Live Brucellosis Vaccines
Comparative Studies of Immunobiological Characteristics of Live Brucellosis Vaccines
Tech Area / Field
8 Project completed
Senior Project Manager
Melnikov V G
Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov
- All-Russian Research Veterinarian Institute, Russia, Tatarstan, Kazan
- United States Department of Interior National Park Service, USA, WY, Yellowstone\nUnited States Department of Agriculture / Agricultural Research Service, USA, MD, Beltsville\nTexas A&M University, USA, TX, College Station
The objective of this proposal is selection of the safest and most effective vaccines for prophylaxis against brucellosis of bison.
Brucellosis is one of the most dangerous infectious diseases, which greatly damages livestock breeding economically.
According to the data of the FAO/WHO Joint Expert Committee (Report 6, 1986), brucellosis in livestock occurs the world wide. The Brucella genus is devided into 6 species (B. abortus, B. melitensis, B. suis, B. neotome, B. ovis, and B. canis), which in turn are sub-categorized according to their biological properties into biovars.
Within the Russian Federation, brucellosis has been detected in practically all forms of farm and domestic animals (i.e., cattle, sheep, goats, swine, reindeer, noble deer, horses, camels, yaks, buffalo, zebu, and dogs). Bovine brucellosis is of the greatest concern due to its epizootic and economic impacts.
Immunoprophylaxis is the most promising method for combating brucellosis. A wide array of live and inactivated brucellosis vaccines has been developed. The live brucellosis vaccine obtained from the attenuated agglutinogenic B. abortus 19 strain has received the most acceptance and has been widely used in the former USSR, the USA, and many other countries of the world. A considerable deficiency of these vaccines is the stimulation of antibodies in blood of immunized animals, which complicates the diagnosis and determination of the epizootic status of Brucella exposed animals. These antibodies are detected in serological tests accepted for brucellosis diagnosis.
Many brucellosis vaccines obtained from live and inactivated dissociated Brucella strains have been proposed and developed with the goal of eliminating these deficiencies.
Presently among the dissociated Brucella strains, the live strain B. aborts 82 is being used in several countries of the CIS. The other Russian live vaccines prepared from reduced-agglutinogenic strains have undergone laboratory and production testing. They include the Russian B. aborts 82-PS and B. aborts 75/79- AB, and US live vaccines obtained from inagglutinogenic B. abortus RB-51 and rfbK strain.
The vaccines from the aforementioned strains possess different immunological and anti-epizootic effectiveness. No comparative studies have been conducted with all these vaccines to determine the safest and most effective vaccine for brucellosis prophylaxis.
Spreading of Brucella abortus in free-ranging bison (Bison bison) in Yellowstone National Park is a significant wildlife health issue in the United States. Evidence of the disease was first observed in Yellowstone bison in 1917. Currently, an estimated 40% of Yellowstone bison have positive serologic titers to brucellosis. Although brucellosis has minimal population level effects on free-ranging bison, the perceived threat of transmission of B. abortus from bison to cattle has made this a contentious issue, particularly because B. abortus has been nearly eradicated elsewhere in the United States.
A joint management plan to reduce the risk of transmission of brucellosis from bison to cattle and to conserve free-ranging bison was developed in the USA. One component of the joint management plan is an in-park program to remotely deliver a safe and effective brucellosis vaccine to vaccine-eligible bison. The vaccination program will not involve intensive management practices and will not be accompanied by a traditional test and slaughter program. Therefore, use of a highly efficacious vaccine is a critical component for successful control of the disease.
Brucella abortus strain RB-51 is a live vaccine approved for use in the United States to protect cattle from brucellosis. On-going research suggests that RB-51 is safe for use on immature bison and that efficacy will likely be, at best, similar that observed in cattle (about 75% efficacy). Although RB-51 is adequate for application to bison in the short-term, it would be preferable to have a brucellosis vaccine that is more effective than RB-51 and is safe for all ages, sexes, and pregnancy status bison urther, improvements in vaccine delivery systems and diagnostic techniques would also be beneficial to management of bison brucellosis.
