Toxigence of Anthrax Vaccine Strains

UDC 579.62 hps://doi.org/10.31548/ujvs2020.03.009 TOXIGENCE OF ANTHRAX VACCINE STRAINS

G. A. ZAVIRIYHA, Candidate of Agricultural Sciences hps://orcid.org/0000-0002-46028477 "State Center for Innovave Biotechnology", Kyiv, Ukraine Е-mail: [email protected] U. N. YANENKO, Candidate of Veterinary Sciences hps://orcid.org/ 0000-0001-5678-3356 "State Center for Innovave Biotechnology", Kyiv, Ukraine Е-mail: [email protected], N. I. KOSYANCHUK, Candidate of Veterinary Sciences, Associate Professor Department of Veterinary Hygiene Named Aer Professor A. K. Skorokhodko hps://orcid.org/ 0000-0002- 3055-8107 Naonal University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine Е-mail: [email protected]

Abstract. The arcle presents the results of the studying of anthrax strains and Bacillus anthracis-like strains on the formaon of toxins. We found that anthrax vaccine strains acvely produce exotoxins to the culture fluid. The amount of specific protein is different under the same incubaon condions and depends on the individual characteriscs of the microorganism populaon, because of this, different ters of the toxin are registered. Strain B. anthracis K-79 Z (vaccine) with the same number of planng microbial cells and growing on the same culture medium and at the same temperature produces by two orders of magnitude more exotoxin than strains B. anthracis Tsenkovsky II IBM 92Z (virulent), B. anthracis Stern 34F2, (vaccine), B. anthracis 55 (vaccine), B. anthracis SB (vaccine), B. anthracis Tsenkovsky I (vaccine, аpathogenic). The amount of exotoxin may change if the pH of the medium changes. The acvity of exotoxin producon, when the pH changes, depends on the characteriscs of the anthrax strain. Keywords: anthrax, exotoxin, exotoxin producon

Introduction forms spores. The infectious dose of the pathogen is only 1–10 spores. They fall Anthrax is a part of the moﬆ dan- victim to the blood of a sick animal or a gerous infectious diseases common to corpse. The pathogen forms spores that, humans and animals. This disease is once in the soil, can persiﬆ for more than common in all mammals, moﬆ often 100 years. After reaching a favourable among cattle (large and small) and hors- environment, the spores become active es. The causative agent is the gram-posi- in 1–6 days, some spores may intensify tive bacterium Bacillus anthracis, which after 60 days or more (Cizauskas et al.,

