(Kh. Kh. Abdullin)
95
Pathomorphology and Pathomorphological Diagnosis
(A. V. Alculov)
102
The Blood Picture of Animals Stricken With Anthrax
(G. I. Romanov)
109
Treatment of Sick Ani.mals
(S. G. Kolesov, G. I. Romanov)
114
Immunity
(S. G. Kolesov, et al.) .......................I
121
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Anthrax in Pigs
(N. G. Ipatenko) 154
- Veterinary-Sanitary Measures
(Yu. I. Boykov) 175
Biological Soil Decontamination
(V. V. Arkhipov) 210
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ANNOTATION
: This book describes one of the most dangerous diseases of animals and man. Brief
_ information from the history of the disease and its occurrence in our country and
in other countries of the world is presented. T'he pathogen, the disease symptoms,
and the pathoanatomical changes observed in sick animals are described.
Much attention is devoted to the problems associated with treating sick animals,
immunity, and veterinary and sanitary measures implemented at farms when anthrax
appears.
- INTRUDUCTION
Anthrax is one of the dangerous diseases with an acute course. In the past it
inflicted tremendous losses upon animal husbandry, and it often evoked mass illness
among people.
There were no effective ways to contirol this disease, and it was only after the
pathogen was discovered that i.ntensive work began on questions of the microbiology,
- epizootiology, and diagnosis of anthrax. Scientists answered them successfully
at the end of the past century and in the first half of the present century.
Especially great changes in the methods for controlling infection occurred after
acquisition of anthrax vaccines, owing to which a possi.bility arose for preventing
the disease. The vaccines were proposed by L. Pasteur (1881) and L. S. Tsenkovskiy
_ (1883). Immunity was studied and disinfection methods to be used with anthrax were
developed in parallel.
As of today the questions noted above have been studied sufficiently well, but before
we can provide fully conclusive arsswers to them, we must do further work to reveal,
record, and eliininate infection foci in the soil. Mass animal vaccination alone
would not insure total elimination of the disease, since the threat of arisal of
_ anthrax among unvaccinated animals would constantly arise.
Despite the complexity of the measures being implemented to control anthrax, its
- epizootics have been eliminated from our coimtry. The present problem is to
eliminate.known anthrax foci and discover those yet unrevealed.
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Scientific research and practical efforts o� the last 15 years showed that the great
and difficult task of era(a.1cating anthrax--a dangerous infection of the community of
man and animals, may be complete3 suc;:essfully through joint implementation of
measures by veterinary and medical services.
The main ways to eliminatP anthrax are to vaccinate animals in areas harboring this
disease, reveal and study infection foci in the soil and decontaininate them, and
study the ways of the pathogen's spread, as well as a number of cther problems.
This book describes anthrax control miethods proposed by domestic and foreign
scientists. In addition ~o Known methods, it presents new ones being used in m~cro-
biology as well as in the diagnosis of the disease, in the treatment of patients,
and in disinfection. Theoretical aspe^ts having to do with microbiology, epizoo�
- tiology, pathogenesis, immunity, and a numher of othe-r problems are illwninated as
well.
The book is i.ntended for scientists studying anthrax, and for practical workers
employed by sovkhoz and kolkhoz veterinary services and diagnostic laboratories.
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HISTORICAL SURiIEY
Anthrax has been known to mankind as an infectious disease of animals since ancier.t
times. It was diaqnosed in many countries of the world where the geographic condi-
tions, soil, and temperature favored survival and developaent of the pathogen in
the environment. The spread of anthrax in those distant times was promoted by
development of states and nations, by migrations of people and animals over great
- distances, by growth in the strength of ties between states, and by wars. A disease
with symptoms recalling anthrax ?.s even described in the Bible. It was observed
among animals as well as people as a carbuncular disease.
A disease having clinical signs recalliny anthrax was also observed in Ancient
Greece. Thus in the "Iliad" (Book 1) Homer describes a disease that arose in the
Greek encampment during the seiqe of Troy in 1218 B.C.; it first infected mules and
dogs, and then people. A similar disease is mentioned in the works nf Hippocrates
- (460-377 B.C.). According to him the island of Aegina suffered very strongly from
" this disease in 1295 B.C., as did the Thessalian city of Kranok in 430 B.C.
Nor was anthrax a rare phenomenon in Ancient Rome. Many historians ana poets of
this country described it under various names. Plutarch wrote that violent out-
breaks occurring during the time of Romulus killed animals and people very quickly.