During the work within the proposed project seven live brucellosis vaccines, including three known and patented domestic vaccines, from the following strains will be studied:
1. B. abortus 82 SR-form (Russia). The strain was discovered by Professor K.M. Salmakov (All-Russian Research Veterinary Institute, ARVI, Kazan). This strain possesses reduced agglutinogenic properties. The vaccine produced from this strain was placed into veterinary practice by the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation in 1988 for specific prophylaxis against brucellosis in cattle. This strain possesses sufficient immunological and anti-epizootic effectiveness. It can be used for immunization and re-vaccination of young and adult cattle. However during its initial application in pregnant cattle, post-vaccination complications such as abortions may occur, therefore, cattle are vaccinated after parturition. Complications, as a rule, do not occur in animals having strain 82 induced immunological protection. Immunity enhancement may be achieved by re-vaccination of cows every 1-2 years.
2. B. abortus 82- PS SR-form. (Russia) is a penicillin susceptible variant of the vaccine strain 82. It is non-abortogenic and possesses reduced agglutinogenic properties. The strain was discovered by Professor K.M Salmakov and G.A. Belozerova, Dr. Sci (Vet) (ARVI, Kazan). The vaccine from this strain was tested under field conditions with permission of the Veterinary Department in the Ministry of Agriculture of the Russian Federation. The final conclusion on its immunogenic and anti-epizootic properties has not yet to be determined.
3. B. abortus 75/79-AB, RS-form (Russia). It is non-abortogenic and possesses sufficient immunogenic and reduced agglutinogenic properties. It was discovered by a group of authors of the Altai NIVS and the All-Russian Research Institute for Controls, Standardization and Certification of Biological Preparations (ASRCI, Moscow) (I.P.Nikiforov, K.V.Shumilov, et al) by means of purposeful selection by culture, agglutinogenic and immunogenic properties from B. abortus 75/79-A. This culture was isolated by I.P Nikiforov during bacteriological examination of pathological materials (supramammary lymph nodes) of a cow previously vaccinated with the strain 82 vaccine. With permission of the Veterinary Department of the Ministry of Agriculture of the Russian Federation, the live vaccine from B. abortus KB 75/79-AB strain was tested under field conditions in the Altaisky Krai and Astrakhan regions. The "Privolzhskaya Biofabrika" has been producing live vaccines since 1997.
4. B. abortus RB-51, R- form (USA), non-agglutinogenic. It was discovered by G. Schurig, and the USDA principal scientific investigator is Dr. Steve Olsen. The RB-51 vaccine strain is being used in the USA for prophylaxis of brucellosis in cattle and bison.
5. B. abortus rfbK, R- form (USA). The discoverers of the rfbK vaccine are Professors Thomas A. Ficht and Leslie G. Adams (Texas A&M University, College of Veterinary and Medicine, Texas A&M University, College Station,TX). The rfbK vaccine has been evaluated by Adams and Ficht in cattle under laboratory conditions and found to be slightly more protective than S19. The rfbK vaccine has been evaluated in over 15,000 cattle under field conditions in the Republic of South Africa where it was found to be safe and efficacious.
6. B. abortus RB-51 expressing antigen 85A of Mycobacterium avium paratuberculosis (USA). The discoverer of this vaccine is Dr. Gerhardt Schurig in collaboration with Drs. S. Olsen and D. Hunter.
7. B. abortus 19, S- form, agglutinogenic. It is the standard USDA vaccine strain, which was discovered in the 1940's by Cotton in the USA. Reference series of this vaccine are stored at the ASRCI. A lyophilized live vaccine is being produced at the "Shelkovosky Biocomplex". The strain will be used as a control in experiments for comparative study of various Brucella vaccine strains.
Comparative studies will be conducted on a large number of laboratory guinea pigs. The following principal tests will be performed during the testing of brucellosis vaccines:
1. Phenotyping of morphological characteristics of the cultures.
2. Determination of the culture-and-biochemical properties and typing of the strains (CO2 demand, H2S formation, growth capability in media added stains, penicillin and erythritol; phagotyping, agglutinability, dissociation, etc.)