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2014; Hendricks et al., 2014; Shadomy gen causes 45–90 % of mortality even et al., 2016; Chugh, 2019). with timely treatment with antimicrobial For this reason, the area contaminat- drugs (Mourez, 2004). High mortality is ed with anthrax spores is a factor in the associated with the rapid development of transmission of the disease indefinitely. bacterial infection and the action of tri- During its ﬆay in the soil, the an- partite toxin (Crowe et al., 2010). thrax pathogen periodically reﬆores its Both field and vaccine ﬆrains of the population, thus maintaining the infec- anthrax pathogen have the ability to pro- tivity of the territory. To reproduce its duce toxins. B. anthracis has two main population and mass, B. anthracis in- virulence factors: poly-γ-D-glutamic fects animals or humans, and with their acid capsule and tripartite toxin (Mock corpses, it re-enters the soil and closes & Fouet, 2001). The capsule plays an the natural cycle of its biology. antiphagocytic role and allows bacte- Humans can become infected by an- ria to avoid absorption by macrophages thrax via respiratory syﬆem (inhalation), (Zwartouw & Smith, 1956). The tripar- gaﬆrointeﬆinal tract, and through the skin tite toxin contains protective antigen, le- (this is the moﬆ common form and 95% thal factor, and oedema factor (Stanley manifeﬆs as a malignant puﬆule). The in- et al., 1960: Stanley et al., 1961). These haled form of anthrax is the moﬆ severe, toxins act on the innate and adaptive im- as it often causes septicemia and menin- mune syﬆem and affect the subsequent gitis with high mortality and the higheﬆ consequences of the disease (Tourni- risk of man-made spread. The gaﬆrointes- er et al., 2009; Moayeri et al., 2015). tinal form of anthrax occurs when con- Field and vaccine ﬆrains clearly differ suming meat and meat products of poor in the lethality and the amount of tox- quality (Glomski et al., 2007; Hashemi et in production. Field ﬆrains produce lit- al., 2015; Owen et al., 2015; Vieira et al., tle toxin during in vitro cultivation, but 2017; Mwakapeje et al., 2018). they are more aggressive (predominate Between 2,000 and 100,000 cas- lethal and oedematous fractions). Vac- es of anthrax are reported worldwide cine ﬆrains produce more toxin, which each year. This disease remains a major is dominated by a protective fraction. health threat in Africa, the Middle Eaﬆ, All anthrax vaccines develop anti- South America, Central Asia, and Haiti toxic immunity in a vaccinated animal, (Doğanay & Eşel, 2008; Abboud et al., but the ability of anthrax microbes to 2009; Liu et al., 2020). Anthrax spores produce an exotoxin is not the same. The can be used as a target for bioterrorism. ﬆress and duration of immunity (immune In 2001, spores of the pathogen were protection of vaccinated animals) depend mailed to exceptional individuals in the on this property, so ﬆudies of the toxige- United States, which caused 22 cases nicity of anthrax ﬆrains are relevant. of cutaneous, inhalation, and meninge- al anthrax and 5 deaths (Abrol, 2016; Analysis of recent sources Walsh et al., 2018). and publications Stability of spores for many years, un- pretentiousness to the conditions of cul- Exotoxins of the anthrax pathogen tivation leads to human disease or death as biotechnological objects are relevant – all this makes anthrax a primary patho- for many scientiﬆs in different countries gen of the NIAID category. The patho- (Friebe et al., 2016).

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Studying the protective factor of Material and methods of research the antigen and the mechanism of formation have shown that the protective To conduct the experiment we took factor may be a new means for con- lyophilized museum cultures of anthrax trolling cytotoxicity (Kintzer et al., vaccine ﬆrains and Bacillus anthracis-like 2009) and ﬆudying the complex action ﬆrains: B. anthracis Tsenkovsky II IBM of exotoxins as toxic complexes (Lacy 92 Z (virulent), B. anthracis K-79 Z, et al., 2002). Studying the mechanism B. anthracis Sterne 34F2, B. An- of action of a protective exotoxin on a thracis 55, B. anthracis SB, B. anthracis cell againﬆ the background of the le- Tsenkovsky I, B. anthracoides 67, thal and oedematous toxin are relevant B. Subtilis 17, B. cereus 8035 (virulent), (Chaudhary et al., 2012; Slater et al., which are ﬆored in collection of micro- 2013). The use of protective antigen organisms in the “State Center for Inno- of the anthrax pathogen as an inhibi- vative Biotechnology”. The methods for tor of breaﬆ cancer cell development ﬆudying vaccine ﬆrains described by has shown the prospects for the devel- S. G. Kolesov, generally accepted in mi- opment and application of new thera- crobiological practice, were used (1976). peutic approaches in the fight againﬆ The agglutination reaction and the tumours (Felix et al., 2020). concentration of the spore suspension Particular attention is paid to the for each ﬆrain were determined in colo- ﬆudying of the epizootic situation and ny forming units (CFU) by scientific and disease monitoring worldwide. Cre- methodological recommendations “Lab- ating a unified surveillance syﬆem, oratory diagnosis of anthrax in animals, ﬆrengthening cross-sectoral coordina- detection of pathogens from pathological tion on prevention and control, biolog- and biological material, raw materials ical and social ﬆrategies for anthrax of animal origin and environmental ob- prevention focuses scientiﬆs’ atten- jects” (Skrypnyk et al., 2015). Statiﬆical tion on endemic regions (Sahoo et al., processing of the obtained research re- 2020). Improving anthrax vaccines for sults was performed using the program humans and animals is an important “Microsoft Excel 2011” for Windows. ﬆage in preventing infection. We are The titer of microorganisms toxige- conducting research to find new ap- nicity in the causative agent of anthrax proaches to the use of antibiotics in the and Bacillus anthracis-like ﬆrains was schemes of anthrax vaccines. There is a determined using the disc precipitation queﬆion of disposable vaccine creation reaction (DPR) by the method improved (Clark & Wolfe, 2020). by A. I. Zaviryukha (Zaviryukha & Ste- The development of vaccines, which panyuk, 1978). Precipitated anthrax preserve natural antigenic ﬆructures serum RP № 3905-14-0403-08, man- that mimic natural infection, and the ufactured by Kherson State Biological ﬆudying of the effects of antibodies that Factory, was used for the ﬆudy. neutralize toxins are an important re- Bacterial cells of vaccine ﬆrains search area (Dumas et al., 2020). and Bacillus anthracis-like ﬆrains were The purpose is to ﬆudy the prop- grown on ﬆandard culture media: broth erties of toxins formation by anthrax and meat-peptone agar, and injection of pathogens and Bacillus anthracis-like 5 and 10 % gelatine at pH 7.2 – 7.6 and ﬆrains. a temperature of 37.0  1.0 0 C.

