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PROPHYLAXIS AND THERAPY OF ANTHRAX

V.G. Popov, G.Ya. Scherbakov, L.I. Marinin, N.A. Staritsyn, A.V. Stepanov

State Research Center for Applied Microbiology, the Ministry of Health Russian Federation

The purpose of this work is to generalize the literary data on a question about prophylaxis and treatment of anthrax, to designate the prospects of further researches.

Present-day epidemiological conditions on the anthrax is considered by domestic and foreign scientists as strained and non having the tendency to stabilization /1,2/. This situation have the great number of the reasons. First, anthrax spores are widely distributed in the nature, they have high stability to adverse environment factors. Secondly, B.anthracis utilization in purpose of bioterror weapon is ascertained as the obvious fact /3/. Thirdly, cultural-morphological variability of B.anthracis is too complicated to identification and, accordingly, in due time to adequately react on epidemiological situation. At last, wrong points of view on medical tactics at the decision of questions for emergency prophylaxis and treatment to antibiotic resistant strains are exist. There is a low professional standard of the available medical personnel to work with especially dangerous infections. All of these are don't allows to reach desirable result in prophylaxis and therapy of anthrax. Application of various vaccines for Anthrax prophylaxis give some efficiency, but is not an absolute guarantee from lethal outcomes /4, 5/.

Search of new means of prophylaxis and treatment of the Anthrax and development of new approaches in this direction are very actual. Now the principle of application of antibiotics is put in a basis of treatment of all forms of the Anthrax in a combination with geterological specific globulin /6/. The patients with easy current of disease are appointed antibiotics penicillinic and tetracycline lines within 5-7 days. The treatment by antibiotics to middle heavy and heavy current patients of the Antrax should be combined with introduction of pathogenetic means and specific globulin. In this case penicillin must be entered both intravenously and intramuscularly besides apply tetracycline preparations.

Globulin is entered to the patient only in absence of the increased sensitivity of him. Very heavy current of the Anthrax and a sepsis demand intensive complex therapy. Gamma globulin is entered to patient without definition of sensitivity of him on a background of prednisolon /6/.

There are some data in the literature on treatment of versions of visceral forms of the Anthrax (enteric, pulmonary), anthrax meningitis by chloramfenicol, gentamycin, cristapen and other reserve antibiotics /7/. However, at late introduction, at a stage toxemia expressed, theses not always rendered medical action. The infection proceeds extremely sharply and is transient.

One of possible directions of search of alternative means in treatment and preventive maintenance Anthrax infections can be use of specific bacteriophage preparations and antibodies for intravenous introduction.

Application of phages at a number of infectious diseases is used in the medical purposes more than hundred years /8, 9/. However, there are only some works on B.anthracis phagotherapy were published during the period with 1922 for 1933. Interest to this direction has considerably decreased with occurrence of antibiotics.

Besides, the positive medical effect at B.anlhracis phagotherapy was observed not always. One of the reasons of the first failures is an insufficient level of scrutiny of the nature of bacterial viruses. Another is casual, empirical choice of phages for the medical purposes. It is necessary to note also, that many researchers did not take into account methodical nuances of phages cultivation and features of their storage.

Research of biological and biochemical properties of phages, in particular molecular aspects receptor specificity, can provide a new direction in phagotherapy. In this case the main question is concern to development of phage preparations including phages with different receptor specificity, and also to separate phage elements responsible for inhibition of molecular mechanisms of cellular functions. Using in this case the appropriate phages (the cell completely ceases to function) more effectively in comparison with other antimicrobic agents. A bright example using of such approach began the recent opening which has shown, that recombinant lysine, received from scale of phage B.anlhracis, it is possible to use fiber for specific inhibition of germination and spore formation //. Occurrence of the increased number sequences bacteriophage genomes and biocomputer science allows to predict with some degree of reliability of function of some phage genes.

Not less important, than antibiotic therapy can be the using of chemically pure preparations of specific antibodies. At present time so-called preparations of the directed action are widely enough used: anti grippe, anti staphylococcal, anti tetanus, against tick encephalitis, anti diphtheria, etc. /10/. The whole direction was abroad generated concerning application, a dosage, ways of use of antibodies for intravenous introduction /11/.

However intravenous introduction heterogeneous Anthrax globulin is impossible because of anaphylactic reaction occurrence. At treatment of some diseases bacterial etiology apply a preparation consisting from F (ab) 2 - F (ab) - fragments of antibody G. It is more effective JgG as to a lesser degree suppresses immune system, having, during too time, high-grade ability to contact bacterial antigens. Collateral reactions to its introduction completely are absent, and biological activity is higher /12/. Our researches with Anthrax infections confirm these data.