3. Analysis of antigenic properties of the vaccines.
4. Examination for residual virulence, harmlessness, stability, contagiousness, abortogenicity, persistence, etc.
5. Characterization of immunogenic activity of the brucellosis vaccines in guinea pigs.
To conduct work on the Project three American vaccinal strains will be used for comparative investigations, and when the Project is over, one of the most efficient domestic vaccinal strains will be transferred to the USA to vaccinate bison.
Currently accepted vaccines possess insufficient immunogenicity and do not provide effective protection against associated Brucella infections. In addition, among the other considerable disadvantages of these vaccines is their reactogenicity, short-term immunity, and allergizing effects. In this connection, enhancing protective immunogenic properties of vaccines is considered to be an urgent problem of modern immunology.
Application of adjuvants, i.e. substances capable of enhancing specific immune responses to the introduced antigens, is considered as a possible mechanism to enhance the vaccine efficacy [36, 35]. Recently, much of the research has been focused on the development of new adjuvants to potentiate both humoral and cellular immunity with minimal or no side effects. Adjuvants are substances with different compositions and properties, e.g. proteins, lipids, chemical compounds, natural substances of plant and animal origin, as well as immunomodulators, e.g. metal salts, etc. All of them influence the different types of immunity thus affecting immune system holistically. Mounting evidence is reported over the latest years on the immunomodulating effects (direct and indirect) of a number of new and generally accepted medicinal preparations, e.g. glucocorticosteroides, non-steroid anti-inflammatory preparations, eubiotics, antibiotics, enzymes and their compositions, amino acids, microelements, hormones, antihistamine preparations, etc.
Increasing knowledge about the influence of cytokines on different cells of immune system has become available, and researchers are keen to determine the potential use of many cytokines to serve as adjuvants .
POLYOXYDONIUM is a novel preparation with immunotropic activity. It belongs to the class of water-soluble derivatives of heteroceptic polyamines . This class of polymeric compounds has no analogues anywhere in the world. With respect to the chemical structure, it is a co-polymer of N-oxide-1.4-ethyleneperasine and (N-carboxyethyle)-1.4-ethylenperasynium bromide with molecular mass of 80 kD . The preparation will be obtained from the State Research Centre (SRC)-Institute for Immunology at the Ministry of Health of the Russian Federation.
In in vitro experiments with cells of the phagocytic system, monocytes and neutrophils, these serve as targets for POLYOXYDONIUM which enhances the neutrophil’s ability to phagocytize and kill bacterial cells [Pinegin B.V., 2000]. When interacting with peripheral blood mononuclear cells, POLYOXYDONIUM induces synthesis of the other endogenic cytokines, such as tumor necrosis factor -alfa (TNF-a) and interleukin 1 beta (IL-1 b), and increases cytotoxicity of NK-cells. In vivo POLYOXYDONIUM has more complicated effects and many side effects on the immune system. Taking into account that the development of any immune process begins with the cells of monocytic-and-macrophagic system, and that cytokines produced by these cells possess pleiotropic effects, POLYOXYDONIUM increases their functional activity and leads to the activation of both cellular and humoral immunity. Combined use POLYOXYDONIUM with low doses of antigens induces increases of 5-10 fold in the synthesis of antibodies.
Very favorable adjuvant features of POLYOXYDONIUM have been expressed under the trials with the developed conjugated brucellosis antigen-polymer vaccine (BAPV). Under the optimal conditions for synthesis and correlation between polymer and antigen (1:3), the BAPV vaccine has protective activity equal to the commercial vaccine “Abortox” and the vaccines derived from the strains B. abortus 19 and 82 .
The GALAVYTE adjuvant was developed in Sechenov Medical Academy (Moscow) where it was found to have a direct effect on immune system via the macrophage system [44, 45]. GALAVYTE is aminophtalasine sodium salt, which possesses immunomodulation and anti-inflammatory features . In addition, GALAVYTE possesses the ability to restore the oppressed phagocytic function of macrophages and neutrophils, and consequently, anti-infection protective effects. Antigen-presenting macrophages’ function can be restored and NK-cells and CD8*-cytotoxic lymphocytes are activated by GALAVYTE.