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From microbial colonies grown on agar, Results of the research smears were prepared and ﬆained according and their discussion to Gram, Mikhin, Olt, Rebiger, Romanows- ky-Giemsa, the length and width of micro- Inveﬆigated extracellular anthrax bial cells were measured with a micrometre, toxins, which were obtained by the meth- and their shape and the presence of spores od mentioned above. The results of the were determined. The shape and physical examination by the reaction of Ascoli characteriﬆics of the colony were ﬆudied. and the DPR are presented in Table 1. Antigens were prepared by autoclav- Our data indicate that the virulent ing from the microbial mass of each ﬆrain, ﬆrain B. anthracis Tsenkovsky IІ IBM which was inveﬆigated in the Ascoli reac- 92Z and the avirulent B. anthracis tion and in the DPR with anthrax serum K-79Z produce extracellular toxin out- for precipitation. We prepared antigens side the cell membrane in a liquid nu- from the culture fluid of vaccine ﬆrains, trient medium. The toxin reacts with which were filtered through a bacterial fil- antibodies of the precipitating anthrax ter and examined by the DPR and Ascoli. serum in the precipitation reaction. The control was antigens made from However, the amount of exotoxin in the bacterial mass and ﬆerile filtrate of cul- culture fluid of virulent and avirulent ture fluids of anthracoid microorgan- ﬆrains is not the same. We noted the isms B. Anthracoides 67, B. Subtilis 17, ability to produce exotoxin at a titer of B. cereus 8035 (virulent). 1:32 in vaccine ﬆrains of anthrax.

1. The results of ﬆudying the ﬆerile filtrates of culture fluids of microbes of the genus Bacillus, which were examined by the Ascoli reaction and the disk precipitation reaction Ascoli DPR dilution of culture fluid Strain n n 1:2 1:4 1:8 1:1 1:3 1:6 1:2 1:4 1:8 1:3 1:6 1:16 1:128 1:128 В. аnthracis ++++++++++++++++ К-79 Z В. anthracis Tsenkovsky IІ ++++----++++---- ІВМ-92 Z B. anthracis ++++++- -++++++- - Sterne 34F2 B. anthracis 55 + + ++++- -++++++- - B. anthracis SВ ++++++- -++++++- - B. anthracis +++++- - -+++++- - - Tsenkovsky I В. cereus 8035 ------B. anthracoides 67 ------B. subtilis 17 ------

Note: n – native ﬂuid; + positive reaction; – negative reaction.