= Dionysius of Halicarnassus and Titus Livius asserted that it wrecked cruel havoc
amonq farm animals and caused the death of large numbers of people in 463, 453, and
452 B.C.. Titus Livius reported a disease which arose first among animals and then
infected people in 433, 428, 400 and 175 B.C..: Pliny the Elder (Gaius Secundus)
- mentioned a carbuncular epidemic in Italy in 163 B.C., and he believed that the
_ disease had been imported there with woolen goods from the Narbonne district
(Southern Gaul); Cato, Var�ro, and Lucretius called the disease sacred, or volatile,
fire (ignis sacer). It was also described by Aulus Cornelius Celsus, who lived at
the end of the lst centuzy B.C. and the beginning of the lst century A.D. (S. F.
Khotovitskiy, 1831).
In his poem "Georgics" (37-30 B.C.), the RQman�poet Virgil accurately descri.bed a
disease observed among animals of different species and man, leaving no doubt that
the reference was to anthrax.
It is also mentioned in other ancient writings. There are several mcntions of
anthrax i.n the lOth century collection of veterinary writings entitled "Hippiatrica".
In the opinion of some authors it is also descri.bed in the "Quadruped Medicine"
attributed to the 11th century (D. M. Klemm, W. R. Klemm, 1959). -
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The first reports of anthrax on Russian territory may be found in the Nikonovskaya -
Chronicle (979 A.D.), which indicates that many doriestic and wild animals as well
as people died. The Tverskaya Chronicle (the entry for 1158) offers evidence of
the death of horses, cattle, and people in Novgorod. An epizootic recurred in this _
city in 1204.
In 1284 epizootics caused ttle death of farm animals not only in Russia but also in
Poland, Lithuania, and other neighboring countries. Many horscs, cattle, and
sheep fell. Anthrax was probably among the diseases causing these animal deaths.
J. M. Koropov presents interesting data from Russian chronicles in the book
"Istoriya veterinarii v SSSR" [History of Veteri.nary Medicine in the USSR] (1954).
Thus major epizootics were observed among animals of different species and epidc -ics
occurred among people in 1309, 1374, 1375, 1443, and 1448. In 1640 the disease
appeared among people, its origin being associated with removal of skins from
deceased aniunals. The same thing happened again in 1643 in the vicinity of the
city of Ostashkov.
In connection with the violent epizootic in 1640, a Czarist ukaze prohibited the
removal of skins from sick and fallen animals, and required their carcasses to be
buried deep in the ground. It was decreed that violators were to be whipped ab-
- solutely without mercy. Hence it follows that by this time, the infectiousness
of the disease and the danger of human contact with the skins and hides of animals
dying from or stricken with anthrax were well known in Russia.
By as early as the mid-1350's quarantines were introduced :.ato the major trading
cities of Italy as a means for isolating foreign vessels, people, and goods arriving
- in the country for the first time. Beginning with the 16th century quarantine
me3sures began to be implemented in many countries of Europe, to include Russia.
However, despite the steps taken, anthrax continued to spread, and to cause con-
siderable losses. It is evident from descriptions written by Kirkh in 1658 that
the disease brake out among cattle and then among people in 1617. About 60,000
persons diec7 ~S. M. Derizhanov, 1935).
There are ir..dications that anthrax spread through Germany and France in 1712; through
Poland, Silesia, and Saxony in 1726; through many European countries in 1756; through
Finland in 1758 and 1774; through Sweden in 1774. According to a report by S. F.
Khotovitskiy it caused the death of 85,000 horses in Siberia in 1781.
~ It is interesting that even in 1831, concluding his hi.storic survey, S. F. Khoto-
vitskiy wrote in the book "O sibirskoy yazve" (On Anthrax] that with time, the
clinical signs and manifestations of "ogneVik" (anthrax) underwent change: In
ancient times it supposedly affected people more, in the Middle Ages it manifested
itself in the form of leprosy, and in the present more animals fall ill with it,
~ and people are rarely affected. That such notions are mistaken is obvious. 'i"here-
fore in this historical survey we attempted to cite only those sources which provide
descriptions of illness observed simultaneously in different species of animals and
- people, which is typical of anthrax.
But all these sources were notha.ng more than passing references to cases of disease,
in which case the authors very often described carbuncular and other epizootics with
the general terms iqnis sacer, ignis volatilis, and ignis S. Antonii, treating
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diseases such as human and cattle pl-aque, anthrax, and rabies as all the same.
- Therefore m3ny reports by authors writing in ancient times and in the Middle Ages
shoulc3 be treated very cautiously. It was not until the late 17th and early 18th
centuries that the first scientific works appeared, in which the symptoms of the
disease, its causes, and its occurrence were descxibed, and recommendations en the
treatment of sick animals were even offered. One of the first such works is that
of Ramazzini, who descri.bed an epizootic that dominated almost all of Italy in 1690-
1.691. He was the first to distinguish carbuncular diseases from cattle plague, but
he did not isolate anthrax as a separate nosological unit. This was first done
by the Russian physicians Abram Eshke and Plikita Nozhevshchikov employed by the
IGolyvan'-Voskressensk plants of Altayskiy Kray. They presented detailed information
on anthrax to the medical board. In his work, A. Eshke (1758) described symptoms
of the disease in people, he pointed out its similarity to disease in animals,
- and he noted its seasonality. In 1762 N. G. Nozhevshchikov ma.de a detailed study
_ of the sy.nptoms of the disease and the c;,nditions promoting its arisal.