High immunomodulating properties have been shown by TUMOUR NECROSIS FACTOR-BETA (TNF-b) - a cytotoxicity modulator, produced by T-lymphocytes . TNF- b possesses immunostimulating activity, and regulates cytokine synthesis, as well as proliferation and differentiation of Т and B-lymphocyte processes, enhances immunoglobulin secretion, and stimulates development of the humoral immune response to heterological antigens. TNF- b enhances functional leukocyte activity, possesses anti-infection activity, i.e. eliminates virus-infected cells and has an anti-parasitic action. TNF- b causes increasing expression of MHC (major histocompatibility complex) molecules on cell membranes which is of great importance for antimicrobial immunity [48, 49]. Preliminary experiments on the use of TNF- b with brucellosis vaccines enhanced the immunogenicity.
Several promising adjuvants have been tested at the All-Russian Research Veterinarian Institute Institute (ARVI, Kazan). They include XYMEDON, which has an activating effect on immunocompetent cells, and DILUENT OF LYOPHILIZED LIVE BRUCELLOSIS VACCINE, which inhibits RNA activity, causes short-term inhibition of phagocytic activity of macrophages, thus facilitating better dissemination of vaccinal strain cells in host’s body. Additionally, POTASSIUM TYOSULPHATE enhances the synthesis of specific antybodies and improves the functional activity of T-and B-lymphocytes, etc.
In the course of the project implementation, 10 different adjuvants, including 8 developed and patented by Russian researchers, 1 developed in the USA, namely, RIBI-ADJUVANT, and 1 developed by Latvian scientists – LARIFAN, will be obtained and tested. These adjuvants were selected in accordance with mechanism of their effect on the immune system as well as with the application method. The optimal immunizing dose with respect to small lab animals (guinea pigs) will be determined for each adjuvant. Study of the influence of adjuvants under test on the vaccinal process formation, as well as of magnitude and duration of the acquired with their help immunity will be conducted on the basis of parameters of humoral, cellular, and mucosal immunity, as well as phagocytosis and pathomorphological analysis of internal organs of the immunized animals. Study of their application efficiency for enhancing brucellosis vaccine protective features in acute experiments involving challenge of the vaccinated lab animals by virulent Brucella culture is considered to be a crucial criterion in selection of adjuvants. Analysis of the obtained results will be done using statistic methods for investigation (Student's criterion, etc).
As a result of the conducted screening of known adjuvants one most effective preparation will be selected and transferred to the USA with recommendations on its application to vaccinate bison.
To date, veterinary experts throughout the world apply injection method for animal vaccination against brucellosis. However, the given method demands a direct contact with animals and, sometimes, their fixation (limitation of mobility). Application of this method for vaccination of wildlife animals, in particular, bison is rather difficult. We consider vaccination of animals with the use of feed additives containing vaccine component to be the optimal method. The proposed method suggests selection of optimal carrier of the vaccinal strain providing for aerosol generation due to breathing out air under animals’ feeding and able-to-access in upper respiratory tract and alimentary canal, and adhesive to mucous membranes. When feeding, the vaccine fixed on a carrier will be partially delivered with air breathed into nasal cavity and upper respiratory tract while the major part will get into gastrointestinal tract. At that, antigen effect on mucous membranes will enhance formation of local immunity thus providing for a barrier for infection. Taking into account that there are a lot of lymphatic nodes in organs of respiratory and gastrointestinal systems, antigen getting into them is expected to cause general immune response.
Vaccinal preparation as a feed additive will allow using the way of animal immunization adequate to the natural way of infection. At that, easy way of the vaccine delivery and low cost of the vaccination is of great importance. The method for ballistic vaccine delivery, which will be developed within the framework of the given project, could provide the mentioned advantages as well.