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Saprophytic spore-forming bacilli of Colonies that grown on agar were B. anthracoides 67, B. subtilis 17, B. ce- seeded in a liquid nutrient medium – reus 8035, and B. subtilis 17 do not pro- meat broth with different pH values – 6.5; duce a specific toxin, and therefore their 8.5; 7.5. They were incubated in a ther- filtrates of culture fluids do not react by moﬆat for 48 hours at 370 C. Then we fil- precipitation with antibodies of precipi- tered the bacterial mass through bacterial tated anthrax serum. filters type F5. The received filtrates were We inveﬆigated the impact of pH on the checked according to DSTU 4483:2005. production of exotoxin by anthrax ﬆrains, We performed the DPR with culture which began with the production of bacte- fluid filtrates to determine the anthrax rial masses with an identical number of mi- protein titer. The results of the examina- crobial cells – 30.2–30.8 × 106 CFU. tion are presented in Table 2.

2. Eﬀect of pH on exotoxin production by anthrax vaccine ﬆrains, М ± m, n = 10

Number of grown Exotoxin titer № Strain pH of the medium microbial cells, CFU in the DPR 6.5 8 В. аnthracis 7.5 64 1. 30.5 × 106 К-79 Z 8.5 32 M ± m 34.66 ± 16.22** 6.5 4 В. anthracis 7.5 8 2. Tsenkovsky 30.7 × 106 IІ IBM 92 Z 8.5 4 М ± m 5.33 ± 1.33** 6.5 8 B. anthracis 7.5 32 3. 30.2 × 106 Sterne 34F2 8.5 16 М ± m 18.66 ± 7.05** 6.5 8 B. anthracis 7.5 32 4. 30.8 × 106 55 8.5 16 М ± m 18.66 ± 7.05** 6.5 8 B. anthracis 7.5 32 5. 30.5 × 106 SB 8.5 16 М ± m 18.667 ± 7.05** 6.5 8 B. anthracis 7.5 16 6. 30.2 × 106 Tsenkovsky I 8.5 8 М ± m 10.66 ± 2.66** Note: * P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.001.