Among foreign researchers, the first to offer a scientific description of anthrax
was Moran in France. In 1766 he submitted a report on this disease to the
Academy of Sciences in Paris (12).
- In 1769 Fournier distinguished anthrax as a separate nosological unit.
Other works providing detailed descriptions of anthrax appeared as well. In the
1770's P. S. Pallas observed and described it in the Urals, along the Irtysh River,
in the vicinity of Ishim, and in the Kirghiz steppes. In 1780 M. Tomassen received
a decoration from the Dijon Academy for his research on this disease.
S. S. Andreyevskiy (1786-1789) contributed much labor to the study of anthrax.
Following a 3-year expeditian into the Chelyabinsk District of the Ural'sk Vice-
regency, iz 1788 he presented the medical board his essay "On Anthrax" containing
a detailed map of the disease's occurrence, a description of the signs of the
_ diseases, and illustrations providing animpression of the size and internal structure
- of carbuncles appearing on animals and man (9). In this same work the author de-
scribed an experiment he performed upon himself in the presence of Doctor V.
Zhukovskiy, Chelyabinsk Governor Shveygofen an.i Judge Olovyannikov, in which he
demonstrated the infectiousness of the disease and its identity to that observed
in animals and man.
By this experiment S. S. Andreyevskiy wanted to confirm the infectiousness of the
disease and its identity to that observed in animals and man.
Moreover S. S. Andreyevskiy is credited with the main role in defining the disease,
which he named Siberian ulcer after the place in which he studied it, and in de-
scribing the methods of the disease's prevention and treatment.
Many other researchers continued to study anthrax subsequently. In 1790 it was de-
scribed by I. Peterson, and in 1792 M. L. Gamaleya published his book. V. Kozlov
_ (1795), V. Zhukovskiy (1795), and Pinyayev (1797) submitted essays to the medical
- board in which they indicated a number of interesting ideas they had come up with.
Thus M. L. Gamaleya reported the possibility for transmission of infection from sick
to healthy animals by blood-sucking insects; V. Kozlov suggested the hypothesis that
tiny invisible living beings were the cause of disease.
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It was the 19th century. Anthrax epizoatics continued to rage, and they were re-
corded in different countries almost every year. Especially large outbreaks were
abserved in West Europe in 1.803, 1807, i811, 1822, and 1834. According to estimates
by (Delafan) more than 284,000 sheep were killed by this disease in 1843 just in the
small province of (Bos') (in Orleans) (P. Lyubimov, 1867).
It is believed that anthrax was brouqht into the USA by the first settlers from
, Europe. Major outbreaks were noted there among domestic animai.s and people in
- 1835, 1851, and 1884.
In Russiar anthrax epizootics were registered several times in the period from 1807
to 1857. For example many deer, and even wolves feeding on carcasses, died in
- Lapland in 1823. The disease also spread among cattle, sheep, and people. Dea',s
- among just damesticated deer alone exceeded 5,000.(13).
This situation alarmed veterinary specialists studying anthrax. Books were published
by F. Uden (1807), Fr. Geyrot (1807), and Ellizen (1808). In 1823 Barthelemy
managed to demonstrate the disease's contagiousness. S. F. Khotovitskiy's book
"On Anthrax" was published in 1831, F. Shkinskiy's book "On Siberian Fire Disease"
was published in 1832, and V. I. V:,evolodc�v's "Experience in Studying Epidemic
- Diseases Among Domestic Animals" was published in 1846. These books provide an
exhaustive description of the disease.
In 1850 Davaine and Rayer found thin, cylindrical, motionless rods in the blood of
aniunals stricken and killed by anthrax. In 1855 Pollender r.eported that back in
1849, when he autopsied cows that had been killed by this disease, he found their
blood to contain "rod-shaped, very thin bodies, dense in appearance and completely
straight and motionless."
Professor F. Brauel' of the Derptskiy (presently Tartu) Russian Veterinary School
conducted research confirmi.ng the existence of these rod-shaped bodies, and he
described them in 1857-1858. As a result of numerous blood innoculations he
- also managed to experitnPntally infect different animals. In 1863 Davaine proved
the capability blood containing anthrax bacilli had for causing disease. He
established that rod-shaped elements appear in the blood of sick rabbits 5 hours
= prior to death at the earliest, and that it is only from this moment that the blood
- acquires the capability for evoking illness in other animals.