In this connection it is planned to assess the immunity in lab animals following oral, nasal, oral-and-nasal, and injection immunization by the vaccine against brucellosis; select the optimal carrier of the vaccinal strain providing for aerosol generation due to air exchange volume under animals’ feeding and able-to-access in upper respiratory tract and alimentary canal, and adhesive to mucous membranes; determine optimal conditions for oral-and-nasal vaccination in the experiments with lab animals conducted on the basis of immunological control dependent on antigen distribution in respiratory tract and alimentary canal, and on frequency of immunization; compare the levels of local and general immune response; conduct trials of ballistic method for vaccine delivery and assess indices of post-vaccinal immunity.
Early detection of brucellosis agent with the use of highly sensitive and specific methods is an important condition for enhancing the efficiency of brucellosis specific prophylaxis. Typically in laboratory diagnosis several tests, namely, bacteriologic culture, microscopy, and serologic are accepted. Thus, to diagnose acute and subacute brucellosis, bacteriologic culture test is applied, which takes rather long time (up to 45 days). Blood culture is variable for isolation of Brucella from hemocultures (it is known that the frequency of B. melitensis isolation from blood is 62-90%, and B. abortus – 5-15%), and negative results of bacteriological culture is not adequate for a confirmed negative brucellosis diagnosis. The tests for brucellosis serodiagnosis yield variable results at different stages of disease, and there is not one among them which does not give cross reactions with Yersinia enterocolitica 0:9 serogroup. Application of live attenuated vaccines for brucellosis vaccinoprophylaxis in animals and humans (with respect to epidemiological indices) confuses the diagnosis, as there are no absolute differential tests for post-vaccinal and post-infection reactions. Therefore, the final brucellosis diagnosis is based on the results of epidemiological analysis, as well as laboratory diagnosis, and clinical signs of the disease [59, 60].
PCR has broad application for detection of Brucella at a high sensitivity when the causal agent is at a low level of concentration in biological specimens as well as with environmental samples for short time periods (6-8 hours). A considerable advantage of the method is the possibility to detect atypical forms of microorganisms in material contaminated with foreign microflora .
Placenta, lymph nodes, fetal abomasal contents, fetal lung, amnionic/allantoic fluids, milk and blood serum serve as diagnostic specimens for PCR. Most published papers appear to focus on the development of highly efficient, simple and fast techniques for DNA chromosome extraction from Brucella in tissues and blood serum samples for the PCR-diagnosis of brucellosis.
To monitor Brucella abortus dissemination in bison inhabiting the Greater Yellowstown Area (GYA), it may be advantageous to use urine and feces as diagnostic samples. We have not identified any published papers related to the use of PCR for Brucella detection in urine and feces. There is only a single publication related to the use of PCR for Brucella detection in urine.
The Russian Epidemiology Institute the Russian Research Anti-Plague Institute “Microb” and the “Niarmedic” commercial firm have developed test-systems intended for the PCR diagnosis of brucellosis blood samples. Nevertheless, in order to apply PCR for Brucella detection directly in urine and fecal samples, it will be necessary to achieve maximal sensitivity and specificity of this test.
In this connection, at the first stage of the work it is envisaged to test (taking Brucella strains from the collection as an example) various commercial test-systems intended for diagnosis of brucellosis with the help of widely known PCR-method, as well as to compare their specificity and sensitivity.
It should be noted that the potential for PCR diagnosis depends in many respects on effectiveness of the method for nucleic acids (NA) extraction from the causal agent presented in diagnostic specimens (blood cells, blood sera, urine, gastric juices, mucous, feces, or punctates of internal organs). Different methodical approaches will be used for isolation and purification of NA obtained from microorganisms and histological materials, ranging from physical factors exposure (as boiling of test sample for 5-15 minutes) to traditional methods for DNA extraction, using highly effective lysing and denaturing agents both separately and in combination, including proteinase K, proteinase K in combination with SDS, lysozyme and proteinase K, lysozyme in combination with SDS, ionic (SDS) or non-ionic detergents (TritonX-100), NaOH with pH HCl neutralization, NaOH in combination with SDS, often following deproteinization by phenol and chloroform, precipitation of NA from aqueous phase by 2 or 2.5 ethanol aliquots over NaCl or potassium acetate, and by redissolving precipitated NA in distilled water [56, 63-69]. Generally, most techniques for NA isolation intended for their high quality purification are used in research practice, nevertheless, they possess a number of disadvantages for PCR diagnosis as they are multi-stage, labor and time consuming, and require expensive reagents (enzymes and detergents) and toxic organic solvents (phenol or chloroform).