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Indicators of Table 2 show that at differ- The Perspective: we plan to continue the ent pH of the medium, the amount of specif- ﬆudying of the toxigenic properties of an- ic protein in the ﬆrains increased differently thrax ﬆrains in order to obtain an exotoxin (1:8–1:64 – B anthracis K-79Z, 1:8–1:32 that will be used in the development of new – B. anthracis Sterne 34F2, B. аnthracis 55, drugs againﬆ anthrax in humans and animals. and B. anthracis SB, 1:8–1:16 – B. аnthracis Tsenkovsky I). At the same cultivation conditions, a high titer of specific anthrax References protein showed the exotoxin B. anthracis Abboud, N., De Jesus, M., Nakouzi, A., Cordero, K-79Z – 1:64 at pH 7.5. Strains B. anthracis R. J., Pujato, M., Fiser, A., ... & Casadevall, A. Sterne 34F2, B. anthracis 55, and B. an- (2009). Idenficaon of linear epitopes in thracis SB at pH 7.5 produced exotoxin at Bacillus anthracis protecve angen bound the same level, and it was 1:32. Also, in these by neutralizing anbodies. Journal of Bio- ﬆrains, the same amount of exotoxin was ob- logical Chemistry, 284(37):25077-25086. served when cultured in a medium at a pH of doi: 10.1074/jbc.M109.022061. 6.5, which was 1:8. When the pH was raised Abrol, S. (2016). Countering Bioterrorism to 8.5, the exotoxin purge of these ﬆrains Threat to India: employing global best was at 1:16. This indicates similar characteris- pracces and technology as force mul- tics of these vaccine ﬆrains and the effect of plier. India Quarterly, 72(2):146-162. pH on the production of exotoxin. doi: 10.1177/0974928416637934. Chaudhary, A., Hilton, M. B., & Seaman, S. Conclusions and future (2012). TEM8/ANTXR1 blockade inhibits perspectives of the study pathological angiogenesis and potenates tumoricidal responses against mulple Vaccine ﬆrains of anthrax actively cancer types. Cancer Cell, 21(2):212-216. produce exotoxins in the culture fluid. doi: 10.1016/j.ccr.2012.01.004. The amount of specific protein under the Chugh, T. (2019). Bioterrorism: Clinical and same incubation conditions is different public health aspects of anthrax. Current and depends on the individual character- Medicine Research and Pracce, 9(3):110- iﬆics of the microorganism populations. 111. doi: 10.1016/j.cmrp.2019.05.004. The B. anthracis K-79 Z ﬆrain with Cizauskas, C. A., Bellan, S. E., Turner, W. C., Vance, R. E., the same number of microbial cells and & Getz, W. M. (2014). Frequent and seasonally under cultivation in the same culture variable sublethal anthrax infecons are accom- medium and temperature conditions pro- panied by short-lived immunity in an endemic duces by two orders of magnitude more system. Journal of Animal Ecology, 83(5):1078- exotoxin than B. anthracis Tsenkovsky 1090. doi: 10.1111/1365-2656.12207. IІ IBM 92Z, B. аnthracis Sterne 34F2, Clark, A., & Wolfe, D. N. (2020). Current State of B. anthracis 55, B. anthracis SB and Anthrax Vaccines and Key R&D Gaps Mov- B. anthracis Tsenkovsky I. ing Forward. Microorganisms. 8(5):651. Vaccine ﬆrains of anthrax, which we doi: 10.3390/microorganisms8050651. ﬆudied, produce exotoxin when the pH Crowe, S. R., Ash, L. L., & Engler, R. J. M. (2010). of the medium. Its amount varies de- Select human anthrax protecve angen pending on the pH of the medium. The epitope–specific anbodies provide pro- activity of exotoxin production when tecon from lethal toxin challenge. The the pH changes depends on the charac- Journal of Infecous Diseases, 202(2):251- teriﬆics of the anthrax ﬆrain. 260. doi: 10.1086/653495.

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Г. А. Завірюха, У. М. Яненко, Н. І. Кос’янчук (2020). ТОКСИГЕННІСТЬ ВАКЦИН- НИХ ШТАМІВ СИБІРКИ. Ukrainian Journal of Veterinary Sciences, 11(3): 85–92, hps://doi.org/10.31548/ujvs2020.03.009 Анотація. У статті представлені результати дослідження штамів сибірки та антракоїдів щодо утворення токсинів. Ми виявили, що вакцинні штами сибірки активно продукують екзотоксини в культуральну рідину. Кількість специфічного білка різна за однакових умов інкубації й залежить від індивідуальних особливостей популяції мікроорганізмів, через що реєструються різні титри токсину. Штам B. anthracis K-79Z (вакцина) з однаковою кількістю посіяних мікробних клітин і за вирощування на одному й тому ж поживиному середовищі та за однакової температури виробляє на два порядки більше екзотоксину, ніж штами B. anthracis Tsenkovsky II IBM 92Z (вірулентний), B. anthracis Sternе 34F2, (вакцина), B. anthracis 55 (вакцина), B. anthracis SB (вакцина), B. anthracis Tsenkovsky I (вакцина, патогенна). Кількість екзотоксину може змінюватися внаслідок зміни рН середовища. Активність продукування екзотоксину внаслідок зміни рН середовища залежить від характеристик штаму сибірки. Ключові слова: сибірка, екзотоксин, продукція екзотоксину

92 | ISSN 2663-967X Український часопис ветеринарних наук Vol. 11, № 3, 2020