R. Koch reported in 1876 that the formations under examination were microorganisms
which reproduced by division and which were capable of developing on nutrient media.
He also proved that such cultures could evoke anthrax in animals.
Louis Pasteur performed siunilar research in 1877. He obtained a pure culture of
anthrax pathogen, studiad its development i.n artificial nutrient media, and
established that culture filtrate was harmless and that the infectious element
could survive in soil for long periods of time. In 1881 he created anthrax vaccines
1 and 2, which was an outstanding achievement.
A culture of anthrax microbes was obtained in Russia for the first time by the
prominent scientist V. K. Vysokovich in 1882, and in 1883 L. S. Tsenkovskiy pre-
pared anthrax vaccines 1 and 2 and successfully corapleted sheep vaccination experi-
ments, thus initiating brilliant research on prevention of anthra.r among agricultural
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ani.mals by vaccination. Despite this, anthrax continued to arise both in Russia
and in many countries of the world in the latter half of the 19th and start of the
20th eentury.
Nocard and Leclainche (19) reported siqnificant spread of anthrax and considerable
losses in animal husbandry due to it in France, Italy, Great Britain, Germany,
Spain, Belgium, and elsewhere. They also cited data on the accurrence of anthrax
in Australia and in American and Asian countries.
According to V. F. Nagorskiy 643,303 animals caught anthrax in 60 provinces of
European Russia from 1885 to 1899; of these, 517,924 head died. During the same
_ period 50,473 animals fell ill in 79 districts of Geraany; of these, 46,898 head
died.

According to N. A. Mikhin (1942) from 32,000 to 60,000 animals caught anth.rax each
year from 1900 to 1912, which is an annual average of 47,976 head. Sizable anthrax
epizootics also broke out among reindeer in the country's north.
Anthrax was also highly prevalent in the country during the first years of Soviet
- rule, whxch elicited great concern. It was in this connection that the RFSFR Soviet
of Peoples Commissars published a decree on 10 October 1927 ordering the RFSFR
Peaples Commissariat of Agriculture to develop measures to control it.
A group of scientific and practical veterinary specialists was created in compliance
with a directive of the RE'SFR Peoples Commissariat of Agriculture. In 1928-1929
J this group implemented a complex of ineasures against anthrax in the Belgorod District
of the Central Chernozem Province. Their work was published in the journal
PRAKTICHESKAYA VF;TERZNARZYA, No 11-12, 1929. The obtained results were extensively
employed in measures against anthrax throughout the entire country, which dramati-
cally reduced the disease's occurrence. For example by 1950 enzootic and epizootic
_ outbreaks of anthrax were completely eliminated.
Following rehabilitation of the war-devastated national economy, the principal
measures for controlling anthrax included mass vaccination of an.Lmals and implemen-
- tation of veterinary and sanitary tneasures. As a result in the last 26 years (from
,1947 to 1973) the number of susceptible locations in the countxy decreased by a
factor of 21.33, the number of animals falling ill dropped by a factor of 20.57,
and the number of animals dying decreased by a factor of 20.1.
However, the possibility that sporadic cases of this infection may occur does exist.
This can be explained by the biological features of the pathogen, by its ability
to survive in soil for long periods of time, and by our still-inadequate knowledge
of former cencers of infection.
In some countries of E;urope anthrax arises today only sporadically, and it apparent-
ly offers no major threat. However, in some countries it arises periodically in
the form of epizootic outbreaks. Accordi.ng to data of the International Epizootic
Bureau, in 1959-1964 7,871 anthrax outbreaks were registered in Greece, 1,837 were
registered in Italy, and 1,215 were reqistered in Spain. In Great Britain, 1,215
outbreaks of animal anthrax were registered in 1952, and 609 were registered in
1953 (according to a report on the work of the veterinary service).
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Anthrax outbreaks have also been registered in Asian countries--in Iran, Turkey,
India, Syria, and elsewhere. Anthrax is also abserved in American counltYies.
Sporadic cases of anthrax arise vearly in the United States of America. Neverthe-
- less data of the International Epizootic IIureau show that there has been a clear
tendency for the disease to decline in the period from 1968 to 1972.
BIBLIOGRAPHY
1. Vsevolodov, V. I., "Opyt ucheniya o poval'nykh boleznyakh mezhdu domashnimi
zhivotnymi" [EXPerience in Studying Epidemic Diseases Among Domestic Animals"],
St. Petersburg, 1846.
2. Gamaleya, M. L., "0 sibirskoy yazve i o yeye narounom lechenii" [On Anthrax _-,d
on Its Folk Treatment], PERM', 1792.