Isolation of NA of the causal agent from diagnostic material increases the vulnerability of the PCR due to very low levels of Brucella concentration in specimen, the risk of NA-matrix losses increases, particularly when many processing straps are used, and/or a deproteinization step is used. These factors may reduce the sensitivity of the PCR diagnosis rather than false-positive results, which can be avoided by good laboratory practices.
Moreover, additional difficulties in case of molecular diagnosis of infectious diseases (using PCR) may arise when extraction of NA must be done with microorganisms having a cell wall, especially those having resistance to chemical and physical factors. It is known that Brucella organisms, in contrast to other Gram-negative bacteria, have an outer membrane of their cell wall with a strong interaction between its components (proteins and lipids), NA, and with lipopolysaccaride and peptidoglican. Brucella cell wall structures features conditions, which limits its lysis during DNA isolation. Some researchers have faced this problem when searching optimal procedures of rapid extraction of DNA from Brucella intended for PCR diagnosis.
Commercial test-systems for PCR diagnosis of brucellosis are genus-specific and do not make possible the differentiation of the species, biovars and Brucella strains. Taking this into consideration, the project also envisages improvement of diagnostic test-system for detection of brucellosis agent by the PCR using primers based on IS6501 nucleotide sequence [77-79]. This test-system might provide differentiation between species and biovars.
During the implementation of the project, it is expected:
- to develop techniques for preparing Brucella matrix DNA obtained from bison urine and feces, for PCR-diagnosis;
- to improve performance of DNA amplification using commercial test-systems for Brucella specific diagnosis;
- to develop diagnostic test-systems to differentiate species and biovars by the PCR;
- to obtain experimental data on detection of Brucella from GYA bison urine and fecal samples using PCR, traditional bacteriological culture and serological methods.
The team of the project scientists includes researchers and experts from the RCT&HRB and ARVI (Kazan).
The RCT&HRB researchers have extensive expertise in the area of this project, being authors of research on adjuvants, isolation and purification of antigens, cytokines, and other immunomodulators, as well as in study of immunoregulating activity of preparations in vitro and in vivo, and development of novel and improvement of existing vaccines and methods for infective agent detection for the needs of public health protection and veterinary.
The ARVI is a large scientific center at the Veterinary Department of the Ministry of Agriculture of the Russian Federation. Comparative research studies on the immunobiological properties of the various brucellosis vaccines will be conducted by highly qualified specialists who have extensive scientific and practical experience in controlling brucellosis in farm animals, including the research and development of improved methods of diagnosis and specific prophylaxis for brucellosis.
To fulfil the stages of the proposed research of this project, scientists and experts from the All Russian Research Institute for Controls, Standardization and Certification (ASRCI, Moscow) who are experienced in creating the National Collection of Industrial Brucella Vaccine Strains and testing of new Brucellosis strains and their deposition, as well as testing new brucellosis vaccines and diagnostic reagents, will be committed to this project. The researchers from the ASRCI are the originators of a number of brucellosis vaccines, including the live strain B. abortus 75/79-AB vaccine.
Implementation of the proposed project will contribute a significantly to solution of one of the most urgent social problems related to redirection of the former biological weapon scientists’ efforts to basic and applied investigations within the international scientific society in the field of biology, immunology, and medicine for peaceful purposes.
Foreign collaborators will participate in the project implementation by discussing the work plan and the approaches for its implementation, as well as in exchange of information, in joint symposia and workshops, writing and reviewing scientific publications, providing patent information on the project, and assisting in identifying financial support for the project personnel participating in international meetings.