In a series of experiences according to biological properties of antibodies of various classes it is shown, that in Jg G fractions of whey of immune laboratory animals contain antibodies to protective antigen В.anthracis /13/. Antibodies of this class ingibate suppress development dispute and spore formation, causes lyses of vegetative cells (tab. 1, 2). Jg M activates phagocytosis of sprouting spores. Opsoniesed effect of JgM concerning vegetative and incapsuleted spores is not marked (tab. 3, 4, 5 /14/). At research of biological activity of antibodies in vivo on rabbits it is established, that intravenous introduction gomological preparations Jg G, F (ab) 2 - fragments in 10 % of concentration 1 hour prior to infect animals, and also in early terms after infection (1, 4, 8, 12, 16, 20, 24 hours) renders the expressed protective effect.

Table 1. Inhibition influence of immunity whey and Anthrax Antibodies on germination dispute B. anthracis

Parameter,
%

The control Immune whey Gamma globulin
Immunoglobulins of the not immune rabbits Immunoglobulins of the immune rabbits
    heated not heated   JgG JgA JgM JgG JgA JgA
J 46±1 56±4 60±4 59±4 45±1 46±2 45±2 60±1 45±2 49±5
N 68±3 49±3 43±3 44±3 70±3 68±6 60±6 43±2 70±6 62±1
SJ - 28 37 35 <1 1 <1 37 <1 9

Notes:

  1. J - degree of spore dispute;
  2. N- quantity of spore dispute;
  3. SJ- inhibition a degree designed on the formula SJ= 100 -N test / N control 100%;
  4. In the given table the average meanings (importance) (M ± m) given 3-5 experiences are given, each experience is executed in 3 times;
  5. The initial concentration of immunoglobulins, scale of a globulin (designed on the contents of fiber) is equal to their contents whey, the concentration of preparations in nutritious environment(Wednesday) made 10 %.

Table 2. Comparative estimation of inhibition action of antibodies on B. anthracis spore formation

Researched preparation Concentration of a preparation
mg/ml
The contents of capsule substance, mcg/mg
of the dry rest of crates
Inhibition activity,
UA/mg of fiber
    Total Glutamine polypeptide fraction on capsule substance on a glutamine polypeptide fraction
whey 156,0 63,1±5,8 41,7±6,7 0,2 0,3
gamma globulin 10,0 45,4±4,7 26,73,9 5,9 6,5
Jg G 6,3 28,6±3,1 21,2±1,7 11,8 11,5
Control 156,0 110,0±7,0 75,6±10,1 0 0

Table 3. Results of opsonophagocitary reaction in the attitude of spore dispute

The opsonin factor

A statistical parameter parameters of a phagocytes
  In immune system in non immune system
  Phago-cytosis number, % Phagocytosis index ОR,% Phagocytosis number, %
Phagocytosis index
OR,%
Whey 89±3,2
<0,05
7,81±0,31
<0,05
37 <0,05 78,2±1,2
<0,05
4,81±0,2
>0,05
4
<0,05
JgG 62,2±8,1
>0,05
6,52±0,51
>0,05
1
>0,05
71,8±2.1
>0,05
4,80±0,32
>0,05
0
>0,05
JgA 63,9±11,2
>0,05
7,51±1.20
>0,05
8
>0,05
71,9±1,3
>0,05
5,01±0,19
>0,05
0
>0,05
JgM 94,3±4,1
<0,05
8,58±0,31
<0,05
45 <0,05 70,9±2,3
>0,05
5,41±0,32
>0,05
5
>0,05
Control 78,8±2,5 <0,05 5.60±0,28
>0,05
0 69,9±2,1 5,21±0,44 0

Notes:

  1. OR - opsonin action of environments(Wednesdays) designed on the formula OR=100-Fc\Fо х 100%, where Fc - activity of a phagocytosis in the control, Fо - activity of a phagocytosis in experience;
  2. The initial concentration of immunoglobulins designed on the contents of fiber is equal to their contents whey, the concentration of preparations in nutritious environment made 10 %.

Table 4. Results of opsonophagocitary reaction in vegatative crates

The opsonin factor

parameters of a phagocytosis
  In immune system In non immune system
  Phagocytosis number, % Phagocytosis index Phagocytosis number, % Phagocytosis index
whey 29,4±5,1 3,08±0,14 31,2±4.1 3,12±0,31
JgG 18,2±8,1 3,05±0,18 26,2±7,2 3,08±0,18
JgA 16,3±9,2 3,25±0,11 19,3±4,1 3,21±0,43
JgM 19,3±8,1 3,19±0,22 22,1±6,4 3,11±0,21
Control 29,2±7,4 3,07±0,41 21,1±4.8 3.32±0.41