3. j,Qmmr, Ye. M., VETERINARNYY VESTNIK, Khar'kOV, NO 3, 4, 5r 1882, p 105. _
4. Zenuner, Ye. M., VETi:RIDIARNYY VESTNIK, Khar'kov, No 6, 1882, p 1�
5. Koropov, V. M., "Istoriya veterinarii v SSSR" [History of Veterinary Medicine
in the USSR], Moscow, Sel'khozgiz, 1954. i-
6. Lebedev, N. D., PRAKTICHESKAYA VETERINA1uYA, No 11-12, 1929, p 1962.
7. Liviy, Tit., "Ri.mskaya istoriya ot osnovaniya goroda" [The History of Rome
Since the City's Founding], Moscora, 1899.
8. Nagorskiy, V. F., "AVN," Book 7(Appendix), 1902.
� 9. Palkin, B. N. , ZHMEI Vol 7, 1951, p 81.
10. Peterson, I., "Kratkoye opisaniye bolezni v Rpssii, nazyvayemoy vetryanoy i
sibirskoy yazvoy" [A Brief Description of a Disease in Russia Called Wind and -
Siberian Ulcer], Tobol'sk, 1790.
- 11. Publiy Vergiliy Maron, "Sel'skiye poemy Bukoliki, Georgiki" [pastoral Poems of
the Bucolics and Georgics], Moscow, 1934.
12. Rozen'yer, L. A., "Sibirskaya yazva u cheloveka" [Anthrax in Man] Kishinev,
1948.
13. Khotovitskiy, S. F., "O sibirskoy yazve" [On Anthrax], St. Petersburg, 1831.
~ 14. Tsion, R. A., PRAKTICHESKP,YA VE`FERINARIYA, No 11-12, 1929, p 978-
15. Shkinskiy, F., "0 si.birskom ognevike" [On Siberian Fever] , St. Petersburg, -
1832.
16. Brauell, F., "Archiv. f. path. Anat. und Phys.," 1858, p 4.
17. Davain, C. I., C.R. ACAD. SCI., Vol 57, No 4, 1863, p 220�
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18. Koch, R., COHN'S BEITRAGE 2UR BIOIAGIE DER PFLANZEN, Vol 2, 1876, p 276.
19. Noc:ard, E., and Leclainche, E. ,"Mikrobnyye bolezni zhivotn�lkh" [Microbial
Diseases of Animals] , St. Petersburg, 1906.
~
20. Pasteur, L. , Chamberland, Ch. Ed. , aI1d ROUX, E., C.R. ACAD. SCI., Vol 92, 1881,
p 429.
21. Pollender, F. A. A., VIERT. F. GERICHTL. U. OFF. MED. BERLIN, Vol 8, 1855,
- p 103.
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BIOLOGY OF THE PATHOGEN
Anthrax bacillus was discovered microscopically for the first time by Pollender i;.
1849. In 1850 the French scientists Devain and Rayer discovered filamentous,
motionless bodies in tl?e blood of sheep stricken and killed by anthrax. The same
sort of bodies were observed in Russia among dead animals by Professor F. Brauel'
of the Derptskiy Veterinary School (1857). He was one of the first to discover
_ bacilli in the blood of a person who died from anthrax, and to experimentally re-
- produce the typical disease in animals by infecting them with blood containing
microscopically visible bacilli (vibrios).
However, the significance of these bacilli remain unc3.arified for a long period of
time, and it was not until 1863 that Davain canclusively established the role of
these formations as anthrax pathogens. Thus 1863 is said to be the official date of
discovery of anthrax bacillus. The rod-shaped bodies described by this scholar were
named anthrax bacteria (Bacteri des charbonneus).
(Delyafon) attempted to obtain a culture of the possible anthrax pathogen back in
- 1860, but it was not until 1876 that pure cultures of anthrax bacillus could be
isolated, first by R. Koch and then by L. Pasteur. Using these cultures, they re-
produced the disease in animals independently of one another.
Research by R. Koch in 1876 and by L. Pasteur in 1877 proved that vegetative cells
of the anthrax microbe are capable of forming spores.
In 1888 Serafini discovered that anthrax bacilli formed capsules.
In Russia, an anthrax microbe culture was first obtained by V. K. Vysokovich in 1882.
In subsequent years the biology of anthrax pathogen was studied by L. S. Tsenkovskiy,
I. N. Lange, P. N. Andreyev, N. A. Pokshishevskiy, N. A. Mikhin, S. N. Vyshelesskiy,
R. A. Tsion, M. V. Revo, Ya. Ye. K,olyakov, F. A. Terent'yev, and many other
scientists.
The anthrax pathogen--BaciZZus anthrac2s (F. Kon, 1872) is classifed as order
Eubacterial2s, family Bacillaceae, and genus and subgenus BaeiZZus. This genus
contains about 25 aerobia and facul tative- anaerobic bacteria reacting positively to
catalase.