Table 5. Phagocytosis by normal macrophages spore dispute, sensibilised by whey or immunoglobulins of the immunity rabbits

The opsonin factor Parameters of a phagocytosis
  Phagocytosis number, % Phagocytosis index OR,%
whey 91,2±4,1
<0,05
7,62±0,32
<0,05
49
<0,05
JgG 69,3±3,2
>0,05
4,21±0,38
>0,05
0
>0,05
JgA 71,1±2,8
>0,05
4,82±0,
>0,05
0
>0,05
JgM 96,6±3,1
<0,05
8,20±0,31
<0,05
55
<0,05
control 67,1±4,2 5.60±0,28
>0,05
0

The resulted data are important to development of specific antibodies for intravenous introduction. The resulted data testify to an ambiguous biological role of separate classes of antibodies in system of specific immunity at the Siberian ulcer, and can be used further by development сывороточных preparations for emergency preventive maintenance and therapy.

Approaches to creation of similar preparations are various. It can be reception of specific preparations from whey (plasma) of blood immunized people. The given direction is connected to complexity of reception of initial raw material with high credits of specific antibodies. The second direction of works - reception monoclonal antibodies with the set specificity to separate antigenes В. anthracis at various stages of their development with the help of modern biotechnology, is in our opinion more perspective.

The sharp increase in number of laboratories abroad, making monoclonal antibodies with the help of hybrid technology, is the certificate of advantage of use monoclonal antibodies as tool for research in comparison with standard preparations of antibodies of the person. As an example it is possible to result works on reception of cellular lines and their clones which develop antibodies of the person to tetanic anatoxin and to toxin. The similar result can have scientific and practical value for receptions Anthrax antibodies. The analysis, the data of the literature and results of own researches allow us to make the following conclusion.

For preventive maintenance and treatment Anthrax infections the alive, chemical, combined vaccines, antibiotics and scale use globulin. However occurrence antibiotic-resistant straines of В. anihrticia and real threat of their application as potential agents of bioterrorism can result in heavy consequences. Commercial anti Anthrax gamma globulin and others geterological whey preparations have sensibility properties and are not effective at late their introduction at a stage expressed toxemia. Taking into account the given literatures and the resulted results on biological properties of specific antibodies, it is expedient to carry out researches on development complex anti Anthrax preparation on the basis of specific bacteriophages, monoclonal antibodies and antibiotics for emergency preventive maintenance and treatment of the Siberian ulcer.

The choice of a direction of researches, finally, depends on qualification of experts, scientific and technical base and financing of works.

LITERATURE:

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2. Cherkasskii B.L. Anthrax in С modern world // Problems of epidemiology and zoonosis. М., 1975.
3. Karkishenko N.N. Medicinal prophylaxis.- М: 2001- 752 pp.
4. Marinin L.I., Onishchenko G.G. et al. Microbiological diagnosis of Anthrax. М.: VNUNMMZRF, 1999,-224 pp.
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6. Instruction and methods on laboratorial diagnostics, prophylaxis and medical treatment of Anthrax.-М., 1982.-61 pp.
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9. Barrow P.A. and I.S. Sootliill - Bacieriophage Therapy and renewed assessment of the Potential.//Trends Microbial. 1997. v.5 pp.268-271.
10. Onishchenko G.G., Aleshkina V.A., Afanas'eva S.S. et al. Immune preparation and perspectives of their use in infectology. М.: GOUVUNMCMZZF, 2002, -607 pp.
11. Berkman S.A., Lee M.L., Gall R.P. Clinical uses of intravenosus immunoglobulins // Annals of international medicine. - 1990. - vol119, N4. - P. 278-292.
12. Sedlacek H.H., Gronski P., Hofstaetter et al. The biological properties of immunoglobulin G and its split products [F(ab)2 and Fab] // Klin Wochenschr., - 1983. - vol.61.- N13. - pp.723-735.
13. Kravets I.D., Popov V.G., Galiev A.H. et al. Method for receiving the antibody diagnostic preparation to Anthrax antigen from commercial precipitate whey. // Immunology - 1982. - № З.- p. 52-54.
14. Ashmarin I.P., Tarumov V.S., Popov V.G. et al. Opsonin activity of Anthrax whey immunoglobulines -Laboratorial work. 1980, №5 p. 257-420.
15. Kennetf R.G., Mac-Kern J., Behtol K.B. Monoclonal antibodies. М., Medicine. 1983.-416 pp.
16. Haber E., KrauseR.M. (eds) Antibodies of human diagnosis and Therapy. New York: Raven Press, 1977.
17. Zurawski V. Jr., Uber E., Black P.H. Production of antibody to tetanus toxoid by continous human lymphobeastoid cell eines. Science. - 1978.

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