The following species are most closely associated with anthrax bacillus: Bac. esreus
sive Bac. anthracoides sive Bac. pseudoanthrrzeis (waxy bacillus), Bac. cereus var.
rmjeo2des sive Bac. rmyeoides (ront-like bacillus), Bac. megaterizpn (cabbage bacillus),
subtiZis sive Bac. mesenterieus Trevisan (grass bac*llus, also referred to in
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the literature as potato bacillus), and Bcte. ptoniZus sive Bac. mesenteric,~u8 Chester
(the latter synonym is often used for potato bacillus).
They are all saprophytes with the exception of Bac. cereu8, which synthesizes an
active enzyme of pathogenicity, lecithinase, and which is capable of e liciting
dietary toxicosis.
Bae. anthrac2s is a rather large (from 1-1.3 to 3-10 u) Gram-positive, nonmotile rod
forming capsules and spores. It is encountered in three forms: a vegetative form--
cells of varying size (encapsulated and nonencapsulated), a spore form--spores en-
cased .within a well expressed exosporiwn, and a spore form in which t.he spores exist
in isolation from one another.
Unstained bacterial cells in preparations maiie from the blood and tissues of animals
stricken or killed by anthrax may have the form of homogeneous transparent rods with
_ slightly rounded ends.
The rods lie separately, or they are joined together into short chains. The number
of cells in a chain of highly virulent strains does not exceed three, while tYzere
may be more with strains exhibiting low residual virulence.
Lipoprotein granules, located subterminally and terminally for the udost part, have
been discovered in the bacteria.
The morphology of unstained bacteria from cultures grown in solid or liquid nutrient
media is similar to that described above; however, in this case the cells always
form more or less long chains.
Rods contained in smears from cultures of strains that exhibit typical growth as a
flaky precipitate in liquia nutrient media arrange themselves in long chains
(Figure 1). Bacteria from cultures exhibiting atypical diffuse growth in liquid
media form short chains.
The cells of varying length recall cylinders with perfectly rounded margins. The
_ surface of the cell wall is uneven.
In stained preparations, the ends of rods forming chains face one another; they are
straight and closely clipped. Free ends, meanwhile, are slightly rounded. Some-
times the chains look like bamboo reeds, the clipped ends of the microbial cells
are sometimes dented inward, and they are syumetrically thickened at their points
of attachment. Such forms are encountered among bacteria synthesizing a capsule
- when growing in media containing proteins, or among bacteria reproducing in the
animal body.
The bacterial cell has a nucleus. The first differentiated nucleoid (nuclear
_ apparatus) was observed in anthrax bacillus by F. Ya. Kitayev (1922), who defined
it as a chromidial apparatus. He noted concurrently that the nuclear element takes
- part in division of vegetative cells, and that it is seen in genni.nating spores.
Later the existence of aformed nucleus in anthrax bacillus was confirmed by Flewett
(1948).
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e
Figure 1. Vegetative Cells From Broth Culture: Luminescent
microscopy. ML-2 microscope. NINF-10. Objective
95 x1.25; ocular photo 012.5 x.
In 1952 M. N. Meysel' and L. V. Mirolyubova reported that the nucleus of vaccine
anthrax strains has the form of helical strands occupying the central part of the
cell and arranged along its axi.s.
According to Chatterjee and Williams (1962) the chromatin bodies of cells in young
cultures were long, continuous formations located centrally. Both continuous and
dividing nucleoids were present in mature cells. Long chromatin bodies in cells
in 24-hour cult.ires arranged themselves as large complexes consisting of spherical
formations.
Using cytochemical tests, the above authors concluded that RNP, is associated with
the cytoplasm of the bacterial cell whiZe DNA is associated with the nucleoids, and
- that anthrax bacillus possesses a differentiated, discrete nucleoid.
G. V. Dunayev (1967, 1972) also discovered a differentiated nucleoid in vegetative
cells from Tsenkovskiy and STI-1 No 2 vaccine strains fixed and stained by the
- classical Pekarskiy-Robinou method.
Vegetative anthrax bacillus cells cantain a clearly outlined nucleoid in all phases
of development of the bacterial culture.
- The nuclear structure is revealed especially clearly when luminescent and phase-
contrast microscopy are combined.
Mesosomes (equivalent to mitochondria) have also been discovered in anthrax bacillus.
These organoids are polyfunctional. Z'he membrane-mesosome system in bacteria is
not only responsible for oxidative phosphorylation, electron transport, and the
di- and tricarboxylic acid cycles, but it is also involved in protein synthesis
(G. I. Burd, 1967; Rose, 1968).
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The mesosomes of vegetative anthrax bacillus cells are clearly outlined, bright
yellow-green granules in direct contact with the cytoplasmic membrane.
Various inclusions are seen in the cytoplasm of vegetative anthrax bacillus cells.
Volutin granules staining metachromatically may be found at the poles of the bacteria
and sometimes at their center when they are stained by Loeffler's method, which in-
volves holding the bacteria for some time in methylene blue solution. Staining with
Sudan black reveals lipid granules, which are especially abundant during spore forma-
' tion. They are encountered in spore-forming aerobic bacilli of all species, in-
cluding among saprophytes.
Hochkiss' cytochemical reaction to polysaccharides reveals small glycogen granules.
However, they are rarely encountered among both vegetative cells and bacteria ini-
tiating spore formation.
The Ultrastructure of Vegetative Microbial Cells
- Roth and Williams (1963, 1964) were the first to report the submicroscopic structure
of vegetative anthrax bacillus cells. Studying ultrathin spleen sections from mice
- infected by anthrax pathogen, they discovered elements of a discrete nucleoid i.n
vegetative cells of this microbe.
The fine structure of virulent (No 66, No 2222) and vaccine (Tsenkovskiy's vaccine
No 2, STI-1, Stern's) strains of anthrax bacilli had been studied in sufficient
detail owing to the research of I. B. Pavlova and L. N. Kats (1966), A. A. Avakyan,
T. B. Pavlova, L. N. Kats, and Ye. N. Levina (1967), I. I. Belokonov and G. V.
Dunayev (1968), I. I. Belokonov (1970), T. A. Trzhetsetskaya and A. V. Kulikovskiy
(1972), and Moberly, Shafa, and Gerhardt (1966).
0
The cell wall of anthrax bacillus seen in ultrathi.n sections is 360-400 A, and it
consists of three layers: two osmiophilic layers each 30-40 A thick, and one osmio-
phobic layer 300-320 X thick. However, this wall structure is not always noted.
The wall consists more frequently of an internal osmiophilic layer of greater
density and an outer, moderately dense layer. Tt:e outer layer of the wall often
transforms into fibrillar structures that spread over the entire surface of the cell
(Figure 2). It is hypothesized that these osmi.ophilic fibrillar formations are
capsule remnants.
'1'he cytoplasmic membrane is smooth or somewhat twisted. Its trilaminar structure
can be revealed only in some portions of the cell. It is more noticeable in lysed
cells. Usually its outer layer is in firm contact with the cell wall, giving the
appearance of a single-layered formation (see Fig;ire 2).
As a result of invaginational growth, the cytoplasmic membrane forms projections
into the cytoplasm, differing in form, size, structure, and location, and described
as intracytoplasmic membranous structures (mesosomes).
The thickness of the membranes in these formations is 80-100 A. They are usually
_ simple invaginations of -the cytoplasmic membrane and appear as coils, ovals, and
uneven lines. In many bacteria, the intracytoplasmic membranous structures penetrate
- into the zone of the nucleoid, and on occasion they are bound to it.
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Figure 2. Ultrathin Anthrax Bacillus Section: KC--cell wall;
Liki--cytoplasmic membrane; OCK--fibrillar structures of
the cell wall; MC--membranous structures; H--nucleoid;
P--ribosomes. x65,000.
The nucleoid appears as an extensive osmiophobic zone in the central part of the
cell, and it is not sharply delimited from the cytoplasm. The bulk of the nucleoid
consists of an irregular network of fibrils 25-50 A thick distributed uniformly
throughout its entire area.
The cytoplasm has a fine-grained structure, and it consists of 150-200 A granules,
similar in size to bacterial ribosomes. Ri.bosomes are often arranged in chains,
forming polyribosomes. Clearly outlined vacuoles are encountered in the cytoplasm
of many anthrax bacillus cells (Figure 3). They are often large, and enclosed by
a membrane. Shape is imparted to the vacuoles by a membrane with polyri.bosomes on
its outer side, which is especially noticeable in lysed cells. The vacuoles often
concentrate near the nucleoid. In all probability the vacuoles form owing to
_ dissolution of lipid inclusions, mainly poly-S-oxybutyric acid granules (Gerhardt,
1967), during fixation and dehydration.
Cell division involves forntiation of a transverse septum, as a result of which two
individuals of equal size form. Creation of the transverse septum begins with
invagination of the cytoplasmic membrane, with simultaneous inclusion of the cell
wall in this process. As a result small syametrically arranged identations
oriented in the direction of the cytoplasm form.
The next act of division often begins before cells have sepaxated following previous
division. This leads to formation of streptobacilli. 7'he length of individual
bacteria in the chains often varies.
The ultrastructre of anthrax bacillus exhibits unique features during toxin bio-
synthesis and secretion when cultures are grown in special media (G. V. Dunayev,
1972).
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Figure 3. Ultrathin Anthrax Bacillus Section: B--vacuoles. x55,000.
y A culture in its exponential growth phase consists mainly of entire cells. The
thickness of the cell wall of such bacteria is 330-350 A, and the outer layer under-
gces a transition into clearly pronounced fibrillar structures. Z'he cytoplasmic
membrane is a single-layered formation 80-110 A thick with a convoluted outline.
A high accumulation of osmi.ophilic masses can be noted within the zone of the
nucleoid. Intracytoplasmic channels are revealed in some areas of the bacterial
cells; they differ in their morphology from the usual type of inembrane structures
- observed in microbes of this species: They are straight, short, and 110-120 JL wide,
while membrane thickness is 75-80 A. These channels pass through the cell wall and
communicate with the outside environment.
Individual cells with a lysed protoplasm but with well preserved membrane structures
are also encountered. Localized areas of lysed cell wall up to 375 A in size are
often revealed in such cells. Bacterial cells actively secreting toxin are observed
in the exponential growth phase.(F'igure 4). Inclusions of compact osmiophilic `
' particles are noted in the cytoplasm of such cells.
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R 1
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Figure 4. Ultrathin Sections of Anthrax Bacillus Cells Actively
Secreting Toxin. X39,000. ,
Outside the cells, the osmiophilic particles are contained in an optically less
dense substance, also produced by secreting bacteria. As the particles get farther
away from the microbial cells, they grow constantly larger, and the distance between
them increases.
Intense development of the ribosome apparatus and the membrane-mesoson,:e apparatus
is noted among bacterial cells synthesizi.ng toxin; a close relationship is si.mul-
taneously established between them, and intracytoplasmic membrane structures
penetrate into the nucleoid zone.
When the microbial population is in its stable growth phase, cells are encountered
with individual portions of the cell wall destroyed (local lysis), and it is
through these breaks that a structure taking the form of fibrillar-granular osmio-
philic material contained in the cytoplasm is secreted. There are no noticeable
- differences in the fine structure of vegetative cells of virulent and vaccine
anthrax strains.
Microbe Staining Methods
- Anthrax pathogen is stained intensively by alcohol-water solutions of the principal
aniline dyes broadly employed in bacterial staining. Smears and impressions of the
tissues of dead ani.mals and blood smears stain very well with Loeffler's methylene
blue.
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Culture preparations may be stained in 1-2 minutes by a 1:10 dilution of fuchsin
(Pfeiffer's alcohol-water solution) and Loeffler's methylene blue.
This bacillus (the vegetative form) reacts positively to Gram's stain. Negatively
staining cells are encountered in very young and in very old cultures. This microbe
does not have any specific tinctorial features inherent to it alone.
Capsule Formation
The Structure of the Capsule and Ita Biological Significance
Anthrax bacillus fornLS capsules in the body of a sick animal or when it is grown in
artificial nutrient media containing a large concentration of native protein. In-
tense capsule formation occurs during growth in both liquid and solid seram nutrient
media.
In Gladstone-Fields medium, encapsulated rods appear after the third hour of incuba-
tion (A. V. Mashkov and V. P. Bodisko, 1958), and by the 14th-16th hours practically
the entire culture consists of them. Then begins intensified diffusion of capsule
substance from the surface of the cell into the surrounding medium.
QCI [State Control Znstitute of IIiomedical Preparations] protein medium is an
elective substrate offering the conditions for capsule biosynthesis by anthrax
bacillus. The phenomenon of capsule formation is clearly noted in anthrax bacilli
also in their growth xn coagulated equine blood serum by Shaffer's method, as well
as in serum agar, especially in a C02 atmosphere.
Capsule synthesi.s is rather intensively pronounced when capsule-forming strains are
grown in proteiri media used to obtaan protective antigen.
In this case capsule formation begins after 2,5 hours of growth, and it may be well
pronounced in 6-hour cultures. Cells encased in a capsule are detected in 24-hour
cultures as well.
However, in addition to encapsulated cells,
revealed in both the exponential and stable
forming vaccine strain cultures (Figure 5).
different genetic characteristics, and main
polypeptide, are encountered in populations
bacillus.
bacteria devoid of capsules are also
growth phases of virulent and capsule-
This is evidence that mutants with
Ly withAUt the capsule glutamic acid
of different strains of anthrax
In addition to native prot:i.n, an alkaline environment and presence of C02 promote
capsule formation in culture (Sterne, 1937; Chu, 1952).
_ The influence of carbon dioxide upon the activity of some bacterial mitochondrial
enzymes was established owing to research by Eastin and Thorne (1963). This research
shawed that the demand virulent and avirulent strains of anthrax bacillus exhibit _
for C02 during formation of capsule polypeptide differs: The former need it in .
higher concentrations, while the latter can also synthesize capsule polypeptide in _
an atmosphere devoid of C02.
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